Vaccine Constructs And Uses Thereof Against Staphylococcus Infections

Abstract

There is provided a fusion construct of formula (I): X-A-linker-B-Z (I) wherein: (1) A and B are identical or different and are independently: (a) a polypeptide comprising a SACOL0029 polypeptide as set forth in any one of the sequences depicted in FIG. 24 (SEQ ID NOs: 5 and 121 to 131), a SACOL264 polypeptide (SEQ ID NO: 185), a SACOL0442 polypeptide as set forth in any one of the sequences depicted in FIG. 22D (SEQ ID NOs: 29 and 82 to 92), a SACOL0718 polypeptide (SEQ ID NO: 186), a SACOL0720 polypeptide as set forth in any one of the sequences depicted in FIGS. 23I-J (SEQ ID NOs: 11 and 109 to 120), a SACOL1353 polypeptide (SEQ ID NO: 187), a SACOL1416 polypeptide (SEQ ID NO: 188), a SACOL1611 polypeptide (SEQ ID NO: 189), a SACOL1867 polypeptide as set forth in any one of the sequences depicted in FIG. 25D (SEQ ID NOs: 152 to 164), a SACOL1912 polypeptide (SEQ ID NO: 43), a SACOL1944 polypeptide (SEQ ID NO: 190), a SACOL2144 polypeptide (SEQ ID NO: 191), a SACOL2365 polypeptide (SEQ ID NO: 192), a SACOL2385 polypeptide (SEQ ID NO: 50) or a SACOL2599 polypeptide (SEQ ID NO: 193), based on the gene nomenclature from the Staphylococcus aureus COL (SACOL) genome set forth in NCBI Reference Sequence NC_002951.2; (b) a polypeptide encoded by a gene from a same operon as a gene encoding the polypeptide of (a); (c) a polypeptide comprising an immunogenic fragment of at least 13 consecutive amino acids of (a) or (b); (d) a polypeptide comprising an amino acid sequence at least 60% identical overall to the sequence of the polypeptide of any one of (a) to (c); or (e) a polypeptide comprising an immunogenic variant comprising at least 13 consecutive amino acids of any one of (a) to (c); (2) the linker is an amino acid sequence of at least one amino acid or is absent; (3) X is absent or is an amino acid sequence of at least one amino acid; and (4) Z is absent or is an amino acid sequence of at least one amino acid. Also provided are compositions and kits comprising the fusion and uses of these fusions, compositions and kits.

Description

CROSS REFERENCE TO RELATED APPLICATIONS

[0001] This patent application is a divisional patent application of U.S. patent application Ser. No. ______, now U.S. patent Ser. No. ______, which a national stage entry of International Patent Application No. PCT/CA2017/051253, filed Oct. 20, 2017, which claims priority to U.S. Provisional Patent Application No. 62/411,120, filed Oct. 21, 2016, the contents of which are herein incorporated in their entirety.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

[0002] N.A.

FIELD OF THE INVENTION

[0003] The present Invention relates to vaccine constructs and uses thereof against Staphylococcus infections. More specifically, the present invention is concerned with vaccine constructs combining antigens and their uses against Staphylococcus infections such as bovine intramammary infections (IMI).

REFERENCE TO SEQUENCE LISTING

[0004] Pursuant to 37 C.F.R. 1.821(c), a sequence listing is submitted herewith as an ASCII compliant text file named SEQUENCE LISTING USP62411120_ST25, created on Oct. 5, 2017 and having a size of 278 kilobytes. The content of the aforementioned file is hereby incorporated by reference in its entirety.

BACKGROUND OF THE INVENTION

[0005] Bovine mastitis is the most frequent and costly disease for dairy producers and Staphylococcus aureus is considered to be the transmittable bacterium that is the most often responsible for the development of the disease (Sears ef al., 2003). Staphylococcal IMIs, which may lead to mastitis, are difficult to treat and frequent relapses are common (Sandholm ef al., 1990).

[0006] The development of vaccines for the prevention and control of S. aureus IMIs has been extensively investigated although no formulation has demonstrated protective efficacy to date. This is probably because of inadequate vaccine targets (Middleton, 2008; Middleton 2009), high diversity among strains capable of provoking mastitis (Buzzola, 2007; Kerro-Dego, 2006; Middleton, 2008) or the failure to elicit an appropriate immune response (Bharathan, 2011; Ferens, 2000; Fowler, 2014; Proctor, 2012). It is increasingly understood that immunity solely based on vaccine-induced antibodies may be important but is however insufficient for inducing protection against S. aureus (Middleton 2008; Middleton 2009). It appears that cell mediated immunity (CMI) based on Th1 and Th17 type responses may be necessary to complete the protection (Fowler, 2014: Lin, 2009; Proctor, 2012: Spellberg, 2012).

[0007] Bacterial susceptibility to antibiotics in vitro is a poor predictor of therapeutic efficacy in chronically infected cows (Owens ef al., 1997). Although infections that follow treatment of mastitis can be due to newly acquired strains, they are often the result of the persistence of the original infective organism (Sandholm ef al., 1990; Myllys ef al., 1997). Existing therapies thus often fail to eliminate the infection and it would be highly desirable to find novel approaches to prevent or treat staphylococcal IMI.

[0008] A lack of vaccine efficacy and protective ability has been noted for commercially available S. aureus vaccines (Middleton, 2008). Thus, it would be highly desirable to use highly efficient S. aureus antigens that are known to be expressed during IMI as vaccine components for protection against IMI and mastitis.

[0009] The present invention seeks to meet these and other needs.

[0010] The present description refers to a number of documents, the content of which is herein incorporated by reference in their entirety.

SUMMARY OF THE INVENTION

[0011] In a first aspect, the present invention provides fusion polypeptides displaying increased immunogenicity and their use as vaccine against staphylococcal IMI.

[0012] A characteristic of staphylococcal such as S. aureus IMI is the ability of S. aureus to persist within host cells. In particular. S. aureus small colony variants (SCVs) do not generally generate invasive infections and can be internalized in host cells. In a further aspect therefore, the present invention provides live-attenuated S. aureus strains for vaccine purposes based on the phenotypic aspects of SCVs to provide an immune response against such strains and increase the vaccine protective efficacy.

[0013] In an aspect, the present invention provides the following items:

[0014] Item 1: A fusion construct of formula (I): X-A-linker-B-Z (I) wherein: (1) A and B are identical or different and are independently: (a) a polypeptide comprising a SACOL0029 polypeptide as set forth in any one of the sequences depicted in FIG. 24 (SEQ ID NOs: 5 and 121 to 131), a SACOL0264 polypeptide (SEQ ID NO: 185), a SACOL0442 polypeptide as set forth in any one of the sequences depicted in FIG. 22D (SEQ ID NOs: 29 and 82 to 92), a SACOL0718 polypeptide (SEQ ID NO: 186), a SACOL0720 polypeptide as set forth in any one of the sequences depicted in FIGS. 23I-K (SEQ ID NOs: 11 and 109 to 120), a SACOL1353 polypeptide (SEQ ID NO: 187), a SACOL1416 polypeptide (SEQ ID NO: 188), a SACOL1611 polypeptide (SEQ ID NO: 189), a SACOL1867 polypeptide as set forth in any one of the sequences depicted in FIG. 25D (SEQ ID NOs: 152 to 164), a SACOL1912 polypeptide (SEQ ID NO: 43), a SACOL1944 polypeptide (SEQ ID NO: 190), a SACOL2144 polypeptide (SEQ ID NO: 191), a SACOL2365 polypeptide (SEQ ID NO: 192), a SACOL2385 polypeptide (SEQ ID NO: 50) or a SACOL2599 polypeptide (SEQ ID NO: 193), based on the gene nomenclature from the Staphylococcus aureus COL (SACOL) genome set forth in NCBI Reference Sequence NC_002951.2; (b) a polypeptide encoded by a gene from a same operon as a gene encoding the polypeptide of (a); (c) a polypeptide comprising an immunogenic fragment of at least 13 consecutive amino acids of (a) or (b); (d) a polypeptide comprising an amino acid sequence at least 60% identical overall to the sequence of the polypeptide of any one of (a) to (c); or (e) a polypeptide comprising an immunogenic variant comprising at least 13 consecutive amino acids of any one of (a) to (c); (2) the linker is an amino acid sequence of at least one amino acid or is absent; (3) X is absent or is an amino acid sequence of at least one amino acid; and (4) Z is absent or is an amino acid sequence of at least one amino acid.

[0015] Item 2: The construct of item 1, wherein (1) (a) is a polypeptide comprising a SACOL0029 polypeptide as set forth in any one of the sequences depicted in FIG. 24 (SEQ ID NOs: 5 and 121 to 131), a SACOL0442 polypeptide as set forth in any one of the sequences depicted in FIG. 22D (SEQ ID NOs: 29 and 82 to 92), a SACOL0720 polypeptide as set forth in any one of the sequences depicted in FIGS. 231-K (SEQ ID NOs: 11 and 109 to 120), or a SACOL1867 polypeptide as set forth in any one of the sequences depicted in FIG. 25D (SEQ ID NOs: 152 to 164).

[0016] Item 3: The construct of item 2, wherein at least one of A and B is (a) a polypeptide comprising a SACOL0029 polypeptide as set forth in any one of the sequences depicted in FIG. 24 (SEQ ID NOs: 5 and 121 to 131); (b) a polypeptide encoded by a gene from a same operon as a gene encoding the polypeptide of (a); (c) a polypeptide comprising an immunogenic fragment of at least 13 consecutive amino acids of (a) or (b); (d) a polypeptide comprising an amino acid sequence at least 60% identical overall to the sequence of the polypeptide of any one of (a) to (c); or (e) a polypeptide comprising an immunogenic variant comprising at least 13 consecutive amino acids of any one of (a) to (d); and the other one of A and B is (a') a polypeptide comprising a SACOL1867 polypeptide as set forth in any one of the sequences depicted in FIG. 25D (SEQ ID NOs: 152 to 164); (b') a polypeptide encoded by a gene from a same operon as a gene encoding the polypeptide of (a'); (c') a polypeptide comprising an immunogenic fragment of at least 13 consecutive amino acids of (a') or (b'); (d') a polypeptide comprising an amino acid sequence at least 60% identical overall to the sequence of the polypeptide of any one of (a') to (c'); or (e') a polypeptide comprising an immunogenic variant comprising at least 12 consecutive amino acids of any one of (a') to (d').

[0017] Item 4: The construct of item 2, wherein at least one of A and B is (a) a polypeptide comprising a SACOL0442 polypeptide as set forth in any one of the sequences depicted in FIG. 22D (SEQ ID NOs: 29 and 82 to 92); (b) a polypeptide encoded by a gene from a same operon as a gene encoding the polypeptide of (a); (c) a polypeptide comprising an immunogenic fragment of at least 13 consecutive amino acids of (a) or (b); (d) a polypeptide comprising an amino acid sequence at least 60% identical overall to the sequence of the polypeptide of any one of (a) to (c); or (e) a polypeptide comprising an immunogenic variant comprising at least 13 consecutive amino acids of any one of (a) to (d); and the other one of A and B is (a') a polypeptide comprising a SACOL0720 polypeptide as set forth in any one of the sequences depicted in FIGS. 23I-K (SEQ ID NOs: 11 and 109 to 120); (b') a polypeptide encoded by a gene from a same operon as a gene encoding the polypeptide of (a'); (c') a polypeptide comprising an immunogenic fragment of at least 13 consecutive amino acids of (a') or (b'); (d') a polypeptide comprising an amino acid sequence at least 60% identical overall to the sequence of the polypeptide of any one of (a') to (c'); or (e') a polypeptide comprising an immunogenic variant comprising at least 12 consecutive amino acids of any one of (a') to (d').

[0018] Item 5: The construct of item 2, wherein A and B are identical or different and are (a) a polypeptide comprising a SACOL0720 polypeptide as set forth in any one of the sequences depicted in FIGS. 23I-K (SEQ ID NOs: 11 and 109 to 120); (b) a polypeptide encoded by a gene from a same operon as a gene encoding the polypeptide of (a); (c) a polypeptide comprising an immunogenic fragment of at least 13 consecutive amino acids of (a) or (b); (d) a polypeptide comprising an amino acid sequence at least 60% identical overall to the sequence of the polypeptide of any one of (a) to (c); or (e) a polypeptide comprising an immunogenic variant comprising at least 13 consecutive amino acids of any one of (a) to (d).

[0019] Item 6: The construct of any one of items 1-2 and 4, wherein said immunogenic fragment (d) comprises one or more of the following amino acid sequences:

TABLE-US-00001 (SEQ ID NO: 34) KDTINGKSNKSRNW; and (SEQ ID NO: 1) KDGGKYTLESHKELQ

[0020] Item 7: The construct of item 6, wherein said immunogenic fragment (d) comprises one or more of the following amino acid sequences:

TABLE-US-00002 (SEQ ID NO: 30) STQNSSSVQDKQLQKVEEVPNNSEKALVKKLYDRYSKDTINGKSNKSRNWVYSERPLN ENWRIHLEGTYTVAGRVYTPKRNITLNKEWTLKELDHIIRFAHISYGLYMGEHLPKGNI VINTKDGGKYTLESHKELQKDRENVKINTADIK NVTFKLVKSVNDIEQV; (SEQ ID NO: 32) DKQLQKVEEVPNNSEKALVKKLYDRYSKDTINGKSNKSRNWWSERPLNENQVRIHLEG TYTVAGRVYTPKRNIT LNKEWTLKELDHIIRFAHISYGLYMGEHLPKGNIVINTK; and (SEQ ID NO: 33) DKQLQKVEEVPNNSEKALVKKLYDRYSKDTINGKSNKSRNWVYSERPLNENQVRIHLE GTYTVAGRVYTPKRNITLNKEWTLKELDHIIRFAHISYGLYMGEHLPKGNIVINTKDGGK YTLESHKELQKDRENVKINTADIKNVTFKLVKSV NDIEQV.

[0021] Item 8: The construct of any one of items 1-2 and 4-5, wherein said immunogenic fragment (d) comprises one or more of the following amino acid sequences:

TABLE-US-00003 (SEQ ID NO: 21) QFGFDLKHKKDALA; (SEQ ID NO: 22) TIKDQQKANQLAS; (SEQ ID NO: 23) KDINKIYFMTDVDL; and (SEQ ID NO: 24) DVDLGGPTFVLND.

[0022] Item 9: The construct of item 8, wherein said immunogenic fragment (d) comprises one or more of the following amino acid sequences:

TABLE-US-00004 (SEQ ID NO: 12) RASLSSEIKYTAPHDVTIKDQQKANQLASELNNQKIPHFYNYKEVIHTKLYKDNLFDVK AKEPYNVTITSDKYIPNTD LKRGQADLFVAEGSIKDLVKHKKHGKAIIGTKKHHVNIKLRKDINKIYFMTDVDLGGPT FVLNDKDYQEIRKYTKAK HIVSQFGFDLKHKKDALALEKAKNKVDKSIETRSEAISSISSLTG; (SEQ ID NO: 13) ASLSSEIKYTAPHDVTIKDQQKANQLASELNNQKIPHFYNYKEVIHTKLYKDNLFDVKA KEPYNVTITSDKYIPNTDL KRGQADLFVAEGSIKDLVKHKKHGKAIIGTKKHHVNIKLRKDINKINKIYFMTDVDLGGPTF VLNDKDYQEIRKYTKAKHI VSQFGFDLKHKKDALALEKAKNKVDKSIETRSEAISSISSLTG; (SEQ ID NO: 14) ASLSSEIKYTAPHDVTIKDQQKANQLASELNNQKIPHFYNYKEVIHTKLYKDNLFDVKA KEPYNVTITSDKYIPNTDL KRGQADLFVAEGSIKDLVKHKKHGKAIIGTKKHHVNIKLRKDINKIYFMTDVDLGGPTF VLNDKDYQE; (SEQ ID NO: 15) KDINKIYFMTDVDLGGPTFVLNDKDYQEIRKYTKAKHIVSQFGFDLKHKKDALA; (SEQ ID NO: 16) KDINKIYFMTDVDLGGPTFVLNDKD; (SEQ ID NO: 17) KDINKIYFMTDVDLGGPTFVLNDKDY; (SEQ ID NO: 19) KD INKIYFMTDVDLGGPTFVLN D; (SEQ ID NO: 18) SQFGFDLKHKKDALA; and (SEQ ID NO: 20) KHIVSQFGFDLKHKKDALA.

[0023] Item 10: The construct of any one of items 1-3, wherein said immunogenic fragment (cl) comprises one or more of the following amino acid sequences:

TABLE-US-00005 (SEQ ID NO: 165) PYNGWSFKDATGF; (SEQ ID NO: 167) AHPNGDKGNGGIYK; (SEQ ID NO: 169) SISDYPGDEDISVM; (SEQ ID NO: 172) RGPKGFNFNENVQA; (SEQ ID NO: 175) QFESTGTIKRIKDN; and (SEQ ID NO: 178) GNSGSPVLNSNNEV.

[0024] Item 11: The construct of item 10, wherein said immunogenic fragment (d) comprises the following amino acid sequence:

TABLE-US-00006 (SEQ ID NO: 39) TQVKDTNIFPYNGWSFKDATGFVIGKNTIITNKHVSKDYKVGDRITAHPNGDKGNGGIY KIKSISDYPGDEDISVM NIEEQAVERGPKGFNFNENVQAFNFAKDAKVDDKIKVIGYPLPAQNSFKQFESTGTIKRI KDNILNFDAYIEPGNSG SPVLNSNNEVIGWYGGIGKIGSEYNGAVYFTPQIKDFIQKHIEQ.

[0025] Item 12: The construct of any one of items 1 to 11, wherein the linker comprises at least four identical or different amino acids selected from the group consisting of glycine, serine, alanine, aspartate, glutamate and lysine.

[0026] Item 13: The construct of any one of items 1 to 12, wherein the linker comprises (GGGGS)n (SEQ ID NO: 67), (ERKYK)n (SEQ ID NO: 61); or (EAAAK)n (SEQ ID NO: 63), wherein n=1 to 5.

[0027] Item 14: The construct of any one of items 1 to 13, wherein said X comprises a polyhistidine of 6 to 10 amino acids.

[0028] Item 15: The construct of any one of items 1 to 13, wherein said X is absent.

[0029] Item 16: The construct of any one of items 1 to 15, wherein said Z is absent.

[0030] Item 17: An isolated nucleic acid molecule encoding the construct defined in any one of items 1 to 16.

[0031] Item 18: A vector comprising the isolated nucleic acid defined in item 17.

[0032] Item 19: A host cell comprising the vector defined in item 18.

[0033] Item 20: The cell of item 19, which is a live attenuated form of Staphylococcus aureus.

[0034] Item 21: The cell of item 20, wherein the live attenuated form of Staphylococcus aureus has a stabilized small colony variant (SCV) phenotype.

[0035] Item 22: The cell of item 21, wherein the live attenuated form of Staphylococcus aureus having a stabilized SCV phenotype is a .DELTA.hemB.DELTA.720 S. aureus.

[0036] Item 23: A composition comprising: (A) at least one of the constructs defined in any one of items 1 to 16; at least one of the nucleic acid molecules defined in item 17; at least one of the vectors defined in item 18; or at least one of the cells defined in any one of items 19 to 22; and (B) (i) the polypeptide defined in any one of items 1 to 11; (ii) a live attenuated Staphylococcus aureus; (iii) a pharmaceutically acceptable excipient; (iv) an adjuvant; or (v) a combination of at least two of (i) to (iv).

[0037] Item 24: The composition of item 23, wherein the live attenuated form of Staphylococcus aureus expresses: (a) a polypeptide comprising a SACOL0029 polypeptide as set forth in any one of the sequences depicted in FIG. 24 (SEQ ID NOs: 5 and 121 to 131), a SACOL0264 polypeptide (SEQ ID NO: 185), a SACOL0442 polypeptide as set forth in any one of the sequences depicted in FIG. 22D (SEQ ID NOs: 29 and 82 to 92), a SACOL0718 polypeptide (SEQ ID NO: 186), a SACOL0720 polypeptide as set forth in any one of the sequences depicted in FIGS. 231-K (SEQ ID NOs: 11 and 109 to 120), a SACOL1353 polypeptide (SEQ ID NO: 187), a SACOL1416 polypeptide (SEQ ID NO: 188). SACOL1611 (SEQ ID NO: 189), a SACOL1867 polypeptide as set forth in any one of the sequences depicted in FIG. 25D (SEQ ID NOs: 152 to 164), a SACOL1912 polypeptide (SEQ ID NO: 43). SACOL1944 (SEQ ID NO: 190), a SACOL2144 polypeptide (SEQ ID NO: 191), a SACOL2365 polypeptide (SEQ ID NO: 192), a SACOL2385 polypeptide (SEQ ID NO: 50) or a SACOL2599 polypeptide (SEQ ID NO: 193) based on the gene nomenclature from the Staphylococcus aureus COL (SACOL) genome set forth in NCBI Reference Sequence NC_002951.2; (b) a polypeptide encoded by a gene from a same operon as a gene encoding the polypeptide of (a); (c) a polypeptide comprising an immunogenic fragment of at least 13 consecutive amino acids of (a) or (b); (d) a polypeptide comprising an amino acid sequence at least 60% identical overall to the sequence of the polypeptide of any one of (a) to (c); or (e) a polypeptide comprising an immunogenic variant comprising at least 13 consecutive amino acids of any one of (a) to (c).

[0038] Item 25: The composition of item 23 or 24, wherein the live attenuated form of Staphylococcus aureus has a stabilized small colony variant (SCV) phenotype.

[0039] Item 26: The composition of any one of items 23 to 25, wherein the adjuvant comprises alum, an oil (e.g., emulsified oil, mineral oil), saponin (e.g., Quil-A.TM.), cyclic-diguanosine-5'-monophosphate (c-di-GMP), polyphosphasine, indolicidin, pathogen-associated molecular patterns (PAMPS) or a combination of at least two thereof.

[0040] Item 27: A method for preventing and/or treating a Staphylococcal intramammary infection (IMI) in a mammal, said method comprising administrating to said mammal an effective amount of the construct defined in any one of items 1 to 16: of the nucleic acid molecule defined in item 17; of the vector defined in item 18; of the cell defined in any one of items 19 to 22; or of the composition defined in any one of items 23 to 26.

[0041] Item 28: The method of item 27, wherein said Staphylococcal IMI is caused by one or more Staphylococcus aureus strains.

[0042] Item 29: The method of item 27 or 28, wherein said mammal is a cow.

[0043] Item 30: A use of an effective amount of (i) the construct defined in any one of items 1 to 16; (ii) the nucleic acid molecule defined in item 17; (iii) the vector defined in item 18; of the cell defined in any one of items 19 to 22; (iv) the composition defined in any one of items 23 to 26; or (v) a combination of at least two of (i) to (iv), for preventing and/or treating a Staphylococcal intramammary infection (IMI) in a mammal.

[0044] Item 31: The use of item 30, wherein said Staphylococcal IMI is caused by one or more Staphylococcus aureus strains.

[0045] Item 32: The use of item 30 or 31, wherein said mammal is a cow.

[0046] Item 33: The construct defined in any one of items 1 to 16; the nucleic acid molecule defined in item 17; the vector defined in item 18; the cell defined in any one of items 19 to 22; the composition defined in any one of items 23 to 26 or a combination of at least two thereof, for use in the prevention and/or treatment of a Staphylococcal intramammary infection (IMI) in a mammal.

[0047] Item 34: The construct, nucleic acid molecule, vector, cell, composition or combination of item 33, wherein said Staphylococcal IMI is caused by one or more Staphylococcus aureus strains.

[0048] Item 35: The construct, nucleic acid molecule, vector, cell or composition of item 33 or 34, wherein said mammal Is a cow.

[0049] Item 36: A kit for preventing and/or treating a Staphylococcal intramammary infection (IMI) in a mammal comprising: (A) (i) at least one of the constructs defined in any one of items 1 to 16; (ii) at least one of the nucleic acid molecules defined in item 17; (iii) at least one of the vectors defined in items 18; (iv) at least one of the cells defined in any one of items 19 to 22; or (v) a combination of at least two of (i) to (iv), and (B) (i) the polypeptide defined in any one of items 1 to 11; (ii) a live attenuated Staphylococcus aureus; (iii) a pharmaceutically acceptable excipient; (iv) an adjuvant; (v) instructions for using the kit for preventing and/or treating a Staphylococcal intramammary infection (IMI) in a mammal; or (vi) a combination of at least two of (i) to (v).

BRIEF DESCRIPTION OF THE DRAWINGS

[0050] In the appended drawings:

[0051] FIGS. 1A-D, shows serum total IgG (FIG. 1A), lgG1 (FIG. 1B), and lgG2 (FIG. 1C) titers for the vaccinated (9) and placebo (0.10) cows for each antigen of the vaccine, namely SACOL0029, SACOL0442. SACOL0720, SACOL1867, SACOL1912, and SACOL2385, four weeks after the second immunization (just before the experimental infection). In FIG. 1D, the lgG2/lgG1 ratio is represented for the vaccinated cows. In FIGS. 1A, B and C, open circles (o) represent data for the vaccinated cows, black squares (.box-solid.) represent data for the placebo cows. Each symbol represents the titer for one cow. Horizontal lines represent the medians: dashed lines represent the medians for the vaccinated cows while continuous lines represent the medians for the placebo cows. In FIGS. 1A, B and C, titers for the vaccinated cows are higher than the titers for the placebo cows (P<0.0001). In FIG. 1 D, symbols represent the ratio lgG2/lgG1 for each cow. Horizontal lines represent the medians. The different letters show statistical differences. ***. P<0.001.

[0052] FIG. 2, shows antigen dependent proliferation of blood CD4+ cells from the vaccinated cows (9) and placebo cows (10) four weeks after the second immunization for each antigen. Each symbol represents the percentage of CD4+ cells that have proliferated for each cow after a week of incubation with the positive control (ConA) or each antigen, namely SACOL0029, SACOL0442, SACOL0720, SACOL1867. SACOL1912, and SACOL2385. Open circles (0.0) represent data for the vaccinated cows, black squares (.box-solid.) represent data for the placebo cows. Horizontal lines represent the medians: dashed lines represent the medians for the vaccinated cows while continuous lines represent the medians for the placebo cows. Statistical analysis: Mixed procedure of SAS. The symbol * shows the statistical differences between the vaccinated and the placebo groups for antigens SACOL0029. SACOL0442. SACOL0720 and SACOL1912 (*, P<0.05). In addition, the proliferation of CD8+ cells was similar for the vaccinated and placebo cows for all antigens with the exception of the antigen SACOL0720 for which higher proliferation of the CD8+ cells was observed for the vaccinated cows (data not shown).

[0053] FIG. 3, shows experimental S. aureus intramammary infections in dairy cows. Four weeks and 4 days after the second immunization, 63 Colony Forming Unit (CFU) of S. aureus were infused into 3 of the 4 quarters of the vaccinated (9) and placebo cows (10) at the evening milking (day 1, arrow in FIG. 3). Aseptic milk samples were taken at morning milking and Somatic Cell Counts (SCC) were determined by Valacta (Ste-Anne-de-Bellevue, QC). Open circles (o) and the dashed line represent data for the vaccinated cows, while the black squares (.box-solid.) and the continuous line represent data for the placebo cows. Each open circle represents the mean of SCC for all the infected quarters of the vaccinated cows (27) while each square represents the mean of SCC for all the infected quarters of the placebo cows (30 quarters). Over the challenge period, somatic cell counts in milk were found to be significantly lower for the vaccinated cows than for the placebo cows (***: PO.001).

[0054] FIGS. 4A-C. FIG. 4A shows the correlation between CFU and the SCC for each cow, and FIG. 4B shows the correlation between serum IgG1 titer against SACOL0442 and SCC for each cow. Each symbol represents data for one cow. In FIG. 4A, it represents the mean of SCC and CFU for the 3 infected quarters from the beginning to the end of the infection. In FIG. 4B, it represents the mean of SCC for the 3 infected quarters from the beginning to the end of the infection and the serum lgG1 titer against SACOL0442 four weeks after the second immunization and just before the experimental infection. Open circles represent data for the vaccinated cows and black squares represent data for the placebo cows. There is a strong correlation between the SCC/ml and the CFU/ml and a negative correlation between the SCC and the lgG1 titer against SACOL0442. In FIG. 4C, the correlation between SCC or CFU relative to milk lgG2 titer against SACOL0029 is shown for each cow ten days after experimental infection (6 weeks after the second immunization). Each symbol represents data for one cow ten days after the experimental infection. Aseptic milk samples were taken at morning milking and the viable counts of S. aureus were determined by 10-fold dilutions and plating on tryptic soy agar (TSA) plates while SCC were determined by Valacta (Ste-Anne-de-Bellevue, QC). SCC and CFU data for each cow is the mean of the data for the 3 infected quarters ten days after the experimental infection. Milk samples for the determination of milk lgG2 titers are the mix of an equivalent volume of milk from the 4 quarters of each cow 10 days after the experimental infection (6 weeks after the second immunization). Black squares (.box-solid.) represent data for the placebo cows, open circles (o) represent data for the vaccinated cows.

[0055] FIG. 5, shows serum total IgG titers for the vaccinated cows for each antigen of a vaccine comprising the fused antigens SACOL0029 and SACOL1867 (shown as SACOL0029-1867) and the antigens SACOL0442 and SACOL0720. In the ELISA, the targeted antigens were SACOL0029-1867, SACOL0029, SACOL1867, SACOL0442 and SACOL0720. Each open circle represents the titer four weeks after the second immunization for each of the 11 cows whereas each black diamond represents the preimmune titer. Horizontal lines represent the medians: solid line for the preimmune serums, dotted line for the samples taken four weeks after immunization. Titers for the vaccinated cows are higher than the titers of the preimmune serums (**, P<0.01; ***, P<0.001 for the other antigens tested).

[0056] FIG. 6 shows serum total IgG titers against the SACOL1867 antigen of mice immunised with the fusion protein (SACOL0029-1867; fusion), a combination of the separate proteins (SACOL0029+SACOL1867; combination), the SACOL0029 protein only (0029) or SACOL1867 protein only (1867), in equivalent molar quantities. Open circles (o ) represent data for preimmune titers, black squares (.box-solid.) represent data for the immune titers. For the preimmune titers, preimmune sera were mixed equally between the 5 mice of each immunization group to obtain a preimmune pool titer, represented by one open circle per group. For the immune titers, each square symbol represents the titer for one mouse. Horizontal lines represent the medians: black lines represent the medians for the immune serums while dashed lines (and the open circle) represent the medians for the preimmune serums pool. Titers for the vaccinated mice in the fusion, combination and 1867 groups are higher than the titers for the preimmune mice (P<0.001), and the titers of the mice that received SACOL0029 monovalent antigen only were not found to be significantly different from the titers of the preimmune pool against SACOL1867. Statistical significance between immune titers of fusion group versus the combination and the two monovalent vaccines groups is shown (***: P<0.001).

[0057] FIG. 7. Serum total IgG (as measured by O.D. 450 nm in the ELISA assay) directed against a B-cell epitope sequence KDGGKYTLESHKELQ (SEQ ID NO: 1) contained in a fragment of the amino acid sequence of SACOL0442 (GEHLPKGNIVINTKDGGKYTLESHKELQKDRENVKINTAD. SEQ ID NO: 2), and obtained from mice immunized with either a fusion of peptides encoded from SACOL0442 and SACOL0720 KDGGKYTLESHKELQEAAAKEAAAKKDINKIYFMTDVDLGGPTFVLND (SEQ ID NO: 3) (Group 1) or the peptide KDGGKYTLESHKELQ (SEQ ID NO: 1) encoded from SACOL0442 (Group 2). Each group was composed of 4 animals (n=4) that were injected two times with equimolar amounts of the sequence KDGGKYTLESHKELQ (SEQ ID NO: 1) (corresponding to 100 .mu.g of KDGGKYTLESHKELQEAAAKEAAAKKDINKIYFMTDVDLGGPTFVLND (SEQ ID NO: 3) for Group 1 and 31.25 .mu.g of KDGGKYTLESHKELQ (SEQ ID NO: 1) for Group 2) at a 2-week interval, and sera were prepared from blood harvested one week after the last injection. The ELISA assay was carried out with serum samples diluted 100 000 times and a fragment from SACOL0442 (GEHLPKGNIVINTKDGGKYTLESHKELQKDRENVKINTAD, (SEQ ID NO: 2)) was used as the target antigen containing the peptide epitope KDGGKYTLESHKELQ (SEQ ID NO: 1). Individual data are expressed as circles on the graph and the medians by bars. The difference between groups was found statistically significant (P<0.0286, Kuskal-Wallis test. GraphPad Prism.TM. 7.00).

[0058] FIGS. 8A-B. Deletion of hemB in ATCC29213 and .DELTA.720 strains of Staphylococcus aureus. (FIG. 8A) The hemB gene in the wild-type (WT) strain ATCC29213 and its isogenic mutant .DELTA.720 was deleted by homologous recombination and replacement with an ermA cassette to create the mutant strains .DELTA.hemB and .DELTA.720.DELTA.hemB, respectively. Thick lines and numbers denote the PCR-amplified regions depicted in B for parental (1) and hemB deleted (2) strains. (FIG. 8B) PCR products of the WT strain and its isogenic .DELTA.hemB mutant (similar results were obtained with .DELTA.720 and .DELTA.720.DELTA.hemB strains).

[0059] FIGS. 9A-C, show influence of S. aureus .DELTA.hemB, .DELTA.720, and .DELTA.hemB.DELTA.720 mutations on MAC-T cell infectivity. MAC-T cells were infected with each of the four strains for 3 h, then were incubated with lysostaphin an additional 30 min (t=3 h), 12 h or 24 h and lysed for measurement of viable intracellular bacteria (CFU). (FIG. 9A) Relative recovery of the initial inoculum found within cells at t 3 h for .DELTA.720, and (FIG. 9B) for .DELTA.hemB.DELTA.720 mutants. Results are normalized according to that obtained for ATCC 29213 (WT) or .DELTA.hemB, respectively, and are expressed as means with SD (**, P.ltoreq.0.01; ***, P.ltoreq.0.001; unpaired f test). (FIG. 9C) Means and SD of intracellular CFUs for WT and mutants at 12 h (left) and 24 h (right). A two-way ANOVA and Tukey's multiple comparisons test was used (*: P<0.05; ***: P<0.001). All values indicate the mean of three independent experiments, each performed in triplicate.

[0060] FIG. 10, shows persistence of S. aureus ATCC 29213 (WT) and isogenic mutants within MAC-T cells over time. MAC-T cells were infected with each of the four strains for 3 h, then were incubated with lysostaphin an additional 30 min. 12 h or 24 h and lysed for measurement of intracellular bacteria (CFU). Intracellular bacterial CFUs are expressed as the percentage of the initial inoculum after being transformed in base 10 logarithmic values (Log 10 CFU/ml). Values indicate the mean of three independent triplicate experiments with standard deviations.

[0061] FIGS. 11A-B, shows viability of MAC-T cells infected by S. aureus ATCC 29213 (WT) and isogenic mutants. MAC-T cells were infected with each of the four strains for 3 h, then were incubated with lysostaphin for 12 h (FIG. 11 A) or 24 h (FIG. 11 B). MTT viability assays were then performed with a method described in Kubica et al., 2008. The results are reported as percent viability relative to uninfected cells and are expressed as the mean with SD of three independent experiments done in triplicate. Statistical significance with (0) symbol are compared to the WT (Two-way ANOVA and Tukey's multiple comparisons test: * or .PHI.: P<0.05; **: P<0.01: ***: P<0.001; .PHI..PHI..PHI..PHI.: P<0.0001).

[0062] FIG. 12. Shows murine IMIs with the parental (WT) and .DELTA.hemB.DELTA.720 (.DELTA..DELTA.) strains. Mice were infected as previously described and glands harvested at the indicated hour (h) or day (D) after infection. Each column represents the median value of bacterial CFU counts for a group of glands, and ranges are indicated by bars. A minimum of six glands per group were used excepted for the WT strain at D7 (2 glands: only one mouse survived). Mortality of mice at specific time points is indicated by arrows. The asterisk indicates the clearance of .DELTA.hemB.DELTA.720) from glands (below the detection limit of 10 CFU/gland).

[0063] FIG. 13. Double mutant (.DELTA.720.DELTA.hemB) stimulates neutrophil influx in mammary glands to similar levels compared to WT in the first 24 hours following infection. Mice were infected as described in materials and methods, and a control group (PBS) of mice received a sterile PBS injection. Glands were harvested at indicated times, homogenized and kinetically assayed for MPO activity as described in materials and methods. Each dot represents MPO Units for one gland, which is shown as raw values adjusted by gram of gland. Means are represented by thick lines.

[0064] FIG. 14. Visual Inflammation of the large R4 and L4 mammary glands 24 h after mouse IMI with S. aureus ATCC 29213 (WT) and the double mutant .DELTA.720.DELTA.hemB (.DELTA..DELTA.). Mice were infected as described in materials and methods, and control group (PBS) mice received a sterile PBS injection. Pictures show glands that were harvested after 24 h. In each panel, the R4 (left) and L4 (right) glands are shown.

[0065] FIG. 15A. Neutrophil infiltration goes back to normal levels after clearance of the double mutant .DELTA.720.DELTA.hemB. Mice were infected as described in materials and methods, and a control group (PBS) of mice received a sterile PBS injection. Glands were harvested at the indicated times, homogenized and kinetically assayed for MPO activity as described in materials and methods. Columns represent means of MPO Units of a group of 6 glands (4 for the PBS control) adjusted by gram of gland, and error bars illustrate standard deviation. Statistical significance between the Day 4 and 12 groups post infection is shown by (.PHI.) symbol. One-Way ANOVA and Tukey's multiple comparison tests were used (.PHI..PHI.: P<0.01; NS: No significant difference between groups).

[0066] FIGS. 15B-C. Immunization of mice with the live-attenuated double mutant (.DELTA.720.DELTA.hemB) stimulates a strong humoral response against S. aureus bovine mastitis isolates of commonly found spa types. Mice were immunized as previously described: serums were collected before priming immunization (Preimmune) and ten days after the boost immunization (Immune). FIG. 15B. IgG titers rise with increasing doses of the live-attenuated strain .DELTA.720G.DELTA.hemB each dot represents the total IgG titer of one mouse against a .DELTA.720GA/iem8 whole cell extract. Medians are represented by thick lines for Immune titers and dashed lines for Preimmune titers. Titers were compared to their corresponding preimmune titers (Two-way ANOVA and Tukey's multiple comparisons test: ****: P<0.0001). FIG. 15C. Immunization with the live-attenuated mutant M20GLhemB confers IgG titers against components that are shared by mastitis strains of commonly found spa types. Each dot represents the total IgG titer of one mouse against the whole cell extract of the indicated strain. Medians are represented by thick lines for Immune titers and dashed lines for Preimmune titers. All immune titers were compared to their corresponding preimmune titer (P<0.0001) and between clinical strains (Two-way ANOVA and Sidak's multiple comparisons test: NS: no significant difference).

[0067] FIG. 16, shows total serum IgG titers against SACOL0029-1867 fusion protein of mice immunised with the protein mix (composed of 5 g of the antigens SACOL0029, SACOL0442, SACOL0720, and the SACOL0029-1867 fusion), 10 CFU of the attenuated live strain .DELTA.720.DELTA.hemB alone or a combination of the protein mix and the .DELTA.720.DELTA.hemB strain. Open circles (o) represent data for preimmune titers, black squares (.box-solid.) represent data for the immune titers. Each symbol represents the titer for one mouse. Horizontal lines represent the medians: black lines represent the medians for the immune serums while dashed lines represent the medians for the preimmune serums. Titers for the vaccinated mice in the protein mix group and combination group are higher than the titers for the preimmune mice (P<0.001). Statistical significance between immune titers of combination versus the two other vaccinated mice groups is shown (**: P<0.01).

[0068] FIG. 17, shows total serum IgG titers against SACOL0029 of mice immunised with the protein mix (composed of 5 Mg of the antigens SACOL0029, SACOL0442, SACOL0720, and the SACOL0029-1867 fusion), 105 CFU of the attenuated live strain .DELTA.720.DELTA.hemB alone or a combination of the protein mix and the .DELTA.720.DELTA.hemB strain. Open circles (o) represent data for preimmune titers, black squares (.box-solid.) represent data for the immune titers. Each symbol represents the total IgG titer for one mouse. Horizontal lines represent the medians: black lines represent the medians for the immune serums while dashed lines represent the medians for the preimmune serums. Statistical significance between immune and preimmune titers of the three mice groups is shown (**: P<0.01).

[0069] FIG. 18, shows total serum IgG titers against the staphylococcal surface protein ClfA for mice immunised with the SACOL0029, SACOL0442, SACOL0720, and SACOL0029-1867 protein mix, 10.sup.5 CFU of the attenuated live strain .DELTA.720.DELTA..LAMBDA..beta..pi.7.beta. alone or a combination of the protein mix and Lll b emB. Open circles (o) represent data for preimmune titers, black squares (.box-solid.) represent data for the immune titers. Each symbol represents the titer for one mouse. Horizontal lines represent the medians: black lines represent the medians for the immune serums while dashed lines represent the medians for the preimmune serums. Statistical significance between preimmune titers and immune titers is shown (*: P<0.05).

[0070] FIG. 19, below shows serum ratio of lgG2a/lgG1 titers against the SACOL0029-1867 fusion polypeptide for mice immunised with the protein mix, or the combination of the protein mix and the attenuated N2QNiemB live strain.

[0071] FIG. 20, shows serum ratios against the SACOL0029 antigen. Open squares (.box-solid.) represent data for preimmune titers, black squares (.box-solid.) represent data for the immune titers. Each symbol represents the titer ratio for one mouse. Horizontal lines represent the medians. Statistical significance between the protein mix group versus combination group ratios is shown (*: P<0.05; **: P<0.01).

[0072] FIGS. 21A-J. I. SACOL0029 polynucleotides (full length sequence SEQ ID NO: 4) and polypeptides (full length, fragment(s) and variant(s) sequences SEQ ID NOs: 5 to 9). Selected epitopes are shown shaded and/or bolded; II. SACOL0720 polynucleotides (full length sequence SEQ ID NO: 10) and polypeptides (full length, fragments and variant(s) sequences SEQ ID NOs: 11 to 27). Selected epitopes are shown shaded; Ill. SACOL0442 polynucleotides (full length sequence SEQ ID NO: 28) and polypeptides (full length, fragments and variant(s) sequences SEQ ID NOs: 29 to 36 and 1). Selected epitopes are shown shaded; IV. SACOL1867 polynucleotides (full length sequence SEQ ID NO: 37) and polypeptides (full length, fragment(s) and variant(s) sequences SEQ ID NOs: 38 to 41). Selected epitopes are shown shaded. Predicted transmembrane (enzim.hu/hmmtop/html/submit.html) domain shown bolded; V. SACOL1912 polynucleotide (full length sequence SEQ ID NO: 42) and polypeptides (full length and variant(s) sequences SEQ ID NOs: 43 to 44). Selected epitopes are shown shaded (see e.g., SEQ ID NOs: 45-48); VI. SACOL2385 polynucleotide (full length SEQ ID NO: 49) and polypeptides (full length and variant(s) sequences SEQ ID NOs: 50 to 51). Selected epitopes are shown shaded (see e.g., SEQ ID NOs: 52-53); VII. Fusions: (i) SACOL0029-1867 fusion polynucleotide sequences (SEQ ID NOs: 54 and 56) and polypeptide sequences (SEQ ID NOs: 55, 57-58). In the polynucleotide and polypeptide sequences, the double underlined sequence, if any, is that of the polyhistidine, the italicized sequence is the sequence of the SACOL0029 fragment, the single underlined sequence is the sequence of the linker and the bolded sequence is the sequence of the SACOL1867 fragment; (ii) SACOL0720-720 fusion polypeptide sequence (SEQ ID NO: 27 In the polypeptide sequence, the double underlined sequence, if any, is that of the polyhistidine, the italicized sequences are the sequences of the SACOL0720 fragments and the single underlined sequence is the sequence of the linker; (iii) SACOL0442-720 fusion polypeptide sequence (SEQ ID NO: 3). In the polynucleotide and polypeptide sequences, the double underlined sequence, if any, is that of the polyhistidine, the italicized sequence is the sequence of the SACOL0442 fragment, the single underlined sequence is the sequence of the linker and the bolded sequence is the sequence of the SACOL0720 fragment; and VIII. Sequences of linkers (SEQ ID NOs: 59 to 70)

[0073] FIGS. 22A-D I. Multiple polynucleotide sequences (SEQ ID NOs: 71-72, 28, 73 to 81) alignment for full length SACOL0442 and orthologues; II. Multiple polypeptide sequences (SEQ ID NOs: 29 and 82 to 92) alignment for full length SACOL0442, orthologues and consensus sequences derived therefrom are presented. In these sequences, "*" denotes that the residues in that column are identical in all sequences of the alignment, ":" denotes that conserved substitutions have been observed, and "." denotes that semi-conserved substitutions have been observed. Consensus sequences derived from these alignments are also presented wherein X is any amino acid. In the polypeptide sequences, selected epitopes are shown shaded (see e.g., SEQ ID NOs: 1, 34, 93-97).

[0074] FIGS. 23A-K I. Multiple polynucleotide sequences (SEQ ID NOs: 98 to 104, 10, and 105 to 108) alignment for full length SACOL0720 and orthologues; H. Multiple polypeptide sequences (SEQ ID NOs: 11 and 109 to 120) alignment for full length SACOL0720, orthologues, and consensus sequences derived therefrom are presented. In these sequences, "*" denotes that the residues in that column are identical in all sequences of the alignment, ":" denotes that conserved substitutions have been observed, and "." denotes that semi-conserved substitutions have been observed. Consensus sequences derived from these alignments are also presented wherein X is any amino acid. In the polypeptide sequences, selected epitopes are shown shaded (see e.g., SEQ ID NOs: 22, 19 and 21).

[0075] FIG. 24 Multiple polypeptide sequences (SEQ ID NOs: 5 and 121 to 131) alignment for full length SACOL0029, orthologues, and consensus sequences derived therefrom are presented. In these sequences, "*" denotes that the residues in that column are identical in all sequences of the alignment, ":" denotes that conserved substitutions have been observed, and "." denotes that semi-conserved substitutions have been observed. Consensus sequences derived from these alignments are also presented wherein X is any amino acid. In the polypeptide sequences, selected epitopes are shown shaded (see e.g., SEQ ID NOs: 132-139). Bolded epitope identified by BCPred.TM.. Shaded epitopes identified by AAp predictions.

[0076] FIGS. 25A-D. I-Multiple polynucleotide sequences (SEQ ID NOs: 140 to 151) alignment for full length SACOL1867 and orthologues; II-Multiple polypeptide sequences (SEQ ID NOs: 152 to 164) alignment for full length SACOL1867, orthologues, and consensus sequences derived therefrom are presented. In these sequences, "*" denotes that the residues in that column are identical in all sequences of the alignment. ":" denotes that conserved substitutions have been observed, and "." denotes that semi-conserved substitutions have been observed. Consensus sequences derived from these alignments are also presented wherein X is any amino acid. In the polypeptide sequences, selected epitopes are shown shaded (see e.g., SEQ ID NOs: 165-180) and the end of the signal peptide domain and/or transmembrane domain is marked with a line (separates signal peptide and/or transmembrane domain from secreted form).

[0077] FIG. 26. 1--polynucleotide sequence (SEQ ID NO: 181) for full length SACOL1715 (hemB); and 11-amino acid sequence (SEQ ID NO: 182) for full length SACOL1715 (hemB).

[0078] FIG. 27. 1--polynucleotide sequence (SEQ ID NO: 183) for full length ClfA (NWMN_0756, newman); and 11-amino acid sequence (SEQ ID NO: 184) for full length ClfA.

DESCRIPTION OF ILLUSTRATIVE EMBODIMENTS

[0079] The present invention showed that a fusion of two antigens created an unexpected synergy in the immune response.

[0080] In addition, the present invention also stabilized the SCV phenotype of a Staphylococcus via hemB

(complete deletion thus impairing the possibility of reversion to an invasive phenotype (Tuchscherr, 2011)) enabling its use as a vaccine delivery system. HemB is coding for the HemB protein/monomer, which combines to create the porphobilinogen synthase or aminolevulinate dehydratase enzyme [EC 4.2.1.24]. In addition, further attenuation was brought about by inactivation of an antigen of the present invention, namely gene SACOL0720, which has been previously shown to be important for S. aureus in cationic peptide resistance (Falord, 2012; Kawada-Matsuo, 2011; Meehl, 2007) and in vivo during IMI (Allard, 2013). Hence, this attenuated double mutant strain expressing constructs of the present invention is usable for immunization and protection against IMIs.

General Definitions

[0081] Headings, and other identifiers, e.g., (a), (b). (i). (ii), etc., are presented merely for ease of reading the specification and claims. The use of headings or other identifiers in the specification or claims does not necessarily require the steps or elements be performed in alphabetical or numerical order or the order in which they are presented.

[0082] In the present description, a number of terms are extensively utilized. In order to provide a clear and consistent understanding of the specification and claims, including the scope to be given such terms, the following definitions are provided.

[0083] The use of the word "a" or "an" when used in conjunction with the term "comprising" in the claims and/or the specification may mean "one" but it is also consistent with the meaning of "one or more", "at least one", and "one or more than one".

[0084] Throughout this application, the term "about" is used to indicate that a value includes the standard deviation of error for the device or method being employed to determine the value. In general, the terminology "about" is meant to designate a possible variation of up to 10%. Therefore, a variation of 1, 2, 3, 4, 5, 6, 7, 8, 9 and 10% of a value is included in the term "about". Unless indicated otherwise, use of the term "about" before a range applies to both ends of the range.

[0085] As used in this specification and claim(s), the words "comprising" (and any form of comprising, such as "comprise" and "comprises"). "having" (and any form of having, such as "have" and "has"), "including" (and any form of including, such as "includes" and "include") or "containing" (and any form of containing, such as "contains" and "contain") are inclusive or open-ended and do not exclude additional, un-recited elements or method steps.

[0086] As used herein, the term "consists of" or "consisting of" means including only the elements, steps, or ingredients specifically recited in the particular claimed embodiment or claim.

Polypeptides, Nucleic Acids and Delivery Systems

[0087] As used herein, the term "vaccine" refers to any compound/agent ("vaccine component"), or combinations thereof, capable of inducing/eliciting an immune response in a host and which permits to treat and/or prevent an infection and/or a disease. Therefore, non-limiting examples of such agent include proteins, polypeptides, protein/polypeptide fragments, immunogens, antigens, peptide epitopes, epitopes, mixtures of proteins, peptides or epitopes as well as nucleic acids, genes or portions of genes (encoding a polypeptide or protein of interest or a fragment thereof) added separately or in a contiguous sequence such as in nucleic acid vaccines, and the like.

[0088] In an aspect of the present invention, there is provided a fusion construct of formula I:

X-A-linker-B-Z (formula (I),

[0089] Wherein A and B are identical or different and are each independently an antigenic polypeptide (i.e. native, fragment or variant thereof) of the present invention.

[0090] In a specific embodiment, A and/or B is (a) a polypeptide comprising a SACOL0029 polypeptide as set forth in any one of the sequences depicted in FIG. 24 (SEQ ID NOs: 5 and 121 to 131), a SACOL0264 polypeptide (SEQ ID NO: 185), a SACOL0442 polypeptide as set forth in any one of the sequences depicted in FIG. 22D (SEQ ID NOs: 29 and 82 to 92), a SACOL0718 polypeptide (SEQ ID NO: 186), a SACOL0720 polypeptide as set forth in any one of the sequences depicted in FIGS. 231-J (SEQ ID NOs: 11 and 109 to 120), a SACOL1353 polypeptide (SEQ ID NO: 187), a SACOL1416 polypeptide (SEQ ID NO: 188), SACOL1611 (SEQ ID NO: 189), a SACOL1867 polypeptide as set forth in any one of the sequences depicted in FIG. 25D (SEQ ID NOs: 152 to 164), a SACOL1912 polypeptide as set forth in FIG. 21G-V (SEQ ID NO: 43), SACOL1944 (SEQ ID NO: 190), a SACOL2144 polypeptide (SEQ ID NO: 191), a SACOL2365 polypeptide (SEQ ID NO: 192), a SACOL2385 polypeptide as set forth in VI on FIG. 21H (SEQ ID NO: 50) or a SACOL2599 polypeptide (SEQ ID NO: 193). In a specific embodiment, the above polypeptide (a) is the secreted or extracellular fragment of the polypeptide defined above. Transmembrane domains can be predicted using, for example, the software TMpredrM (ExPASy) ch.embnet.org/software/TMPRED_form.html, psort.org/psortb/index.html enzim.hu/hmmtop/html/submit.html and/or Sign/P 4.1 (cbs.dtu.dk/services/SignalP). TMpred.TM. and Signal/P 4.1 predicted extracellular domain for: SACOL0720: AA 310-508; SACOL0442 AA 36 to 203. Enzim predicted a transmembrane domain SACOL1867 (1-40) so that extracellular domain was: AA 41-239 while psort.org/psortb/index.html predicted that SACOL1867 was an extracellular protein. Since the above-mentioned transmembrane and/or signal peptide domains are putative, the present invention encompasses cases where the antigens presented herein (e.g., SACOL1867) have or not a signal peptide and/or transmembrane domain and encompasses the corresponding extracellular fragments. In an embodiment, the above-mentioned polypeptide is a polypeptide normally secreted or expressed at the surface of the bacteria (e.g., Staphylococcus aureus).

[0091] The Genbank.TM. accession numbers for S. aureus genes listed herein and their encoded antigenic polypeptides encompassed by the present invention are depicted in Table I below:

TABLE-US-00007 TABLE I Genbank .TM. accession numbers for the IMI-associated S. aureus genes and encoded polypeptides described herein Gene name Gene ID No. GenBank .TM. protein No. SACOL0029 3236748 YP_184940.1 (SEQ ID NO: 5) SACOL0100 3236858 YP_185004.1 SACOL0101 3236840 YP_185005.1 SACOL0105 3236844 YP_185009.1 SACOL0148 3236734 YP_185048.1 SACOL0154 3238707 YP_185054.1 SACOL0204 3236774 YP_185103.1 SACOL0205 3236775 YP_185104.1 SACOL0264 3236683 YP_185159.1 WP_000570071 (SEQ ID NO: 185) SACOL0442 3236485 YP_185332.1 (SEQ ID NO: 29) SACOL0461 3236475 YPJ85351.1 SACOL0608 3236353 YP_185493.1 SACOL0660 3238251 YP_185544.1 SACOL0688 3236721 YP_185570.1 SACOL0690 3236723 YP_185572.1 SACOL0704 3236241 YP_185586.1 SACOL0718 3236599 YP_185600.1 WP_000985996 (SEQ ID NO: 186) SACOL0720 3236600 YPJ85601.1 (SEQ ID NO: 11) SACOL0829 3238649 YP_185703.1 SACOL1054 3236163 YPJ85919.1 SACOL1142 3236098 YP_186005.1 SACOL1145 3237661 YP_186008.1 SACOL1320 3236394 YP_186175.1 SACOL1353 3236077 YP_186206.1 WP_000603968 (SEQ ID NO: 187) SACOL1416 3236563 YP_186268.1 WP_000548932 (SEQ ID NO: 188) SACOL1611 3236575 YPJ86451.1 WP_001095260 (SEQ ID NO: 189) SACOL1637 3238018 YP_186477.1 SACOL1680 3238476 YP_186520.1 SACOL1781 3236594 YPJ86614.1 SACOL1812 3238705 YP_186645.1 SACOL1867 3236101 YP_186695.1 (SEQ ID NO: 38) SACOL1912 3236086 YP_186737.1 (SEQ ID NO: 43) SACOL1944 3237515 YP_186769.1 WP_000149064 (SEQ ID NO: 190) SACOL2092 3238693 YP_186907.1 SACOL2144 3237436 YP_186957.1 WP_000908177 (SEQ ID NO: 191) SACOL2169 3237416 YPJ86981.1 SACOL2171 3237418 YP_186983.1 SACOL2321 3238070 YP_187128.1 SACOL2325 3238483 YP_187132.1 SACOL2342 3235997 YP_187148.1 SACOL2365 3238203 YP_187170.1 WP_000827000 (SEQ ID NO: 192) SACOL2379 3237628 YP_187183.1 SACOL2385 3238646 YP_187189.1 (SEQ ID NO: 50) SACOL2599 3237186 YP_187390.1 AAW38600 (SEQ ID NO: 193)

[0092] Consensuses derived from the alignments of certain the above listed polypeptides are presented in FIGS. 21-25. In specific embodiment of these consensuses, each X in the consensus sequences (e.g., consensuses in FIGS. 21-25) is defined as being any amino acid, or absent when this position is absent in one or more of the orthologues presented in the alignment. In specific embodiment of these consensuses, each X in the consensus sequences is defined as being any amino acid that constitutes a conserved or semi-conserved substitution of any of the amino acid in the corresponding position in the orthologues presented in the alignment, or absent when this position is absent in one or more of the orthologues presented in the alignment. In FIGS. 21-25, conservative substitutions are denoted by the symbol ":" and semi-conservative substitutions are denoted by the symbol In another embodiment, each X refers to any amino acid belonging to the same class as any of the amino acid residues in the corresponding position in the orthologues presented in the alignment, or absent when this position is absent in one or more of the orthologues presented in the alignment. In another embodiment, each X refers to any amino acid in the corresponding position of the orthologues presented in the alignment, or absent when this position is absent in one or more of the orthologues presented in the alignment. In a specific embodiment, A and/or B is a polypeptide satisfying any one of these consensuses or a fragment thereof.

[0093] Conservative amino acid mutation may include addition, deletion, or substitution of an amino acid; a conservative amino acid substitution is defined herein as the substitution of an amino acid residue for another amino acid residue with similar chemical properties (e.g., size, charge, or polarity). Such a conservative amino acid substitution may be a basic, neutral, hydrophobic, or acidic amino acid for another of the same group (see e.g., Table II below). By the term "basic amino acid" it is meant hydrophilic amino acids having a side chain pK value of greater than 7, which are typically positively charged at physiological pH. Basic amino acids include histidine (His or H), arginine (Arg or R), and lysine (Lys or K). By the term "neutral amino acid" (also "polar amino acid"), it is meant hydrophilic amino acids having a side chain that is uncharged at physiological pH, but which has at least one bond in which the pair of electrons shared in common by two atoms is held more closely by one of the atoms. Polar amino acids include serine (Ser or S), threonine (Thr or T), cysteine (Cys or C), tyrosine (Tyr or Y), asparagine (Asn or N), and glutamine (Gin or Q). The term "hydrophobic amino acid" (also "non-polar amino acid") is meant to include amino acids exhibiting a hydrophobicity of greater than zero according to the normalized consensus hydrophobicity scale of Eisenberg (1984). Hydrophobic amino acids include proline (Pro or P), isoleucine (He or I), phenylalanine (Phe or F), valine (Val or V), leucine (Leu or L), tryptophan (Trp or W), methionine (Met or M), alanine (Ala or A), and glycine (Gly or G). "Acidic amino acid" refers to hydrophilic amino acids having a side chain pK value of less than 7, which are typically negatively charged at physiological pH. Acidic amino acids include glutamate (Glu or E), and aspartate (Asp or D).

[0094] A semi-conserved amino acid replaces one residue with another one that has similar steric conformation, but does not share chemical properties. Examples of semi-conservative substitutions would include substituting cysteine for alanine or leucine; substituting serine for asparagine; substituting valine for threonine; or substituting proline for alanine.

[0095] The Table II below indicates which amino acid belongs to each amino acid class.

TABLE-US-00008 Class Name of the amino acids Aliphatic Glycine, Alanine, Valine, Leucine, Isoleucine Hydroxyl or Sulfur/ Serine, Cysteine, Selenocysteine, Threonine, Selenium-containing Methionine Cyclic Proline Aromatic Phenylalanine, Tyrosine, Tryptophan Basic Histidine, Lysine, Arginine Acidic and their Amide Aspartate, Glutamate, Asparagine, Glutamine

[0096] The similarity and identity between amino acid or nucleotide sequences can be determined by comparing each position in the aligned sequences. Optimal alignment of sequences for comparisons of similarity and/or identity may be conducted using a variety of algorithms, for example using a multiple sequence alignment program/software well known in the art such as ClustalW.TM., SAGA.TM., UGENE.TM. or T-coffee.TM.. Examples of multiple sequence alignments are described in the examples below and depicted in FIGS. 21 A to 25.

Gene Operon

[0097] In another embodiment, A and/or B is (b) a polypeptide encoded by a gene from a same operon as a gene encoding the polypeptide of (a) as defined above. For example, SACOL0718 is a gene from the same operon as SACOL0720.

Fragment

[0098] In another embodiment, A and/or B Is (c) a polypeptide comprising an Immunogenic fragment of at least 13 consecutive amino acids of (a) or (b) as defined above.

[0099] An Immunogenic fragment of a protein/polypeptide Is defined as a part of a protein/polypeptide which is capable of inducing/eliciting an immune response in a host. In an embodiment, the immunogenic fragment is capable of eliciting the same immune response in kind, albeit not necessarily in amount, as the protein/polypeptide. An immunogenic fragment of a protein/polypeptide preferably comprises one or more epitopes of said protein/polypeptide. An epitope of a protein/polypeptide is defined as a fragment of said protein/polypeptide of at least about 4 or 5 amino acids in length, capable of eliciting a specific antibody and/or an immune cell (e.g., a T cell or B cell) bearing a receptor capable of specifically binding said epitope. Two different kinds of epitopes exist: linear epitopes and conformational epitopes. A linear epitope comprises a stretch of consecutive amino acids. A conformational epitope is typically formed by several stretches of consecutive amino acids that are folded in position and together form an epitope in a properly folded protein. An immunogenic fragment as used herein refers to either one, or both, of said types of epitopes. In an embodiment where immunogenic fragments are used alone (i.e. not fused in a larger polypeptide construct (e.g., fusion with other antigenic fragment)), the immunogenic fragment of a protein/polypeptide comprises at least 16 amino acid residues. In a further embodiment, the immunogenic fragment comprises at least 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, or 160 consecutive amino acids of the native protein/polypeptide. In a specific embodiment, the fragment has at least 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, or 50 or more consecutive amino acids of the native protein/polypeptide. In an embodiment where the at least one immunogenic fragment forms part of a larger polypeptide construct (e.g., fusion with other antigenic polypeptide, fragment or variant thereof), the immunogenic fragment comprises at least 13 consecutive amino acid residues of the polypeptide. Without being so limited, fragments encompassed by the present invention comprise immunogenic fragments of at least 13 consecutive amino acids of SACOL029 as shown in FIG. 21 (and corresponding fragments in SACOL029 orthologues (e.g., depicted in FIG. 24); of SACOL0442 as shown in FIG. 21 (and corresponding fragments in SACOL0442 orthologues (e.g., depicted in FIG. 22); of SACOL0720 as shown in FIG. 21, (and corresponding fragments in SACOL0720 orthologues (e.g., depicted in FIG. 23); and of SACOL1867 as shown in FIG. 21 (and corresponding fragments in SACOL1867 orthologues (e.g., depicted in FIG. 25). In another embodiment, fragments encompassed by the present invention include immunogenic fragments comprising at least one epitope of antigenic proteins/polypeptides of the present invention (polypeptide (a) defined above). In another embodiment, fragments encompassed by the present invention include immunogenic fragments comprising at least one epitope as depicted (shaded) in any one of the antigenic proteins/polypeptides depicted in any one of FIGS. 21 to 25. Without being so limited, epitopes in a sequence may be predicted with softwares such as BCPred.TM., AAP.TM., FBCPred.TM. and ABCPred.TM..

[0100] In an embodiment, the above-mentioned immunogenic fragment comprises a sequence that is conserved (i.e. identical) in at least two different strains of Staphylococcus aureus. In further embodiments, the above-mentioned immunogenic fragment comprises a sequence that is conserved (i.e. identical) in at least 3, 4, 5, 6, 7, 8, 9 or 10 different strains of Staphylococcus aureus. In another embodiment, the above-mentioned strains of Staphylococcus aureus are COL, RF122, NCTC 8325, JH1, JH9, Newman, Mu3, Mu50, USA300-FPR3757, N315, MW2 or MSSA476. In an embodiment, the above-mentioned strains of Staphylococcus aureus are associated with bovine mastitis (e.g., RF122).

Variants

[0101] In another embodiment, the above-mentioned polypeptide, or a polypeptide substantially identical to said polypeptide, is expressed in at least two different strains of Staphylococcus aureus. Substantially identical as used herein refers to polypeptides having at least 60% of identity, in embodiments at least 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% of identity in their amino acid sequences. In further embodiments, the polypeptides have at least 60%, 65%, 70%, 71%, 72%, 73%, 74%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% of identity in their amino acid sequences with other polypeptides to which they are compared.

[0102] In another embodiment. A and/or B is (d) a polypeptide comprising an amino acid sequence at least 60% identical overall to the sequence of the polypeptide of any one of (a) to (c) defined above. In other embodiments, the amino acid is at least 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to (a) (e.g., over their full length). In further embodiments, the amino acid is at least 60%, 65%, 70%, 71%, 72%, 73%, 74%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% to (a). For example, antigens orthologues presented in alignments of FIGS. 21-25 are not identical but present a certain identity with the antigens or fragments to which they are compared. Consensuses presented in these FIGs embody such percent identities.

[0103] In another embodiment, A and/or B is (e) a polypeptide comprising an immunogenic variant comprising at least 13 consecutive amino acids of any one of (a) to (d). An immunogenic variant of a protein/polypeptide is defined as a part of a protein/polypeptide which is capable of inducing/eliciting an immune response in a host. As will be understood by the person of ordinary skill, agents (proteins/polypeptides, fragments thereof) having non-naturally occurring modifications (e.g., immunogenic variants) and which are capable of inducing an immune response specific for the unmodified agent (e.g., capable of inducing the production of antibodies capable of recognizing the unmodified agent) are also within the scope of the term "vaccine component". For example, the vaccine components of the present invention can be modified to enhance their activity, stability, and/or bioavailability, and/or to reduce their toxicity. Conservative amino acid substitutions may be made, like for example replacement of an amino acid comprising an acidic side chain by another amino acid comprising an acidic side chain, replacement of a bulky amino acid by another bulky amino acid, replacement of an amino acid comprising a basic side chain by another amino acid comprising a basic side chain, and the like. A person skilled in the art is well able to generate variants of a protein/polypeptide. This is for instance done through screening of a peptide library or by peptide changing programs. An immunogenic variant according to the invention has essentially the same immunogenic properties of said protein in kind, not necessarily in amount. An immunogenic variant of a protein/polypeptide of the invention may for instance comprise a fusion protein and/or chimeric protein. For example, the biological function of a protein identified herein predicted to be an exotoxin, enterotoxin or superantigen (e.g., SACOL0442) could potentially interfere with the mammalian immune system and antibody production, and/or show some toxicity in the host. Although such interference was not observed when the SACOL0442 polypeptide was used in combination with for example SACOL0720 during immunization, it may be useful to modify the protein or polypeptide used for vaccination so that the biological activity of the exotoxin is decreased. For such a purpose, it is possible to inactivate the exotoxin with chemicals (e.g., formaldehyde). It is also possible to use molecular biology techniques to delete or mutate the putative region(s) involved in exotoxin activity without losing immunogenicity (Chang ef a/., 2008). Another example is the conjugation or mixture of amino acid-based components with nucleic acids (e.g., genes or portions of genes added separately or in a contiguous sequence) carbohydrates such as those found in microbial polysaccharide capsules or biofilms. Other examples of variants include antigens described herein or fragments thereof comprising at either of their N or C terminus or inserted within their antigen sequence, an oligopeptide useful for purification (e.g., affinity purification) or useful as a spacer or linker. Examples of oligopeptides useful for affinity purification include polyhistidine tags (e.g., 6-10 histidine residues including or not RGS tags (e.g. HHHHHH, RGSHHHHHH, or RGSHHHHHGS). The his-tag may also be followed by an amino acid sequence suitable to facilitate a removal of the polyhistidine-tag using endopeptidases. The "X" and/or "Z" segments as recited in formula (I) also may comprise such oligopeptide useful for purification and/or sequence suitable to facilitate removal of such oligopeptide useful for purification.

[0104] In specific embodiments, the immunogenic fragment comprises at least one epitope of the polypeptide (a). Without being so limited, in certain embodiments, the immunogenic fragment comprises at least one epitope of the polypeptide (a) as depicted (shaded) in the sequences presented in FIGS. 21-25, In other specific embodiments, the variants are as disclosed in FIGS. 21-25.

Linker

[0105] Insertion of linkers between fusion protein domains can increase bioactivity by augmenting distance between domains alleviating potential repulsive forces between different segments (e.g., antigenic fragments) of the construct resulting in improved and/or restored protein folding. Different sequences of polypeptide linkers can be used and arm known to have distinct properties, such as flexible, rigid or cleavable linkers. The present invention encompasses the use of any such linkers including any one of those listed in Chen et, al. Adv Drug Deliv Rev. (2013), 65(10):1357-69 for example. Examples herein provide illustrations of specific linkers that were used (i.e. GGGGSGGGGSGGGGS (SEQ ID NO: 60), ERKYK (SEQ ID NO: 61), or and EAAAKEAAAK (SEQ ID NO: 62)), i.e. flexible linker structures, rich in small hydrophilic amino acids that maintain distance between the two connected domains and improve their folding.

[0106] In another specific embodiment, the Fc comprises a CH2 domain, a CH3 domain and a hinge region. In another specific embodiment, the Fc is a constant domain of an immunoglobulin selected from the group consisting of lgG-1, lgG-2, lgG-3, lgG-3 and lgG-4. In another specific embodiment, the Fe is a constant domain of an immunoglobulin lgG-1.

[0107] Linkers may be included between contiguous antigens of the fusion (e.g., 1 linker in fusion comprising

two antigens, 2 linkers in fusions comprising three antigens, three linkers in fusions comprising four antigens, etc.). In fusions where large protein domains are used, linker may be larger and may comprise a fragment crystallizable region (Fc).

[0108] In a specific embodiment, the linker is an amino acid sequence of at least one amino acid or is absent. In a specific embodiment, the linker comprises at least three (at least 4, 5, 6, 7, 8, 9 or 10) amino acids selected from the group consisting of glycine, serine, alanine, aspartate, glutamate and lysine. In a specific embodiment, the linker is (EAAAK)n (SEQ ID NO: 63); (GGGGS)n (SEQ ID NO: 67); or (XPXPXP)n (SEQ ID NO: 69) wherein x is any amino acid; wherein n is any one of 1 to 5, more specifically 1, 2, 3.4 or 5; EAAAKEAAAK (SEQ ID NO: 62); EAAAKEAAAKEAAAK (SEQ ID NO: 64); GGGGS (SEQ ID NO: 67): GGGGSGGGGS (SEQ ID NO: 68); GGGGSGGGGSGGGGS (SEQ ID NO: 60); XPXPXP (SEQ ID NO: 69), wherein x is any amino acid; XPXPXPXPXPXP (SEQ ID NO: 70), wherein x is any amino acid; ERKYK (SEQ ID NO: 61); ERKYKERKYK (SEQ ID NO: 65); ERKYKERKYKERKYK (SEQ ID NO: 66). In a more specific embodiment, the linker is GGGGSGGGGSGGGGS (SEQ ID NO: 60), ERKYK (SEQ ID NO: 61), or EAAAKEAAAK (SEQ ID NO: 62).

N and C Terminal of Construct

[0109] X and Z are each independently absent or an amino acid sequence of at least one amino acid. Without being so limited, they may be one or more of amino acids resulting from cloning strategy, amino acids used to facilitate purification of the construct (e.g. polyhistidine), amino acids suitable to facilitate a removal of the purification-tag using endopeptidases. In specific embodiments, where the fusion construct comprises three or more antigen polypeptides, any one of X and/or Z may also include the sequence of a further antigen (antigen C, antigen D, etc.) and, optionally that of at least one further linker. Such embodiments wherein X and/or Z comprise one or more further antigen(s) and optionally linker(s), could be more specifically illustrated as e.g., formula (II) or (III) as follows X'-C-linkeri-A-linker2-B-Z' (II) when the fusion comprises at least 3 antigens; or X'-C-linkeri-A-linker2-B-linker3-D-Z' (III) when the fusion comprises at least 4 antigens. In both formula (II) and (III) X', Z', linker{circumflex over ( )} linker, and, the case being, linker, are identical or different and are independently defined as are X, Z and linker in formula (I) defined herein.

[0110] Hence, in specific embodiments, the fusion construct comprises 2, 3, 4 or more antigen polypeptides (and, the case being further linkers). In a more specific embodiment, and without being so limited the fusion construct may be SACOL0029_SACOL0442; SACOL0029_SACOL0720; SACOL0029_SACOL1867; SACOL0029_SACOL0720_SACOL1867; SACOL0029_SACOL1867_SACOL0442; SACOL0029_SACOL0720_SACOL0442; SACOL0442_SACOL0029_SACOL0720; SACOL0442_SACOL0029_SACOL1867; SACOL0442_SACOL1867_SACOL0720; SACOL0720_SACOL0442_SACOL1867; or SACOL0029_SACOL1867_SACOL0720_SACOL0442, or any of the foregoing constructs wherein the antigen polypeptides are in any other order.

Combination

[0111] The constructs of the present invention may be used as sole immunogenic component of a composition (e.g., vaccine) of the present invention or in combination with one or more further fusion construct(s), immunogenic polypeptide(s), fragment(s) or variant(s) thereof and/or live attenuated bacteria (e.g., S. aureus) (expressing or not fusion constructs and/or polypeptide(s), fragment(s) orvariant(s) thereof).

[0112] The one or more fusion constructs may be any immunogenic fusion construct including a further fusion construct as defined above (see e.g., Example 14).

[0113] The one or more immunogenic polypeptide(s), fragment(s) or variant(s) thereof for use in compositions of the present invention may be any polypeptide(s), fragment(s) or variant(s) that contribute to the immunogenicity of the compositions of the present invention as defined herein. Without being so limited, such polypeptide(s), fragment(s) or variants) includes (a) a polypeptide comprising a SACOL0029 polypeptide as set forth in any one of the sequences depicted in FIG. 24 (SEQ ID NOs: 5 and 121 to 131), a SACOL0264 polypeptide (SEQ ID NO: 185), a SACOL0442 polypeptide as set forth in any one of the sequences depicted in FIG. 22D (SEQ ID NOs: 29 and 82 to 92), a SACOL0718 polypeptide (SEQ ID NO: 186), a SACOL0720 polypeptide as set forth in any one of the sequences depicted in FIGS. 231-K (SEQ ID NOs: 11 and 109 to 120), a SACOL1353 polypeptide (SEQ ID NO: 187), a SACOL1416 polypeptide (SEQ ID NO: 188), SACOL1611 (SEQ ID NO: 189), a SACOL1867 polypeptide as set forth in any one of the sequences depicted in FIG. 25D (SEQ ID NOs: 152 to 164), a SACOL1912 polypeptide (SEQ ID NO: 43), a SACOL1944 polypeptide (SEQ ID NO: 190), a SACOL2144 polypeptide (SEQ ID NO: 191), a SACOL2365 polypeptide (SEQ ID NO: 192), a SACOL2385 polypeptide (SEQ ID NO: 50) or a SACOL2599 polypeptide (SEQ ID NO: 193); (b) a polypeptide encoded by a gene from a same operon as a gene encoding the polypeptide of (a); (c) a polypeptide comprising an immunogenic fragment of at least 13 consecutive amino acids of (a) or (b); (d) a polypeptide comprising an amino acid sequence at least 60% identical overall to the sequence of the polypeptide of any one of (a) to (c); or (e) a polypeptide comprising an immunogenic variant comprising at least 13 consecutive amino acids of any one of (a) to (c), as defined above. Without being so limited, any such polypeptide(s), fragment(s) or variant(s) encompasses those included in compositions (e.g., vaccines #1 to #8) exemplified in Examples 1 to 14 and 21-26.

Live Attenuated Bacteria

[0114] The live attenuated bacteria [e.g. S. aureus) for use in compositions of the present invention may be independent from the fusions constructs and/or polypeptide(s), fragment(s) or variant(s) thereof of the present invention, or be the vessel for (i.e. may express) such fusion constructs and/or polypeptide(s), fragment(s) or variants) thereof of the present invention.

[0115] Without being so limited, as illustrated herein, useful live attenuated bacteria in the context of combinations of the present invention include Staphylococcus [e.g., aureus) bacteria having at least one gene contributing to virulence (e.g., .DELTA.720) or contributing to fitness in the host (e.g., a metabolic gene) mutated or deleted. Without being so limited, such gene may be any one of the genes identified in Novick 2003, Novick 2008, or Maresso and Schneewind 2008.

[0116] In a further embodiment, the live attenuated bacteria may be further attenuated by having a stabilized SCV phenotype. As used herein the terms "SCV phenotype" refers to bacteria having a dysfunctional oxidative

metabolism causing a slow growth, an alteration in the expression of virulence factors, and an ability to be internalized in host cells. As used herein the term stabilized SCV phenotype is used to denote an SCV strain retaining the SCV phenotype i.e. unable to produce invasive revertants (i.e., a reversion to the normal growth phenotype). Such stabilized SCV S. aureus may be produced by mutating or deleting any one of the genes (e.g., .DELTA.hemB) listed in Table III below. Without being limited, the present invention encompasses the use of the stabilized SCV S. aureus exemplified in Examples 15 to 25. Mutation as used herein includes a substitution, a deletion and/or an insertion of one or more nucleotides that prevents expression of the polypeptide encoded by a gene of the present invention or that prevents expression of a functional polypeptide. In a preferred embodiment, the mutation prevents expression of the polypeptide. In another specific embodiment, the two mutations in the same attenuated live or inactivated strain of S. aureus are a deletion or an insertion. It is expected that a mutated strain of S. aureus having a mutation at any position of one of the genes of the present invention that prevents expression of the polypeptide can be used as an attenuated live vaccine in accordance with the present invention. Attenuated live vaccines, i.e. vaccines comprising the bacterium according to the invention in a live attenuated form, have the advantage over inactivated vaccines that they best mimic the natural way of infection. In addition, their replicating abilities allow vaccination with low amounts of bacteria; their number will automatically increase until it reaches the trigger level of the immune system. From that moment on, the immune system will be triggered and will finally eliminate the bacteria. A minor disadvantage of the use of live attenuated bacteria however might be that inherently there is a certain level of virulence left. This need not be a real disadvantage as long as the level of virulence is acceptable, i.e. as long as the vaccine at least decreases the bacterial infection (e.g., IMI) symptoms. Of course, the lower the remaining virulence of the live attenuated vaccine is, the less influence the vaccination has on weight gain during/after vaccination.

TABLE-US-00009 TABLE III Genbank .TM. accession numbers for S. aureus genes associated with SCV phenotype (Kahl, 2014) Gene Name GenBank .TM. Gene ID No. GenBank .TM. Protein No. hemB 3238571 (SACOL1715) AAW36820.1 WP_000667126.1 Gl: 446589780 EG4.2.1.24 menB 3236546 (SACOL1052) AAW36517.1 WP_000526687.1 Gl: 446448832 EC: 2.2.1.9 thyA 3238178 (SACOL1462) AAW36663.1 WP_000667126.1 Gl: 446589780 EC.2.1.1.45 fusA 3236183 (SACOL0593) AAW37703.1 Gl: 57285609 FusE (gene: 3238328 (SACOL2224) AAW37099.1 rplF) Gl: 57285005 relA (relA2) 3238211 (SACOL1689) AAW36795.1 Gl: 57284701 EC: 2.7.6.5 cspB 3238398 (SACOI2731) AAW37379.1 Gl: 57285285 hemH 3236274 (SACOI1888) AAW36901.1 Gl: 57284807 EC: 4.99.1.1 ctaA 3237823 (SACOL1124) AAW38004.1 Gl: 57285910

Nucleic Acids

[0117] The nucleic acid of the present invention preferably comprises a nucleotide sequence that encodes one or more proteins/polypeptides noted above (or fragments thereof) operably linked to regulatory elements needed for gene expression, such as a promoter, an initiation codon, a stop codon, enhancers, and a polyadenylation signal. Regulatory elements are preferably selected that are operable in the species to which they are to be administered. In specific embodiments, the nucleic acid is as depicted in FIGS. 21 to 25.

[0118] Within the context of the present invention is the in vivo administration of a nucleic acid of the invention to a mammal so that one or more proteins/polypeptides (or a fragment thereof) of interest is/are expressed in the mammal (e.g., nucleic acid vaccine, DNA or RNA vaccine).

Delivery Systems

[0119] The nucleic acid of the present vaccine can be "naked" DNA or can be operably incorporated in a vector. Nucleic acids may be delivered to cells in vivo using methods well known in the art such as direct injection of DNA, receptor-mediated DNA uptake, viral-mediated transfection or non-viral transfection and lipid-based transfection, all of which may involve the use of vectors. Direct injection has been used to introduce naked DNA into cells in vivo (see e.g., Acsadi ef al. (1991) Nature 332:815-818; Wolff ef al. (1990) Science 247:1465-1468). A delivery apparatus (e.g., a "gene gun") for injecting DNA into cells in vivo may be used. Such an apparatus may be commercially available (e.g., from BioRad). Naked DNA may also be introduced into cells by complexing the DNA to a cation, such as polylysine, which is coupled to a ligand for a cell-surface receptor (see for example Wu, G, and Wu. C. H. (1988) J. Biol. Chem. 263: 14621; Wilson ef al. (1992) J. Biol. Chem. 267: 963-967; and U.S. Pat. No. 5,166,320). Binding of the DNA-ligand complex to the receptor may facilitate uptake of the DNA by receptor-mediated endocytosis. A DNA-ligand complex linked to adenovirus capsids which disrupt endosomes, thereby releasing material into the cytoplasm, may be used to avoid degradation of the complex by intracellular lysosomes (see for example Curiel ef al. (1991) Proc. Natl. Acad. Sci. USA 88: 8850; Cristiano ef al. (1993) Proc. Natl. Acad. Sci. USA 90:2122-2126).

[0120] Useful delivery vectors include biodegradable microcapsules, immuno-stimulating complexes (ISCOMs) or liposomes, and genetically engineered attenuated live vectors such as cells, viruses or bacteria.

[0121] Liposome vectors are unilamellar or multilamellar vesicles, having a membrane portion formed of lipophilic material and an interior aqueous portion. The aqueous portion is used in the present invention to contain the polynucleotide material to be delivered to the target cell. It is generally preferred that the liposome forming materials have a cationic group, such as a quaternary ammonium group, and one or more lipophilic groups, such as saturated or unsaturated alkyl groups having about 6 to about 30 carbon atoms. One group of suitable materials is described in European Patent Publication No. 0187702, and further discussed in U.S. Pat. No. 6,228,844 to Wolff et al., the pertinent disclosures of which are incorporated by reference. Many other suitable liposome-forming cationic lipid compounds are described in the literature. See, e.g., L. Stamatatos, et al., Biochemistry 27:3917 3925 (1988); and H. Eibl, et al., Biophysical Chemistry 10:261 271 (1979). Alternatively, a microsphere such as a polylactide-coglycolide biodegradable microsphere can be utilized. A nucleic acid construct is encapsulated or otherwise complexed with the liposome or microsphere for delivery of the nucleic acid to a tissue, as is known in the art.

[0122] Preferred viral vectors include Bacteriophages, Herpes virus, Adenovirus, Polio virus, Vaccinia virus, defective retroviruses, adeno-associated virus (AAV) and Avipox. Methods of transforming viral vector with an exogenous DNA construct are also well described in the art.

[0123] See Sambrook and Russell, above.

[0124] As indicated above, the nucleic acid (e.g., DNA or RNA) may be incorporated in a host such as a host cell in vitro or ex vivo (e.g., an immune cell such as a dendritic cell) or, as indicated above, in an attenuated microbial host (e.g., attenuated S. aureus, SCV, etc., see e.g., Examples 25-26 for instance) by transfection or transformation, and the transfected or transformed cell or microorganism, which expresses the polypeptide (e.g. fusion of multiple antigens or fragments therefor and/or single antigens or fragments thereof) of interest, may be administered to the subject. Following administration, the cell will express the protein or polypeptide of interest (or a variant or fragment thereof) in the subject, which will in turn lead to the induction of an immune response directed against the protein, polypeptide or fragment thereof.

[0125] The use of attenuated live bacteria to immunize and/or to deliver specific constructs or antigen mixture of the present invention represents an interesting approach to improve immune responses (Griffiths and Khader, 2014). Live attenuated organisms that mimic natural infection stimulate the immune system in a powerful manner, eliciting broad and robust immune responses that produce both serum and mucosal antibodies, and effector and memory T cells which act synergistically to protect against disease (Detmer and Glenting, 2006; Kollaritsch et al, 2000; Pasetti et al., 2011). Examples of suitable attenuated live bacterial vectors include S. aureus, Salmonella typhimurium, Salmonella typhi, Shigella, Bacillus, Lactobacillus, Bacille Calmette-Guerin (BCG), Escherichia coli, Vibrio cholerae, Campylobacter, or any other suitable bacterial vector, as is known in the art. Methods of transforming live bacterial vectors with an exogenous DNA construct are well described in the art. See, for example. Joseph Sambrook and David W. Russell, Molecular Cloning, A Laboratory Manual, 3rd Ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (2001). The present invention encompasses the use of a composition comprising an attenuated live bacterium (e.g., .DELTA.hemB.DELTA.720 S. aureus expressing the construct of the present invention as sole immunogenic component or in combination with other attenuated live bacteria each expressing another polypeptide, fragment or variant of the present invention (e.g., SACOL0442. SACOL0720 or fragments or variants thereof).

Compositions

[0126] The polypeptides, nucleic acids and delivery systems (e.g., host cells comprising said nucleic acids or vectors) described herein can be formulated into compositions. As used herein, the term "pharmaceutically acceptable" refers to vaccine components (e.g., excipients, carriers, adjuvants) and compositions that are physiologically tolerable and do not typically produce an allergic or similar untoward reaction, such as gastric upset, dizziness and the like, when administered to a subject. Preferably, as used herein, the term "pharmaceutically acceptable" means approved by regulatory agency of the federal or state government or listed in the U.S. Pharmacopeia or other generally recognized pharmacopeia for use in animals, and in humans. The term "excipient" refers to a diluent, carrier, or vehicle with which the vaccine components of the present invention may be administered. Sterile water or aqueous saline solutions and aqueous dextrose and glycerol solutions may be employed as carriers, particularly for injectable solutions.

[0127] In an embodiment, the agent of the present invention is administered in combination with an adjuvant or immunostimulant. Suitable adjuvant or immunostimulant that may improve the efficacy of components to raise an immune response include but is not limited to oils (e.g., mineral oils, emulsified oil such as MONTANIDE.TM. or EMULSIGEN.TM.-D), metallic salts (e.g., alum, aluminum hydroxide or aluminum phosphate), cationic peptides (Bowdish et al., 2005; Hancock, et al., 2000) such as indolicidin, a cationic peptide produced by the cow's immune cells (Falla et al., 1996), natural and artificial microbial components (e.g., bacterial liposaccharides. Freund's adjuvants, muramyl dipeptide (MDP), cyclic-diguanosine-5'-monophosphate (c-di-GMP), pathogen-associated molecular patterns (PAMPS) such as surface polysaccharides, lipopolysaccharides, glycans, peptidoglycan or microbial DNA (e.g., CpG), plant components such as saponins (e.g., Quil-A.TM.), and/or one or more substances that have a carrier effect (e.g., bentonite, latex particles, liposomes, ISCOM.TM., DNA and polyphosphazine (PCPP) copolymers). Immunization with synthetic nanoparticles (such as those made from a biodegradable synthetic polymer like poly(D, L-lacticco-glycolic acid)) containing antigens plus ligands that signal through TLR to stimulate proinflammatory cytokines is also possible (Kasturi et al, 2011).

[0128] Vaccine components of the invention may be administered in a pharmaceutical composition. Pharmaceutical compositions may be administered in unit dosage form. Any appropriate route of administration may be employed, for example, parenteral, subcutaneous, intramuscular, intramammary, intracranial, intraorbital, ophthalmic, intraventricular, intracapsular, intraarticular, intraspinal, intracisternal, intraperitoneal, intranasal, aerosol, or oral administration. Examples of specific routes of administration include parenteral, e.g., intravenous, intradermal, subcutaneous, intramammary; oral (e.g., inhalation); transdermal (topical); transmucosal, and rectal administration.

[0129] Conventional pharmaceutical practice may be employed to provide suitable formulations or compositions to administer such vaccine components with or without adjuvants to subjects. Methods well known in the art for making pharmaceutical compositions and formulations arm found in, for example, Remington: The Science and Practice of Pharmacy, (20.sup.rd ed.) ed. A. R. Gennaro A R., 2000, Lippincott: Philadelphia. Formulations for parenteral administration may, for example, contain excipients, sterile water, or saline, polyalkylene glycols such as polyethylene glycol, miglyol, oils of vegetable origin, or hydrogenated napthalenes. Biocompatible, biodegradable lactide polymer, lactide/glycolide copolymer, or polyoxyethylene-polyoxypropylene copolymers may be used to control the release of the compounds. Other potentially useful parenteral delivery systems for compounds of the invention include ethylenevinyl acetate copolymer particles, osmotic pumps, implantable infusion systems, and liposomes. Formulations for inhalation or intramammary injection may contain excipients, for example, lactose, or may be aqueous solutions containing, for example, polyoxyethylene-9-lauryl ether, miglyol, glycocholate and deoxycholate, or may be oily solutions (e.g., paraffin oil) for administration in the form of nasal drops, or as a gel.

[0130] Therapeutic formulations may be in the form of liquid solutions or suspension; for oral administration, formulations may be in the form of tablets or capsules; and for intranasal formulations, in the form of powders, nasal drops, or aerosols. Solutions or suspensions used for parenteral, intradermal, intramammary or subcutaneous application can include the following components: a sterile diluent such as water for injection, saline solution, fixed oils (e.g., paraffin oil), polyethylene glycols, glycerin, propylene glycol, miglyol or other synthetic solvents; antibacterial agents such as benzyl alcohol or methyl parabens; antioxidants such as ascorbic acid or sodium bisulfite; chelating agents such as ethylenediaminetetraacetic acid; reducing agents such dithiothreitol, buffers such as acetates, citrates or phosphates and agents for the adjustment of tonicity such as sodium chloride or dextrose. The pH can be adjusted with acids or bases, such as hydrochloric acid or sodium hydroxide. The parenteral preparation can be enclosed in ampoules, disposable syringes or multiple dose vials made of glass or plastic.

[0131] Pharmaceutical compositions suitable for injectable use include sterile aqueous solutions (where water soluble) or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersion. For intravenous or intramammary administration, suitable carriers include physiological saline, bacteriostatic water, Cremophor.TM. EL.TM. (BASF. Parsippany. N.J.) or phosphate buffered saline (PBS).

[0132] Oral compositions generally Include an Inert diluent or an edible carrier. They can be enclosed In gelatin capsules or compressed Into tablets or feed. For the purpose of oral vaccine administration, the active components can be Incorporated with excipients and used In the form of tablets, troches, capsules or In feed. Pharmaceutically compatible binding agents, and/or adjuvant materials can be Included as part of the composition. The tablets, pills, capsules, troches and the like can contain any of the following Ingredients, or compounds of a similar nature: a binder such as microcrystalline cellulose, gum tragacanth or gelatin; an excipient such as starch or lactose, a disintegrating agent such as alginic acid, Primogel.TM., or corn starch; a lubricant such as magnesium stearate or sterotes; a glidant such as colloidal silicon dioxide; a sweetening agent such as sucrose or saccharin; or a flavoring agent such as peppermint, methyl salicylate, or orange flavoring.

[0133] For administration by Inhalation, the vaccine components are delivered In the form of an aerosol spray from pressured container or dispenser which contains a suitable propellant, e.g., a gas such as carbon dioxide, or a nebulizer.

[0134] Systemic administration can also be by transmucosal or transdermal means. For transmucosal or transdermal administration, penetrants appropriate to the barrier to be permeated are used in the formulation. Such penetrants are generally known in the art, and include, for example, for transmucosal administration, detergents, bile salts, and fusidic acid derivatives. Transmucosal administration can be accomplished through the use of nasal sprays or suppositories. For transdermal administration, the active compounds are formulated into ointments, salves, gels, or creams as generally known in the art.

[0135] Liposomal suspensions (including liposomes targeted to specific cell types) can also be used as pharmaceutically acceptable carriers.

[0136] The pharmaceutical compositions may also contain preserving agents, solubilizing agents, stabilizing agents, wetting agents, emulsifiers, sweeteners, colorants, odorants, salts for the variation of osmotic pressure, buffers, coating agents or antioxidants. They may also contain other therapeutically valuable agents.

[0137] Intravenous, intramuscular, subcutaneous, intramammary or oral administration is a preferred form of use. The dosages in which the components of the present invention are administered in effective amounts depend on the nature of the specific active ingredient, the host and the requirements of the subject and the mode of application.

Microbial Targets

[0138] Polypeptides, nucleic acids and delivery systems of the present invention may be used as antimicrobial agents against Staphylococcal infections including those causing intramammary infection (IMI). In a preferred embodiment, the Staphylococcal infections are caused by Staphylococcus aureus.

Methods of Immunizing with Polypeptides, Nucleic Acids, Vectors, Cells, Compositions and Delivery Systems

[0139] Encompassed by the methods, uses, pharmaceutical compositions and kits of the present invention is passive and active immunization.

[0140] Passive immunization is the injection of antibodies or antiserum, previously generated against the pathogen (or antigens described herein), in order to protect or cure a recipient animal of an infection or future infection. Protection fades over the course of a few weeks during which time the active immunization with polypeptides, nucleic acids or delivery systems (e.g., as described above) will have time to generate a lasting protective response. Serum for passive immunization can be generated by immunization of donor animals using the polypeptides, nucleic acids or delivery systems, as described herein. This serum, which contains antibodies against the antigens, can be used immediately or stored under appropriate conditions. It can be used to combat acute infections (e.g., IMI) or as a prophylactic (Tuchscherr ef a/., 2008). Use of antibodies or serums in a passive immunization can be combined with other agents such as an antibiotic to increase the cure rate of an infection currently in progress or to increase protection against an imminent infection.

[0141] Active immunization is administration of the polypeptides, nucleic acids or delivery systems as described herein to a subject.

[0142] The components identified in accordance with the teachings of the present invention have a prophylactic and/or therapeutic value such as they can be used to raise an immune response to prevent and/or combat diseases or conditions, and more particularly diseases or conditions related to microbial infections.

[0143] The terms "prevent/preventing/prevention" or "treat/treating/treatment" as used herein, refer to eliciting the desired biological response, i.e., a prophylactic and therapeutic effect, respectively in a subject. In accordance with the present invention, the therapeutic effect comprises one or more of a decrease/reduction in the severity, intensity and/or duration of the microbial infection (e.g., staphylococcal infection) or any symptom thereof following administration of the polypeptide, nucleic acid or delivery system (agent/composition of the present invention) of the present invention when compared to its severity, intensity and/or duration in the subject prior to treatment or as compared to that/those in a non-treated control subject having the infection or any symptom thereof. In accordance with the invention, a prophylactic effect may comprise a delay in the onset of the microbial infection (e.g., staphylococcal infection) or any symptom thereof in an asymptomatic subject at risk of experiencing the microbial infection (e.g., staphylococcal infection) or any symptom thereof at a future time; or a decrease/reduction in the severity, intensity and/or duration of a microbial infection (e.g., staphylococcal infection) or any symptom thereof occurring following administration of the agent/composition of the present invention, when compared to the timing of their onset or their severity, intensity and/or duration in a non-treated control subject (i.e. asymptomatic subject at risk of experiencing the microbial (e.g., bacterial) infection (e.g., staphylococcal infection) or any symptom thereof); and/or a decrease/reduction in the progression of any preexisting microbial infection (e.g., staphylococcal infection) or any symptom thereof in a subject following administration of the agent/composition of the present invention when compared to the progression of microbial infection (e.g., staphylococcal infection) or any symptom thereof in a non-treated control subject having such preexisting microbial infection (e.g., staphylococcal infection) or any symptom thereof. As used herein, in a therapeutic treatment, the agent/composition of the present invention is administered after the onset of the microbial infection (e.g., staphylococcal infection) or any symptom thereof. As used herein, in a prophylactic treatment, the agent/composition of the present invention is administered before the onset of the microbial infection (e.g., staphylococcal infection) or any symptom thereof or after the onset thereof but before the progression thereof.

[0144] As used herein, "decrease" or "reduction" of microbial infection (e.g., staphylococcal infection) or any symptom thereof refers to a reduction in a symptom of at least 10% as compared to a control subject (a subject not treated with the agent/composition present invention), in an embodiment of at least 20% lower, in a further embodiment of at least 30% lower, in a further embodiment of at least 40% lower, in a further embodiment of at least 50% lower, in a further embodiment of at least 60% lower, in a further embodiment of at least 70% lower, in a further embodiment of at least 80% lower, in a further embodiment of at least 90% lower, in a further embodiment of 100% (complete inhibition).

[0145] As used herein, the term "symptom" in reference to a staphylococcal infection refers to any staphylococcal infection symptom such as pain, inflammation, fever, vomiting, diarrhea, fatigue muscle aches, anorexia, dehydration, low blood pressure, cellulitis, impetigo, boil and scalded skin syndrome. More particularly, in reference to a staphylococcal IMI, a staphylococcal IMI symptom refers for example to visual abnormalities in milk (e.g., such as a watery appearance, flakes, clots, malodourous, presence of blood), redness of the udder, swelling in the udder, tenderness in the udder, elevated rectal temperature (>39.0.degree. C.), anorexia, decreased rumen motility and fatigue. An increase in milk somatic cell counts (SCC) is another staphylococcal IMI. Milk somatic cells include white blood cells such as leukocytes or neutrophils as well as epithelial cells. It is generally agreed that a SCC of >200,000/mL may represent a staphylococcal IMI symptom or is indicative of a staphylococcal IMI.

Dosage

[0146] Toxicity or efficacy of vaccine components to elicit an immune response can be determined by standard procedures in cell cultures or experimental animals. The dose ratio between toxic and immune stimulatory effects can be measured. Components that exhibit large ratios are preferred. While components that exhibit toxic side effects may be used, care should be taken to design a delivery system in order to minimize potential damage to cells and, thereby, reduce side effects.

[0147] Data obtained from cell culture assays and laboratory animal studies can be used in formulating a range of dosage for use in large animals and humans. The dosage of such components lies preferably within a range of administered concentrations that include efficacy with little or no toxicity. The dosage may vary within this range depending upon the dosage form employed and the route of administration utilized.

[0148] Any suitable amount of the pharmaceutical composition may be administered to a subject. The dosages will depend on many factors. Typically, the amount of active ingredient contained within a single dose will be an amount that effectively prevents, or treats IMI without inducing significant toxicity. The skilled artisan will appreciate that certain factors may influence the dosage required to effectively raise an immune response in a subject. Moreover, the therapeutically effective amount of the antigens (e.g., fusion construct) of the present invention may require a series of doses. In general, an amount of about 0.01 mg-500 mg of antigens including the fusion construct per dose, come into consideration. In a specific embodiment, an amount of about 0.1 mg-1 mg of antigens including the fusion construct per dose, come into consideration. Generally, one, two or three doses of the vaccine may favor optimal development of immunity. The time between two doses may be as short as three or four weeks but it may be preferred to separate the priming dose (first dose) and the booster dose (second dose) by five, six, seven, eight, nine or ten weeks before stimulating the immune system with the booster shot. A subsequent booster shot (a recall shot) may also be optimal to provide a sustainable immunity. This recall could for example occur every half year (6 months), yearly, every two years, every three or every five years.

[0149] "Sample" or "biological sample" refers to any solid or liquid sample isolated from a live being. In a particular embodiment, it refers to any solid (e.g., tissue sample) or liquid sample isolated from a mammal, such as milk, a biopsy material (e.g., solid tissue sample), blood (e.g., plasma, serum or whole blood), saliva, synovial fluid, urine, amniotic fluid and cerebrospinal fluid. Such sample may be, for example, fresh, fixed (e.g., formalin-, alcohol- or acetone-fixed), paraffin-embedded or frozen prior to analysis of the infectious agent's expression level.

Patients

[0150] As used herein the term "subject" or "patient" refers to an animal, preferably a mammal such as but not limited to a human, cow (e.g., heifer, multiparous, primiparous, calf), goat, sheep, ewe, ass, horse, pig, chicken, cat, dog, etc. who is the object of treatment, observation or experiment. In a specific embodiment, it is a cow (e.g., at risk of experiencing staphylococcal (e.g., IMI) infection).

[0151] As used herein the terms "subject at risk of experiencing a staphylococcal infection (e.g., staphylococcal infection (e.g., IMI) or any symptom thereof at a future time" refers to a mammal (e.g., a cow (e.g., heifer, multiparous, primiparous, calf), goat, sheep) that is used for milk or meat production.

[0152] In an embodiment, the above-mentioned mammal is a cow.

Method of Detection

[0153] Examples of methods to measure the amount/level of selected proteins/polypeptides include, but are not limited to: Western blot, immunoblot, enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA), immunoprecipitation, surface plasmon resonance, chemiluminescence, fluorescent polarization, phosphorescence, immunohistochemical analysis, matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry, microcytometry, microarray, microscopy, flow cytometry, and assays based on a property of the protein including but not limited to DNA binding, ligand binding, interaction with other protein partners or enzymatic activity.

[0154] In an embodiment, the amount of the polypeptide/protein within the methods of the present invention is detected using antibodies that are directed specifically against the polypeptide/protein. The term "antibody" as used herein encompasses monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), and antibody fragments, so long as they exhibit the desired biological activity or specificity. "Antibody fragments" comprise a portion of a full-length antibody, generally the antigen binding or variable region thereof. Interactions between antibodies and a target polypeptide are detected by radiometric, colorimetric, or fluorometric means. Detection of antigen-antibody complexes may be accomplished by addition of a secondary antibody that

is coupled to a detectable tag, such as for example, an enzyme, fluorophore, or chromophore.

[0155] Methods for making antibodies are well known in the art. Polyclonal antibodies can be prepared by immunizing a suitable subject (e.g., rabbit, goat, mouse, or other mammal) with the polypeptide/protein of interest or a fragment thereof as an immunogen. A polypeptide/protein "fragment" "portion" or "segment" is a stretch of amino acid residues of at least about 5, 7, 10, 14, 15, 20, 21 or more amino acids of the polypeptide noted above. The antibody titer in the immunized subject can be monitored over time by standard techniques, such as with an enzyme linked immunosorbent assay (ELISA) using immobilized exosomal marker polypeptide or a fragment thereof. At an appropriate time after immunization, e.g., when the antibody titers are highest, antibody-producing cells can be obtained from the animal, usually a mouse, and can be used to prepare monoclonal antibodies by standard techniques, such as the hybridoma technique originally described by Kohler and Milstein (1975) Nature 256: 495-497, the human B cell hybridoma technique (Kozbor et al. (1983) Immunol. Today 4: 72), the EBV-hybridoma technique (Cole et al. (1985) in Monoclonal Antibodies and Cancer Therapy, ed. Reisfeld and Sell (Alan R. Liss. Inc., New York, N.Y.), pp. 77-96) or trioma techniques. The technology for producing hybridomas is well known (see generally Coligan et al., eds. (1994) Current Protocols in Immunology, John Wiley & Sons, Inc., New York, N.Y.).

[0156] Alternatively, to preparing monoclonal antibody-secreting hybridomas, a monoclonal antibody can be identified and isolated by screening a recombinant combinatorial immunoglobulin library (e.g., an antibody phage display library) with a polypeptide or a fragment thereof to thereby isolate immunoglobulin library members that bind the polypeptide.

[0157] Kits for generating and screening phage display libraries are commercially available (e.g., the Pharmacia Recombinant Phage Antibody System.TM., Catalog No. 27-9400-01; and the Stratagene SurfZAP.TM. Phage Display Kit, Catalog No. 240612).

[0158] Furthermore, antibodies directed against one or more of the polypeptides/proteins described herein may be obtained from commercial sources.

[0159] The use of immobilized antibodies specific for the polypeptides/proteins is also contemplated by the present invention and is well known by one of ordinary skill in the art. The antibodies could be immobilized onto a variety of solid supports, such as magnetic or chromatographic matrix particles, the surface of an assay place (such as microtiter wells), pieces of a solid substrate material (such as plastic, nylon, paper), and the like. An assay strip could be prepared by coating the antibody or a plurality of antibodies in an array on solid support. This strip could then be dipped into the test sample and then processed quickly through washes and detection steps to generate a measurable signal, such as a colored spot.

[0160] The analysis of a plurality (2 or more) of polypeptides/proteins may be carried out separately or simultaneously with one test sample. Several polypeptides/proteins may be combined into one test for efficient processing of a multiple of samples.

[0161] The analysis of polypeptides/proteins could be carried out in a variety of physical formats as well. For example, the use of microtiter plates or automation could be used to facilitate the processing of large numbers of test samples. Alternatively, single sample formats could be developed to facilitate immediate treatment and diagnosis in a timely fashion. Particularly useful physical formats comprise surfaces having a plurality of discrete, addressable locations for the detection of a plurality of different analytes. Such formats include protein microarrays, or "protein chips" (see, e.g., Ng and Hag. J. Cell Mol. Med. 6: 329-340, 2002) and capillary devices.

[0162] In an embodiment, the above-mentioned level of expression is determined by measuring the level of expression of a mRNA transcribed from said one or more genes.

[0163] Methods to determine nucleic acid (mRNA) levels are known in the art, and include for example polymerase chain reaction (PCR), reverse transcriptase-PCR (RT-PCR), SAGE, quantitative PCR (q-PCR). Southern blot. Northern blot, sequence analysis, microarray analysis, detection of a reporter gene, or other DNA/RNA hybridization platforms. For RNA expression, preferred methods include, but are not limited to: extraction of cellular mRNA and Northern blotting using labeled probes that hybridize to transcripts encoding all or part of one or more of the nucleic acids encoding the protein/polypeptide of this invention; amplification of mRNA expressed from one or more of the nucleic acids encoding the proteins/polypeptides of this invention using specific primers, polymerase chain reaction (PCR), quantitative PCR (q-PCR), and reverse transcriptase-polymerase chain reaction (RT-PCR), followed by quantitative detection of the product by any of a variety of means; extraction of total RNA from the biological sample, which is then labeled and used to probe cDNAs or oligonucleotides encoding all or part of the nucleic acids encoding the proteins/polypeptides of this invention, arrayed on any of a variety of surfaces.

Kits

[0164] The present invention also encompasses kits comprising the components of the present invention. For example, the kit can comprise one or more components. The components can be packaged in a suitable container and device for administration. The kit can further comprise instructions for using the kit.

[0165] The present invention also provides a kit or package comprising reagents useful for administering one or more construct, polypeptide, nucleic acid, vector, host, compositions of the present invention, or a combination of at least two thereof, to a subject in need thereof for treating and/or preventing Staphylococcal IMI. Such kit may further comprise, for example, instructions for the prevention and/or treatment of Staphylococcal IMI, containers, reagents useful for performing the methods. The kit may further include where necessary agents for reducing background interference in a test, agents for increasing signal, software and algorithms for combining and interpolating marker values to produce a prediction of clinical outcome of interest, apparatus for conducting a test, calibration curves and charts, standardization curves and charts, and the like.

MODE(S) FOR CARRYING OUT THE INVENTION

[0166] The present invention is illustrated in further details by the following non-limiting examples.

Example 1: Materials and Methods for Vaccine Including SACOL0029, SACOL0442, SACOL0720, SACOL1867, SACOL1912 and SACOL2385 (Vaccine #1)

[0167] Production of the antigens. Six antigens that are highly expressed during S. aureus bovine intramammary infection were selected for inclusion in a first bovine vaccine (Vaccine #1). These antigens are:

SACOL0029 (GenBank.TM. accession No.: YPJ84940.1) (SEQ ID NO: 5), SACOL0442 (YPJ85332.1) (SEQ ID NO: 29), SACOL0720 (YPJ85601.1) (SEQ ID NO: 11), SACOL1867 (GenBank.TM. accession No.: YPJ86695.1) (SEQ ID NO: 38), SACOL1912 (GenBank.TM. accession No.: YPJ86737.1) (SEQ ID NO: 43), and SACOL2385 (GenBank.TM. accession No.: YPJ87189.1) (SEQ ID NO: 50). His-tagged recombinant proteins of SACOL0029, SACOL1867, SACOL1912, and SACOL2385 were engineered and produced by GenScript, Inc. (Piscataway, N.J.). His-tagged recombinant proteins of SACOL0442 and SACOL0720 were engineered and produced using QIA expression technology (pQE30 plasmid) from Qiagen Inc. (Mississauga. ON, Canada), according to the manufacturers' recommendations. See, FIG. 21 I to VI for the his-tagged sequences of the antigens. Examples 2-5 and FIG. 1 relate to this vaccine #1.

[0168] Immunization of dairy cows. Nineteen healthy multiparous Holstein cows in mid-lactation were housed in a level II biosafety barn at the Dairy and Swine Research and Development Centre of Agriculture and Agri-Food Canada (Sherbrooke, QC). Cows were randomly divided into 2 groups: one group (10 cows) received saline (placebo group); the other group (9 cows) received the vaccine #1 (vaccinated group). The vaccine was composed of 300 pg of each of six antigens (SACOL0029, SACOL0720, SACOL1867, SACOL1912, and SACOL2385) combined with Emulsigen.TM.-D (MVP Technologies, Omaha, Neb.), CpG ODN 2007 (TCGTCGTTGTCGTTTTGTCGTT (SEQ ID NO: 194), a pathogen-associated molecular pattern (PAMP), VIDO, Saskatoon, SW) and the cationic peptide indolicidin (ILPWKWPWWPWRR (SEQ ID NO: 195), used to induce the cow's immune response. (Chemprep Inc., Miami, Fla.). Two immunizations were performed 10 weeks apart, subcutaneously in the neck. No adverse side effects were observed. Blood from the caudal vein and milk samples were taken before the first immunization (preimmune serums) and then every two weeks for the detection of total IgG, IgG1 and lgG2. Larger volumes of blood from the jugular vein (150 ml was taken before the first immunization and 14 weeks after the first immunization (i.e., 4 weeks after the second immunization) for peripheral blood mononuclear cells (PBMCs) isolation and analysis of the cellular immune responses.

[0169] Detection of total IgG, lgG1 and lgG2 by ELISA. Detection of total IgG, IgG1 and lgG2 against each of the antigens in serum and milk was performed as previously described with some modifications (Ster et al., Vef. Immunol. Immunopathol. (2010), 136: 311-318). Nunc MaxiSorp.TM. 96-well plates (Thermo Fisher Scientific Inc., Rochester, N.Y.) were coated with the test antigen (5 .mu.g/mL diluted in carbonate/bicarbonate buffer, Sigma Aldrich, Oakville, ON) and incubated overnight at 37.degree. C. The plates were then saturated with the PBS containing 0.5% gelatin (BD. Franklin Lakes, N.J.) for 1 h at 37.degree. C. One hundred microliters of two-fold serial dilutions of the sera in PBS containing 0.5% gelatin and 0.1% Tween.TM. 20 were loaded into the plates and incubated for 1 h at 37.degree. C. The plates were washed three times with PBS containing 0.1% Tween.TM. 20. One hundred microliters of horseradish peroxidase (HRP)-conjugated secondary antibody were added to the plate. The secondary antibodies used were a goat anti-bovine IgG (Jackson ImmunoResearch Laboratories Inc., West Grove, Pa.), a sheep anti-bovine lgG1 (AbD Serotec, Raleigh, N.C.) or a sheep anti-bovine lgG2 (AbD Serotec), diluted 1/50.000 1/20,000 and 1/20,000 respectively in PBS containing 0.5% gelatin and 0.1% Tween.TM. 20. After 1 h of incubation at 37.degree. C. followed by 3 washes, peroxidase activity was detected with 3,3',5,5'-tetramethylbenzidine (TMB) reagent (KPL Inc., Gaithersburg, Md.) according to the manufacturer's recommendations.

[0170] Detection of total IgG, lgG1 and lgG2 in milk was carried out using the same procedure with few modifications. Milk samples were diluted into PBS containing 0.5% gelatin. The sheep anti-bovine lgG2 was diluted 1/10.000 into PBS containing 0.5% gelatin and 0.1% Tween.TM. 20.

[0171] Evaluation of the cellular immune response. PBMCs were isolated from jugular vein blood and labelled with carboxyfluoroscein diacetate, succinimidyl ester (CFDA-SE; Molecular Probes Inc., Eugene, Oreg.) as previously described (Loiselle et al., J. Dairy. Sci. (2009), 92:1900-1912). At the end of the CFDA-SE labelling procedure, the PBMCs were suspended in RPMI medium containing 5% FBS and 1.times. antibiotic/antimycotic (.DELTA.5955, Sigma Chemical Aldrich). The PBMCs (5.times.10.sup.6 cells per well) were stimulated with the mitogen concanavalin A (ConA; positive control; Sigma Aldrich) at a final concentration of 1 .mu.g/mL, or each antigen (5 .mu.g per well) and incubated for 7 days at 37.degree. C. As a negative control, the PBMCs were incubated without any mitogen. Stimulations were performed in duplicate (Ster et al., 2010).

[0172] The proliferation of CD4+ and CD8+ cells was evaluated after incubation with the different mitogens. The cells were centrifuged at 300*g for 5 min, suspended in PBS containing 0.5% BSA. The mouse anti-bovine CD8 coupled with Alexa Fluor.TM. 647 (diluted 1/20. AbD Serotec) and the mouse anti-bovine CD4 coupled with rPE (diluted 1/20. AbD Serotec) were then added. After 20 min of incubation on ice, the cells were washed three times with PBS containing 0.5% BSA. The cells were then suspended in PBS with 0.5% formaldehyde. The percentages of the proliferative populations were determined by flow cytometry on a BD FACS Canto II flow cytometer using the BD FACS Diva software.

[0173] Experimental S. aureus in IMI in dairy cows. Before their use in experimental IMI, the relationship between the absorbance of the bacterial cultures (.DELTA.600 nm) and CFU was determined. The day of the challenge, a volume of the overnight culture of S. aureus in Mueller Hinton broth (MHB; BD) was transferred to 200 mL of fresh MHB to obtain an .DELTA.600 nm of 0.1 and subsequently grown at 35.degree. C. until the .DELTA.600 nm reached a value corresponding to 10.sup.8 CFU/mL in the exponential phase of growth. The strain to be used in this experimental infection (CLJ08-3) had previously been characterized in the co-inventor's lab (Allard et al., Vet. Microbiol. (2013) 162: 761-770). For intramammary infusions, bacteria were routinely diluted in sterile PBS (Sigma Aldrich) to obtain approximately 50 CFU in 3 mL. In this experiment, the inoculum was plated on TSA and found to contain 63 cfu in 3 mL.

[0174] Somatic cell count (SCC) determinations and bacterial analysis of aseptic quarter milk samples were carried out prior to experimental IMI to ensure that all cows were free of IMI. Experimental infusion of mammary quarters with bacteria was performed in three (randomly chosen) of the four quarters of each cow after the evening milking according to a procedure previously described (Petitclerc et al., J. Dairy. Sci. (2007), 90: 2778-2787) with few modifications. Briefly, before inoculation, teats were scrubbed with gauze soaked in 70% ethanol. Teats were allowed to air-dry before intramammary infusion of 3 mL of bacterial suspension (containing 63 CFU) into three of the four quarters. Immediately after infusion, all quarters were thoroughly massaged and teats were dipped in an iodophore-based teat sanitizer. Disposable gloves were worn throughout the procedure and disinfected before proceeding to the next animal. All quarters infused with S. aureus became infected and all cows showed clinical signs (inflammation, and/or poor milk appearance) of mastitis at some time during the first few days after infusion of S. aureus.

[0175] Evaluation of the S. aureus viable counts after experimental infections. Aseptic milk samples were taken before the morning milking three times a week during the 3 first weeks following the experimental infection and then twice a week for the 2 remaining weeks. After foremilk was discarded and the teats were disinfected with 70% ethanol, a 10-mL milk sample was aseptically collected in a 50-mL sterile vial for each individual quarter. Milk samples were serially diluted and 100 .mu.i, of each dilution were plated on both tryptic soy agar (Becton Dickinson) and mannitol salt agar plates (Becton Dickinson) for CFU determinations and S. aureus identification. Plates were then incubated for 24 h at 35.degree. C. before the colonies were counted. The dilutions that showed between 30 and 300 colonies were used to calculate the bacterial concentration. Each dilution was plated in duplicate.

[0176] Evaluation of the somatic cell counts. At the same frequency as for aseptic milk samples, milk was harvested using individual quarter milking units at morning milking and weighed for the determination of quarter milk production. A non-aseptic 50-mL sample was also taken from each quarter milking units for the determination of the SCC by a commercial laboratory (Valacta Inc., Ste-Anne-de-Bellevue, QC, Canada). The milking units were thoroughly washed and disinfected with an iodine-based germicide detergent (K.O. Dyne.RTM., GEA Farm Technologies, Westmoreland, N.Y.) between their uses on each cow. All other materials in contact with milk were disinfected with 70% ethanol.

[0177] Statistical analysis. Statistical analyses of the experimental infection data were performed using the MIXED procedure of SAS (SAS Institute Inc., Cary, N.C.) as repeated measurements. For the analysis of SCC and CFU, data were Iog10 transformed prior to analysis.

[0178] Statistical analysis of the antibody titers and of the correlation between CFU and SCC was performed using GraphPad Prism.TM. v6.05.

[0179] Ethics statement. All animal experiments were approved by the Agriculture and Agri-Food Canada local institutional animal care committee and conducted in accordance with the guidelines of the Canadian Council on Animal Care.

Example 2: Serum Total IgG1 Titers Following Vaccination--Vaccine #1

[0180] Recombinant His-tagged antigens for SACOL0029 (GenBank.TM. accession No.: YPJ84940.1) (SEQ ID NO: 5), SACOL0442 (SEQ ID NO: 29), SACOL0720 (SEQ ID NO: 11), SACOL1867 (GenBank.TM. accession No.: YPJ86695.1) (SEQ ID NO: 38), SACOL1912 (GenBank.TM. accession No.: YPJ86737.1) (SEQ ID NO: 43), and SACOL2385 (GenBank.TM. accession No.: YPJ87189.1) (SEQ ID NO: 50), were prepared and administered to healthy cows as described in Example 1 (Production of the antigens and Immunization of dairy cows). Nine dairy cows received the vaccine and 10 cows received saline (placebo). Total serum IgG, lgG1 and lgG2 titers were detected as described in Example 1 (Detection of total IgG, lgG1 and lgG2 by ELISA).

[0181] As expected, and as shown in FIGS. 1B-C, immunization induced an increased production of antigen-specific serum lgG1 and lgG2 for the vaccinated group in comparison to the placebo group. Interestingly, the lgG2/lgG1 ratio was 1 for SACOL0442 (see FIG. 1D), which is an indication of a balanced Th1/Th2 immune response to this antigen. For the antigens SACOL0029, SACOL0720, SACOL1912 and SACOL2385, the lgG2/lgG1 ratio is significantly lower than the ratio for SACOL0442 which indicated that these antigens induced mostly an lgG1 antibody response via the Th2 pathway.

Example 3: Antigen Dependent Proliferation of Blood CD4+ and CD8+ Cells Following Vaccination-Vaccine #1

[0182] Antigen dependent proliferation of blood CD4+ and CD8+ cells from the vaccinated cows (9) and placebo cows (10) was evaluated as described in Example 1 (Evaluation of the cellular immune response) four weeks after the second immunization (just before the experimental infection) for each antigen. The results for CD4+ cells are shown in FIG. 2, in which each symbol represents the percentage of CD4+ cells that have proliferated for each cow after a week of incubation with the positive control (ConA) or each antigen. Open circles (o) represent data for the vaccinated cows, black squares (.box-solid.) represent data for the placebo cows. Horizontal lines represent the medians: dashed lines represent the medians for the vaccinated cows while continuous lines represent the medians for the placebo cows.

[0183] The symbol * shows the statistical differences between the vaccinated and the placebo groups for antigens SACOL0029, SACOL0442, SACOL0720 and SACOL1912 (*, PO.05).

[0184] In addition, the proliferation of CD8+ cells was similar for the vaccinated and placebo cows for all antigens with the exception of the antigen SACOL0720 for with higher proliferation of the CD8+ cells was observed for the vaccinated cows (data not shown). Induction of CD8+ cells also seemed to be important for the resolution of the infection (Riollet et al., 2001; Burton and Erskine, 2003). The vaccine was able to stimulate both cellular (CD8+) and humoral (CD4+) immune response. The vaccine #1 with its different antigens leads to a balanced immune response.

Example 4: Protection Effect of the Vaccine as Evaluated by Following the Evolution of Somatic Cell Counts (SCC)--Vaccine #1

[0185] Experimental S. aureus IMI infection in dairy cows were carried out and evaluated as described in Example 1 (Experimental S. aureus IMI in dairy cows, Evaluation of the S. aureus viable counts after experimental infections, Evaluation of the somatic cell counts and Statistical analysis). Four weeks and 4 days after the second immunization, 63 CFU of S. aureus were infused into 3 of the 4 quarters of the vaccinated (9) and placebo cows (10) at the evening milking (day 1, arrow in FIG. 3). Aseptic milk samples were taken at morning milking and SCC was determined by Valacta (Ste-Anne-de-Bellevue, QC). The results are shown FIG. 3, in which open circles (o) and the dashed line represent data for the vaccinated cows, while the black squares (.box-solid.) and the continuous line represent data for the placebo cows. Each open circle represents the mean of SCC for all the infected quarters of the vaccinated cows (27) while each square represents the mean of SCC for all the infected quarters of the placebo cows (30 quarters).

[0186] Over the challenge period. SCC in milk were found to be significantly lower for the vaccinated cows than for the placebo cows (***; P<0.001), indicating less inflammation and a better control of the infection in the vaccinated cows.

Example 5: Correlation Between SCC or the Viable Counts of S. aureus (CFU) Relative to Serum or Milk IgG Titers Against Specific Antigens--Vaccine #1

[0187] As shown in FIGS. 4A-B, SCC were positively correlated to S. aureus CFU of the challenge period (FIG. 4A, r=0.82, P<0.0001) and negatively correlated to the serum lgG1 titer against SACOL0442 measured prior to the infection (FIG. 4B, r=-0.49, P<0.05). Vaccination thus had reduced this criterium of inflammation induced by the challenge. A similar analysis was performed with the samples collected at day 10. The same correlations were observed as previously obtained but at this particular time point SCC and S. aureus CFU also correlated to the milk lgG2 titer against SACOL0029 (FIG. 4C, r=-0.48, P<0.05 and r=-0.58, P<0.05, respectively). These results show that more than one antigen is involved in the immune response against the infection.

Example 6: Materials and Methods for Vaccine Including SACOL0442, SACOL0720, and a Fusion Between SACOL1867 and SACOL0029--Vaccine #2

[0188] Production of the antigens. Four antigens that are highly expressed during S. aureus bovine intramammary infection were selected for inclusion in vaccine #2. The antigens are polypeptides encoded by: SACOL0029 (GenBank.TM. accession No.: YP_184940.1) (SEQ ID NO: 5). SACOL1867 (GenBank.TM. accession No.: YPJ86695.1) (SEQ ID NO: 38). SACOL0442 (SEQ ID NO: 29), and SACOL0720 (SEQ ID NO: 11). The SACOL0029 and SACOL1867 antigens were included in the form of a fusion. His-tagged recombinant proteins of SACOL0720 and SACOL0029-1867 were engineered and produced by GenScript. Inc. (Piscataway, N.J.). A his-tagged recombinant protein of SACOL0442 was engineered and produced using QIA expression technology (pQE30 plasmid) from Qiagen Inc. (Mississauga. ON. Canada), according to the manufacturers' recommendations. (see FIGS. 21D-E and I, and items II, III and VII for SACOL0720. SACOL0442, and SACOL0029-1867 his-tagged sequences). The surface protein ClfA (SEQ ID NO: 184), was also additionally produced by using the QIA expression vector by cloning the clfA gene from S. aureus ATCC 25904. The latter recombinant protein was not part of the vaccine composition but was used in ELISA assays to determine IgG titers of sera against other S. aureus proteins such as ClfA.

[0189] The vaccine was composed of 300 .mu.g of each of 3 antigens (SACOL0442 and SACOL0720 as defined in Example 1 and the fusion SACOL0029-1867) and with Emulsigen.TM.-D (MVP Technologies, Omaha, Nebr.). CpG ODN 2007 (i.e. Tc.theta.T06 .theta.T06THT6T06.pi. (SEQ ID NO: 194) (IDT, Coralville, Iowa)), and indolicidin (ILPWKWPWWPWRR (SEQ ID NO: 195, GenScript, Piscataway, N.J., Chemprep Inc., Miami, Fla.) (vaccine #2).

[0190] Immunization of dairy cows. Eleven healthy multiparous Holstein cows in mid-lactation were housed in a level II biosafety barn at the Dairy and Swine Research and Development Centre of Agriculture and Agri-Food Canada (Sherbrooke, QC). Cows received the vaccine #2 (vaccinated group). Two immunizations were performed 10 weeks apart, subcutaneously in the neck. No adverse side effects were observed. Blood from the caudal vein and milk samples were taken before the first immunization (preimmune serums) and then every week for the detection of total IgG.

[0191] Detection of total IgG by ELISA. Detection of total IgG against each of the antigens in serum was performed as previously described with some modifications (Ster et al., Vet. Immunol. Immunopathol. (2010), 136: 311-318). Nunc MaxiSorp.TM. 96-well plates (Thermo Fisher Scientific Inc., Rochester, N.Y.) were coated with the test antigen (5 g/mL diluted in carbonate/bicarbonate buffer. Sigma Aldrich, Oakville, ON) and incubated overnight at 37.degree. C. The plates were then saturated with the PBS containing 0.5% gelatin (BD, Franklin Lakes, N.J.) for 1 h at 37.degree. C. One hundred microliters of two-fold serial dilutions of the sera in PBS containing 0.5% gelatin and 0.1% Tween.TM. 20 were loaded into the plates and incubated for I h at 37.degree. C. The plates were washed three times with PBS containing 0.1% Tween.TM. 20. One hundred microliters of horseradish peroxidase (HRP)-conjugated secondary antibody were added to the plate. The secondary antibodies used were a goat anti-bovine IgG (Jackson ImmunoResearch Laboratories Inc., West Grove, Pa.) diluted 1/1000,000 in PBS containing 0.5% gelatin and 0.1% Tween.TM. 20. After 1 h of incubation at 37.degree. C. followed by 3 washes, peroxidase activity was detected with 3,3',5,5'-tetramethylbenzidine (TMB) reagent (KPL Inc., Gaithersburg, Md.) according to the manufacturer's recommendations.

Example 7: The Fusion of Antigens Induces High Antibody Titers--Vaccine #2

[0192] FIG. 5 shows serum total IgG titers for the vaccinated cows for each antigen of the vaccine (including the fused antigens SACOL0029 and SACOL1867 (labeled SACOL0029-1867 on FIG. 5). Each open circle represents the titer four weeks after the second immunization for each cow (just before the experimental infection) whereas each black diamond represents the preimmune titer. Horizontal lines represent the medians: solid line for the preimmune serums, dotted line for the samples taken four weeks after immunization. Titers for the vaccinated cows are higher than the titers of the preimmune serums (**, P<0.01; ***, P<0.001 for the other antigens tested).

[0193] FIG. 5 thus shows that the vaccine composed of three separate antigens, including a fusion peptide, induces a strong immune response in the cows. Furthermore, FIG. 5 surprisingly shows that the fused antigens SACOL0029 and SACOL1867 (fusion SACOL0029-SACOL1867) raised antibody titers that were above those raised by each of the antigen alone (Compare FIG. 1A vs. FIG. 5) and that such fused antigens provide an additional benefit to the vaccine.

[0194] More particularly, when administered individually in a vaccine, the titers of immune cows against SACOL0029 and SACOL1867 reached 3200 and 51200, respectively (FIG. 1A), whereas when these antigens were administered as a fusion, the titers of immune cows reached 12800 and 409600, respectively (FIG. 5), showing that the fusion create an unexpected synergy in the immune response.

Example 8: Materials and Methods for Vaccine Comprising SACOL0029, SACOL1867, and a Fusion Between SACOL1867 and SACOL0029--Vaccine #3

[0195] Production of the antigens. Three antigens derived from two genes that are highly expressed during S. aureus bovine intramammary infection were selected for inclusion in a vaccine. These antigens are: SACOL0029 (GenBank.TM. accession No.: YP_184940.1) (SEQ ID NO: 5), SACOL1867 (GenBank.TM. accession No.: YP_186695.1) (SEQ ID NO: 38) and a fusion between SACOL1867 and SACOL0029 (GenBank.TM. accession No.: YPJ84940.1) (SEQ ID NO: 5). His-tagged recombinant proteins of SACOL0029, SACOL1867 and SACOL0029-1867 were engineered and produced by GenScript, Inc. (Piscataway, N.J.). (see FIGS. 21A, F and I, items I. IV and VII for SACOL0029, SACOL1867, and SACOL0029-1867 his-tagged sequences).

[0196] Immunization of mice. The immunogenic properties of recombinant S. aureus proteins encoded by the SACOL0029, SACOL1867 genes and a fusion of SACOL0029 and SACOL1867 were evaluated in mice. Four groups of mice received the exact equimolar quantity of proteins, either in a monovalent form (SACOL0029 or SACOL1867) or in a multivalent form (the fusion SACOL0029-1867 or SACOL0029 together with SACOL1867 in combination), were compared.

[0197] In brief, the theoretical molecular weight of each amino acid sequence corresponding to the entire fusion or to the SACOL0029 or SACOL1867 portion of the fusion were calculated using the ExPASy.TM. Bioinformatic resource portal (http://web.expasy.org/cgi-bin/compute_pi/pi_tool).

[0198] Five micrograms of the fusion were administered to one group of mice. The corresponding molar quantity of 5 .mu.g of the SACOL0029-1867 fusion was determined to be 168.55 pmol, in regard to its theoretical molecular weight. An amount of 1.15 .mu.g and 3.69 .mu.g of SACOL0029 and SACOL1867, respectively was administered in two other groups of mice in order to provide 168.55 pmol of each antigen, respectively. The last group of mice received the combination of the two individual antigens (168.55 pmol of each).

[0199] For the preparation of the immunization doses, SACOL0029, SACOL1867 and the SACOL0029-1867 fusion polypeptides were individually mixed and suspended in PBS to obtain the final equimolar quantity of each antigenic dose in a volume of 100 .mu.l. Twenty CD-1 female mice were randomly divided into 4 groups: group A (5 mice) received 5 .mu.g of the SACOL0029-1867 fusion protein (Fusion); group B (5 mice) received 1.15 .mu.g of SACOL0029 and 3.69 .mu.g of SACOL1867 (Combination): group C (5 mice) received 1.15 .mu.g of SACOL0029 (0029) and group D received 3.69 .mu.g of SACOL1867 (1867). The CD-1 mice were immunized by two subcutaneous injections in the neck two weeks apart. No adverse side effects were observed during the totality of the experimental immunization period. Blood samples were taken just before the first priming injection (preimmune serums) and ten days after the boost immunization (immune serums). The blood aliquots were allowed to clot at room temperature for an hour, centrifuged at 10,000 g for 10 min at 4.degree. C. The sera were harvested and kept at -20.degree. C. until subsequent analysis.

[0200] Detection of total IgG by ELISA. Detection of serum total IgG against SACOL0029 and SACOL1867 recombinant proteins was performed as previously described with some modifications (Ster et al., Vet. Immunol. Immunopathol. (2010), 136: 311-318). Nunc MaxiSorp.TM. 96-well plates (Thermo Fisher Scientific Inc., Rochester, N.Y.) were coated with 75 .mu.I of each of the test antigen (6.67 .mu.g/mL diluted in carbonate/bicarbonate buffer. Sigma Aldrich, Oakville, ON) and incubated overnight at room temperature. The plates were then saturated with PBS containing 5% skim milk powder for 1 h at 37.degree. C. One hundred microliters of four-fold serial dilutions of the sera in PBS containing 3% milk and 0.025% Tween.TM. 20 were loaded into the plates and incubated for 1 h at 37.degree. C. The plates were washed three times with PBS containing 0.05% Tween.TM. 20. One hundred microliters of horseradish peroxidase (HRP)-conjugated secondary antibody were then added to the plate. The secondary antibody used was a commercial goat anti-mouse IgG (Jackson ImmunoResearch Laboratories Inc., West Grove, Pa.), diluted 1/5000 in PBS containing 3% milk and 0.025% Tween.TM. 20. After 1 h of incubation at 37.degree. C. followed by 3 washes, peroxidase activity was detected with the addition of one hundred microliters of 3,3',5,5'-tetramethylbenzidine (TMB) reagent (KPL Inc., Gaithersburg, Md., according to the manufacturer's recommendations.

[0201] Statistical analysis Statistical analysis of the antibody titers and of the correlation was performed using GraphPad Prism.TM. v6.05.

Example 9: The Fusion of Antigens Induces Significantly Higher Antibody Titers Compared to Monovalent Antigens or a Combination of Antigens--Vaccine #3

[0202] FIG. 6 shows that an antigen (SACOL1867) included in a fusion polypeptide (i.e., the SACOL0029-1867 fusion protein) can induce a strong and specific antibody immune response against that specific antigen (SACOL1867), and, more importantly, that this response can be significantly higher than that obtained with a monovalent form of this antigen (SACOL1867 administered alone) or a multivalent combination of individual polypeptides that are part of the fusion (combination of SACOL1867 plus SACOL0029). Thus, in addition to the advantage of generating an immune response against multiple polypeptidic targets, such fused antigens also provide the additional benefit of greatly improving the antibody titers against those targets.

Example 10: Materials and Methods for Vaccines Including SACOL0720 Fragments) and/or SACOL442 Fragment(s)--Vaccines #4-6

[0203] Production of the antigens. Peptides and amino acid fragments of 15 to 50 amino acids in length and derived from sequences SACOL0442 and/or SACOL0720 were selected based on the presence of B-cell epitopes. Fusions of peptide epitopes were also designed in which an amino acid linker (for example, EAAAKEAAAK (SEQ ID NO: 62), or ERKYK (SEQ ID NO: 61) or KDYERKYKKHIVS (SEQ ID NO: 196)) joined the various epitopes. Peptides and amino acid fragments were synthesized by Biomatik, Inc. (Cambridge. ON). Upon receipt, lyophilised peptides and amino acid fragments were suspended in sterile water at a concentration of 5 mg/mL and stored at -80.degree. C. until day of use.

[0204] Immunization of mice. Peptides and amino acid fragments were used as antigens for immunization of mice. For the preparation of the immunization doses, each peptide and amino acid fragment or a combination of such were mixed and suspended in PBS containing 20% of the EMULSIGEN.RTM.-D oil-in-water emulsion adjuvant to obtain a final dose of 100 .mu.g of polypeptide per dose, unless otherwise specified. CD-1 female mice were randomly divided into different groups of 3 to 4 animals. Mice were immunized by two subcutaneous injections in the neck two weeks apart. No adverse side effects were observed during the totality of the experimental period. Blood samples were taken just before the first priming injection (preimmune serums) and ten days after the boost immunization (immune serums). The blood aliquots were allowed to clot at room temperature for an hour, and then centrifuged at 10.000 g for 10 min at 4.degree. C. The sera were harvested and kept at -20.degree. C. until subsequent analysis.

[0205] Detection of total IgG by ELISA. Detection of serum total IgG, against specific amino acid sequences found in the antigens used for the immunization of mice, was performed as previously described with some modifications (Ster et al., Vet. Immunol. Immunopathol. (2010), 136: 311-318). Nunc MaxiSorp.TM. 96-well plates (Thermo Fisher Scientific Inc., Rochester, N.Y.) were coated with 100 .mu.I of each of the target amino acid sequences diluted at a final concentration of 5 .mu.c/.GAMMA..eta.i, in carbonate/bicarbonate buffer (Sigma Aldrich. Oakville, ON) and incubated overnight at room temperature. The plates were then saturated with PBS containing 5% skim milk powder for 1 h at 37.degree. C. One hundred microliters of four-fold or two-fold serial dilutions of the sera in PBS containing 1% milk and 0.025% Tween.TM. 20 were loaded into the plates and incubated for 1 h at 37.degree. C. The plates were then washed three times with PBS containing 0.05% Tween.TM. 20. One hundred microliters of horseradish peroxidase (HRP)-conjugated secondary antibody were then added to the plate. The secondary antibody used was a goat anti-mouse IgG (Jackson ImmunoResearch Laboratories Inc., West Grove, Pa.), diluted 1/5000 in PBS containing 1% milk and 0.025% Tween.TM. 20. After 1 h of incubation at 37.degree. C. followed by 3 washes with PBS Tween.TM. 20 and a final wash with PBS, peroxidase activity was detected with 3,3',5,5'-tetramethylbenzidine (TMB) reagent (KPL Inc., Gaithersburg, Md.) according to the manufacturer's recommendations.

[0206] Statistical analysis. Statistical analysis of the antibody titers and optical densities was performed using GraphPad Prism.TM. v6.05.

Example 11: Immune Response Against a Fusion of Peptides that Includes Epitopes Encoded from Sequences SACOL0442 and SACOL0720--Vaccine #4

[0207] A fusion of peptide epitopes encoded from SACOL0442 and SACOL0720 was used to vaccinate mice (n=4). The sequence of the fusion of peptides was KDGGKYTLESHKELQEAAAKEAAAKKDINKIYFMTDVDLGGPTFVLND (SEQ ID NO: 3) (vaccine #4), where the linker is italicized and the different epitopes are identified in bold characters. The epitopes were KDGGKYTLESHKELQ (SEQ ID NO: 1) encoded from SACOL0442, KDINKIYFMTDVDL (SEQ ID NO: 23) encoded from SACOL0720, and DVDLGGPTFVLND (SEQ ID NO: 24) also encoded from SACOL0720. The IgG antibodies from the sera harvested from the animals were able to bind amino acid fragments comprising B-cell epitopes from either SACOL0442 (i.e. KDGGKYTLESHKELQ (SEQ ID NO: 1)) and/or SACOL0720 (i.e. QFGFDLKHKKDALA (SEQ ID NO: 21); KDINKIYFMTDVDL (SEQ ID NO: 23), DVDLGGPTFVLND (SEQ ID NO: 24)) in ELISA assays with antibody titers of 1/6400 or higher. The fusion of peptides used for immunization and the amino acid fragments or polypeptides used as antibody targets in ELISA assays are shown in Table III below. In this table, the epitopes are in bold and the linker sequence is italicized.

TABLE-US-00010 TABLE III Polypeptide vaccine and antibody response targets Fusion of peptides used for vaccination KDGGKYTLESHKELQEAAAKEAAAKKDINKIYFMTDVDLGGPTFVLND (SEQ ID NO: 3) Peptides and polypeptides targets bound by IgG from vaccinated mice in an ELISA assay KDGGKYTLESHKELQEAAAKEAAAKKDINKIYFMTDVDLGGPTFVLND (SEQ ID NO: 3) (fusion of peptides); GEHLPKGNIVINTKDGGKYTLESHKELQKDRENVKINTAD (SEQ ID NO: 2) (fragment encoded by SACOL0442); KDINKIYFMTDVDLGGPTFVLNDKDYERKYKKHIVSQFGFDLKHKKDALA (SEQ ID NO: 271) (variant comprising fragments encoded by SACOL0720) SACOL0442 (SEQ ID NO: 55) (i.e. polyhistidine version shown in FIG. 21 E, item II); SACOL0720 (SEQ ID NO: 25) (i.e. polyhistidine version shown in FIG. 21 D, item III);

[0208] All antibody targets shown above were bound in an ELISA assay by IgG from mice vaccinated with the fusion antigen above.

[0209] This demonstrates that a fusion of peptide epitopes encoded by both SACOL0442 and SACOL0720 can be used to immunize and elicit an immune response in a mammal. The obtained immune response includes the production of antibodies that recognize SACOL0442 or SACOL0720, amino acid fragments or variants encoded from either SACOL0442 or SACOL0720.

Example 12: A Fusion of Multiple Epitopes Used as an Antigen in Immunizations Significantly Enhances the Immune Response Against a Single Epitope--Vaccine #4

[0210] A fusion of peptide epitopes encoded from SACOL0442 and SACOL0720 was used to vaccinate mice (n=4). The sequence of the fusion of peptides was KDGGKYTLESHKELQEAAAKEAAAKKDINKIYFMTDVDLGGPTFVLND (SEQ ID NO: 3), where the linker is italicized and the different epitopes are identified in bold characters. The epitopes were KDGGKYTLESHKELQ (SEQ ID NO: 1) encoded from SACOL0442, KDINKIYFMTDVDL (SEQ ID NO: 23) encoded from SACOL0720, and DVDLGGPTFVLND (SEQ ID NO: 24) also encoded from SACOL0720. Another group of mice (n=4) was immunized with the single peptide epitope KDGGKYTLESHKELQ (SEQ ID NO: 1), encoded from SACOL0442.

[0211] Sera were collected from animals and tested for the presence of IgG antibodies directed toward an amino acid fragment encoded from SACOL0442 (GEHLPKGNIVINTKDGGKYTLESHKELQKDRENVKINTAD) (SEQ ID NO: 2), which contains the peptide epitope KDGGKYTLESHKELQ (SEQ ID NO: 1).

[0212] As shown on FIG. 7, immunization with the fusion of three peptide epitopes (one encoded from SACOL0442 and two encoded from SACOL0720) significantly increased the antibody production against an amino acid fragment encoded from SACOL0442 (GEHLPKGNIVINTKDGGKYTLESHKELQKDRENVKINTAD) (SEQ ID NO: 2), which contains the peptide epitope KDGGKYTLESHKELQ (SEQ ID NO: 1), compared to the antibody level obtained when only using the peptide epitope KDGGKYTLESHKELQ (SEQ ID NO: 1) as antigen for immunization.

Example 13: Immune Response Against a Polypeptide Fragment of 50 Amino Acids Encoded a Variant of SACOL0720--Vaccine #5

[0213] A group of mice (n=3) was vaccinated with a 50-amino acid peptide fragment (KDINKIYFMTDVDLGGPTFVLNDKDYERKYKKHIVSQFGFDLKHKKDALA (SEQ ID NO: 27)) (vaccine #5) containing B-cell epitopes (bold characters) encoded from the sequence SACOL0720, more specifically epitopes KDINKIYFMTDVDL (SEQ ID NO: 23), DVDLGGPTFVLND (SEQ ID NO: 24) and QFGFDLKHKKDALA (SEQ ID NO: 21). The overall sequence of KDINKIYFMTDVDLGGPTFVLNDKDYERKYKKHIVSQFGFDLKHKKDALA (SEQ ID NO: 27) vary from the native sequence of SACOL0720 by four amino acids in the region linking the epitopes DVDLGGPTFVLND (SEQ ID NO: 24) and QFGFDLKHKKDALA (SEQ ID NO: 21). Vaccine #5 can thus be considered being a variant fragment of SACOL0720 or a fusion of epitopes from SACOL0720, which are spaced by linker ERKYK (SEQ ID NO: 61).

[0214] Sera were tested for the presence of IgG antibodies directed toward a fragment of the native protein encoded by SACOL0720 (SEQ ID NO: 25) (i.e. polyhistidine version shown in FIG. 21 D, item II). Both mice vaccinated with the peptide fragment corresponding to the variant sequence of amino acids (or fusion SACOL0720-720) produced antibodies that recognized epitopes in the original sequence of amino acids in an ELISA assay with titers of 1/6400 or higher.

[0215] This demonstrates that an amino acid fragment that comprises epitopes encoded from sequence SACOL0720 can elicit an immune response in a mammal. This also further demonstrates that a variant of the native sequence has the capacity to stimulate the immune system against the original fragment sequence containing B-cell epitopes.

Example 14: Immune Response Against a Combination of Fusions (Peptide Fusion 0442-0720 and Polypeptide Fusion 0029-1867)--Vaccine #6

[0216] A fusion of peptide epitopes encoded from SACOL0442 and SACOL0720 (see sequence in FIG. 211, Item VII-fusions) was combined to a polypeptide fusion containing sequences of SACOL0029 and SACOL 1867 (see sequence in FIG. 21H, Item VII) and was used to vaccinate mice (vaccine #6).

[0217] For the preparation of the immunization doses, the peptide fusion 0442-0720 and the polypeptide fusion 1867-0029 were mixed and suspended in PBS containing 20% of the EMULSIGEN.RTM.-D oil-in-water emulsion adjuvant to obtain a final dose of 100 .mu.g and 5 .mu.g per dose of the peptide fusion (0442-0720) and the polypeptide fusion (0029-1867), respectively. CD-1 female mice (n=3) were immunized by three subcutaneous injections in the neck. The first two injections were made one week apart and the third injection 3 weeks after the second one. No adverse side effects were observed during the totality of the experimental period. Blood samples were taken just before the first priming injection (preimmune serums) and fourteen days after the last boost immunization (immune serums). The blood aliquots were allowed to clot at room temperature for an hour, and then centrifuged at 10,000 g for 10 min at 4.degree. C. The sera were harvested and kept at -20.degree. C. until subsequent analysis.

[0218] The IgG antibodies from the sera harvested from the animals were able to bind amino acid fragments comprising epitopes from either SACOL0442 or SACOL0720 or to polypeptide SACOL0029 or SACOL1867 in ELISA assays with antibody titers of 1/6400 or higher. The fusion of peptides and polypeptides used for immunization and the polypeptides or amino acid fragments used as antibody targets in ELISA assays are shown in the Table IV below.

TABLE-US-00011 TABLE IV Mixed polypeptide fusion vaccine and antibody response targets A mixture of the fusion of peptides 0442-0720 and polypeptide fusion 0029-1867 was used for vaccination (vaccine #6) The epitopes are in bold and the linker sequence is italicized 0442-0720: KDGGKYTLESHKELQEAAAKEAAKKDINKIYFMTDVDLGGPTFVLND (SEQ ID NO: 3) 0029-1867: SACOL0029-GGGGSGGGGSGGGGS-SACOL1867 (SEQ ID NO: 55) Peptides and polypeptides bound by IgG from vaccinated mice in an ELISA assay GEHLPKGNIVINTKDGGKYTLESHKELQKDRENVKINTAD (fragment encoded by SACOL0442) (SEQ ID NO: 2) (see sequence in FIG. 211, Item VII-fusions); KDINKIYFMTDVDLGGPTFVLNDKDYERKYKKHIVSQFGFDLKHKKDALA (fragment encoded by SACOL0720) (SEQ ID NO: 271) (see sequence in FIG. 211, item VII-fusions); SACOL1867 (SEQ ID NO: 40) (see his-tagged sequence in FIG. 21 F, Item IV); SACOL0029 (SEQ ID NO: 8) (see his-tagged sequence in FIG. 21A, item I);

[0219] This demonstrates that a combination of fusions (e.g., peptide fusion 0442-0720 mixed with the polypeptide fusion 0029-1867) can be used to immunize and elicit an immune response in a mammal. The obtained immune response includes the production of antibodies that recognize amino acid sequences encoded from either SACOL0442 or SACOL0720 or SACOL0029 or SACOL1867.

Example 15: Materials and Methods for Attenuated Live Mutant

[0220] Bacterial strains and growth conditions. Strains used in Examples 15-25 are listed in Table V. S. aureus ATCC 29213 and its isogenic mutant .DELTA.720 were previously described (Allard ef al. 2013). Except otherwise stated, S. aureus strains were grown in tryptic soy broth (TSB) and agar (TSA) (BD, ON, Canada), and Escherichia coli DH5a were grown in LB and LBA medium (BD). Whenever required, ampicillin (IOO.mu.c/i.eta.I) (Sigma, Oakville. Ontario, Canada), chloramphenicol (20 .mu.g ill) (ICN Biomedicals, Irvine, Calif.), and erythromycin (10 .mu.g/ml) (Sigma) were added to agar plates. For the immunological tests, four different bovine mastitis isolates were selected corresponding to some of the predominant S. aureus spa types found in Canadian dairy herds and elsewhere in the world (Veh ef al., 2015; Mitra ef al., 2013). Strain SHY97-3906 (spa t529) was isolated from a case of clinical bovine mastitis that occurred during the lactation period, and CU08-3 (spa t359) was originally isolated from a cow with persistent mastitis at dry-off (Allard ef al., 2013). Strains Sa3151 (spa t13401) and Sa3181 (spa t267) were obtained from the Canadian Bovine Mastitis and Milk Quality Research Network (CBMMQRN) Mastitis Pathogen Culture Collection (Universite de Montreal, Faculte de medecine veterinaire, St-Hyacinthe, QC, Canada), and were isolated from cases of subclinical intramammary infections.

TABLE-US-00012 TABLE V Strains and plasmids used in Examples 15-25 Strain or plasmid Relevant details Source or reference Strains S. aureus RN4220 Derivative of 8325-4, acceptor of foreign DNA Kreiswirth ef a/. (1983) -- ATCC29213 Wild Type, SACOL0720 (vraG) positive, American Type Culture normal phenotype .DELTA.720 SACOL0720 (vraG) transposon insertion Allard et al. (2013) isogenic mutant of ATCC29213 .DELTA.hemB hemB::EW.sup.r, isogenic mutant of ATCC29213, As described herein SCV phenotype .DELTA.720.DELTA.hemB hemB::EWr, isogenic mutant of .DELTA.720, SCV As described herein phenotype E. coli SHY97-3906 isolated from a case of (spa t529) clinical bovine mastitis CLJ08-3 (spa isolated from a cow with t359) persistent mastitis at dry-off Sa3151 (spa CBMMQRN) Pathogen t13401) Culture Collection Sa318l (spa t267) CBMMQRN) Pathogen Culture Collection DH5a lacZDM15) hsdR17 recA1 endA1 gyrA96 thil Invitrogen (ON, Canada) relA1 Plasmids pBT2 Shuttle vector, temperature-sensitive, AprCmr Bruckner (1997) PBT-E pBT2 derivative, inserted ErmA cassette As described herein pBT2 derivative, for hemB deletion; pBT-EhemB Ap.sup.rCm.sup.rEm.sup.r As described herein

[0221] Cell culture conditions. An established bovine mammary epithelial cell (BMEC) line, MAC-T (Huynh ef a/., 1991), was used as a cell culture model of infection. The MAC-T cells were routinely cultured and maintained in Dulbecco's modified Eagle's medium (DMEM) containing 10% heat-inactivated fetal bovine serum (FBS), supplemented with 5 .mu.g/ml insulin (Roche Diagnostics Inc., Laval, Canada) and 1 Mg/ml hydrocortisone (Sigma), and incubated at 37.degree. C. in a humidified incubator with 5% C02. Cell culture reagents were purchased from Wisent (St-Bruno, QC, Canada).

[0222] DNA manipulations. Recommendations from the manufacturers of kits were followed for genomic DNA isolation (Sigma), plasmid DNA isolation (Qiagen, ON, Canada), extraction of DNA fragments from agarose gels (Qiagen) and purification of PCR products and of digested DNA fragments (Qiagen). An additional treatment of 1 h with lysostaphin (Sigma) at 200 .mu.g/ml was used to achieve efficient lysis of S. aureus cells in genomic and plasmid DNA isolations. Primers (IDT.RTM. Integrated DNA Technologies; Coraville, Iowa, USA) were designed to add restriction sites upstream and downstream of the amplified products. PCRs were performed using the Taq DNA Polymerase (NEB, Pickering, ON, Canada) for routine PCR or the Q5 high fidelity DNA Polymerase (NEB) for cloning, and cycling times and temperatures were optimized for each primer pair. Plasmid constructs were generated using E, coli DH5a (Invitrogen, Burlington, ON, Canada), restriction enzymes (NEB), and the T4 DNA ligase (NEB). Plasmid constructs were validated by restriction digestion patterns and DNA sequencing before electroporation in S. aureus RN4220 (Kreiswirth ef al., 1983) and in final host strains. Plasmids used in Examples 15-25 are listed in Table V above.

[0223] Generation of live attenuated S. aureus strain .DELTA.720 and .DELTA.hemB.

[0224] An isogenic hemB mutant of the ATCC 29213 strain was constructed, in which the hemB gene was deleted and replaced by the insertion of an emrA cassette by homologous recombination. S. aureus ATCC 29213 mutant for gene SACOL0720 (.DELTA.720) was generated using the TargeTron.TM. Gene Knockout System (with the TargeTron.TM. Vector pNL9164 (Sigma-Aldrich Canada Ltd.) (Chen et al., 2007) for disruption of bacterial genes by insertion of group II introns (fragment size of approx. 2 Kb as previously described (Allard et al., 2013) between nucleotide 803 and 804 in S. aureus ATCC29213. The manufacturer protocols and recommendations were followed.

[0225] Generation of .DELTA.hemB .DELTA.720. To achieve a second mutation in gene hemB in order to obtain a SCV phenotype in the .DELTA.720 mutant genetic background, another strategy was used: the temperature-sensitive pBT2-hemB:emrA (pBT-E:hemB) was used in a strategy previously described (Mitchell et al., 2008), with some modifications. Briefly, the pBT-E plasmid was constructed by the insertion of an ermA cassette between Xbal and Sail sites of temperature-sensitive shuttle vector pBT2 (Bruckner, 1997). The flanking regions of gene hemB (SACOL1715) DNA fragments were amplified from S. aureus ATCC 29213 and were cloned on both sides of the ermA cassette into the plasmid pBT-E. The plasmid was then transferred for propagation into S. aureus RN4220 (res-). After bacterial lysis with lysostaphin (200 Mg/ml for 1 h at room temperature), plasmid DNA was isolated and used to transform ATCC 29213 and .DELTA.720 by electroporation. For plasmid integration and mutant generation, bacteria were first grown overnight at 30.degree. C. with 10 .mu.g/ml of erythromycin and a 1 .mu.g/ml hemin supplementation (Sigma-Aldrich, ON, Canada). Bacteria were then diluted 1:1000 and grown overnight at 42.degree. C. with 2.5 .mu.g/ml of erythromycin and 1 .mu.g/ml hemin. This step was repeated twice. Finally, bacteria were diluted 1:1000 and grown overnight at 42.degree. C. without antibiotics. Mutants with the inactivated hemB gene were selected as resistant to erythromycin and sensitive to chloramphenicol, together with an SCV phenotype that can be complemented (i.e., reversion to the normal growth phenotype) by a 5 Mg/ml hemin supplementation on agar plates. The deletion of hemB in the ATCC 29213 (i.e., .DELTA.hemB) and .DELTA.720 (i.e., .DELTA.hemB.DELTA.720) strains was confirmed by PCR (see FIGS. 8A and B).

[0226] Hemin supplementation in broth culture. To evaluate the capacity of hemin to restore optimal growth kinetics of S. aureus .DELTA.hemB and the double mutant .DELTA.72.DELTA.hemB, overnight bacterial cultures were diluted to an .DELTA..sub.600 nm of approximately 0.1 in culture tubes containing fresh BHI supplemented with hemin (Sigma) added at various concentrations. The A.sub.600 nm of cultures was monitored at different points in time during the incubation period at 35.degree. C. (225 rpm).

[0227] S. aureus Infection of Bovine Mammary Epithelial Cells (BMECs).

[0228] MAC-T BMECs were used for the characterization of intracellular infectivity and persistence of ATCC 29213 (WT) and its isogenic mutants. Forty-eight hours before infection, 1.times.10.sup.5/ml MAC-T cells were seeded on treated 24-well plates (Corning) to obtain 30% confluence. Monolayers were grown to confluence under 10% CO.sub.2 at 37.degree. C. Six hours prior to infection, monolayers were washed with DMEM and incubated with invasion medium (IM) (growth medium without antibiotics containing 1% heat-inactivated FBS). Overnight bacterial cultures were diluted 1:20 in fresh TSB and grown to mid-logarithmic growth phase, then washed with PBS and diluted in IM to a multiplicity of infection of 10. Invasion was achieved by incubating monolayers with bacteria for 3 h. Monolayers were then washed with DMEM and incubated with IM containing 20 .mu.g/ml lysostaphin to kill extracellular bacteria. The use of lysostaphin to kill extracellular normal and SCV S. aureus was previously validated in cell invasion assays (Moisan ef a/., 2006 and Tuchscherr ef a/, 2011). The treatment was allowed for 30 min to determine CFUs at 3 h of infection, or for an additional 12 or 24 h. Then, following extensive washing with Dulbecco's Phosphate-Buffered Saline (DPBS), monolayers were detached with trypsinization and lysed with 0.05% Triton X-100 and PBS was added to obtain a final I X concentration. The lysate was serially diluted and plated on TSA for CFUs determination.

[0229] BMECs viability and metabolic activity assay. To determine the cytotoxic damage inflicted by S. aureus ATCC 29213 (WT) and its isogenic mutants on MAC-T cells, the MTT cell metabolic activity assay that measures the reduction of 3-[4,5-dimethylthiazol-2-yl]-2,5 diphenyl tetrazolium bromide (MTT) into an insoluble formazan product in viable cells, was performed. The assay followed the method of Kubica ef a/. (Kubica ef a/, 2008) with some modifications. Briefly, S. aureus infection of cells was achieved as described in the persistence assay, but instead of lysis after 12 h or 24 h, cells were incubated with 100 .mu.I of MTT reagent (5 mg/ml) (Sigma) in DPBS for 2 h at 37.degree. C. Following this, an acidic solvent solution of 16% SDS and 40% PMF, pH 47, was added to lyse the cells and solubilize the crystals of formazan overnight. The samples were read using an Epoch microplate reader (Biotek Instruments Inc.) at a wavelength of 570 nm. All assays were performed in triplicate, and control wells with uninfected cells (high viability control) or lysed WT infected cells (bacteria background control; treated with 0.05% triton X-100 for 10 min before MTT addition) were included to each plate. The level of metabolic activity was calculated using the following formula:

(absorbance of the sample-background control)/high control)*100.

[0230] Virulence in the mouse mastitis model. The mouse mastitis model of infection is based on that previously described (Brouillette, 2005; Brouillette, 2004). All the experiments performed with mice were approved by the ethics committee on animal experimentation of the Faculte des sciences of the Universite de Sherbrooke and were conducted in accordance with the guidelines of the Canadian Council on Animal Care.

Briefly, one hour following removal of 12-14 day-old offspring, lactating CD-1 mice (Charles River Laboratories) were anesthetized with ketamine and xylazine at 87 and 13 mg/kg of body weight, respectively, and mammary glands were inoculated under a binocular. Mammary ducts were exposed by a small cut at the near ends of teats and a 100 .mu.l-bacterial suspension containing 10.sup.2 CFUs in endotoxin-free phosphate-buffered saline (PBS, Sigma) was injected through the teat canal using a 32-gauge blunt needle. Two glands (fourth on the right [R4] and fourth on the left [L4] from head to tail) were inoculated for each animal. Mammary glands were aseptically harvested at the indicated times, weighed and visually evaluated for inflammation. Bacterial burden was evaluated after mechanical tissue homogenization in PBS, serial dilutions, and plating on agar for CFU determination. In a second experiment, homogenized glands were conserved for protein extraction for myeloperoxidase (MPO) activity enzymatic assays.

[0231] Mammary Gland Protein Extraction.

[0232] Total protein extraction from mammary glands was performed by an optimized method previously described (Pulli ef a/., 2013), with some modifications. Mammary tissues were homogenized in a buffer containing a final concentration of potassium phosphate of 50 mM, pH 6.0, and hexadecyltrimethylammonium bromide (CTAB) 50 mM (Sigma). The samples were then sonicated, freeze-thawed in liquid nitrogen, and centrifuged at 2000 g for 15 min at 4.degree. C. Finally, the fat layer was removed by aspiration, and supernatants were saved for a final centrifugation of 15 min at 15 000 g, to discard every cellular debris. Supernatants were distributed in aliquots and kept at -80.degree. C. until use for the enzymatic assays or protein concentration determination as measured by the bicinchoninic acid method (BCA) Protein Assay Kit (Thermo-Scientific).

[0233] MPO Activity Assay.

[0234] Neutrophil recruitment in mammary tissues was measured by quantification of MPO enzyme activity by the o-dianisidine-H.sub.2O.sub.2 method, modified for microplates (Bradley, RD, and Rothstein. GPPC, 1982). In a 96-well microplate, 10 .mu.I of tissue extraction supernatants were incubated with a solution of o-dianisidine hydrochloride (0.0.167 mg/mL) (Sigma) and 0.0005% H.sub.2O.sub.2 (Sigma) in 50 mM CTAB phosphate buffer 50 mM, pH 6.0. The MPO activity was measured kinetically with intervals of 15 s over a period of 5 min in an Epoch microplate reader at 460 nm. A Unit of MPO was considered as the amount of enzyme that degrades 1 .mu.mol of H.sub.2O.sub.2/min at 25.degree. C., assuming an absorption coefficient of 11.3 mM.sup.-1 cm.sup.-1 at 460 nm for o-dianisidine (Zhang ef a/., 2004). Results were expressed as units of MPO per g of gland.

[0235] Mouse immunizations with the live attenuated mutant .DELTA.720.DELTA.hemB. The immunogenic properties of the attenuated strain .DELTA.720.DELTA.hemB administered as a live vaccine were evaluated in mice. In preliminary studies, the mice well tolerated intramuscular and subcutaneous (SC) injections of the attenuated strain. The doses of 10.sup.6, 10.sup.7 and 10.sup.8 CFUs and the SC route were selected for subsequent experiments. For the preparation of bacterial inoculum, S. aureus .DELTA.720.DELTA.hemB colonies previously grown on BHIA plates were washed twice in ice cold PBS and suspended in PBS containing 15% glycerol, then aliquoted and kept at -80.degree. C. until subsequent use. The viable bacterial counts in the inoculum preparation was validated by serial dilution plating on BHIA. CD-1 mice were randomly divided into 3 groups: group 1 (n=3) received a dose of 10.sup.6 CFUs; group 2 (n=3), 10.sup.7 CFUs, and group 3 (n=3), 10.sup.8 CFUs. Mice were immunized by two subcutaneous injections of bacteria in PBS (100 .mu.l), in the neck, two weeks apart. Blood samples were taken just before the priming injection (preimmune serums) and ten days after the boost immunization (immune serums). Blood aliquots were allowed to clot at room temperature for an hour and then centrifuged at 10,000 g for 10 min at 4.degree. C. The serums were collected and kept at -20.degree. C. until subsequent analysis.

[0236] Preparation of S. aureus Cell Extracts.

[0237] Preparation of S. aureus whole cell extracts was done as previously described with some modifications (Asli ef a/., 2016). Briefly, overnight bacterial cultures were diluted 1/1000 in fresh BHI broth, and then incubated at 35.degree. C. (225 rpm) until an .DELTA.600 nm of -0.8 was reached. Bacterial cells were centrifuged and pellets were washed in ice-cold PBS twice and suspended with the addition of 5 ml of PBS per ml of pellet. Bacterial suspensions were first treated with lysostaphin (Sigma) (100 .mu.g/ml of pellet) for 1 h at 37.degree. C., and then 3 .mu.g of protease inhibitor cocktail (Sigma), 8 .mu.g of RNAse A (Sigma) and 8 .mu.g of DNAse (Qiagen) per ml of pellet were added to the suspension. After 30 min at room temperature, cells were mechanically disrupted by 3 to 4 passages in a SLM Aminco.TM. French Pressure cell disrupter, and then centrifuged at 12,000.times.g and 4.degree. C. for 10 min to remove unbroken cells. Supernatant was collected and total protein concentration was determined as previously described with the BCA Protein Assay Kit.

[0238] Detection of Mouse Total IgG by ELISA.

[0239] Detection of serum total IgG against the .DELTA.720.DELTA.hemB vaccination strain and each of the bovine IMI isolates was performed to demonstrate and measure the systemic humoral response generated by the immunization of mice. For target antigens. Nunc MaxiSorp.TM. 96-well plates (Thermo Fisher Scientific Inc., Rochester, N.Y.) were coated with 100 .mu.l of each of the whole S. aureus cell extracts (10 Mg/ml diluted in carbonate/bicarbonate buffer, Sigma), and incubated overnight at room temperature. The plates were then saturated with PBS containing 5% skim milk powder for 1 h at 37.degree. C., followed by a second blocking step with an addition of 5% porcine serum to prevent unspecific S. aureus protein A interactions. One hundred microliters of two-fold serial dilutions of the sera in the dilution buffer (PBS with 2% milk, 5% porcine serum and 0.025% Tween.TM. 20) were loaded into the plates and incubated for 1 h at 37.degree. C. Plates were then washed three times with PBS containing 0.05% Tween.TM. 20, and loaded with 100 .mu.I of horseradish peroxidase (HRP)-conjugated goat anti-mouse IgG (Jackson ImmunoResearch Laboratories Inc., West Grove, Pa.) diluted 1/5000 in the dilution buffer. After 1 h of incubation at 37.degree. C. followed by 3 washes, peroxidase activity was detected with 3,3',5,5'-tetramethylbenzidine (TMB) reagent (KPL Inc., Gaithersburg, Md.) according to the manufacturer's recommendations.

[0240] Statistical analysis. Statistical analyses were carried out with the GraphPad Prism.TM. software (v.6.02). Intracellular bacterial CFUs and bacterial CFUs/g of gland (IMI in mice) were transformed in base 10 logarithm values before being used for statistical analyses. Statistical tests used for the analysis of each experiment and significance are specified in the figure legends.

Example 16: Construction of Strain S. aureus ATCC 29213 .DELTA.720, .DELTA.hemB and .DELTA.720.DELTA.hemB

[0241] Live attenuated organisms that mimic natural infection stimulate the immune system in a powerful manner, eliciting broad and robust immune responses that produce both serum and mucosal antibodies, and effector and memory T cells which act synergistically to protect against disease (Detmer, 2006; Kollaritsch, 2000; Pasetti, 2011).

[0242] A mutation in gene SACOL0720 was shown to alter the virulence of S. aureus in experimental IMI infections in the cow (Allard et al., 2013).

[0243] Further live-attenuated strains were prepared for vaccine purposes based on the phenotypic aspects of S. aureus SCVs. SCVs do not generally generate invasive infections (i.e. additional attenuation) and can be internalized in host cells and therefore will stimulate the cell-mediated immune response in addition to the humoral immune response.

[0244] A stable S. aureus SCV was first created through the deletion of the hemB gene (.DELTA.hemB) (see Example 15, Generation of live attenuated S. aureus strain .DELTA.720 and .DELTA.hemB). Further attenuation of this SCV was then achieved by inactivation of gene SACOL0720 (.DELTA.720) (see Example 15. Generation of .DELTA.hemB.DELTA.720).

[0245] After infection of MAC-T bovine mammary epithelial cells, the double mutant (.DELTA.720 .DELTA.hemB) significantly showed lower internalization and cell destruction compared to that seen with .DELTA.hemB and .DELTA.720, respectively.

Example 17: Strain S. aureus .DELTA.hemB.DELTA.720 is Attenuated in MAC-T Cells

[0246] The infectivity of ATCC 29213 (WT), .DELTA.720, .DELTA.hemB and .DELTA.hemB.DELTA.720 strains were then compared in intracellular persistence assays using MAC-T cells. By comparing the three mutant strains to their isogenic WT parent, distinct effects of mutations in genes hemB and SACOL0720 were observed. A short 3-h incubation of bacteria with cell monolayers followed by the addition of lysostaphin to eliminate extracellular bacteria demonstrated high levels of internalization into MAC-T cells for both WT and .DELTA.hemB strains, based on the recovery of viable intracellular bacteria (CFUs) (FIGS. 9A and B). The single .DELTA.720 mutant however showed significantly less (P.ltoreq.0.01) internalization compared to its parental WT strain (FIG. 9A). The reduction in internalization seen in .DELTA.720 was even more pronounced when comparing the double mutant .DELTA.hemB.DELTA.720 to .DELTA.hemB, with a 10-fold reduction of inoculum recovery in this 3-h internalization assay (P<0.001, FIG. 9B). This initial reduction of internalized bacterial load was still apparent 12 and 24 h post invasion (PI) for the double mutant strain .DELTA.hemB.DELTA.720 (FIG. 9C), as illustrated by the 1-log.sup.10 reduction of CFU/ml at both time points compared to that observed for .DELTA.hemB (P<0.001). The difference in initial intracellular bacterial loads between the single .DELTA.720 mutant and WT strains (FIG. 9A) gradually vanished with longer incubation times (FIG. 9C), as both strains did not well persist in MAC-T cells (FIG. 10). On the opposite, intracellular CFUs recovered for the single .DELTA.hemB strain was significantly higher compared to that recovered for the three other strains at 24 h PI (FIG. 9C. P.ltoreq.0.001 against all). Overall and as expected for the SCV phenotype, the .DELTA.hemB strain showed a higher intracellular persistence than any other strain over time (FIG. 10). These results suggest that the .DELTA.720 mutation mainly reduces the internalization process into MAC-T cells. Results further demonstrate that the .DELTA.hemB.DELTA.720 mutant is still capable of internalization and persistence into BMECs but at a much lower degree than that seen with the single .DELTA.hemB mutant.

[0247] The .DELTA.hemB.DELTA.720 and .DELTA.hemB SCVs cause low BMEC disruption. As reported above, .DELTA.hemB and .DELTA.hemB.DELTA.720 SCV strains showed a greater persistence over time in MAC-T cells, as illustrated by their sustained viability at 12 and 24 h PI in comparison with WT and .DELTA.720 strains (FIGS. 9C and 10). The percentage of the inoculum recovered from cells stayed nearly the same from 0 to 24 h after lysostaphin addition, both for the double and single hemB mutants, with a slight increase at 12 h, indicating intracellular growth (FIG. 10). Both strains started to decrease at a slow rate after this time of infection. However, the apparent reduction of intracellular CFUs for the WT and .DELTA.720 strains was concomitant with the visual observation of increasing damage to cell monolayers over time, in comparison to that observed with strains of the SCV phenotypes.

[0248] MAC-T Cells Viability was Also Evaluated Following Infection by Each of the Four Strains Studied.

[0249] MAC-T cell viability was evaluated by the MTT method (Kubica et al., 2008). Results show that both SCV strains (.DELTA.hemB and .DELTA.hemB.DELTA.720) caused significantly less MAC-T killing in this assay in contrast to the WT and .DELTA.720 strains. When compared to .DELTA.hemB, the WT strain nearly reduced by half the viability of cells at 12 h (FIG. 11 A: WT: 25.4%; .DELTA.hemB: 48.4%). This difference was still apparent at 24 h (FIG. 11 B: 16.3% vs. 34.5%, respectively), even if the bacterial load was 10 times higher for the .DELTA.hemB mutant (FIG. 9C). The MAC-T cells were more damaged by .DELTA.hemB than by the double mutant .DELTA.720.DELTA.hemB but the difference was only significant at 24 h (P<0.01). When compared directly to the WT strain, the double mutant .DELTA.720.DELTA.hemB sustained epithelial cells viability 2.3 times more at 12 h (FIG. 11 A) and 2.7 times more at 24 h (FIG. 11B) (12 and 24 h: P<0.0001). Therefore, the greater intracellular persistence of both SCVs strains compared to the WT and .DELTA.720 strains over time (FIG. 10) was likely to be attributed to a lower toxicity of the SCVs to MAC-T cells (FIG. 11). Taken together, results from the BMEC infection assays provided evidence of an additive effect of both .DELTA.hemB and .DELTA.720 mutations for the attenuation of the WT strain.

Example 18: Strain S. aureus .DELTA.hemB.DELTA.720 is Attenuated in a Mouse IEM Model

[0250] To attest attenuation of .DELTA.hemB.DELTA.720 in an in vivo model of infection, the virulence of the double mutant was evaluated and compared to the WT strain in a murine IMI model (Brouillette and Malouin, 2005). For both strains, the exponential phase of infection took place mainly within the first 12 h post-infection, while the maximal bacterial burden was reached at 24 h for the double mutant and 48 h (day 2 [D2]) for the WT strain (FIG. 12). At 24 h, the double mutant showed a reduction of 1.9 log.sup.10 in mean CFU/g of gland compared to WT (P<0.05). Also after 24 h, the mutant bacterial burden showed a constant decline until complete bacterial clearance was reached at day 12 (shown by the asterisk on FIG. 12). In contrast, the parental WT strain provoked severe invasive infections compared to the mutant, killing 3 of 9 remaining mice at day 2 and 2 of 3 mice at day 7 (FIG. 12; arrows) before glands could be harvested for those groups. Mice surviving the WT infection maintained high viable counts (9 log.sup.10 CFU/g of gland) at day 7, an approximate 5 log.sup.10 difference in bacterial burden compared to the double mutant. These results clearly demonstrate a markedly reduced capacity of strain .DELTA.hemB.DELTA.720 to multiply and survive in the mammary gland. The .DELTA.hemB.DELTA.720 double mutant is therefore strongly attenuated in a mouse intramammary infection (IMI) model and is efficiently cleared from mammary glands.

[0251] The attenuated strain .DELTA.hemB.DELTA.720 appears ideal for vaccination purposes and for intracellular delivery of antigens. Indeed, the low and temporary internalization of .DELTA.hemB.DELTA.720 should help stimulation of cell-mediated immunity, a component of the immune response that is important for defense against S. aureus (Fowler and Proctor, 2014).

Example 19: Inflammatory Response to .DELTA.720.DELTA.hemB and WT Strains Following IMI

[0252] To monitor the inflammatory response (immune response) of the mice to infections with WT and mutant strains, neutrophil infiltration in glands was evaluated by the MPO enzymatic activity of total protein extracts of gland homogenates. MPO activity in biological samples has previously been strongly correlated with absolute number of neutrophils (Xia, 1997), and is hence an adequate marker. During the first hours after infection, neutrophil recruitment followed similar profiles for the double mutant and WT infected glands (FIG. 13), with exponential intensification of apparent neutrophil infiltration from 12 h to 24 h post infection coinciding with bacterial growth albeit with a certain delay. The absolute numbers of polymorphonuclear cells in relation with the bacterial load in mammary glands was previously shown to not always peak at the same time (Brouillette, 2005). No significant difference in MPO activity could be observed at 6, 12 and 24 h between glands infected by mutant and WT strains (FIG. 13). This equivalence in apparent neutrophil infiltration did not however correlate with the visual observation of inflammation at 24 h, at which point the WT infection generated extensive redness of infected glands in comparison to the double mutant (photographs of FIG. 14). On the contrary, mutant infected glands were not visually altered on the macroscopic level compared to PBS controls. The disparity between visual assessment of inflammation and neutrophil infiltration results could be attributed to the differences in bacterial loads (FIGS. 9A-C) and the cytotoxic activity of the WT strain (FIG. 11), and could be coherent with the highly invasive and disseminative capacity of the strain via toxins and enzymes expression. Hence, these results indicate that neutrophil recruitment in the glands infected by the mutant strain was equivalent to that seen with the WT strain and that this was sufficient to allow a subsequent decline and clearance of the mutant bacterial loads.

[0253] Lastly, to confirm strain safety, and to assess that this inflammatory response was not consequent to an inadmissible reactogenic strain, MPO activity was monitored in M2QkhemB infected glands 4 and 12 days after infection. The level of activity was then compared to levels obtained with PBS injected mice. As illustrated in FIG. 15, the apparent neutrophil presence in mutant infected glands was still high 4 days after infection, with MPO activity ranging from 8 to 21 Units/g of gland. Besides, gland involution, the process by which the lactating gland returns to a morphologically near pre-pregnant state, is ordinarily associated with neutrophilic recruitment that allows phagocytosis of apoptotic cells during the remodelling of tissue (Stein, 2007). In the days following infection in this model, mice glands are already in that normal state of modification, as indicated by their rapid shrinking. However, the MPO levels in mutant infected glands went through a substantial decline between day 4 and 12, (P<0.01). MPO levels were then considered to be back to a normal level at day 12 showing no significant difference from that obtained with the PBS-injected mice. The inflammatory response of L720 hemB infected glands goes back to normal levels with bacterial clearance (FIG. 15).

Example 20: Immunization with .DELTA.720.DELTA.hemB Generates a Strong Humoral Response Against Several S. aureus Bovine Intramammary Infection Isolates

[0254] To confirm that immunization with the live .DELTA.720.DELTA.hemB can indeed generate a strong immune response suitable for its use as a putative live vaccine against S. aureus intramammary infections, mice were immunized with different doses of the live vaccine and serum total IgGs were assayed for binding to whole cell extracts of a variety of S. aureus bovine isolates. First, doses of 10.sup.6, 10.sup.7 and 10.sup.8 CFUs, administered subcutaneously in the neck, triggered no adverse effect such as modification of mice behavior or signs of inflammation or necrosis at the immunization site throughout the immunization period. Furthermore, immunization using increasing amounts of the live double mutant ATCC 29213 .DELTA.720.DELTA.hemB yielded increasing titers of systemic IgG antibodies against a whole cell extract of its own antigens (FIG. 15B). The titers of the immune sera were significantly higher than those of the preimmune sera, demonstrating specificity of antibody production against S. aureus antigens present in the live vaccine. Most importantly, immunization using increasing amounts of .DELTA.720.DELTA.hemB also yielded a consequential rise of antibody titers against a variety S. aureus strains isolated from bovine mastitis, including strains from the major spa types found in Canada and elsewhere in the world (FIG. 15C). These results clearly show that (i) immunization with the double mutant can raise an immune response, and that (ii) the strain background (ATCC 29213) share sufficient common features with bovine mastitis strains so that the antibody response also strongly recognizes strains of major spa types.

[0255] Immunization of mice using subcutaneous injections of live .DELTA.720.DELTA.hemB raised a strong humoral response as judged by the high titers of total IgG measured against a whole bacterial cell extract. Also, the vaccine strain .DELTA.720.DELTA.hemB had sufficient common features with bovine mastitis strains so that the antibody response also strongly recognized strains from a variety of common mastitis associated spa types.

[0256] Although this demonstrated that the double mutant background (ATCC 29213) share many common features with bovine mastitis strains, such a double mutant can be created in any desired genetic background if one wishes, notably in any strain that was isolated from bovine mastitis, such as but not limited to S. aureus strain RF122.

[0257] These results show that a SCV strain having some residual intracellular capabilities can allow immune cells recruitment without establishing a severe infection. Such an SCV strain may act as a live-attenuated vaccine that adequately stimulates the immune response to combat pathogens with intracellular abilities.

Example 21: Material and Methods--SACOL0442, SACOL0720, SACOL0029 and a Fusion Between SACOL1867 and SACOL0029+Attenuated Live Bacteria (Vaccine #7)

[0258] Production of the antigens. The production of antigens was performed as described in Example 6, in the section production of the antigens except for the additional presence of the antigen SACOL0029. His-tagged recombinant proteins of SACOL0029 were engineered and produced by GenScript. Inc. (Piscataway, N.J.). (see FIG. 21A, item 1, his-tagged sequence).

[0259] Generation of live attenuated S. aureus strain .DELTA.720.DELTA.hemB. The generation of live attenuated S. aureus strain was performed as described in Example 15 (Generation of .DELTA.hemB.DELTA.720).

[0260] Immunization of mice. The immunogenic properties of recombinant S. aureus proteins encoded by the SACOL0442, SACOL0720, SACOL0029 genes and a fusion of SACOL0029 and SACOL1867 genes in combination, or not, with the live attenuated bacterial strain S. aureus .DELTA.720.DELTA.hemB were evaluated in mice. The mice well tolerated a dose of 10.sup.3, 10.sup.5, 10.sup.6, 10.sup.7 and 10.sup.8 CFU by subcutaneous injections in the neck and intramuscular injections in the thigh. The dose of 10.sup.5 and the subcutaneous route were selected for the following experiments.

[0261] For the preparation of bacterial inoculum. S. aureus .DELTA.720.DELTA.hemB colonies previously grown on BHIA plates were washed twice in ice cold PBS and resuspended in PBS containing 15% glycerol, then aliquoted and kept at -80.degree. C. until subsequent use. To obtain the final mice immunization dose, corresponding to 10 CFU of attenuated bacteria, the frozen inoculum bacterial concentration was evaluated by serial dilution plating on BHIA and then was diluted to a final concentration of 105 CFU/ml in PBS on the day of immunization.

[0262] For the preparation of protein doses. SACOL0029, SACOL0442, SACOL0720, and the SACOL0029-1867 fusion polypeptide were mixed and suspended in PBS to obtain a final dose of 5 g each. CD-1 female mice were randomly divided into 3 groups: group 1 (5 mice) received a mixed protein dose (protein Mix); group 2 (5 mice) received an attenuated bacteria (.DELTA.720.DELTA.hemB) dose (.DELTA.720.DELTA.hemB); group 3 (6 mice) received a combination of mixed proteins and attenuated bacteria (combination). CD-1 female mice were immunized by two subcutaneous injections in the neck two weeks apart. The proteins and bacterial strains doses were diluted in PBS as previously described and administered in a final volume of 100 .mu.I for each group of mice. No adverse side effects were observed during the totality of the experimental immunization period. Blood samples were taken just before the first priming injection (preimmune serums) and ten days after the boost immunization (immune serums). The blood aliquots were allowed to clot at room temperature for an hour, centrifuged at 10,000 g for 10 min at 4.degree. C. The sera were harvested and kept at -20.degree. C. until subsequent analysis.

[0263] Detection of total IgG, lgG1 and lgG2 by EUSA. Detection of serum total IgG, lgG1 and lgG2 against each of the antigens previously used for immunization was performed as previously described with some modifications (Ster et al., Vef. Immunol. Immunopathol. (2010), 136: 311-318). In addition, detection of IgG against staphylococcal surface protein ClfA was performed to demonstrate the supplementary advantages of using a live strain to enhance and balance the immune response against S. aureus. Nunc MaxiSorp.TM. 96-well plates (Thermo Fisher Scientific Inc., Rochester, N.Y.) were coated with 75 .mu.l of each of the test antigen (6.67 .mu.c/.eta..GAMMA. diluted in carbonate/bicarbonate buffer, Sigma Aldrich, Oakville, ON) and incubated overnight at room temperature. The plates were then saturated with PBS containing 5% skim milk powder for 1 h at 37.degree. C. One hundred microliters of four-fold serial dilutions of the sera in PBS containing 3% milk and 0.025% Tween.TM. 20 were loaded into the plates and incubated for 1 h at 37.degree. C. The plates were washed three times with PBS containing 0.05% Tween.TM. 20. One hundred microliters of horseradish peroxidase (HRP)-conjugated secondary antibody were then added to the plate. The secondary antibodies used were a goat anti-mouse IgG, lgG2a and lgG1 (Jackson ImmunoResearch Laboratories Inc., West Grove, Pa.), diluted 1/5000 respectively in PBS containing 3% milk and 0.025% Tween.TM. 20. After 1 h of incubation at 37.degree. C. followed by 3 washes, peroxidase activity was detected with 3,3',5,5'-tetramethylbenzidine (TMB) reagent (KPL Inc., Gaithersburg, Md.) according to the manufacturer's recommendations.

[0264] Statistical analysis. Statistical analysis of the antibody titers and of the correlation was performed using GraphPad Prism.TM. v6.05.

Example 22: The Fusion of Antigens and the Combination with a Live Attenuated S. aureus Strain Induces High Antibody Titers in Mice--Vaccine #7

[0265] The antigens and live attenuated S. aureus strain .DELTA.720.DELTA.hemB are produced as described in Example 21. Mice are immunized and IgGs detected as described in Example 21.

[0266] The results in FIG. 16 show that immunization with the SACOL0029-1867 fusion either when co-administered with other antigens or with a live attenuated strain) induces high and specific antibody responses in mice.

Example 23: The Live Attenuated S. aureus Strain Significantly Improves Antibody Immune Response Against Some Specific Antigens--Vaccine #7

[0267] The antigens and live attenuated S. aureus strain .DELTA.7202hemB are produced as described in Example 21. Mice are immunized and IgGs detected as described in Example 21.

[0268] The results in FIG. 17 show that immunization with the attenuated live strain .DELTA.720.DELTA.hemB significantly increases the production of specific IgG antibodies against the SACOL0029 antigen, in comparison to that obtained with IgG antibodies from mice immunized with the protein mix alone.

Example 24: The Live Attenuated S. aureus Strain Induces Significant Antibody Titers Against Additional Surface Proteins of S. aureus--Vaccine #7

[0269] The antigens and live attenuated S. aureus strain .DELTA.720.DELTA.hemB are produced as described in Example 21. Mice are immunized and IgGs detected as described in Example 21.

[0270] The results in FIG. 18 show that immunization with the attenuated live strain .DELTA.720.DELTA.hemB (alone or when co-administered with polypeptide antigens) significantly increases the production of specific antibodies against the staphylococcal surface protein ClfA, compared to that achieved with the protein mix alone composed of SACOL0029, SACOL0442, SACOL0720, and SACOL0029-1867.

Example 25: The Live Attenuated S. aureus Strain Significantly Balances the Th1/Th2 Immune Response--Vaccine #7

[0271] The antigens and live attenuated S. aureus strain .DELTA.720.DELTA.hemB are produced as described in Example 21.

[0272] Serum lgG2a and IgG1 isotypes against the SACOL0029-1867 fusion protein were detected in serums of vaccinated mice as previously described and the ratio of lgG2a to lgG1 titers of each mouse was determined, lgG2a isotype is associated with the Th1 immune response in mice, whereas lgG1 is a marker for the Th2 response. As described in Example 5, the induction of lgG2 production in cows and the extent of the lgG2 titers in milk significantly correlates with protection of the cows against a challenge with S. aureus, as judged by the levels of the corresponding somatic cells (SCC) or bacterial counts (CFU) in milk of the cows (FIG. 4C).

[0273] The results shown in FIGS. 19 and 20 demonstrate that the attenuated live strain .DELTA.720.DELTA.hemB included in the combination immunization vaccine (.DELTA.720.DELTA.hemB S. aureus administered with SACOL0029, SACOL0442. SACOL0720, and SACOL0029-1867) induces a significantly higher lgG2a/lgG1 antibody ratio against the SACOL0029-1867 fusion and SACOL0029 proteins than that seen with the protein mix immunization (SACOL0029, SACOL0442. SACOL0720, and SACOL0029-1867), resulting in a significantly more balanced Th1/Th2 response.

[0274] Examples 21 to 25 above show that even if a strong antibody response was obtained by the immunization with different antigens (including e.g., SACOL0029-1867 fusion) in a protein mix composition, the immunization of mice with a combination of these antigens with a live attenuated strain significantly improved immune responses against S. aureus, by inducing higher antibody titers against some specific antigens (e.g., SACOL0029), by the production of antibodies against other staphylococcal proteins (e.g., ClfA), and by achieving a more balanced lgG2a/lgG1 ratio, a good marker of a stronger Th1 type response, against the antigens coadministered with the live strain.

Example 26: Expression of Recombinant Proteins in Strain S. aureus .DELTA.hemB.DELTA.720--Vaccines #8, 9, 10 Etc.

[0275] Genes SACOL0442, SACOL0720, SACOL0029, and/or SACOL1867 as well as the fusion (e.g., size of 50 AA or more) of the genes (or of fragments thereof) SACOL0029 and SACOL1867 (SACOL0029-SACOL1867), fusions of fragments (e.g., epitopes) of SACOL720 and/or of SACOL0442 (fusion 720-720) (fusion 442-720) or any other fusion of genes or fragments thereof e.g., SACOL0029-SACOL0442. SACOL0029-SACOL0720, SACOL0029-SACOL0720-SACOL0442. SACOL0029-SACOL0720-SACOL1867. SACOL0029-SACOL1867-SACOL0442 SACOL0442-SACOL0029-SACOL0720, SACOL0442-SACOL0029-SACOL0720, SACOL0442-SACOL1867-SACOL0720, SACOL0720-SACOL0442-SACOL1867, SACOL04029-SACOL1867-SACOL0720-SACOL0442, are cloned in plasmid pCN36 (Charpentier ef a/., 2004) under a constitutive promoter (PblaZ from plasmid pCN40) (Charpentier ef a/., 2004) and expressed in the S. aureus .DELTA.hemB.DELTA.720 strain. Certain protein antigens proposed herein are predicted to be an exotoxin, enterotoxin or superantigen (e.g., SACOL0442) or proteins useful for protection against host defenses (e.g., SACOL0720) and could potentially interfere with the mammalian immune system and antibody production, and/or show some toxicity in the host. Although such interference was not observed with the vaccine composition and formulations described here, it may be useful to modify the protein or polypeptide expressed in the S. aureus .DELTA.hemB.DELTA.720 strain so that the cloned genes do not complement its virulence. For such a purpose, it is possible to use molecular biology techniques to delete or mutate the putative region(s) involved in such protein activity without losing immunogenicity (Chang ef a/., 2008). This is the approach the applicants used to prepare the antigens of vaccine compositions of the present invention.

[0276] Expression of individual recombinant protein products by each of the S. aureus .DELTA.hemB.DELTA.720 strains carrying one of the constructed expression vectors is validated by LC-MS/MS analyses. Briefly, strains grown in BHI with 15 .mu.g/ml tetracycline to mid-logarithmic phase were centrifuged and pellets were inactivated with ethanol. Samples were kept at -20.degree. C. until cell lysis and trypsin digestion procedures. Samples are incubated with lysostaphin and trypsin at 37.degree. C., followed by cell disruption by mechanical homogenization using glass beads and a beadbeater. Lysates are then centrifuged for 25 min at 13 000 rpm at 4.degree. C. in order to remove cell debris, before following with protein digestion with trypsin, reduction and alkylation were done by standard procedures before sample Injection for protein detection using the MRM method of LC-MS/MS.

[0277] Alternatively, recombinant protein expression Is also confirmed by Western blots of bacterial lysates.

Example 27: Mouse Immunization with Attenuated Strains Expressing Antigens--Vaccines #8, Etc.

[0278] CD-1 female mice are vaccinated by two subcutaneous injections two weeks apart. Each of the S. aureus .DELTA.hemB.DELTA.720 strains carrying or not one of the constructed expression vectors, are diluted in saline and administered in a final volume of 100 .mu.l per dose. Group 1 receives a double-mutant strain alone; group 2 receives a double-mutant strain expressing fusion SACOL0029-1867; group 3 receives a double-mutant strain expressing fusion SACOL0029-0442; group 4 receives a double-mutant strain expressing fusion SACOL0029-0720; group 5 receives a mixture of double-mutant strains, one expressing fusion SACOL0029-1867 and the other expressing fusion SACOL0029-0442; group 6 receives a mixture of double-mutant strains, one expressing fusion SACOL0029-1867 and the other expressing fusion SACOL0029-0720; and group 7 receives a mixture of double-mutant strains. Blood samples are taken just before the first injection and twelve days after the second one. The samples are allowed to clot at room temperature for an hour, then centrifuged at 2 000 g for 10 min at 4.degree. C. The supernatants (sera) are harvested and kept at -20.degree. C. until subsequent analysis. Mice are euthanized at day 27 and blood is collected by cardiac puncture. The immune sera are recovered, aliquoted and stored as for the pre-immune sera.

[0279] The immune response to vaccination is evaluated by enzyme-linked immunosorbent assay (ELISA) for the presence of serum polyclonal IgG antibodies directed towards S. aureus whole cells (Wood strain) or specific recombinant proteins. Anti-mouse IgG-HRP (HRP: horseradish peroxidase) is used as a secondary antibody to detect the colorimetric production of 3.3',5,5'-tetramethylbenzidine (TMB) substrate oxidation by peroxidase activity using a spectrophotometer.

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Sequence CWU 1

1

196115PRTStaphylococcus aureus 1Lys Asp Gly Gly Lys Tyr Thr Leu Glu Ser His Lys Glu Leu Gln1 5 10 15240PRTStaphylococcus aureus 2Gly Glu His Leu Pro Lys Gly Asn Ile Val Ile Asn Thr Lys Asp Gly1 5 10 15Gly Lys Tyr Thr Leu Glu Ser His Lys Glu Leu Gln Lys Asp Arg Glu 20 25 30Asn Val Lys Ile Asn Thr Ala Asp 35 40348PRTArtificial sequencesynthetic sequence 3Lys Asp Gly Gly Lys Tyr Thr Leu Glu Ser His Lys Glu Leu Gln Glu1 5 10 15Ala Ala Ala Lys Glu Ala Ala Ala Lys Lys Asp Ile Asn Lys Ile Tyr 20 25 30Phe Met Thr Asp Val Asp Leu Gly Gly Pro Thr Phe Val Leu Asn Asp 35 40 454168DNAStaphylococcus aureus 4atgctagaat ctagagagca attatcagtc gaagaatacg aaacattctt taacagattt 60gataatcaag aatttgattt cgaacgtgaa ttgacacaag atccatattc aaaagtatac 120ttatacagta tagaagacca tatcagaaca tataagatag agaaataa 168555PRTStaphylococcus aureus 5Met Leu Glu Ser Arg Glu Gln Leu Ser Val Glu Glu Tyr Glu Thr Phe1 5 10 15Phe Asn Arg Phe Asp Asn Gln Glu Phe Asp Phe Glu Arg Glu Leu Thr 20 25 30Gln Asp Pro Tyr Ser Lys Val Tyr Leu Tyr Ser Ile Glu Asp His Ile 35 40 45Arg Thr Tyr Lys Ile Glu Lys 50 556165DNAStaphylococcus aureus 6ctagaatcta gagagcaatt atcagtcgaa gaatacgaaa cattctttaa cagatttgat 60aatcaagaat ttgatttcga acgtgaattg acacaagatc catattcaaa agtatactta 120tacagtatag aagaccatat cagaacatat aagatagaga aataa 165754PRTStaphylococcus aureus 7Leu Glu Ser Arg Glu Gln Leu Ser Val Glu Glu Tyr Glu Thr Phe Phe1 5 10 15Asn Arg Phe Asp Asn Gln Glu Phe Asp Phe Glu Arg Glu Leu Thr Gln 20 25 30Asp Pro Tyr Ser Lys Val Tyr Leu Tyr Ser Ile Glu Asp His Ile Arg 35 40 45Thr Tyr Lys Ile Glu Lys 50861PRTArtificial sequencesynthetic sequence 8Met His His His His His His Leu Glu Ser Arg Glu Gln Leu Ser Val1 5 10 15Glu Glu Tyr Glu Thr Phe Phe Asn Arg Phe Asp Asn Gln Glu Phe Asp 20 25 30Phe Glu Arg Glu Leu Thr Gln Asp Pro Tyr Ser Lys Val Tyr Leu Tyr 35 40 45Ser Ile Glu Asp His Ile Arg Thr Tyr Lys Ile Glu Lys 50 55 60966PRTArtificial sequencesynthetic sequence 9Met Arg Gly Ser His His His His His His Gly Ser Leu Glu Ser Arg1 5 10 15Glu Gln Leu Ser Val Glu Glu Tyr Glu Thr Phe Phe Asn Arg Phe Asp 20 25 30Asn Gln Glu Phe Asp Phe Glu Arg Glu Leu Thr Gln Asp Pro Tyr Ser 35 40 45Lys Val Tyr Leu Tyr Ser Ile Glu Asp His Ile Arg Thr Tyr Lys Ile 50 55 60Glu Lys65101889DNAStaphylococcus aureus 10atgaccttta acgagataat atttaaaaat ttccgtcaaa atttatcaca ttatgccatc 60tatctttttt cgttaattac gagtgtagta ttgtatttta gctttgtagc attaaaatac 120gctcataaac taaacatgac agagtcatat ccaattataa aggaaggctc acaagtcgga 180agctactttc tatttttcat cataattgca tttttgttat atgccaatgt gttatttatt 240aaacgacgaa gttatgagct tgcattatat caaacattag gtttatctaa attcaacatt 300atttatatac taatgctcga acaattacta atatttataa ttacggcaat attaggtatt 360attattggta tttttggttc gaaactgtta ttaatgattg tctttacatt attaggaatt 420aaagaaaagg ttccaattat ttttagtttg agggcggtat ttgaaacatt aatgttaatc 480ggtgtcgctt attttttaac atctgctcaa aattttatat tagtgttcaa acaatctatt 540tcacagatgt caaagaataa ccaggttaaa gaaacaaatc ataataaaat tacatttgaa 600gaggttgttt taggcatctt aggtatagta ttgattacca caggatacta tctatctttg 660aacattgttc aatattatga ttctatcggt acacttatgt ttattttatt gtcaactgtg 720attggggcat acttattttt taaaagctct gtttctctag tttttaaaat ggtgaagaag 780tttagaaaag gtgttataag tgtaaatgat gtcatgttct catcatctat tatgtatcgt 840attaagaaaa atgctttttc acttacggtc atggcaatca tttcagcgat tactgtttca 900gttctttgct ttgctgctat aagtagagcg tccttatcaa gtgaaataaa atatactgca 960ccacacgacg ttacaattaa agaccaacaa aaagctaatc aattagcaag tgaattaaac 1020aatcaaaaaa ttcctcattt ttataattat aaagaagtaa ttcatacgaa attgtataaa 1080gataatttat ttgatgtaaa agcgaaagaa ccatacaatg taacaattac tagtgataaa 1140tacatcccta atactgattt gaaacgtggg caagctgatt tatttgtagc ggaaggttct 1200atcaaagatt tagtgaaaca taagaagcat ggtaaggcaa ttataggaac gaaaaaacat 1260catgttaata ttaagttacg taaagatatt aataaaatct attttatgac agatgttgat 1320ttaggtggac caacgtttgt cttaaatgac aaagactatc aagaaataag aaagtataca 1380aaggcaaagc atatcgtctc tcaatttgga ttcgatttga aacataaaaa agatgcttta 1440gcattagaaa aagcgaaaaa taaagttgat aaatctattg aaacaagaag tgaagcgata 1500agctcaatat caagtttaac cggaatatta ttatttgtaa catcattttt aggtattaca 1560ttcttgattg ctgtatgttg cattatatac ataaagcaaa tagatgaaac cgaagatgag 1620ttagagaatt atagtatttt gagaaagctt ggatttacac aaaaagatat ggcaagggga 1680ctaaagttta aaattatgtt taattttggg ttacctttag ttattgcact atcacatgca 1740tattttacat cattagcata tatgaaatta atgggtacaa cgaatcaaat accggttttc 1800atagtaatgg gattatacat ttgtatgtat gctgtttttg cagtgacggc ttataatcat 1860tccaagcgaa cattagacat tccatataa 188911629PRTStaphylococcus aureus 11Met Thr Phe Asn Glu Ile Ile Phe Lys Asn Phe Arg Gln Asn Leu Ser1 5 10 15His Tyr Ala Ile Tyr Leu Phe Ser Leu Ile Thr Ser Val Val Leu Tyr 20 25 30Phe Ser Phe Val Ala Leu Lys Tyr Ala His Lys Leu Asn Met Thr Glu 35 40 45Ser Tyr Pro Ile Ile Lys Glu Gly Ser Gln Val Gly Ser Tyr Phe Leu 50 55 60Phe Phe Ile Ile Ile Ala Phe Leu Leu Tyr Ala Asn Val Leu Phe Ile65 70 75 80Lys Arg Arg Ser Tyr Glu Leu Ala Leu Tyr Gln Thr Leu Gly Leu Ser 85 90 95Lys Phe Asn Ile Ile Tyr Ile Leu Met Leu Glu Gln Leu Leu Ile Phe 100 105 110Ile Ile Thr Ala Ile Leu Gly Ile Ile Ile Gly Ile Phe Gly Ser Lys 115 120 125Leu Leu Leu Met Ile Val Phe Thr Leu Leu Gly Ile Lys Glu Lys Val 130 135 140Pro Ile Ile Phe Ser Leu Arg Ala Val Phe Glu Thr Leu Met Leu Ile145 150 155 160Gly Val Ala Tyr Phe Leu Thr Ser Ala Gln Asn Phe Ile Leu Val Phe 165 170 175Lys Gln Ser Ile Ser Gln Met Ser Lys Asn Asn Gln Val Lys Glu Thr 180 185 190Asn His Asn Lys Ile Thr Phe Glu Glu Val Val Leu Gly Ile Leu Gly 195 200 205Ile Val Leu Ile Thr Thr Gly Tyr Tyr Leu Ser Leu Asn Ile Val Gln 210 215 220Tyr Tyr Asp Ser Ile Gly Thr Leu Met Phe Ile Leu Leu Ser Thr Val225 230 235 240Ile Gly Ala Tyr Leu Phe Phe Lys Ser Ser Val Ser Leu Val Phe Lys 245 250 255Met Val Lys Lys Phe Arg Lys Gly Val Ile Ser Val Asn Asp Val Met 260 265 270Phe Ser Ser Ser Ile Met Tyr Arg Ile Lys Lys Asn Ala Phe Ser Leu 275 280 285Thr Val Met Ala Ile Ile Ser Ala Ile Thr Val Ser Val Leu Cys Phe 290 295 300Ala Ala Ile Ser Arg Ala Ser Leu Ser Ser Glu Ile Lys Tyr Thr Ala305 310 315 320Pro His Asp Val Thr Ile Lys Asp Gln Gln Lys Ala Asn Gln Leu Ala 325 330 335Ser Glu Leu Asn Asn Gln Lys Ile Pro His Phe Tyr Asn Tyr Lys Glu 340 345 350Val Ile His Thr Lys Leu Tyr Lys Asp Asn Leu Phe Asp Val Lys Ala 355 360 365Lys Glu Pro Tyr Asn Val Thr Ile Thr Ser Asp Lys Tyr Ile Pro Asn 370 375 380Thr Asp Leu Lys Arg Gly Gln Ala Asp Leu Phe Val Ala Glu Gly Ser385 390 395 400Ile Lys Asp Leu Val Lys His Lys Lys His Gly Lys Ala Ile Ile Gly 405 410 415Thr Lys Lys His His Val Asn Ile Lys Leu Arg Lys Asp Ile Asn Lys 420 425 430Ile Tyr Phe Met Thr Asp Val Asp Leu Gly Gly Pro Thr Phe Val Leu 435 440 445Asn Asp Lys Asp Tyr Gln Glu Ile Arg Lys Tyr Thr Lys Ala Lys His 450 455 460Ile Val Ser Gln Phe Gly Phe Asp Leu Lys His Lys Lys Asp Ala Leu465 470 475 480Ala Leu Glu Lys Ala Lys Asn Lys Val Asp Lys Ser Ile Glu Thr Arg 485 490 495Ser Glu Ala Ile Ser Ser Ile Ser Ser Leu Thr Gly Ile Leu Leu Phe 500 505 510Val Thr Ser Phe Leu Gly Ile Thr Phe Leu Ile Ala Val Cys Cys Ile 515 520 525Ile Tyr Ile Lys Gln Ile Asp Glu Thr Glu Asp Glu Leu Glu Asn Tyr 530 535 540Ser Ile Leu Arg Lys Leu Gly Phe Thr Gln Lys Asp Met Ala Arg Gly545 550 555 560Leu Lys Phe Lys Ile Met Phe Asn Phe Gly Leu Pro Leu Val Ile Ala 565 570 575Leu Ser His Ala Tyr Phe Thr Ser Leu Ala Tyr Met Lys Leu Met Gly 580 585 590Thr Thr Asn Gln Ile Pro Val Phe Ile Val Met Gly Leu Tyr Ile Cys 595 600 605Met Tyr Ala Val Phe Ala Val Thr Ala Tyr Asn His Ser Lys Arg Thr 610 615 620Ile Arg His Ser Ile62512200PRTStaphylococcus aureus 12Arg Ala Ser Leu Ser Ser Glu Ile Lys Tyr Thr Ala Pro His Asp Val1 5 10 15Thr Ile Lys Asp Gln Gln Lys Ala Asn Gln Leu Ala Ser Glu Leu Asn 20 25 30Asn Gln Lys Ile Pro His Phe Tyr Asn Tyr Lys Glu Val Ile His Thr 35 40 45Lys Leu Tyr Lys Asp Asn Leu Phe Asp Val Lys Ala Lys Glu Pro Tyr 50 55 60Asn Val Thr Ile Thr Ser Asp Lys Tyr Ile Pro Asn Thr Asp Leu Lys65 70 75 80Arg Gly Gln Ala Asp Leu Phe Val Ala Glu Gly Ser Ile Lys Asp Leu 85 90 95Val Lys His Lys Lys His Gly Lys Ala Ile Ile Gly Thr Lys Lys His 100 105 110His Val Asn Ile Lys Leu Arg Lys Asp Ile Asn Lys Ile Tyr Phe Met 115 120 125Thr Asp Val Asp Leu Gly Gly Pro Thr Phe Val Leu Asn Asp Lys Asp 130 135 140Tyr Gln Glu Ile Arg Lys Tyr Thr Lys Ala Lys His Ile Val Ser Gln145 150 155 160Phe Gly Phe Asp Leu Lys His Lys Lys Asp Ala Leu Ala Leu Glu Lys 165 170 175Ala Lys Asn Lys Val Asp Lys Ser Ile Glu Thr Arg Ser Glu Ala Ile 180 185 190Ser Ser Ile Ser Ser Leu Thr Gly 195 20013199PRTStaphylococcus aureus 13Ala Ser Leu Ser Ser Glu Ile Lys Tyr Thr Ala Pro His Asp Val Thr1 5 10 15Ile Lys Asp Gln Gln Lys Ala Asn Gln Leu Ala Ser Glu Leu Asn Asn 20 25 30Gln Lys Ile Pro His Phe Tyr Asn Tyr Lys Glu Val Ile His Thr Lys 35 40 45Leu Tyr Lys Asp Asn Leu Phe Asp Val Lys Ala Lys Glu Pro Tyr Asn 50 55 60Val Thr Ile Thr Ser Asp Lys Tyr Ile Pro Asn Thr Asp Leu Lys Arg65 70 75 80Gly Gln Ala Asp Leu Phe Val Ala Glu Gly Ser Ile Lys Asp Leu Val 85 90 95Lys His Lys Lys His Gly Lys Ala Ile Ile Gly Thr Lys Lys His His 100 105 110Val Asn Ile Lys Leu Arg Lys Asp Ile Asn Lys Ile Tyr Phe Met Thr 115 120 125Asp Val Asp Leu Gly Gly Pro Thr Phe Val Leu Asn Asp Lys Asp Tyr 130 135 140Gln Glu Ile Arg Lys Tyr Thr Lys Ala Lys His Ile Val Ser Gln Phe145 150 155 160Gly Phe Asp Leu Lys His Lys Lys Asp Ala Leu Ala Leu Glu Lys Ala 165 170 175Lys Asn Lys Val Asp Lys Ser Ile Glu Thr Arg Ser Glu Ala Ile Ser 180 185 190Ser Ile Ser Ser Leu Thr Gly 19514146PRTStaphylococcus aureus 14Ala Ser Leu Ser Ser Glu Ile Lys Tyr Thr Ala Pro His Asp Val Thr1 5 10 15Ile Lys Asp Gln Gln Lys Ala Asn Gln Leu Ala Ser Glu Leu Asn Asn 20 25 30Gln Lys Ile Pro His Phe Tyr Asn Tyr Lys Glu Val Ile His Thr Lys 35 40 45Leu Tyr Lys Asp Asn Leu Phe Asp Val Lys Ala Lys Glu Pro Tyr Asn 50 55 60Val Thr Ile Thr Ser Asp Lys Tyr Ile Pro Asn Thr Asp Leu Lys Arg65 70 75 80Gly Gln Ala Asp Leu Phe Val Ala Glu Gly Ser Ile Lys Asp Leu Val 85 90 95Lys His Lys Lys His Gly Lys Ala Ile Ile Gly Thr Lys Lys His His 100 105 110Val Asn Ile Lys Leu Arg Lys Asp Ile Asn Lys Ile Tyr Phe Met Thr 115 120 125Asp Val Asp Leu Gly Gly Pro Thr Phe Val Leu Asn Asp Lys Asp Tyr 130 135 140Gln Glu1451554PRTStaphylococcus aureus 15Lys Asp Ile Asn Lys Ile Tyr Phe Met Thr Asp Val Asp Leu Gly Gly1 5 10 15Pro Thr Phe Val Leu Asn Asp Lys Asp Tyr Gln Glu Ile Arg Lys Tyr 20 25 30Thr Lys Ala Lys His Ile Val Ser Gln Phe Gly Phe Asp Leu Lys His 35 40 45Lys Lys Asp Ala Leu Ala 501625PRTStaphylococcus aureus 16Lys Asp Ile Asn Lys Ile Tyr Phe Met Thr Asp Val Asp Leu Gly Gly1 5 10 15Pro Thr Phe Val Leu Asn Asp Lys Asp 20 251726PRTStaphylococcus aureus 17Lys Asp Ile Asn Lys Ile Tyr Phe Met Thr Asp Val Asp Leu Gly Gly1 5 10 15Pro Thr Phe Val Leu Asn Asp Lys Asp Tyr 20 251815PRTStaphylococcus aureus 18Ser Gln Phe Gly Phe Asp Leu Lys His Lys Lys Asp Ala Leu Ala1 5 10 151923PRTStaphylococcus aureus 19Lys Asp Ile Asn Lys Ile Tyr Phe Met Thr Asp Val Asp Leu Gly Gly1 5 10 15Pro Thr Phe Val Leu Asn Asp 202019PRTStaphylococcus aureus 20Lys His Ile Val Ser Gln Phe Gly Phe Asp Leu Lys His Lys Lys Asp1 5 10 15Ala Leu Ala2114PRTStaphylococcus aureus 21Gln Phe Gly Phe Asp Leu Lys His Lys Lys Asp Ala Leu Ala1 5 102213PRTStaphylococcus aureus 22Thr Ile Lys Asp Gln Gln Lys Ala Asn Gln Leu Ala Ser1 5 102314PRTStaphylococcus aureus 23Lys Asp Ile Asn Lys Ile Tyr Phe Met Thr Asp Val Asp Leu1 5 102413PRTStaphylococcus aureus 24Asp Val Asp Leu Gly Gly Pro Thr Phe Val Leu Asn Asp1 5 1025153PRTArtificial sequencesynthetic sequence 25Met His His His His His His Ala Ser Leu Ser Ser Glu Ile Lys Tyr1 5 10 15Thr Ala Pro His Asp Val Thr Ile Lys Asp Gln Gln Lys Ala Asn Gln 20 25 30Leu Ala Ser Glu Leu Asn Asn Gln Lys Ile Pro His Phe Tyr Asn Tyr 35 40 45Lys Glu Val Ile His Thr Lys Leu Tyr Lys Asp Asn Leu Phe Asp Val 50 55 60Lys Ala Lys Glu Pro Tyr Asn Val Thr Ile Thr Ser Asp Lys Tyr Ile65 70 75 80Pro Asn Thr Asp Leu Lys Arg Gly Gln Ala Asp Leu Phe Val Ala Glu 85 90 95Gly Ser Ile Lys Asp Leu Val Lys His Lys Lys His Gly Lys Ala Ile 100 105 110Ile Gly Thr Lys Lys His His Val Asn Ile Lys Leu Arg Lys Asp Ile 115 120 125Asn Lys Ile Tyr Phe Met Thr Asp Val Asp Leu Gly Gly Pro Thr Phe 130 135 140Val Leu Asn Asp Lys Asp Tyr Gln Glu145 15026158PRTArtificial sequencesynthetic sequence 26Met Arg Gly Ser His His His His His His Gly Ser Ala Ser Leu Ser1 5 10 15Ser Glu Ile Lys Tyr Thr Ala Pro His Asp Val Thr Ile Lys Asp Gln 20 25 30Gln Lys Ala Asn Gln Leu Ala Ser Glu Leu Asn Asn Gln Lys Ile Pro 35 40 45His Phe Tyr Asn Tyr Lys Glu Val Ile His Thr Lys Leu Tyr Lys Asp 50 55 60Asn Leu Phe Asp Val Lys Ala Lys Glu Pro Tyr Asn Val Thr Ile Thr65 70 75 80Ser Asp Lys Tyr Ile Pro Asn Thr Asp Leu Lys Arg Gly Gln Ala Asp 85 90 95Leu Phe Val Ala Glu Gly Ser Ile Lys Asp Leu Val Lys His Lys Lys 100 105

110His Gly Lys Ala Ile Ile Gly Thr Lys Lys His His Val Asn Ile Lys 115 120 125Leu Arg Lys Asp Ile Asn Lys Ile Tyr Phe Met Thr Asp Val Asp Leu 130 135 140Gly Gly Pro Thr Phe Val Leu Asn Asp Lys Asp Tyr Gln Glu145 150 1552750PRTArtificial sequencesynthetic sequence 27Lys Asp Ile Asn Lys Ile Tyr Phe Met Thr Asp Val Asp Leu Gly Gly1 5 10 15Pro Thr Phe Val Leu Asn Asp Lys Asp Tyr Glu Arg Lys Tyr Lys Lys 20 25 30His Ile Val Ser Gln Phe Gly Phe Asp Leu Lys His Lys Lys Asp Ala 35 40 45Leu Ala 5028612DNAStaphylococcus aureus 28atgttcaaaa aatatgactc aaaaaattca atcgtattaa aatctattct atcgctaggt 60atcatctatg ggggaacatt tggaatatat ccaaaagcag acgcgtcaac acaaaattcc 120tcaagtgtac aagataaaca attacaaaaa gttgaagaag taccaaataa ttcagaaaaa 180gctttggtta aaaaacttta cgatagatac agcaaggata caataaatgg aaaatctaat 240aaatctagga attgggttta ttcagagaga cctttaaatg aaaaccaagt tcgtatacat 300ttagaaggaa catacacagt tgctggcaga gtgtatacac ctaagaggaa tattactctt 360aataaagaag ttgtcacttt aaaagaattg gatcatatca taagatttgc tcatatttcc 420tatggcttgt atatgggaga acatttgcct aaaggtaaca tcgtcataaa tacaaaagat 480ggtggtaaat atacattaga gtcgcataaa gagctacaaa aagataggga aaatgtaaaa 540attaatacag ccgatataaa aaatgtaact ttcaaacttg tgaaaagtgt taatgacatt 600gaacaagttt ga 61229203PRTStaphylococcus aureus 29Met Phe Lys Lys Tyr Asp Ser Lys Asn Ser Ile Val Leu Lys Ser Ile1 5 10 15Leu Ser Leu Gly Ile Ile Tyr Gly Gly Thr Phe Gly Ile Tyr Pro Lys 20 25 30Ala Asp Ala Ser Thr Gln Asn Ser Ser Ser Val Gln Asp Lys Gln Leu 35 40 45Gln Lys Val Glu Glu Val Pro Asn Asn Ser Glu Lys Ala Leu Val Lys 50 55 60Lys Leu Tyr Asp Arg Tyr Ser Lys Asp Thr Ile Asn Gly Lys Ser Asn65 70 75 80Lys Ser Arg Asn Trp Val Tyr Ser Glu Arg Pro Leu Asn Glu Asn Gln 85 90 95Val Arg Ile His Leu Glu Gly Thr Tyr Thr Val Ala Gly Arg Val Tyr 100 105 110Thr Pro Lys Arg Asn Ile Thr Leu Asn Lys Glu Val Val Thr Leu Lys 115 120 125Glu Leu Asp His Ile Ile Arg Phe Ala His Ile Ser Tyr Gly Leu Tyr 130 135 140Met Gly Glu His Leu Pro Lys Gly Asn Ile Val Ile Asn Thr Lys Asp145 150 155 160Gly Gly Lys Tyr Thr Leu Glu Ser His Lys Glu Leu Gln Lys Asp Arg 165 170 175Glu Asn Val Lys Ile Asn Thr Ala Asp Ile Lys Asn Val Thr Phe Lys 180 185 190Leu Val Lys Ser Val Asn Asp Ile Glu Gln Val 195 20030168PRTStaphylococcus aureus 30Ser Thr Gln Asn Ser Ser Ser Val Gln Asp Lys Gln Leu Gln Lys Val1 5 10 15Glu Glu Val Pro Asn Asn Ser Glu Lys Ala Leu Val Lys Lys Leu Tyr 20 25 30Asp Arg Tyr Ser Lys Asp Thr Ile Asn Gly Lys Ser Asn Lys Ser Arg 35 40 45Asn Trp Val Tyr Ser Glu Arg Pro Leu Asn Glu Asn Gln Val Arg Ile 50 55 60His Leu Glu Gly Thr Tyr Thr Val Ala Gly Arg Val Tyr Thr Pro Lys65 70 75 80Arg Asn Ile Thr Leu Asn Lys Glu Val Val Thr Leu Lys Glu Leu Asp 85 90 95His Ile Ile Arg Phe Ala His Ile Ser Tyr Gly Leu Tyr Met Gly Glu 100 105 110His Leu Pro Lys Gly Asn Ile Val Ile Asn Thr Lys Asp Gly Gly Lys 115 120 125Tyr Thr Leu Glu Ser His Lys Glu Leu Gln Lys Asp Arg Glu Asn Val 130 135 140Lys Ile Asn Thr Ala Asp Ile Lys Asn Val Thr Phe Lys Leu Val Lys145 150 155 160Ser Val Asn Asp Ile Glu Gln Val 16531116PRTStaphylococcus aureus 31Gln Asp Lys Gln Leu Gln Lys Val Glu Glu Val Pro Asn Asn Ser Glu1 5 10 15Lys Ala Leu Val Lys Lys Leu Tyr Asp Arg Tyr Ser Lys Asp Thr Ile 20 25 30Asn Gly Lys Ser Asn Lys Ser Arg Asn Trp Val Tyr Ser Glu Arg Pro 35 40 45Leu Asn Glu Asn Gln Val Arg Ile His Leu Glu Gly Thr Tyr Thr Val 50 55 60Ala Gly Arg Val Tyr Thr Pro Lys Arg Asn Ile Thr Leu Asn Lys Glu65 70 75 80Val Val Thr Leu Lys Glu Leu Asp His Ile Ile Arg Phe Ala His Ile 85 90 95Ser Tyr Gly Leu Tyr Met Gly Glu His Leu Pro Lys Gly Asn Ile Val 100 105 110Ile Asn Thr Lys 11532115PRTStaphylococcus aureus 32Asp Lys Gln Leu Gln Lys Val Glu Glu Val Pro Asn Asn Ser Glu Lys1 5 10 15Ala Leu Val Lys Lys Leu Tyr Asp Arg Tyr Ser Lys Asp Thr Ile Asn 20 25 30Gly Lys Ser Asn Lys Ser Arg Asn Trp Val Tyr Ser Glu Arg Pro Leu 35 40 45Asn Glu Asn Gln Val Arg Ile His Leu Glu Gly Thr Tyr Thr Val Ala 50 55 60Gly Arg Val Tyr Thr Pro Lys Arg Asn Ile Thr Leu Asn Lys Glu Val65 70 75 80Val Thr Leu Lys Glu Leu Asp His Ile Ile Arg Phe Ala His Ile Ser 85 90 95Tyr Gly Leu Tyr Met Gly Glu His Leu Pro Lys Gly Asn Ile Val Ile 100 105 110Asn Thr Lys 11533159PRTStaphylococcus aureus 33Asp Lys Gln Leu Gln Lys Val Glu Glu Val Pro Asn Asn Ser Glu Lys1 5 10 15Ala Leu Val Lys Lys Leu Tyr Asp Arg Tyr Ser Lys Asp Thr Ile Asn 20 25 30Gly Lys Ser Asn Lys Ser Arg Asn Trp Val Tyr Ser Glu Arg Pro Leu 35 40 45Asn Glu Asn Gln Val Arg Ile His Leu Glu Gly Thr Tyr Thr Val Ala 50 55 60Gly Arg Val Tyr Thr Pro Lys Arg Asn Ile Thr Leu Asn Lys Glu Val65 70 75 80Val Thr Leu Lys Glu Leu Asp His Ile Ile Arg Phe Ala His Ile Ser 85 90 95Tyr Gly Leu Tyr Met Gly Glu His Leu Pro Lys Gly Asn Ile Val Ile 100 105 110Asn Thr Lys Asp Gly Gly Lys Tyr Thr Leu Glu Ser His Lys Glu Leu 115 120 125Gln Lys Asp Arg Glu Asn Val Lys Ile Asn Thr Ala Asp Ile Lys Asn 130 135 140Val Thr Phe Lys Leu Val Lys Ser Val Asn Asp Ile Glu Gln Val145 150 1553414PRTStaphylococcus aureus 34Lys Asp Thr Ile Asn Gly Lys Ser Asn Lys Ser Arg Asn Trp1 5 1035166PRTArtificial sequencesynthetic sequence 35Met His His His His His His Asp Lys Gln Leu Gln Lys Val Glu Glu1 5 10 15Val Pro Asn Asn Ser Glu Lys Ala Leu Val Lys Lys Leu Tyr Asp Arg 20 25 30Tyr Ser Lys Asp Thr Ile Asn Gly Lys Ser Asn Lys Ser Arg Asn Trp 35 40 45Val Tyr Ser Glu Arg Pro Leu Asn Glu Asn Gln Val Arg Ile His Leu 50 55 60Glu Gly Thr Tyr Thr Val Ala Gly Arg Val Tyr Thr Pro Lys Arg Asn65 70 75 80Ile Thr Leu Asn Lys Glu Val Val Thr Leu Lys Glu Leu Asp His Ile 85 90 95Ile Arg Phe Ala His Ile Ser Tyr Gly Leu Tyr Met Gly Glu His Leu 100 105 110Pro Lys Gly Asn Ile Val Ile Asn Thr Lys Asp Gly Gly Lys Tyr Thr 115 120 125Leu Glu Ser His Lys Glu Leu Gln Lys Asp Arg Glu Asn Val Lys Ile 130 135 140Asn Thr Ala Asp Ile Lys Asn Val Thr Phe Lys Leu Val Lys Ser Val145 150 155 160Asn Asp Ile Glu Gln Val 16536171PRTArtificial sequencesynthetic sequence 36Met Arg Gly Ser His His His His His His Gly Ser Asp Lys Gln Leu1 5 10 15Gln Lys Val Glu Glu Val Pro Asn Asn Ser Glu Lys Ala Leu Val Lys 20 25 30Lys Leu Tyr Asp Arg Tyr Ser Lys Asp Thr Ile Asn Gly Lys Ser Asn 35 40 45Lys Ser Arg Asn Trp Val Tyr Ser Glu Arg Pro Leu Asn Glu Asn Gln 50 55 60Val Arg Ile His Leu Glu Gly Thr Tyr Thr Val Ala Gly Arg Val Tyr65 70 75 80Thr Pro Lys Arg Asn Ile Thr Leu Asn Lys Glu Val Val Thr Leu Lys 85 90 95Glu Leu Asp His Ile Ile Arg Phe Ala His Ile Ser Tyr Gly Leu Tyr 100 105 110Met Gly Glu His Leu Pro Lys Gly Asn Ile Val Ile Asn Thr Lys Asp 115 120 125Gly Gly Lys Tyr Thr Leu Glu Ser His Lys Glu Leu Gln Lys Asp Arg 130 135 140Glu Asn Val Lys Ile Asn Thr Ala Asp Ile Lys Asn Val Thr Phe Lys145 150 155 160Leu Val Lys Ser Val Asn Asp Ile Glu Gln Val 165 17037720DNAStaphylococcus aureus 37atgaataaaa atatagtcat taaaagcatg gcagcattag ccattctaac ctcagtaact 60ggaataaatg ctgcagtcgt tgaagagaca caacaaatag caaatgcaga gaagaatgtt 120acgcaagtta aagatacaaa tatttttcca tataatggcg tcgtttcatt taaagatgcg 180acaggttttg taattggaaa aaatacaatt atcaccaata aacatgtatc aaaagattat 240aaagttggcg atagaattac tgcccatcca aacggtgaca aaggaaatgg tggtatatat 300aaaattaaaa gcatttctga ttatccgggt gatgaagaca tctctgtcat gaatattgaa 360gaacaagcag tcgaacgtgg accaaaaggc tttaatttta atgaaaatgt ccaagcattc 420aattttgcga aagatgctaa agttgatgac aaaattaaag ttattggtta cccattacct 480gctcaaaata gttttaaaca gtttgaatct acaggaacta taaaaagaat caaagacaat 540attttaaatt ttgatgcata cattgaaccc gggaattcag gatcaccagt tctaaattct 600aacaatgagg tcataggtgt ggtgtatggc ggtattggaa aaattggttc tgaatataat 660ggtgccgtat actttacgcc tcaaatcaaa gattttattc aaaagcacat tgaacaataa 72038239PRTStaphylococcus aureus 38Met Asn Lys Asn Ile Val Ile Lys Ser Met Ala Ala Leu Ala Ile Leu1 5 10 15Thr Ser Val Thr Gly Ile Asn Ala Ala Val Val Glu Glu Thr Gln Gln 20 25 30Ile Ala Asn Ala Glu Lys Asn Val Thr Gln Val Lys Asp Thr Asn Ile 35 40 45Phe Pro Tyr Asn Gly Val Val Ser Phe Lys Asp Ala Thr Gly Phe Val 50 55 60Ile Gly Lys Asn Thr Ile Ile Thr Asn Lys His Val Ser Lys Asp Tyr65 70 75 80Lys Val Gly Asp Arg Ile Thr Ala His Pro Asn Gly Asp Lys Gly Asn 85 90 95Gly Gly Ile Tyr Lys Ile Lys Ser Ile Ser Asp Tyr Pro Gly Asp Glu 100 105 110Asp Ile Ser Val Met Asn Ile Glu Glu Gln Ala Val Glu Arg Gly Pro 115 120 125Lys Gly Phe Asn Phe Asn Glu Asn Val Gln Ala Phe Asn Phe Ala Lys 130 135 140Asp Ala Lys Val Asp Asp Lys Ile Lys Val Ile Gly Tyr Pro Leu Pro145 150 155 160Ala Gln Asn Ser Phe Lys Gln Phe Glu Ser Thr Gly Thr Ile Lys Arg 165 170 175Ile Lys Asp Asn Ile Leu Asn Phe Asp Ala Tyr Ile Glu Pro Gly Asn 180 185 190Ser Gly Ser Pro Val Leu Asn Ser Asn Asn Glu Val Ile Gly Val Val 195 200 205Tyr Gly Gly Ile Gly Lys Ile Gly Ser Glu Tyr Asn Gly Ala Val Tyr 210 215 220Phe Thr Pro Gln Ile Lys Asp Phe Ile Gln Lys His Ile Glu Gln225 230 23539199PRTStaphylococcus aureus 39Thr Gln Val Lys Asp Thr Asn Ile Phe Pro Tyr Asn Gly Val Val Ser1 5 10 15Phe Lys Asp Ala Thr Gly Phe Val Ile Gly Lys Asn Thr Ile Ile Thr 20 25 30Asn Lys His Val Ser Lys Asp Tyr Lys Val Gly Asp Arg Ile Thr Ala 35 40 45His Pro Asn Gly Asp Lys Gly Asn Gly Gly Ile Tyr Lys Ile Lys Ser 50 55 60Ile Ser Asp Tyr Pro Gly Asp Glu Asp Ile Ser Val Met Asn Ile Glu65 70 75 80Glu Gln Ala Val Glu Arg Gly Pro Lys Gly Phe Asn Phe Asn Glu Asn 85 90 95Val Gln Ala Phe Asn Phe Ala Lys Asp Ala Lys Val Asp Asp Lys Ile 100 105 110Lys Val Ile Gly Tyr Pro Leu Pro Ala Gln Asn Ser Phe Lys Gln Phe 115 120 125Glu Ser Thr Gly Thr Ile Lys Arg Ile Lys Asp Asn Ile Leu Asn Phe 130 135 140Asp Ala Tyr Ile Glu Pro Gly Asn Ser Gly Ser Pro Val Leu Asn Ser145 150 155 160Asn Asn Glu Val Ile Gly Val Val Tyr Gly Gly Ile Gly Lys Ile Gly 165 170 175Ser Glu Tyr Asn Gly Ala Val Tyr Phe Thr Pro Gln Ile Lys Asp Phe 180 185 190Ile Gln Lys His Ile Glu Gln 19540206PRTArtificial sequencesynthetic sequence 40Met His His His His His His Thr Gln Val Lys Asp Thr Asn Ile Phe1 5 10 15Pro Tyr Asn Gly Val Val Ser Phe Lys Asp Ala Thr Gly Phe Val Ile 20 25 30Gly Lys Asn Thr Ile Ile Thr Asn Lys His Val Ser Lys Asp Tyr Lys 35 40 45Val Gly Asp Arg Ile Thr Ala His Pro Asn Gly Asp Lys Gly Asn Gly 50 55 60Gly Ile Tyr Lys Ile Lys Ser Ile Ser Asp Tyr Pro Gly Asp Glu Asp65 70 75 80Ile Ser Val Met Asn Ile Glu Glu Gln Ala Val Glu Arg Gly Pro Lys 85 90 95Gly Phe Asn Phe Asn Glu Asn Val Gln Ala Phe Asn Phe Ala Lys Asp 100 105 110Ala Lys Val Asp Asp Lys Ile Lys Val Ile Gly Tyr Pro Leu Pro Ala 115 120 125Gln Asn Ser Phe Lys Gln Phe Glu Ser Thr Gly Thr Ile Lys Arg Ile 130 135 140Lys Asp Asn Ile Leu Asn Phe Asp Ala Tyr Ile Glu Pro Gly Asn Ser145 150 155 160Gly Ser Pro Val Leu Asn Ser Asn Asn Glu Val Ile Gly Val Val Tyr 165 170 175Gly Gly Ile Gly Lys Ile Gly Ser Glu Tyr Asn Gly Ala Val Tyr Phe 180 185 190Thr Pro Gln Ile Lys Asp Phe Ile Gln Lys His Ile Glu Gln 195 200 20541211PRTArtificial sequencesynthetic sequence 41Met Arg Gly Ser His His His His His His Gly Ser Thr Gln Val Lys1 5 10 15Asp Thr Asn Ile Phe Pro Tyr Asn Gly Val Val Ser Phe Lys Asp Ala 20 25 30Thr Gly Phe Val Ile Gly Lys Asn Thr Ile Ile Thr Asn Lys His Val 35 40 45Ser Lys Asp Tyr Lys Val Gly Asp Arg Ile Thr Ala His Pro Asn Gly 50 55 60Asp Lys Gly Asn Gly Gly Ile Tyr Lys Ile Lys Ser Ile Ser Asp Tyr65 70 75 80Pro Gly Asp Glu Asp Ile Ser Val Met Asn Ile Glu Glu Gln Ala Val 85 90 95Glu Arg Gly Pro Lys Gly Phe Asn Phe Asn Glu Asn Val Gln Ala Phe 100 105 110Asn Phe Ala Lys Asp Ala Lys Val Asp Asp Lys Ile Lys Val Ile Gly 115 120 125Tyr Pro Leu Pro Ala Gln Asn Ser Phe Lys Gln Phe Glu Ser Thr Gly 130 135 140Thr Ile Lys Arg Ile Lys Asp Asn Ile Leu Asn Phe Asp Ala Tyr Ile145 150 155 160Glu Pro Gly Asn Ser Gly Ser Pro Val Leu Asn Ser Asn Asn Glu Val 165 170 175Ile Gly Val Val Tyr Gly Gly Ile Gly Lys Ile Gly Ser Glu Tyr Asn 180 185 190Gly Ala Val Tyr Phe Thr Pro Gln Ile Lys Asp Phe Ile Gln Lys His 195 200 205Ile Glu Gln 21042600DNAStaphylococcus aureus 42atggcaatga actttaaagt ctttgacaat agtcaacttg tagcagaata tgctgctgat 60attattagaa agcaatttaa caataatcct actacaattg caggttttca tttagataca 120gatcaagcgc cagttctaga tgaattaaag aaaaatgttg aaaaacatgc tgttgatttt 180agccaaataa atattttaga ttatgacgat aaaaaatcat atttcgaagc gttaggtgta 240ccagcaggtc aagtttatcc aattgcttat gaaaaagatg caatcgaatt aatcgctgat 300aagattaaaa ctaaagaaaa taaagggaaa ttaacattac aagttgtttc tatcgatgag 360caaggtaagt taaatgttag tattcgtcaa ggactaatgg aagcaagaga aattttctta 420gtagtgacag gtgctaataa acgagatgta gttgaaaaat tatatcaaga aaatggtaaa 480acaagcttcg aaccagccga tttaaaagca catagaatgg taaatgttat tcttgataaa 540gaagcggctg caggtttacc tgaagatgtt aaagcttact ttacgtcacg ctttgcttaa 60043199PRTStaphylococcus aureus 43Met Ala Met Asn Phe Lys Val Phe Asp Asn Ser Gln Leu Val Ala Glu1 5 10

15Tyr Ala Ala Asp Ile Ile Arg Lys Gln Phe Asn Asn Asn Pro Thr Thr 20 25 30Ile Ala Gly Phe His Leu Asp Thr Asp Gln Ala Pro Val Leu Asp Glu 35 40 45Leu Lys Lys Asn Val Glu Lys His Ala Val Asp Phe Ser Gln Ile Asn 50 55 60Ile Leu Asp Tyr Asp Asp Lys Lys Ser Tyr Phe Glu Ala Leu Gly Val65 70 75 80Pro Ala Gly Gln Val Tyr Pro Ile Ala Tyr Glu Lys Asp Ala Ile Glu 85 90 95Leu Ile Ala Asp Lys Ile Lys Thr Lys Glu Asn Lys Gly Lys Leu Thr 100 105 110Leu Gln Val Val Ser Ile Asp Glu Gln Gly Lys Leu Asn Val Ser Ile 115 120 125Arg Gln Gly Leu Met Glu Ala Arg Glu Ile Phe Leu Val Val Thr Gly 130 135 140Ala Asn Lys Arg Asp Val Val Glu Lys Leu Tyr Gln Glu Asn Gly Lys145 150 155 160Thr Ser Phe Glu Pro Ala Asp Leu Lys Ala His Arg Met Val Asn Val 165 170 175Ile Leu Asp Lys Glu Ala Ala Ala Gly Leu Pro Glu Asp Val Lys Ala 180 185 190Tyr Phe Thr Ser Arg Phe Ala 19544206PRTArtificial sequencesynthetic sequence 44Met His His His His His His Met Ala Met Asn Phe Lys Val Phe Asp1 5 10 15Asn Ser Gln Leu Val Ala Glu Tyr Ala Ala Asp Ile Ile Arg Lys Gln 20 25 30Phe Asn Asn Asn Pro Thr Thr Ile Ala Gly Phe His Leu Asp Thr Asp 35 40 45Gln Ala Pro Val Leu Asp Glu Leu Lys Lys Asn Val Glu Lys His Ala 50 55 60Val Asp Phe Ser Gln Ile Asn Ile Leu Asp Tyr Asp Asp Lys Lys Ser65 70 75 80Tyr Phe Glu Ala Leu Gly Val Pro Ala Gly Gln Val Tyr Pro Ile Ala 85 90 95Tyr Glu Lys Asp Ala Ile Glu Leu Ile Ala Asp Lys Ile Lys Thr Lys 100 105 110Glu Asn Lys Gly Lys Leu Thr Leu Gln Val Val Ser Ile Asp Glu Gln 115 120 125Gly Lys Leu Asn Val Ser Ile Arg Gln Gly Leu Met Glu Ala Arg Glu 130 135 140Ile Phe Leu Val Val Thr Gly Ala Asn Lys Arg Asp Val Val Glu Lys145 150 155 160Leu Tyr Gln Glu Asn Gly Lys Thr Ser Phe Glu Pro Ala Asp Leu Lys 165 170 175Ala His Arg Met Val Asn Val Ile Leu Asp Lys Glu Ala Ala Ala Gly 180 185 190Leu Pro Glu Asp Val Lys Ala Tyr Phe Thr Ser Arg Phe Ala 195 200 2054514PRTStaphylococcus aureus 45Asp Ile Ile Arg Lys Gln Phe Asn Asn Asn Pro Thr Thr Ile1 5 104614PRTStaphylococcus aureus 46Tyr Phe Glu Ala Leu Gly Val Pro Ala Gly Gln Val Tyr Pro1 5 104714PRTStaphylococcus aureus 47Ala Asp Lys Ile Lys Thr Lys Glu Asn Lys Gly Lys Leu Thr1 5 104814PRTStaphylococcus aureus 48Gln Glu Asn Gly Lys Thr Ser Phe Glu Pro Ala Asp Leu Lys1 5 1049429DNAStaphylococcus aureus 49atgatattga acttcaatca attcgagaat caaaactttt ttaacggtaa tccaagtgat 60acatttaaag atttaggtaa acaagtattt aattactttt caacaccttc atttgtaacg 120aatatatatg aaacagacga attatattac ttagaagctg aactagcagg tgtaaataaa 180gaagatatta gtatcgattt caataataat acgctcacta ttcaagctac tagaagcgca 240aaatacaaat ctgaacaact cattttagat gagcgtaact tcgaatcatt aatgcgtcaa 300tttgattttg aagctgttga taagcaacat attactgcta gttttgaaaa tgggttatta 360accattacct tgcctaaaat caaaccaagc aatgaaacta cttcatcaac atctattcca 420atttcatag 4295093PRTStaphylococcus aureus 50Met Ile Leu Asn Phe Asn Gln Phe Glu Asn Gln Asn Phe Phe Asn Gly1 5 10 15Asn Pro Ser Asp Thr Phe Lys Asp Leu Gly Lys Gln Val Phe Asn Tyr 20 25 30Phe Ser Thr Pro Ser Phe Val Thr Asn Ile Tyr Glu Thr Asp Glu Leu 35 40 45Tyr Tyr Leu Glu Ala Glu Leu Ala Gly Val Asn Lys Glu Asp Ile Ser 50 55 60Ile Asp Phe Asn Asn Asn Thr Leu Thr Ile Gln Ala Thr Arg Ser Ala65 70 75 80Lys Tyr Lys Ser Glu Gln Leu Ile Leu Asp Glu Arg Asn 85 9051100PRTStaphylococcus aureus 51Met His His His His His His Met Ile Leu Asn Phe Asn Gln Phe Glu1 5 10 15Asn Gln Asn Phe Phe Asn Gly Asn Pro Ser Asp Thr Phe Lys Asp Leu 20 25 30Gly Lys Gln Val Phe Asn Tyr Phe Ser Thr Pro Ser Phe Val Thr Asn 35 40 45Ile Tyr Glu Thr Asp Glu Leu Tyr Tyr Leu Glu Ala Glu Leu Ala Gly 50 55 60Val Asn Lys Glu Asp Ile Ser Ile Asp Phe Asn Asn Asn Thr Leu Thr65 70 75 80Ile Gln Ala Thr Arg Ser Ala Lys Tyr Lys Ser Glu Gln Leu Ile Leu 85 90 95Asp Glu Arg Asn 1005214PRTStaphylococcus aureus 52Asn Gly Asn Pro Ser Asp Thr Phe Lys Asp Leu Gly Lys Gln1 5 105314PRTStaphylococcus aureus 53Glu Asp Ile Ser Ile Asp Phe Asn Asn Asn Thr Leu Thr Ile1 5 1054807DNAArtificial sequencesynthetic sequence 54ctagaatcta gagagcaatt atcagtcgaa gaatacgaaa cattctttaa cagatttgat 60aatcaagaat ttgatttcga acgtgaattg acacaagatc catattcaaa agtatactta 120tacagtatag aagaccatat cagaacatat aagatagaga aaggaggtgg cggttcagga 180ggtggaggat ctggaggcgg tggatcaacg caagttaaag atacaaatat ttttccatat 240aatggcgtcg tttcatttaa agatgcgaca ggttttgtaa ttggaaaaaa tacaattatc 300accaataaac atgtatcaaa agattataaa gttggcgata gaattactgc ccatccaaac 360ggtgacaaag gaaatggtgg tatatataaa attaaaagca tttctgatta tccgggtgat 420gaagacatct ctgtcatgaa tattgaagaa caagcagtcg aacgtggacc aaaaggcttt 480aattttaatg aaaatgtcca agcattcaat tttgcgaaag atgctaaagt tgatgacaaa 540attaaagtta ttggttaccc attacctgct caaaatagtt ttaaacagtt tgaatctaca 600ggaactataa aaagaatcaa agacaatatt ttaaattttg atgcatacat tgaacccggg 660aattcaggat caccagttct aaattctaac aatgaggtca taggtgtggt gtatggcggt 720attggaaaaa ttggttctga atataatggt gccgtatact ttacgcctca aatcaaagat 780tttattcaaa agcacattga acaataa 80755268PRTArtificial sequencesynthetic sequence 55Leu Glu Ser Arg Glu Gln Leu Ser Val Glu Glu Tyr Glu Thr Phe Phe1 5 10 15Asn Arg Phe Asp Asn Gln Glu Phe Asp Phe Glu Arg Glu Leu Thr Gln 20 25 30Asp Pro Tyr Ser Lys Val Tyr Leu Tyr Ser Ile Glu Asp His Ile Arg 35 40 45Thr Tyr Lys Ile Glu Lys Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser 50 55 60Gly Gly Gly Gly Ser Thr Gln Val Lys Asp Thr Asn Ile Phe Pro Tyr65 70 75 80Asn Gly Val Val Ser Phe Lys Asp Ala Thr Gly Phe Val Ile Gly Lys 85 90 95Asn Thr Ile Ile Thr Asn Lys His Val Ser Lys Asp Tyr Lys Val Gly 100 105 110Asp Arg Ile Thr Ala His Pro Asn Gly Asp Lys Gly Asn Gly Gly Ile 115 120 125Tyr Lys Ile Lys Ser Ile Ser Asp Tyr Pro Gly Asp Glu Asp Ile Ser 130 135 140Val Met Asn Ile Glu Glu Gln Ala Val Glu Arg Gly Pro Lys Gly Phe145 150 155 160Asn Phe Asn Glu Asn Val Gln Ala Phe Asn Phe Ala Lys Asp Ala Lys 165 170 175Val Asp Asp Lys Ile Lys Val Ile Gly Tyr Pro Leu Pro Ala Gln Asn 180 185 190Ser Phe Lys Gln Phe Glu Ser Thr Gly Thr Ile Lys Arg Ile Lys Asp 195 200 205Asn Ile Leu Asn Phe Asp Ala Tyr Ile Glu Pro Gly Asn Ser Gly Ser 210 215 220Pro Val Leu Asn Ser Asn Asn Glu Val Ile Gly Val Val Tyr Gly Gly225 230 235 240Ile Gly Lys Ile Gly Ser Glu Tyr Asn Gly Ala Val Tyr Phe Thr Pro 245 250 255Gln Ile Lys Asp Phe Ile Gln Lys His Ile Glu Gln 260 26556825DNAArtificial sequencesynthetic sequence 56atgcaccacc accaccacca cctggaatcc cgtgaacaac tgtccgtcga agaatacgaa 60accttcttta accgctttga taaccaagaa tttgatttcg aacgtgaact gacccaggat 120ccgtattcta aagtgtatct gtacagtatc gaagatcata ttcgcacgta caaaatcgaa 180aaaggcggtg gcggttctgg cggtggcggt agtggcggtg gcggtagcac ccaggtgaaa 240gatacgaata tctttccgta taacggcgtg gtttctttta aagatgcgac cggcttcgtt 300atcggtaaaa acaccatcat cacgaacaaa catgtgagca aagattacaa agttggcgat 360cgtattaccg cccacccgaa tggcgataag ggtaacggcg gtatctacaa aatcaaaagc 420atctctgatt acccgggtga tgaagatatc agcgtgatga atattgaaga acaggcagtt 480gaacgcggcc cgaaaggttt taacttcaat gaaaacgttc aggcgtttaa tttcgcgaaa 540gatgccaaag tggatgataa aatcaaagtt attggctatc cgctgccggc ccagaacagc 600tttaaacagt tcgaatctac cggtacgatc aaacgtatca aagataacat cctgaacttc 660gatgcatata ttgaaccggg caatagtggt agcccggtgc tgaacagtaa caatgaagtt 720attggtgtgg tttatggcgg tatcggcaaa attggtagcg aatacaacgg tgctgtgtat 780tttacgccgc agatcaaaga cttcatccag aaacatatcg aacaa 82557275PRTArtificial sequencesynthetic sequence 57Met His His His His His His Leu Glu Ser Arg Glu Gln Leu Ser Val1 5 10 15Glu Glu Tyr Glu Thr Phe Phe Asn Arg Phe Asp Asn Gln Glu Phe Asp 20 25 30Phe Glu Arg Glu Leu Thr Gln Asp Pro Tyr Ser Lys Val Tyr Leu Tyr 35 40 45Ser Ile Glu Asp His Ile Arg Thr Tyr Lys Ile Glu Lys Gly Gly Gly 50 55 60Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Thr Gln Val Lys65 70 75 80Asp Thr Asn Ile Phe Pro Tyr Asn Gly Val Val Ser Phe Lys Asp Ala 85 90 95Thr Gly Phe Val Ile Gly Lys Asn Thr Ile Ile Thr Asn Lys His Val 100 105 110Ser Lys Asp Tyr Lys Val Gly Asp Arg Ile Thr Ala His Pro Asn Gly 115 120 125Asp Lys Gly Asn Gly Gly Ile Tyr Lys Ile Lys Ser Ile Ser Asp Tyr 130 135 140Pro Gly Asp Glu Asp Ile Ser Val Met Asn Ile Glu Glu Gln Ala Val145 150 155 160Glu Arg Gly Pro Lys Gly Phe Asn Phe Asn Glu Asn Val Gln Ala Phe 165 170 175Asn Phe Ala Lys Asp Ala Lys Val Asp Asp Lys Ile Lys Val Ile Gly 180 185 190Tyr Pro Leu Pro Ala Gln Asn Ser Phe Lys Gln Phe Glu Ser Thr Gly 195 200 205Thr Ile Lys Arg Ile Lys Asp Asn Ile Leu Asn Phe Asp Ala Tyr Ile 210 215 220Glu Pro Gly Asn Ser Gly Ser Pro Val Leu Asn Ser Asn Asn Glu Val225 230 235 240Ile Gly Val Val Tyr Gly Gly Ile Gly Lys Ile Gly Ser Glu Tyr Asn 245 250 255Gly Ala Val Tyr Phe Thr Pro Gln Ile Lys Asp Phe Ile Gln Lys His 260 265 270Ile Glu Gln 27558280PRTArtificial sequencesynthetic sequence 58Met Arg Gly Ser His His His His His His Gly Ser Leu Glu Ser Arg1 5 10 15Glu Gln Leu Ser Val Glu Glu Tyr Glu Thr Phe Phe Asn Arg Phe Asp 20 25 30Asn Gln Glu Phe Asp Phe Glu Arg Glu Leu Thr Gln Asp Pro Tyr Ser 35 40 45Lys Val Tyr Leu Tyr Ser Ile Glu Asp His Ile Arg Thr Tyr Lys Ile 50 55 60Glu Lys Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly65 70 75 80Ser Thr Gln Val Lys Asp Thr Asn Ile Phe Pro Tyr Asn Gly Val Val 85 90 95Ser Phe Lys Asp Ala Thr Gly Phe Val Ile Gly Lys Asn Thr Ile Ile 100 105 110Thr Asn Lys His Val Ser Lys Asp Tyr Lys Val Gly Asp Arg Ile Thr 115 120 125Ala His Pro Asn Gly Asp Lys Gly Asn Gly Gly Ile Tyr Lys Ile Lys 130 135 140Ser Ile Ser Asp Tyr Pro Gly Asp Glu Asp Ile Ser Val Met Asn Ile145 150 155 160Glu Glu Gln Ala Val Glu Arg Gly Pro Lys Gly Phe Asn Phe Asn Glu 165 170 175Asn Val Gln Ala Phe Asn Phe Ala Lys Asp Ala Lys Val Asp Asp Lys 180 185 190Ile Lys Val Ile Gly Tyr Pro Leu Pro Ala Gln Asn Ser Phe Lys Gln 195 200 205Phe Glu Ser Thr Gly Thr Ile Lys Arg Ile Lys Asp Asn Ile Leu Asn 210 215 220Phe Asp Ala Tyr Ile Glu Pro Gly Asn Ser Gly Ser Pro Val Leu Asn225 230 235 240Ser Asn Asn Glu Val Ile Gly Val Val Tyr Gly Gly Ile Gly Lys Ile 245 250 255Gly Ser Glu Tyr Asn Gly Ala Val Tyr Phe Thr Pro Gln Ile Lys Asp 260 265 270Phe Ile Gln Lys His Ile Glu Gln 275 2805945DNAArtificial sequencesynthetic sequence 59ggaggtggcg gttcaggagg tggaggatct ggaggcggtg gatca 456015PRTArtificial sequencesynthetic sequence 60Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser1 5 10 15615PRTArtificial sequencesynthetic sequence 61Glu Arg Lys Tyr Lys1 56210PRTArtificial sequencesynthetic sequence 62Glu Ala Ala Ala Lys Glu Ala Ala Ala Lys1 5 10635PRTArtificial sequencesynthetic sequence 63Glu Ala Ala Ala Lys1 56415PRTArtificial sequencesynthetic sequence 64Glu Ala Ala Ala Lys Glu Ala Ala Ala Lys Glu Ala Ala Ala Lys1 5 10 156510PRTArtificial sequencesynthetic sequence 65Glu Arg Lys Tyr Lys Glu Arg Lys Tyr Lys1 5 106615PRTArtificial sequencesynthetic sequence 66Glu Arg Lys Tyr Lys Glu Arg Lys Tyr Lys Glu Arg Lys Tyr Lys1 5 10 15675PRTArtificial sequencesynthetic sequence 67Gly Gly Gly Gly Ser1 56810PRTArtificial sequencesynthetic sequence 68Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser1 5 10696PRTArtificial sequencesynthetic sequencemisc_feature(1)..(1)Xaa can be any naturally occurring amino acidmisc_feature(3)..(3)Xaa can be any naturally occurring amino acidmisc_feature(5)..(5)Xaa can be any naturally occurring amino acid 69Xaa Pro Xaa Pro Xaa Pro1 57012PRTArtificial sequencesynthetic sequencemisc_feature(1)..(1)Xaa can be any naturally occurring amino acidmisc_feature(3)..(3)Xaa can be any naturally occurring amino acidmisc_feature(5)..(5)Xaa can be any naturally occurring amino acidmisc_feature(7)..(7)Xaa can be any naturally occurring amino acidmisc_feature(9)..(9)Xaa can be any naturally occurring amino acidmisc_feature(11)..(11)Xaa can be any naturally occurring amino acid 70Xaa Pro Xaa Pro Xaa Pro Xaa Pro Xaa Pro Xaa Pro1 5 1071612DNAStaphylococcus aureus 71atgttcaaaa aaaatgactc gaaaaattca attctattaa aatctattct atcgctaggt 60atcatctatg ggggaacatt tggaatatat ccaaaagcag acgcgtcaac acaaaattcc 120ccaagtgtac aagataaaca attccaaaaa gttgaagaag taccaaataa ttcagaaaaa 180gctttggtta aaaaacttta cgatagatac agccaaaata caataaacgg aaaatctaat 240aaatctagga attgggttta ttcagagaga cctttaaatg aaaaccaagt tcgtataaat 300ttagaaggaa catacagagt tgctgataga gtatatacac ctaagagaaa tattactctt 360aataaagaag ttgtcacttt aaaggaattg gatcatatca taagatttgc tcatatttct 420tatggcttat atatgggaga acatttgcct aaaggtaaca tcgtcataaa tacaaaagat 480ggcggtaaat atacattaga gtcgcataaa gagctacaaa aagataggga aaatgtaaaa 540attaatacag ccgatataaa aaatgtaact ttcaaacttg tgaaaagtgt taatgacatt 600gaacaagttt ga 61272612DNAStaphylococcus aureus 72atgttcaaaa aaaatgactc gaaaaattca attctattaa aatctattct atcgctaggt 60atcatctatg ggggaacatt tggaatatat ccaaaagcag acgcgtcaac acaaaattcc 120ccaagtgtac aagataaaca attccaaaaa gttgaagaag taccaaataa ttcagaaaaa 180gctttggtta aaaaacttta cgatagatac agccaaaata caataaacgg aaaatctaat 240aaatctagga attgggttta ttcagagaga cctttaaatg aaaaccaagt tcgtataaat 300ttagaaggaa catacagagt tgctgataga gtatatacac ctaagagaaa tattactctt 360aataaagaag ttgtcacttt aaaggaattg gatcatatca taagatttgc tcatatttct 420tatggcttat atatgggaga acatttgcct aaaggtaaca tcgtcataaa tacaaaagat 480ggcggtaaat atacattaga gtcgcataaa gagctacaaa aagataggga aaatgtaaaa 540attaatacag ccgatataaa aaatgtaact ttcaaacttg tgaaaagtgt taatgacatt 600gaacaagttt ga 61273612DNAStaphylococcus aureus 73atgttcaaaa aatatgactc aaaaaattca atcgtattaa aatctattct atcgctaggt 60atcatctatg ggggaacatt tggaatatat ccaaaagcag acgcgtcaac acaaaattcc 120tcaagtgtac aagataaaca attacaaaaa gttgaagaag taccaaataa ttcagaaaaa

180gctttggtta aaaaacttta cgatagatac agcaaggata caataaatgg aaaatctaat 240aaatctagga attgggttta ttcagagaga cctttaaatg aaaaccaagt tcgtatacat 300ttagaaggaa catacacagt tgctggcaga gtgtatacac ctaagaggaa tattactctt 360aataaagaag ttgtcacttt aaaagaattg gatcatatca taagatttgc tcatatttcc 420tatggcttgt atatgggaga acatttgcct aaaggtaaca tcgtcataaa tacaaaagat 480ggtggtaaat atacattaga gtcgcataaa gagctacaaa aagataggga aaatgtaaaa 540attaatacag ccgatataaa aaatgtaact ttcaaacttg tgaaaagtgt taatgacatt 600gaacaagttt ga 61274612DNAStaphylococcus aureus 74atgttcaaaa aatatgactc aaaaaattca atcgtattaa aatctattct atcgctaggt 60atcatctatg ggggaacatt tggaatatat ccaaaagcag acgcgtcaac acaaaattcc 120tcaagtgtac aagataaaca attacaaaaa gttgaagaag taccaaataa ttcagaaaaa 180gctttggtta aaaaacttta cgatagatac agcaaggata caataaatgg aaaatctaat 240aaatctagga attgggttta ttcagagaga cctttaaatg aaaaccaagt tcgtatacat 300ttagaaggaa catacacagt tgctggcaga gtgtatacac ctaagaggaa tattactctt 360aataaagaag ttgtcacttt aaaagaattg gatcatatca taagatttgc tcatatttcc 420tatggcttgt atatgggaga acatttgcct aaaggtaaca tcgtcataaa tacaaaagat 480ggtggtaaat atacattaga gtcgcataaa gagctacaaa aagataggga aaatgtaaaa 540attaatacag ccgatataaa aaatgtaact ttcaaacttg tgaaaagtgt taatgacatt 600gaacaagttt ga 61275612DNAStaphylococcus aureus 75atgttcaaaa aatatgactc aaaaaattca atcgtattaa aatctattct atcgctaggt 60atcatctatg ggggaacatt tggaatatat ccaaaagcag acgcgtcaac acaaaattcc 120tcaagtgtac aagataaaca attacaaaaa gttgaagaag taccaaataa ttcagaaaaa 180gctttggtta aaaaacttta cgatagatac agcaaggata caataaatgg aaaatctaat 240aaatctagga attgggttta ttcagagaga cctttaaatg aaaaccaagt tcgtatacat 300ttagaaggaa catacacagt tgctggcaga gtgtatacac ctaagaggaa tattactctt 360aataaagaag ttgtcacttt aaaagaattg gatcatatca taagatttgc tcatatttcc 420tatggcttgt atatgggaga acatttgcct aaaggtaaca tcgtcataaa tacaaaagat 480ggtggtaaat atacattaga gtcgcataaa gagctacaaa aagataggga aaatgtaaaa 540attaatacag ccgatataaa aaatgtaact ttcaaacttg tgaaaagtgt taatgacatt 600gaacaagttt ga 61276612DNAStaphylococcus aureus 76atgttcaaaa aatatgactc aaaaaattca atcgtattaa aatctattct atcgctaggt 60atcatctatg ggggaacatt tggaatatat ccaaaagcag acgcgtcaac acaaaattcc 120tcaagtgtac aagataaaca attacaaaaa gttgaagaag taccaaataa ttcagaaaaa 180gctttggtta aaaaacttta cgatagatac agcaaggata caataaatgg aaaatctaat 240aaatctagga attgggttta ttcagagaga cctttaaatg aaaaccaagt tcgtatacat 300ttagaaggaa catacacagt tgctgataga gtatatacac ctaagagaaa tattactctt 360aataaagaag ttgtcacttt aaaggaattg gatcatatca taagatttgc tcatatttct 420tatggcttat atatgggaga acatttgcct aaaggtaaca tcgtcataaa tacaaaagat 480ggcggtaaat atacattaga gtcgcataaa gagctacaaa aagataggga aaatgtaaaa 540attaatacag ccgatataaa aaatgtaact ttcaaacttg tgaaaagtgt taatgacatt 600gaacaagttt ga 61277612DNAStaphylococcus aureus 77atgttcaaaa aatatgactc aaaaaattca atcgtattaa aatctattct atcgctaggt 60atcatctatg ggggaacatt tggaatatat ccaaaagcag acgcgtcaac acaaaattcc 120tcaagtgtac aagataaaca attacaaaaa gttgaagaag taccaaataa ttcagaaaaa 180gctttggtta aaaaacttta cgatagatac agcaaggata caataaatgg aaaatctaat 240aaatctagga attgggttta ttcagagaga cctttaaatg aaaaccaagt tcgtatacat 300ttagaaggaa catacacagt tgctgataga gtatatacac ctaagagaaa tattactctt 360aataaagaag ttgtcacttt aaaggaattg gatcatatca taagatttgc tcatatttct 420tatggcttat atatgggaga acatttgcct aaaggtaaca tcgtcataaa tacaaaagat 480ggcggtaaat atacattaga gtcgcataaa gagctacaaa aagataggga aaatgtaaaa 540attaatacag ccgatataaa aaatgtaact ttcaaacttg tgaaaagtgt taatgacatt 600gaacaagttt ga 61278612DNAStaphylococcus aureus 78atgttcaaaa aatatgactc aaaaaattca atcgtattaa aatctattct atcgctaggt 60atcatctatg ggggaacatt tggaatatat ccaaaagcag acgcgtcaac acaaaattcc 120tcaagtgtac aagataaaca attacaaaaa gttgaagaag taccaaataa ttcagaaaaa 180gctttggtta aaaaacttta cgatagatac agcaaggata caataaatgg aaaatctaat 240aaatctagga attgggttta ttcagagaga cctttaaatg aaaaccaagt tcgtatacat 300ttagaaggaa catacacagt tgctgataga gtatatacac ctaagagaaa tattactctt 360aataaagaag ttgtcacttt aaaggaattg gatcatatca taagatttgc tcatatttct 420tatggcttat atatgggaga acatttgcct aaaggtaaca tcgtcataaa tacaaaagat 480ggcggtaaat atacattaga gtcgcataaa gagctacaaa aagataggga aaatgtaaaa 540attaatacag ccgatataaa aaatgtaact ttcaaacttg tgaaaagtgt taatgacatt 600gaacaagttt ga 61279612DNAStaphylococcus aureus 79atgttcaaaa aatatgactc aaaaaattca atcgtattaa aatctattct atcgctaggt 60atcatctatg ggggaacatt tggaatatat ccaaaagcag acgcgtcaac acaaaattcc 120tcaagtgtac aagataaaca attacaaaaa gttgaagaag taccaaataa ttcagaaaaa 180gctttggtta aaaaacttta cgatagatac agcaaggata caataaatgg aaaatctaat 240aaatctagga attgggttta ttcagagaga cctttaaatg aaaaccaagt tcgtatacat 300ttagaaggaa catacacagt tgctgataga gtatatacac ctaagagaaa tattactctt 360aataaagaag ttgtcacttt aaaggaattg gatcatatca taagatttgc tcatatttct 420tatggcttat atatgggaga acatttgcct aaaggtaaca tcgtcataaa tacaaaagat 480ggcggtaaat atacattaga gtcgcataaa gagctacaaa aagataggga aaatgtaaaa 540attaatacag ccgatataaa aaatgtaact ttcaaacttg tgaaaagtgt taatgacatt 600gaacaagttt ga 61280612DNAStaphylococcus aureus 80atgttcaaaa aatatgactc aaaaaattca atcgtattaa aatctattct atcgctaggt 60atcatctatg ggggaacatt tggaatatat ccaaaagcag acgcgtcaac acaaaattcc 120tcaagtgtac aagataaaca attacaaaaa gttgaagaag taccaaataa ttcagaaaaa 180gctttggtta aaaaacttta cgatagatac agcaaggata caataaatgg aaaatctaat 240aaatctagga attgggttta ttcagagaga cctttaaatg aaaaccaagt tcgtatacat 300ttagaaggaa catacacagt tgctgataga gtatatacac ctaagagaaa tattactctt 360aataaagaag ttgtcacttt aaaggaattg gatcatatca taagatttgc tcatatttct 420tatggcttat atatgggaga acatttgcct aaaggtaaca tcgtcataaa tacaaaagat 480ggcggtaaat atacattaga gtcgcataaa gagctacaaa aagataggga aaatgtaaaa 540attaatacag ccgatataaa aaatgtaact ttcaaacttg tgaaaagtgt taatgacatt 600gaacaagttt ga 61281612DNAStaphylococcus aureus 81atgttcaaaa aatatgactc aaaaaattca atcgtattaa aatctattct atcgctaggt 60atcatctatg ggggaacatt tggaatatat ccaaaagcag acgcgtcaac acaaaattcc 120tcaagtgtac aagataaaca attccaaaaa gttgaagaag taccaaataa ttcagaaaaa 180gctttggtta aaaaactgta cgatagatac agccaaaata caataaacgg aaaatctaat 240aaagctagga attgggttta ttcagagaga cctttaaatg aaaatcaagt tcgcatacat 300ttagaaggta catacagagt tgctgataga gtgtatacac ctaagaggaa cattactctt 360aataaagaag ttgtcacttt aaaagaattg gatcatatca taagatttgc tcatatttct 420tatggcttat atatgggaga acatttgcct aaaggtaaca tcgtcataaa tacaaagaat 480ggcggtaaat atacattaga gtcgcacaaa gagttacaaa agaataggga aaatgtagaa 540attaatactg atgatataaa aaatgtaact ttcgaacttg tgaaaagtgt taatgacatt 600gaacaagttt ga 61282203PRTStaphylococcus aureus 82Met Phe Lys Lys Tyr Asp Ser Lys Asn Ser Ile Val Leu Lys Ser Ile1 5 10 15Leu Ser Leu Gly Ile Ile Tyr Gly Gly Thr Phe Gly Ile Tyr Pro Lys 20 25 30Ala Asp Ala Ser Thr Gln Asn Ser Ser Ser Val Gln Asp Lys Gln Leu 35 40 45Gln Lys Val Glu Glu Val Pro Asn Asn Ser Glu Lys Ala Leu Val Lys 50 55 60Lys Leu Tyr Asp Arg Tyr Ser Lys Asp Thr Ile Asn Gly Lys Ser Asn65 70 75 80Lys Ser Arg Asn Trp Val Tyr Ser Glu Arg Pro Leu Asn Glu Asn Gln 85 90 95Val Arg Ile His Leu Glu Gly Thr Tyr Thr Val Ala Gly Arg Val Tyr 100 105 110Thr Pro Lys Arg Asn Ile Thr Leu Asn Lys Glu Val Val Thr Leu Lys 115 120 125Glu Leu Asp His Ile Ile Arg Phe Ala His Ile Ser Tyr Gly Leu Tyr 130 135 140Met Gly Glu His Leu Pro Lys Gly Asn Ile Val Ile Asn Thr Lys Asp145 150 155 160Gly Gly Lys Tyr Thr Leu Glu Ser His Lys Glu Leu Gln Lys Asp Arg 165 170 175Glu Asn Val Lys Ile Asn Thr Ala Asp Ile Lys Asn Val Thr Phe Lys 180 185 190Leu Val Lys Ser Val Asn Asp Ile Glu Gln Val 195 20083203PRTStaphylococcus aureus 83Met Phe Lys Lys Tyr Asp Ser Lys Asn Ser Ile Val Leu Lys Ser Ile1 5 10 15Leu Ser Leu Gly Ile Ile Tyr Gly Gly Thr Phe Gly Ile Tyr Pro Lys 20 25 30Ala Asp Ala Ser Thr Gln Asn Ser Ser Ser Val Gln Asp Lys Gln Leu 35 40 45Gln Lys Val Glu Glu Val Pro Asn Asn Ser Glu Lys Ala Leu Val Lys 50 55 60Lys Leu Tyr Asp Arg Tyr Ser Lys Asp Thr Ile Asn Gly Lys Ser Asn65 70 75 80Lys Ser Arg Asn Trp Val Tyr Ser Glu Arg Pro Leu Asn Glu Asn Gln 85 90 95Val Arg Ile His Leu Glu Gly Thr Tyr Thr Val Ala Gly Arg Val Tyr 100 105 110Thr Pro Lys Arg Asn Ile Thr Leu Asn Lys Glu Val Val Thr Leu Lys 115 120 125Glu Leu Asp His Ile Ile Arg Phe Ala His Ile Ser Tyr Gly Leu Tyr 130 135 140Met Gly Glu His Leu Pro Lys Gly Asn Ile Val Ile Asn Thr Lys Asp145 150 155 160Gly Gly Lys Tyr Thr Leu Glu Ser His Lys Glu Leu Gln Lys Asp Arg 165 170 175Glu Asn Val Lys Ile Asn Thr Ala Asp Ile Lys Asn Val Thr Phe Lys 180 185 190Leu Val Lys Ser Val Asn Asp Ile Glu Gln Val 195 20084203PRTStaphylococcus aureus 84Met Phe Lys Lys Tyr Asp Ser Lys Asn Ser Ile Val Leu Lys Ser Ile1 5 10 15Leu Ser Leu Gly Ile Ile Tyr Gly Gly Thr Phe Gly Ile Tyr Pro Lys 20 25 30Ala Asp Ala Ser Thr Gln Asn Ser Ser Ser Val Gln Asp Lys Gln Leu 35 40 45Gln Lys Val Glu Glu Val Pro Asn Asn Ser Glu Lys Ala Leu Val Lys 50 55 60Lys Leu Tyr Asp Arg Tyr Ser Lys Asp Thr Ile Asn Gly Lys Ser Asn65 70 75 80Lys Ser Arg Asn Trp Val Tyr Ser Glu Arg Pro Leu Asn Glu Asn Gln 85 90 95Val Arg Ile His Leu Glu Gly Thr Tyr Thr Val Ala Gly Arg Val Tyr 100 105 110Thr Pro Lys Arg Asn Ile Thr Leu Asn Lys Glu Val Val Thr Leu Lys 115 120 125Glu Leu Asp His Ile Ile Arg Phe Ala His Ile Ser Tyr Gly Leu Tyr 130 135 140Met Gly Glu His Leu Pro Lys Gly Asn Ile Val Ile Asn Thr Lys Asp145 150 155 160Gly Gly Lys Tyr Thr Leu Glu Ser His Lys Glu Leu Gln Lys Asp Arg 165 170 175Glu Asn Val Lys Ile Asn Thr Ala Asp Ile Lys Asn Val Thr Phe Lys 180 185 190Leu Val Lys Ser Val Asn Asp Ile Glu Gln Val 195 20085203PRTStaphylococcus aureus 85Met Phe Lys Lys Tyr Asp Ser Lys Asn Ser Ile Val Leu Lys Ser Ile1 5 10 15Leu Ser Leu Gly Ile Ile Tyr Gly Gly Thr Phe Gly Ile Tyr Pro Lys 20 25 30Ala Asp Ala Ser Thr Gln Asn Ser Ser Ser Val Gln Asp Lys Gln Leu 35 40 45Gln Lys Val Glu Glu Val Pro Asn Asn Ser Glu Lys Ala Leu Val Lys 50 55 60Lys Leu Tyr Asp Arg Tyr Ser Lys Asp Thr Ile Asn Gly Lys Ser Asn65 70 75 80Lys Ser Arg Asn Trp Val Tyr Ser Glu Arg Pro Leu Asn Glu Asn Gln 85 90 95Val Arg Ile His Leu Glu Gly Thr Tyr Thr Val Ala Asp Arg Val Tyr 100 105 110Thr Pro Lys Arg Asn Ile Thr Leu Asn Lys Glu Val Val Thr Leu Lys 115 120 125Glu Leu Asp His Ile Ile Arg Phe Ala His Ile Ser Tyr Gly Leu Tyr 130 135 140Met Gly Glu His Leu Pro Lys Gly Asn Ile Val Ile Asn Thr Lys Asp145 150 155 160Gly Gly Lys Tyr Thr Leu Glu Ser His Lys Glu Leu Gln Lys Asp Arg 165 170 175Glu Asn Val Lys Ile Asn Thr Ala Asp Ile Lys Asn Val Thr Phe Lys 180 185 190Leu Val Lys Ser Val Asn Asp Ile Glu Gln Val 195 20086203PRTStaphylococcus aureus 86Met Phe Lys Lys Tyr Asp Ser Lys Asn Ser Ile Val Leu Lys Ser Ile1 5 10 15Leu Ser Leu Gly Ile Ile Tyr Gly Gly Thr Phe Gly Ile Tyr Pro Lys 20 25 30Ala Asp Ala Ser Thr Gln Asn Ser Ser Ser Val Gln Asp Lys Gln Leu 35 40 45Gln Lys Val Glu Glu Val Pro Asn Asn Ser Glu Lys Ala Leu Val Lys 50 55 60Lys Leu Tyr Asp Arg Tyr Ser Lys Asp Thr Ile Asn Gly Lys Ser Asn65 70 75 80Lys Ser Arg Asn Trp Val Tyr Ser Glu Arg Pro Leu Asn Glu Asn Gln 85 90 95Val Arg Ile His Leu Glu Gly Thr Tyr Thr Val Ala Asp Arg Val Tyr 100 105 110Thr Pro Lys Arg Asn Ile Thr Leu Asn Lys Glu Val Val Thr Leu Lys 115 120 125Glu Leu Asp His Ile Ile Arg Phe Ala His Ile Ser Tyr Gly Leu Tyr 130 135 140Met Gly Glu His Leu Pro Lys Gly Asn Ile Val Ile Asn Thr Lys Asp145 150 155 160Gly Gly Lys Tyr Thr Leu Glu Ser His Lys Glu Leu Gln Lys Asp Arg 165 170 175Glu Asn Val Lys Ile Asn Thr Ala Asp Ile Lys Asn Val Thr Phe Lys 180 185 190Leu Val Lys Ser Val Asn Asp Ile Glu Gln Val 195 20087203PRTStaphylococcus aureus 87Met Phe Lys Lys Tyr Asp Ser Lys Asn Ser Ile Val Leu Lys Ser Ile1 5 10 15Leu Ser Leu Gly Ile Ile Tyr Gly Gly Thr Phe Gly Ile Tyr Pro Lys 20 25 30Ala Asp Ala Ser Thr Gln Asn Ser Ser Ser Val Gln Asp Lys Gln Leu 35 40 45Gln Lys Val Glu Glu Val Pro Asn Asn Ser Glu Lys Ala Leu Val Lys 50 55 60Lys Leu Tyr Asp Arg Tyr Ser Lys Asp Thr Ile Asn Gly Lys Ser Asn65 70 75 80Lys Ser Arg Asn Trp Val Tyr Ser Glu Arg Pro Leu Asn Glu Asn Gln 85 90 95Val Arg Ile His Leu Glu Gly Thr Tyr Thr Val Ala Asp Arg Val Tyr 100 105 110Thr Pro Lys Arg Asn Ile Thr Leu Asn Lys Glu Val Val Thr Leu Lys 115 120 125Glu Leu Asp His Ile Ile Arg Phe Ala His Ile Ser Tyr Gly Leu Tyr 130 135 140Met Gly Glu His Leu Pro Lys Gly Asn Ile Val Ile Asn Thr Lys Asp145 150 155 160Gly Gly Lys Tyr Thr Leu Glu Ser His Lys Glu Leu Gln Lys Asp Arg 165 170 175Glu Asn Val Lys Ile Asn Thr Ala Asp Ile Lys Asn Val Thr Phe Lys 180 185 190Leu Val Lys Ser Val Asn Asp Ile Glu Gln Val 195 20088203PRTStaphylococcus aureus 88Met Phe Lys Lys Tyr Asp Ser Lys Asn Ser Ile Val Leu Lys Ser Ile1 5 10 15Leu Ser Leu Gly Ile Ile Tyr Gly Gly Thr Phe Gly Ile Tyr Pro Lys 20 25 30Ala Asp Ala Ser Thr Gln Asn Ser Ser Ser Val Gln Asp Lys Gln Leu 35 40 45Gln Lys Val Glu Glu Val Pro Asn Asn Ser Glu Lys Ala Leu Val Lys 50 55 60Lys Leu Tyr Asp Arg Tyr Ser Lys Asp Thr Ile Asn Gly Lys Ser Asn65 70 75 80Lys Ser Arg Asn Trp Val Tyr Ser Glu Arg Pro Leu Asn Glu Asn Gln 85 90 95Val Arg Ile His Leu Glu Gly Thr Tyr Thr Val Ala Asp Arg Val Tyr 100 105 110Thr Pro Lys Arg Asn Ile Thr Leu Asn Lys Glu Val Val Thr Leu Lys 115 120 125Glu Leu Asp His Ile Ile Arg Phe Ala His Ile Ser Tyr Gly Leu Tyr 130 135 140Met Gly Glu His Leu Pro Lys Gly Asn Ile Val Ile Asn Thr Lys Asp145 150 155 160Gly Gly Lys Tyr Thr Leu Glu Ser His Lys Glu Leu Gln Lys Asp Arg 165 170 175Glu Asn Val Lys Ile Asn Thr Ala Asp Ile Lys Asn Val Thr Phe Lys 180 185 190Leu Val Lys Ser Val Asn Asp Ile Glu Gln Val 195 20089203PRTStaphylococcus aureus 89Met Phe Lys Lys Tyr Asp Ser Lys Asn Ser Ile Val Leu Lys Ser Ile1 5 10 15Leu Ser Leu Gly Ile Ile Tyr Gly Gly Thr Phe Gly Ile Tyr Pro Lys 20 25 30Ala Asp Ala Ser

Thr Gln Asn Ser Ser Ser Val Gln Asp Lys Gln Leu 35 40 45Gln Lys Val Glu Glu Val Pro Asn Asn Ser Glu Lys Ala Leu Val Lys 50 55 60Lys Leu Tyr Asp Arg Tyr Ser Lys Asp Thr Ile Asn Gly Lys Ser Asn65 70 75 80Lys Ser Arg Asn Trp Val Tyr Ser Glu Arg Pro Leu Asn Glu Asn Gln 85 90 95Val Arg Ile His Leu Glu Gly Thr Tyr Thr Val Ala Asp Arg Val Tyr 100 105 110Thr Pro Lys Arg Asn Ile Thr Leu Asn Lys Glu Val Val Thr Leu Lys 115 120 125Glu Leu Asp His Ile Ile Arg Phe Ala His Ile Ser Tyr Gly Leu Tyr 130 135 140Met Gly Glu His Leu Pro Lys Gly Asn Ile Val Ile Asn Thr Lys Asp145 150 155 160Gly Gly Lys Tyr Thr Leu Glu Ser His Lys Glu Leu Gln Lys Asp Arg 165 170 175Glu Asn Val Lys Ile Asn Thr Ala Asp Ile Lys Asn Val Thr Phe Lys 180 185 190Leu Val Lys Ser Val Asn Asp Ile Glu Gln Val 195 20090203PRTStaphylococcus aureus 90Met Phe Lys Lys Asn Asp Ser Lys Asn Ser Ile Leu Leu Lys Ser Ile1 5 10 15Leu Ser Leu Gly Ile Ile Tyr Gly Gly Thr Phe Gly Ile Tyr Pro Lys 20 25 30Ala Asp Ala Ser Thr Gln Asn Ser Pro Ser Val Gln Asp Lys Gln Phe 35 40 45Gln Lys Val Glu Glu Val Pro Asn Asn Ser Glu Lys Ala Leu Val Lys 50 55 60Lys Leu Tyr Asp Arg Tyr Ser Gln Asn Thr Ile Asn Gly Lys Ser Asn65 70 75 80Lys Ser Arg Asn Trp Val Tyr Ser Glu Arg Pro Leu Asn Glu Asn Gln 85 90 95Val Arg Ile Asn Leu Glu Gly Thr Tyr Arg Val Ala Asp Arg Val Tyr 100 105 110Thr Pro Lys Arg Asn Ile Thr Leu Asn Lys Glu Val Val Thr Leu Lys 115 120 125Glu Leu Asp His Ile Ile Arg Phe Ala His Ile Ser Tyr Gly Leu Tyr 130 135 140Met Gly Glu His Leu Pro Lys Gly Asn Ile Val Ile Asn Thr Lys Asp145 150 155 160Gly Gly Lys Tyr Thr Leu Glu Ser His Lys Glu Leu Gln Lys Asp Arg 165 170 175Glu Asn Val Lys Ile Asn Thr Ala Asp Ile Lys Asn Val Thr Phe Lys 180 185 190Leu Val Lys Ser Val Asn Asp Ile Glu Gln Val 195 20091203PRTStaphylococcus aureus 91Met Phe Lys Lys Tyr Asp Ser Lys Asn Ser Ile Val Leu Lys Ser Ile1 5 10 15Leu Ser Leu Gly Ile Ile Tyr Gly Gly Thr Phe Gly Ile Tyr Pro Lys 20 25 30Ala Asp Ala Ser Thr Gln Asn Ser Ser Ser Val Gln Asp Lys Gln Phe 35 40 45Gln Lys Val Glu Glu Val Pro Asn Asn Ser Glu Lys Ala Leu Val Lys 50 55 60Lys Leu Tyr Asp Arg Tyr Ser Gln Asn Thr Ile Asn Gly Lys Ser Asn65 70 75 80Lys Ala Arg Asn Trp Val Tyr Ser Glu Arg Pro Leu Asn Glu Asn Gln 85 90 95Val Arg Ile His Leu Glu Gly Thr Tyr Arg Val Ala Asp Arg Val Tyr 100 105 110Thr Pro Lys Arg Asn Ile Thr Leu Asn Lys Glu Val Val Thr Leu Lys 115 120 125Glu Leu Asp His Ile Ile Arg Phe Ala His Ile Ser Tyr Gly Leu Tyr 130 135 140Met Gly Glu His Leu Pro Lys Gly Asn Ile Val Ile Asn Thr Lys Asn145 150 155 160Gly Gly Lys Tyr Thr Leu Glu Ser His Lys Glu Leu Gln Lys Asn Arg 165 170 175Glu Asn Val Glu Ile Asn Thr Asp Asp Ile Lys Asn Val Thr Phe Glu 180 185 190Leu Val Lys Ser Val Asn Asp Ile Glu Gln Val 195 20092203PRTArtificial sequencesynthetic sequencemisc_feature(5)..(5)Xaa can be any naturally occurring amino acidmisc_feature(12)..(12)Xaa can be any naturally occurring amino acidmisc_feature(41)..(41)Xaa can be any naturally occurring amino acidmisc_feature(48)..(48)Xaa can be any naturally occurring amino acidmisc_feature(72)..(73)Xaa can be any naturally occurring amino acidmisc_feature(82)..(82)Xaa can be any naturally occurring amino acidmisc_feature(100)..(100)Xaa can be any naturally occurring amino acidmisc_feature(106)..(106)Xaa can be any naturally occurring amino acidmisc_feature(109)..(109)Xaa can be any naturally occurring amino acidmisc_feature(160)..(160)Xaa can be any naturally occurring amino acidmisc_feature(175)..(175)Xaa can be any naturally occurring amino acidmisc_feature(180)..(180)Xaa can be any naturally occurring amino acidmisc_feature(184)..(184)Xaa can be any naturally occurring amino acidmisc_feature(192)..(192)Xaa can be any naturally occurring amino acidmisc_feature(201)..(203)Xaa can be any naturally occurring amino acid 92Met Phe Lys Lys Xaa Asp Ser Lys Asn Ser Ile Xaa Leu Lys Ser Ile1 5 10 15Leu Ser Leu Gly Ile Ile Tyr Gly Gly Thr Phe Gly Ile Tyr Pro Lys 20 25 30Ala Asp Ala Ser Thr Gln Asn Ser Xaa Ser Val Gln Asp Lys Gln Xaa 35 40 45Gln Lys Val Glu Glu Val Pro Asn Asn Ser Glu Lys Ala Leu Val Lys 50 55 60Lys Leu Tyr Asp Arg Tyr Ser Xaa Xaa Thr Ile Asn Gly Lys Ser Asn65 70 75 80Lys Xaa Arg Asn Trp Val Tyr Ser Glu Arg Pro Leu Asn Glu Asn Gln 85 90 95Val Arg Ile Xaa Leu Glu Gly Thr Tyr Xaa Val Ala Xaa Arg Val Tyr 100 105 110Thr Pro Lys Arg Asn Ile Thr Leu Asn Lys Glu Val Val Thr Leu Lys 115 120 125Glu Leu Asp His Ile Ile Arg Phe Ala His Ile Ser Tyr Gly Leu Tyr 130 135 140Met Gly Glu His Leu Pro Lys Gly Asn Ile Val Ile Asn Thr Lys Xaa145 150 155 160Gly Gly Lys Tyr Thr Leu Glu Ser His Lys Glu Leu Gln Lys Xaa Arg 165 170 175Glu Asn Val Xaa Ile Asn Thr Xaa Asp Ile Lys Asn Val Thr Phe Xaa 180 185 190Leu Val Lys Ser Val Asn Asp Ile Xaa Xaa Xaa 195 2009314PRTStaphylococcus aureus 93Gln Asn Thr Ile Asn Gly Lys Ser Asn Lys Ser Arg Asn Trp1 5 109414PRTStaphylococcus aureus 94Gln Asn Thr Ile Asn Gly Lys Ser Asn Lys Ala Arg Asn Trp1 5 109515PRTStaphylococcus aureus 95Lys Asn Gly Gly Lys Tyr Thr Leu Glu Ser His Lys Glu Leu Gln1 5 10 159614PRTArtificial sequencesynthetic sequencemisc_feature(1)..(2)Xaa can be any naturally occurring amino acidmisc_feature(11)..(11)Xaa can be any naturally occurring amino acid 96Xaa Xaa Thr Ile Asn Gly Lys Ser Asn Lys Xaa Arg Asn Trp1 5 109715PRTArtificial sequencesynthetic sequencemisc_feature(2)..(2)Xaa can be any naturally occurring amino acid 97Lys Xaa Gly Gly Lys Tyr Thr Leu Glu Ser His Lys Glu Leu Gln1 5 10 15981755DNAStaphylococcus aureus 98atgacagagt catatccaat tattaaggaa ggctcacaag tcggaagcta ctttctattt 60ttcatcataa ttgcattttt gttatatgcc aatgtgttat ttattaaacg acgaagttat 120gagcttgcat tatatcaaac attaggttta tctaaattca acattattta tatactaatg 180ctcgaacaat tactaatatt tataattacg gcaatattag gtattattat tggtattttt 240ggttcaaaac tgttattaat gattgtcttt acattattag gaattaaaga aaaggttcca 300attattttta gtttgagggc ggtatttgaa acattaatgt taatcggtgt cgcttatttt 360ttaacctctg ctcaaaattt tatattagtg ttcaaacaat ctatttcaca gatgtcaaag 420aataaccagg ttaaagaaac aaatcataat aaaattacat ttgaagaggt tgttttaggc 480atcttaggta tagtattgat tatcacagga tactatctat ctttgaacat tgttcaatat 540tatgattcta tcggtatact tatgtttatt ttattgtcaa ctgtgattgg ggcatactta 600ttttttaaaa gctctgtttc tctagttttt aaaatggtga agaagtttag aaaaggtgtt 660ataagtgtaa atgatgtcat gttctcatca tctattatgt atcgtattaa gaaaaatgct 720ttttcactta cggtcatggc aatcatttca gcgattactg tttcagttct ttgctttgct 780gctataagta gagcgtcctt atcaagtgaa ataaaatata ctgcaccaca cgacgttaca 840attaaagacc aacaaaaagc taatcaatta gcaagtgaat taaacaatca aaaaattcct 900catttttata attataaaga agtaattcat acgaaattgt ataaagataa tttatttgat 960gtaaaagcga aagaaccata caatgtaaca attactagtg ataaatatat ccctaatact 1020gatttgaaac gtggacaagc tgatttgttt gtagcggaag gttctatcaa agatttagtg 1080aaacataaga agcatggtaa ggcaattata ggaacgaaaa aacatcatgt taatattaag 1140ttacggaaag atattaataa aatctatttt atgacagatg ttgatttagg tggaccaacg 1200tttgtcttaa atgacaaaga ctatcaagaa ataagaaagt atacaaaagc aaagcatatc 1260gtctctcaat ttggattcga tttgaaacat aaaaaagatg ctttagcatt agaaaaagtg 1320aaaaataaag ttgataaatc tattaaaaca agaagtgaag cgataagctc aatatcaagt 1380ttaaccggaa tattattatt tgtaacatca tttttaggta ttacattctt gattgctgta 1440tgttgcatta tatacattaa gcaaatagat gaaaccgaag atgagttaga gaattatagt 1500atattgagaa agcttggatt tacacaaaaa gatatggcaa ggggactaaa gtttaaaatt 1560atgtttaatt ttgggttacc tttagttatt gcactatcac atgcatattt tacatcatta 1620gcatatatga aattaatggg tacaacgaat caaataccgg ttttcatagt aatgggatta 1680tacatttgta tgtatgctgt ttttgcagtg acggcttata atcattccaa gcgaacaatt 1740agacattcca tataa 1755991755DNAStaphylococcus aureus 99atgacagagt catatccaat tattaaggaa ggctcacaag tcggaagcta ctttctattt 60ttcatcataa ttgcattttt gttatatgcc aatgtgttat ttattaaacg acgaagttat 120gagcttgcat tatatcaaac attaggttta tctaaattca acattattta tatactaatg 180ctcgaacaat tactaatatt tataattacg gcaatattag gtattattat tggtattttt 240ggttcaaaac tgttattaat gattgtcttt acattattag gaattaaaga aaaggttcca 300attattttta gtttgagggc ggtatttgaa acattaatgt taatcggtgt cgcttatttt 360ttaacctctg ctcaaaattt tatattagtg ttcaaacaat ctatttcaca gatgtcaaag 420aataaccagg ttaaagaaac aaatcataat aaaattacat ttgaagaggt tgttttaggc 480atcttaggta tagtattgat tatcacagga tactatctat ctttgaacat tgttcaatat 540tatgattcta tcggtatact tatgtttatt ttattgtcaa ctgtgattgg ggcatactta 600ttttttaaaa gctctgtttc tctagttttt aaaatggtga agaagtttag aaaaggtgtt 660ataagtgtaa atgatgtcat gttctcatca tctattatgt atcgtattaa gaaaaatgct 720ttttcactta cggtcatggc aatcatttca gcgattactg tttcagttct ttgctttgct 780gctataagta gagcgtcctt atcaagtgaa ataaaatata ctgcaccaca cgacgttaca 840attaaagacc aacaaaaagc taatcaatta gcaagtgaat taaacaatca aaaaattcct 900catttttata attataaaga agtaattcat acgaaattgt ataaagataa tttatttgat 960gtaaaagcga aagaaccata caatgtaaca attactagtg ataaatatat ccctaatact 1020gatttgaaac gtggacaagc tgatttgttt gtagcggaag gttctatcaa agatttagtg 1080aaacataaga agcatggtaa ggcaattata ggaacgaaaa aacatcatgt taatattaag 1140ttacggaaag atattaataa aatctatttt atgacagatg ttgatttagg tggaccaacg 1200tttgtcttaa atgacaaaga ctatcaagaa ataagaaagt atacaaaagc aaagcatatc 1260gtctctcaat ttggattcga tttgaaacat aaaaaagatg ctttagcatt agaaaaagtg 1320aaaaataaag ttgataaatc tattaaaaca agaagtgaag cgataagctc aatatcaagt 1380ttaaccggaa tattattatt tgtaacatca tttttaggta ttacattctt gattgctgta 1440tgttgcatta tatacattaa gcaaatagat gaaaccgaag atgagttaga gaattatagt 1500atattgagaa agcttggatt tacacaaaaa gatatggcaa ggggactaaa gtttaaaatt 1560atgtttaatt ttgggttacc tttagttatt gtactatcac atgcatattt tacatcatta 1620gcatatatga aattaatggg tacaacgaat caaataccgg ttttcatagt aatgggatta 1680tacatttgta tgtatgctgt ttttgcagtg acggcttata atcattccaa gcgaacaatt 1740agacattcca tataa 17551001755DNAStaphylococcus aureus 100atgacagagt catatccaat tattaaggaa ggctcacaag tcggaagcta ctttctattt 60ttcatcataa ttgcattttt gttatatgcc aatgtgttat ttattaaacg acgaagttat 120gagcttgcat tatatcaaac attaggttta tctaaattca acattattta tatactaatg 180ctcgaacaat tactaatatt tataattacg gcaatattag gtattattat tggtattttt 240ggttcaaaac tgttattaat gattgtcttt acattattag gaattaaaga aaaggttcca 300attattttta gtttgagggc ggtatttgaa acattaatgt taatcggtgt cgcttatttt 360ttaacctctg ctcaaaattt tatattagtg ttcaaacaat ctatttcaca gatgtcaaag 420aataaccagg ttaaagaaac aaatcataat aaaattacat ttgaagaggt tgttttaggc 480atcttaggta tagtattgat taccacagga tactatctat ctttgaacat tgttcaatat 540tatgattcta tcggtatact tatgtttatt ttattgtcaa ctgtgattgg ggcatactta 600ttttttaaaa gctctgtttc tctagttttt aaaatggtga agaagtttag aaaaggtgtt 660ataagtgtaa atgatgtcat gttctcatca tctattatgt atcgtattaa gaaaaatgct 720ttttcactta cggtcatggc aatcatttca gcgattactg tttcagttct ttgctttgct 780gctataagta gagcgtcctt atcaagtgaa ataaaatata ctgcaccaca cgacgttaca 840attaaagacc aacaaaaagc taatcaatta gcaagtgaat taaacaatca aaaaattcct 900catttttata attataaaga agtaattcat acgaaattgt ataaagataa tttatttgat 960gtaaaagcga aagaaccata caatgtaaca attactagtg ataaatatat ccctaatact 1020gatttgaaac gtggacaagc tgatttgttt gtagcggaag gttctatcaa agatttagtg 1080aaacataaga agcatggtaa ggcaattata ggaacgaaaa aacatcatgt taatattaag 1140ttacggaaag atattaataa aatctatttt atgacagatg ttgatttagg tggaccaacg 1200tttgtcttaa atgacaaaga ctatcaagaa ataagaaagt atacaaaagc aaagcatatc 1260gtctctcaat ttggattcga tttgaaacat aaaaaagatg ctttagcatt agaaaaagtg 1320aaaaataaag ttgataaatc tattaaaaca agaagtgaag cgataagctc aatatcaagt 1380ttaaccggaa tattattatt tgtaacatca tttttaggta ttacattctt gattgctgta 1440tgttgcatta tatacattaa gcaaatagat gaaaccgaag atgagttaga gaattatagt 1500atattgagaa agcttggatt tacacaaaaa gatatggcaa ggggactaaa gtttaaaatt 1560atgtttaatt ttgggttacc tttagttatt gcactatcac atgcatattt tacatcatta 1620gcatatatga aattaatggg tacaacgaat caaataccgg ttttcatagt aatgggatta 1680tacatttgta tgtatgctgt ttttgcagtg acggcttata atcattccaa gcgaacaatt 1740agacattcca tataa 17551011840DNAStaphylococcus aureus 101atgaccttta acgagataat atttaaaaat ttccgtcaaa atttatcaca gctttgtagc 60attaaaatac gcgcataaac taaacatgac agagtcatat ccaattatta aggaaggctc 120acaagtcgga agctactttc tatttttcat cataattgca tttttgttat atgccaatgt 180gttatttatt aaacgacgaa gttatgagct tgcattatat caaacattag gtttatctaa 240attcaacatt atttatatac taatgctcga acaattacta atatttataa ttacggcaat 300attaggtatt attattggta tttttggttc aaaactgtta ttaatgattg tctttacatt 360attaggaatt aaagaaaagg ttccaattat ttttagtttg agggcggtat ttgaaacatt 420aatgttaatc ggtgtcgctt attttttaac ctctgctcaa aattttatat tagtgttcaa 480acaatctatt tcacagatgt caaagaataa ccaggttaaa gaaacaaatc ataataaaat 540tacatttgaa gaggttgttt taggcatctt aggtatagta ttgattacca caggatacta 600tctatctttg aacattgttc aatattatga ttctatcggt atacttatgt ttattttatt 660gtcaactgtg attggggcat acttattttt taaaagctct gtttctctag tttttaaaat 720ggtgaagaag tttagaaaag gtgttataag tgtaaatgat gtcatgttct catcatctat 780tatgtatcgt attaagaaaa atgctttttc acttacggtc atggcaatca tttcagcgat 840tactgtttca gttctttgct ttgctgctat aagtagagcg tccttatcaa gtgaaataaa 900atatactgca ccacacgacg ttacaattaa agaccaacaa aaagctaatc aattagcaag 960tgaattaaac aatcaaaaaa ttcctcattt ttataattat aaagaagtaa ttcatacgaa 1020attgtataaa gataatttat ttgatgtaaa agcgaaagaa ccatacaatg taacaattac 1080tagtgataaa tatatcccta atactgattt gaaacgtgga caagctgatt tgtttgtagc 1140ggaaggttct atcaaagatt tagtgaaaca taagaagcat ggtaaggcaa ttataggaac 1200gaaaaaacat catgttaata ttaagttacg gaaagatatt aataaaatct attttatgac 1260agatgttgat ttaggtggac caacgtttgt cttaaatgac aaagactatc aagaaataag 1320aaagtataca aaagcaaagc atatcgtctc tcaatttgga ttcgatttga aacataaaaa 1380agatgcttta gcattagaaa aagtgaaaaa taaagttgat aaatctatta aaacaagaag 1440tgaagcgata agctcaatat caagtttaac cggaatatta ttatttgtaa catcattttt 1500aggtattaca ttcttgattg ctgtatgttg cattatatac attaagcaaa tagatgaaac 1560cgaagatgag ttagagaatt atagtatatt gagaaagctt ggatttacac aaaaagatat 1620ggcaagggga ctaaagttta aaattatgtt taattttggg ttacctttag ttattgcact 1680atcacatgca tattttacat cattagcata tatgaaatta atgggtacaa cgaatcaaat 1740accggttttc atagtaatgg gattatacat ttgtatgtat gctgtttttg cagtgacggc 1800ttataatcat tccaagcgaa caattagaca ttccatataa 18401021890DNAStaphylococcus aureus 102atgaccttta acgagataat atttaaaaat ttccgtcaaa atttatcaca ttatgccatc 60tatctttttt cattaattac gagtgtagta ttgtatttta gctttgtagc attaaaatac 120gcgcataaac taaacatgac agagtcatat ccaattatta aggaaggctc acaagtcgga 180agctactttc tatttttcat cataattgca tttttgttat atgccaatgt gttatttatt 240aaacgacgaa gttatgagct tgcattatat caaacattag gtttatctaa attcaacatt 300atttatatac taatgctcga acaattacta atatttataa ttacggcaat attaggtatt 360attattggta tttttggttc aaaactgtta ttaatgattg tctttacatt attaggaatt 420aaagaaaagg ttccaattat ttttagtttg agggcggtat ttgaaacatt aatgttaatc 480ggtgtcgctt attttttaac ctctgctcaa aattttatat tagtgttcaa acaatctatt 540tcacagatgt caaagaataa ccaggttaaa gaaacaaatc ataataaaat tacatttgaa 600gaggttgttt taggcatctt aggtatagta ttgattacca caggatacta tctatctttg 660aacattgttc aatattatga ttctatcggt atacttatgt ttattttatt gtcaactgtg 720attggggcat acttattttt taaaagctct gtttctctag tttttaaaat ggtgaagaag 780tttagaaaag gtgttataag tgtaaatgat gtcatgttct catcatctat tatgtatcgt 840attaagaaaa atgctttttc acttacggtc atggcaatca tttcagcgat tactgtttca 900gttctttgct ttgctgctat aagtagagcg tccttatcaa gtgaaataaa atatactgca 960ccacacgacg ttacaattaa agaccaacaa aaagctaatc aattagcaag tgaattaaac 1020aatcaaaaaa ttcctcattt ttataattat aaagaagtaa ttcatacgaa attgtataaa 1080gataatttat ttgatgtaaa agcgaaagaa ccatacaatg taacaattac tagtgataaa 1140tatatcccta atactgattt

gaaacgtgga caagctgatt tgtttgtagc ggaaggttct 1200atcaaagatt tagtgaaaca taagaagcat ggtaaggcaa ttataggaac gaaaaaacat 1260catgttaata ttaagttacg gaaagatatt aataaaatct attttatgac agatgttgat 1320ttaggtggac caacgtttgt cttaaatgac aaagactatc aagaaataag aaagtataca 1380aaagcaaagc atatcgtctc tcaatttgga ttcgatttga aacataaaaa agatgcttta 1440gcattagaaa aagtgaaaaa taaagttgat aaatctatta aaacaagaag tgaagcgata 1500agctcaatat caagtttaac cggaatatta ttatttgtaa catcattttt aggtattaca 1560ttcttgattg ctgtatgttg cattatatac attaagcaaa tagatgaaac cgaagatgag 1620ttagagaatt atagtatatt gagaaagctt ggatttacac aaaaagatat ggcaagggga 1680ctaaagttta aaattatgtt taattttggg ttacctttag ttattgcact atcacatgca 1740tattttacat cattagcata tatgaaatta atgggtacaa cgaatcaaat accggttttc 1800atagtaatgg gattatacat ttgtatgtat gctgtttttg cagtgacggc ttataatcat 1860tccaagcgaa caattagaca ttccatataa 18901031890DNAStaphylococcus aureus 103atgaccttta acgagataat atttaaaaat ttccgtcaaa atttatcaca ttatgccatc 60tatctttttt cattaattac gagtgtagta ttgtatttta gctttgtagc attaaaatac 120gcgcataaac taaacatgac agagtcatat ccaattatta aggaaggctc acaagtcgga 180agctactttc tatttttcat cataattgca tttttgttat atgccaatgt gttatttatt 240aaacgacgaa gttatgagct tgcattatat caaacattag gtttatctaa attcaacatt 300atttatatac taatgctcga acaattacta atatttataa ttacggcaat attaggtatt 360attattggta tttttggttc aaaactgtta ttaatgattg tctttacatt attaggaatt 420aaagaaaagg ttccaattat ttttagtttg agggcggtat ttgaaacatt aatgttaatc 480ggtgtcgctt attttttaac ctctgctcaa aattttatat tagtgttcaa acaatctatt 540tcacagatgt caaagaataa ccaggttaaa gaaacaaatc ataataaaat tacatttgaa 600gaggttgttt taggcatctt aggtatagta ttgattacca caggatacta tctatctttg 660aacattgttc aatattatga ttctatcggt atacttatgt ttattttatt gtcaactgtg 720attggggcat acttattttt taaaagctct gtttctctag tttttaaaat ggtgaagaag 780tttagaaaag gtgttataag tgtaaatgat gtcatgttct catcatctat tatgtatcgt 840attaagaaaa atgctttttc acttacggtc atggcaatca tttcagcgat tactgtttca 900gttctttgct ttgctgctat aagtagagcg tccttatcaa gtgaaataaa atatactgca 960ccacacgacg ttacaattaa agaccaacaa aaagctaatc aattagcaag tgaattaaac 1020aatcaaaaaa ttcctcattt ttataattat aaagaagtaa ttcatacgaa attgtataaa 1080gataatttat ttgatgtaaa agcgaaagaa ccatacaatg taacaattac tagtgataaa 1140tatatcccta atactgattt gaaacgtgga caagctgatt tgtttgtagc ggaaggttct 1200atcaaagatt tagtgaaaca taagaagcat ggtaaagcag ttataggaac gaaaaaacat 1260catgttaata ttaagttgcg gaaagatatt aataaaatct attttatgac agatgttgat 1320ttaggtggac caacgtttgt cttaaatgac aaagactatc aagaaataag aaagtataca 1380aaagcaaagc atatcgtctc tcaatttgga ttcgatttga aacataaaaa agatgcttta 1440gcattagaaa aagtgaaaaa taaagttgat aaatctatta aaacaagaag tgaagcgata 1500agctcaatat caagtttaac cggaatatta ttatttgtaa catcattttt aggtattaca 1560ttcttgattg ctgtatgttg cattatatac ataaagcaaa tagatgaaac cgaagatgag 1620ttagagaatt atagtatttt gagaaagctt ggatttacac aaaaagatat ggcaagggga 1680ctaaagttta aaattatgtt taattttggg ttacctttag ttattgcact atcacatgca 1740tattttacat cattagcata tatgaaatta atgggtacaa cgaatcaaat accggttttc 1800atagtaatgg gattatacat ttgtatgtat gctgtttttg cagtgacggc ttataatcat 1860tccaagcgaa caattagaca ttccatataa 18901041890DNAStaphylococcus aureus 104atgaccttta acgagataat atttaaaaat ttccgtcaaa atttatcaca ttatgccatc 60tatctttttt cattaattac gagtgtagta ttgtatttta gctttgtagc attaaaatac 120gcgcataaac taaacatgac agagtcatat ccaattatta aggaaggctc acaagtcgga 180agctactttc tatttttcat cataattgca tttttgttat atgccaatgt gttatttatt 240aaacgacgaa gttatgagct tgcattatat caaacattag gtttatctaa attcaacatt 300atttatatac taatgctcga acaattacta atatttataa ttacggcaat attaggtatt 360attattggta tttttggttc aaaactgtta ttaatgattg tctttacatt attaggaatt 420aaagaaaagg ttccaattat ttttagtttg agggcggtat ttgaaacatt aatgttaatc 480ggtgtcgctt attttttaac ctctgctcaa aattttatat tagtgttcaa acaatctatt 540tcacagatgt caaagaataa ccaggttaaa gaaacaaatc ataataaaat tacatttgaa 600gaggttgttt taggcatctt aggtatagta ttgattacca caggatacta tctatctttg 660aacattgttc aatattatga ttctatcggt atacttatgt ttattttatt gtcaactgtg 720attggggcat acttattttt taaaagctct gtttctctag tttttaaaat ggtgaagaag 780tttagaaaag gtgttataag tgtaaatgat gtcatgttct catcatctat tatgtatcgt 840attaagaaaa atgctttttc acttacggtc atggcaatca tttcagcgat tactgtttca 900gttctttgct ttgctgctat aagtagagcg tccttatcaa gtgaaataaa atatactgca 960ccacacgacg ttacaattaa agaccaacaa aaagctaatc aattagcaag tgaattaaac 1020aatcaaaaaa ttcctcattt ttataattat aaagaagtaa ttcatacgaa attgtataaa 1080gataatttat ttgatgtaaa agcgaaagaa ccatacaatg taacaattac tagtgataaa 1140tatatcccta atactgattt gaaacgtgga caagctgatt tgtttgtagc ggaaggttct 1200atcaaagatt tagtgaaaca taagaagcat ggtaaagcag ttataggaac gaaaaaacat 1260catgttaata ttaagttgcg gaaagatatt aataaaatct attttatgac agatgttgat 1320ttaggtggac caacgtttgt cttaaatgac aaagactatc aagaaataag aaagtataca 1380aaagcaaagc atatcgtctc tcaatttgga ttcgatttga aacataaaaa agatgcttta 1440gcattagaaa aagtgaaaaa taaagttgat aaatctatta aaacaagaag tgaagcgata 1500agctcaatat caagtttaac cggaatatta ttatttgtaa catcattttt aggtattaca 1560ttcttgattg ctgtatgttg cattatatac ataaagcaaa tagatgaaac cgaagatgag 1620ttagagaatt atagtatttt gagaaagctt ggatttacac aaaaagatat ggcaagggga 1680ctaaagttta aaattatgtt taattttggg ttacctttag ttattgcact atcacatgca 1740tattttacat cattagcata tatgaaatta atgggtacaa cgaatcaaat accggttttc 1800atagtaatgg gattatacat ttgtatgtat gctgtttttg cagtgacggc ttataatcat 1860tccaagcgaa caattagaca ttccatataa 18901051890DNAStaphylococcus aureus 105atgaccttta acgagataat atttaaaaat ttccgtcaaa atttatcaca ttatgccatc 60tatctttttt cgttaattac gagtgtagta ttgtatttta gctttgtagc attaaaatac 120gctcataaac taaacatgac agagtcatat ccaattataa aggaaggctc acaagtcgga 180agctactttc tatttttcat cataattgca tttttgttat atgccaatgt gttatttatt 240aaacgacgaa gttatgagct tgcattatat caaacattag gtttatctaa attcaacatt 300atttatatac taatgctcga acaattacta atatttataa ttacggcaat attaggtatt 360attattggta tttttggttc gaaactgtta ttaatgattg tctttacatt attaggaatt 420aaagaaaagg ttccaattat ttttagtttg agggcggtat ttgaaacatt aatgttaatc 480ggtgtcgctt attttttaac atctgctcaa aattttatat tagtgttcaa acaatctatt 540tcacagatgt caaagaataa ccaggttaaa gaaacaaatc ataataaaat tacatttgaa 600gaggttgttt taggcatctt aggtatagta ttgattacca caggatacta tctatctttg 660aacattgttc aatattatga ttctatcggt acacttatgt ttattttatt gtcaactgtg 720attggggcat acttattttt taaaagctct gtttctctag tttttaaaat ggtgaagaag 780tttagaaaag gtgttataag tgtaaatgat gtcatgttct catcatctat tatgtatcgt 840attaagaaaa atgctttttc acttacggtc atggcaatca tttcagcgat tactgtttca 900gttctttgct ttgctgctat aagtagagcg tccttatcaa gtgaaataaa atatactgca 960ccacacgacg ttacaattaa agaccaacaa aaagctaatc aattagcaag tgaattaaac 1020aatcaaaaaa ttcctcattt ttataattat aaagaagtaa ttcatacgaa attgtataaa 1080gataatttat ttgatgtaaa agcgaaagaa ccatacaatg taacaattac tagtgataaa 1140tacatcccta atactgattt gaaacgtggg caagctgatt tatttgtagc ggaaggttct 1200atcaaagatt tagtgaaaca taagaagcat ggtaaggcaa ttataggaac gaaaaaacat 1260catgttaata ttaagttacg taaagatatt aataaaatct attttatgac agatgttgat 1320ttaggtggac caacgtttgt cttaaatgac aaagactatc aagaaataag aaagtataca 1380aaggcaaagc atatcgtctc tcaatttgga ttcgatttga aacataaaaa agatgcttta 1440gcattagaaa aagcgaaaaa taaagttgat aaatctattg aaacaagaag tgaagcgata 1500agctcaatat caagtttaac cggaatatta ttatttgtaa catcattttt aggtattaca 1560ttcttgattg ctgtatgttg cattatatac ataaagcaaa tagatgaaac cgaagatgag 1620ttagagaatt atagtatttt gagaaagctt ggatttacac aaaaagatat ggcaagggga 1680ctaaagttta aaattatgtt taattttggg ttacctttag ttattgcact atcacatgca 1740tattttacat cattagcata tatgaaatta atgggtacaa cgaatcaaat accggttttc 1800atagtaatgg gattatacat ttgtatgtat gctgtttttg cagtgacggc ttataatcat 1860tccaagcgaa caattagaca ttccatataa 18901061800DNAStaphylococcus aureus 106ttgtatttta gctttgtagc attaaaatac gctcataaac taaacatgac agagtcatat 60ccaattataa aggaaggctc acaagtcgga agctactttc tatttttcat cataattgca 120tttttgttat atgccaatgt gttatttatt aaacgacgaa gttatgagct tgcattatat 180caaacattag gtttatctaa attcaacatt atttatatac taatgctcga acaattacta 240atatttataa ttacggcaat attaggtatt attattggta tttttggttc gaaactgtta 300ttaatgattg tctttacatt attaggaatt aaagaaaagg ttccaattat ttttagtttg 360agggcggtat ttgaaacatt aatgttaatc ggtgtcgctt attttttaac atctgctcaa 420aattttatat tagtgttcaa acaatctatt tcacagatgt caaagaataa ccaggttaaa 480gaaacaaatc ataataaaat tacatttgaa gaggttgttt taggcatctt aggtatagta 540ttgattacca caggatacta tctatctttg aacattgttc aatattatga ttctatcggt 600acacttatgt ttattttatt gtcaactgtg attggggcat acttattttt taaaagctct 660gtttctctag tttttaaaat ggtgaagaag tttagaaaag gtgttataag tgtaaatgat 720gtcatgttct catcatctat tatgtatcgt attaagaaaa atgctttttc acttacggtc 780atggcaatca tttcagcgat tactgtttca gttctttgct ttgctgctat aagtagagcg 840tccttatcaa gtgaaataaa atatactgca ccacacgacg ttacaattaa agaccaacaa 900aaagctaatc aattagcaag tgaattaaac aatcaaaaaa ttcctcattt ttataattat 960aaagaagtaa ttcatacgaa attgtataaa gataatttat ttgatgtaaa agcgaaagaa 1020ccatacaatg taacaattac tagtgataaa tacatcccta atactgattt gaaacgtggg 1080caagctgatt tatttgtagc ggaaggttct atcaaagatt tagtgaaaca taagaagcat 1140ggtaaggcaa ttataggaac gaaaaaacat catgttaata ttaagttacg taaagatatt 1200aataaaatct attttatgac agatgttgat ttaggtggac caacgtttgt cttaaatgac 1260aaagactatc aagaaataag aaagtataca aaggcaaagc atatcgtctc tcaatttgga 1320ttcgatttga aacataaaaa agatgcttta gcattagaaa aagcgaaaaa taaagttgat 1380aaatctattg aaacaagaag tgaagcgata agctcaatat caagtttaac cggaatatta 1440ttatttgtaa catcattttt aggtattaca ttcttgattg ctgtatgttg cattatatac 1500ataaagcaaa tagatgaaac cgaagatgag ttagagaatt atagtatttt gagaaagctt 1560ggatttacac aaaaagatat ggcaagggga ctaaagttta aaattatgtt taattttggg 1620ttacctttag ttattgcact atcacatgca tattttacat cattagcata tatgaaatta 1680atgggtacaa cgaatcaaat accggttttc atagtaatgg gattatacat ttgtatgtat 1740gctgtttttg cagtgacggc ttataatcat tccaagcgaa caattagaca ttccatataa 18001071755DNAStaphylococcus aureus 107atgacagagt catatccaat tataaaggaa ggctcacaag tcggaagcta ctttctattt 60ttcatcataa ttgcattttt gttatatgcc aatgtgttat ttattaaacg acgaagttat 120gagcttgcat tatatcaaac attaggttta tctaaattca acattattta tatactaatg 180ctcgaacaat tactaatatt tataattacg gcaatattag gtattattat tggtattttt 240ggttcgaaac tgttattaat gattgtcttt acattattag gaattaaaga aaaggttcca 300attattttta gtttgagggc ggtatttgaa acattaatgt taatcggtgt cgcttatttt 360ttaacatctg ctcaaaattt tatattagtg ttcaaacaat ctatttcaca gatgtcaaag 420aataaccagg ttaaagaaac aaatcataat aaaattacat ttgaagaggt tgttttaggc 480atcttaggta tagtattgat taccacagga tactatctat ctttgaacat tgttcaatat 540tatgattcta tcggtacact tatgtttatt ttattgtcaa ctgtgattgg ggcatactta 600ttttttaaaa gctctgtttc tctagttttt aaaatggtga agaagtttag aaaaggtgtt 660ataagtgtaa atgatgtcat gttctcatca tctattatgt atcgtattaa gaaaaatgct 720ttttcactta cggtcatggc aatcatttca gcgattactg tttcagttct ttgctttgct 780gctataagta gagcgtcctt atcaagtgaa ataaaatata ctgcaccaca cgacgttaca 840attaaagacc aacaaaaagc taatcaatta gcaagtgaat taaacaatca aaaaattcct 900catttttata attataaaga agtaattcat acgaaattgt ataaagataa tttatttgat 960gtaaaagcga aagaaccata caatgtaaca attactagtg ataaatacat ccctaatact 1020gatttgaaac gtgggcaagc tgatttattt gtagcggaag gttctatcaa agatttagtg 1080aaacataaga agcatggtaa ggcaattata ggaacgaaaa aacatcatgt taatattaag 1140ttacgtaaag atattaataa aatctatttt atgacagatg ttgatttagg tggaccaacg 1200tttgtcttaa atgacaaaga ctatcaagaa ataagaaagt atacaaaggc aaagcatatc 1260gtctctcaat ttggattcga tttgaaacat aaaaaagatg ctttagcatt agaaaaagcg 1320aaaaataaag ttgataaatc tattgaaaca agaagtgaag cgataagctc aatatcaagt 1380ttaaccggaa tattattatt tgtaacatca tttttaggta ttacattctt gattgctgta 1440tgttgcatta tatacataaa gcaaatagat gaaaccgaag atgagttaga gaattatagt 1500attttgagaa agcttggatt tacacaaaaa gatatggcaa ggggactaaa gtttaaaatt 1560atgtttaatt ttgggttacc tttagttatt gcactatcac atgcatattt tacatcatta 1620gcatatatga aattaatggg tacaacgaat caaataccgg ttttcatagt aatgggatta 1680tacatttgta tgtatgctgt ttttgcagtg acggcttata atcattccaa gcgaacaatt 1740agacattcca tataa 17551081890DNAStaphylococcus aureus 108atgaccttta acgagataat atttaaaaat ttccgtcaaa atttatcaca ttatgccatc 60tatctttttt cattaattac gagtgtagta ttgtatttta gctttgtagc attaaaatac 120gctcataaac taaacatgac agagtcatat ccaattataa aggaaggctc acaagtcgga 180agctactttc tatttttcat cataattgca tttttgttat atgccaatgt gttatttatt 240aaacgacgaa gttatgagct tgcattatat caaacattag gtttatctaa attcaacatt 300atttatatac taatgctcga acaattacta atatttataa ttacggcaat attaggtatt 360attattggta tttttggttc gaaactgtta ttaatgattg tctttacatt attaggaatt 420aaagaaaagg ttccaattat ttttagtttg agggcggtat ttgaaacatt aatgttaatc 480ggtgtcgctt attttttaac atctgctcaa aattttatat tagtgttcaa acaatctatt 540tcacagatgt caaagaataa ccaggttaaa gaaacaaatc ataataaaat tacatttgaa 600gaggttgttt taggcatctt aggtatagta ttgattacca caggatacta tctatctttg 660aacattgttc aatattatga ttctatcggt acacttatgt ttattttatt gtcaactgtg 720attggggcat acttattttt taaaagctct gtttctctag tttttaaaat ggtgaagaag 780tttagaaaag gtgttataag tgtaaatgat gtcatgttct catcatctat tatgtatcgt 840attaagaaaa atgctttttc acttacggtc atggcaatca tttcagcgat tactgtttca 900gttctttgct ttgctgctat aagtagagcg tccttatcaa gtgaaataaa atatactgca 960ccacacgacg ttacaattaa agaccaacaa aaagctaatc aattagcaag tgaattaaac 1020aatcaaaaaa ttcctcattt ttataattat aaagaagtaa ttcatacgaa attgtataaa 1080gataatttat ttgatgtaaa atcgaaacaa ccatacaatg taacaattac tagtgataaa 1140tacatcccta gtactgattt gaaacgtggg caagctgatt tgtttgtagc ggaaggttct 1200atcaaagatt tagtgaaaca taagaagcat ggtaaagcag ttataggaac gaaaaaacat 1260catgttaata ttaagttacg taaagatatt aataaaatct attttatgac agatgttgat 1320ttaggtggac caacgtttgt cttaaatgac aaagactatc aagaaataag aaagtataca 1380aaggcaaagc atatcgtctc tcaatttgga ttcgatttga aacataaaaa agatgcttta 1440gcattagaaa aagcgaaaaa taaagttgat aaatctattg agacaagaag tgaagcgata 1500agctcaatat caagtttaac cggaatatta ttatttgtaa catcattttt aggtattaca 1560ttcttgattg ctgtatgttg cattatatac attaagcaaa tagatgaaac cgaagatgag 1620ttagagaatt atagtatatt gagaaagctt ggatttacac aaaaagatat ggcaagggga 1680ctaaagttta aaattatgtt taattttggg ttacctttag ttattgcact atcacatgca 1740tattttacat cattagcata tatgaaatta atgggtacaa cgaatcaaat accggttttc 1800atagtaatgg gattatacat ttgtatgtat gctgtttttg cagtgacggc ttataatcat 1860tccaagcgaa caattagaca ttccatataa 1890109629PRTStaphylococcus aureus 109Met Thr Phe Asn Glu Ile Ile Phe Lys Asn Phe Arg Gln Asn Leu Ser1 5 10 15His Tyr Ala Ile Tyr Leu Phe Ser Leu Ile Thr Ser Val Val Leu Tyr 20 25 30Phe Ser Phe Val Ala Leu Lys Tyr Ala His Lys Leu Asn Met Thr Glu 35 40 45Ser Tyr Pro Ile Ile Lys Glu Gly Ser Gln Val Gly Ser Tyr Phe Leu 50 55 60Phe Phe Ile Ile Ile Ala Phe Leu Leu Tyr Ala Asn Val Leu Phe Ile65 70 75 80Lys Arg Arg Ser Tyr Glu Leu Ala Leu Tyr Gln Thr Leu Gly Leu Ser 85 90 95Lys Phe Asn Ile Ile Tyr Ile Leu Met Leu Glu Gln Leu Leu Ile Phe 100 105 110Ile Ile Thr Ala Ile Leu Gly Ile Ile Ile Gly Ile Phe Gly Ser Lys 115 120 125Leu Leu Leu Met Ile Val Phe Thr Leu Leu Gly Ile Lys Glu Lys Val 130 135 140Pro Ile Ile Phe Ser Leu Arg Ala Val Phe Glu Thr Leu Met Leu Ile145 150 155 160Gly Val Ala Tyr Phe Leu Thr Ser Ala Gln Asn Phe Ile Leu Val Phe 165 170 175Lys Gln Ser Ile Ser Gln Met Ser Lys Asn Asn Gln Val Lys Glu Thr 180 185 190Asn His Asn Lys Ile Thr Phe Glu Glu Val Val Leu Gly Ile Leu Gly 195 200 205Ile Val Leu Ile Thr Thr Gly Tyr Tyr Leu Ser Leu Asn Ile Val Gln 210 215 220Tyr Tyr Asp Ser Ile Gly Thr Leu Met Phe Ile Leu Leu Ser Thr Val225 230 235 240Ile Gly Ala Tyr Leu Phe Phe Lys Ser Ser Val Ser Leu Val Phe Lys 245 250 255Met Val Lys Lys Phe Arg Lys Gly Val Ile Ser Val Asn Asp Val Met 260 265 270Phe Ser Ser Ser Ile Met Tyr Arg Ile Lys Lys Asn Ala Phe Ser Leu 275 280 285Thr Val Met Ala Ile Ile Ser Ala Ile Thr Val Ser Val Leu Cys Phe 290 295 300Ala Ala Ile Ser Arg Ala Ser Leu Ser Ser Glu Ile Lys Tyr Thr Ala305 310 315 320Pro His Asp Val Thr Ile Lys Asp Gln Gln Lys Ala Asn Gln Leu Ala 325 330 335Ser Glu Leu Asn Asn Gln Lys Ile Pro His Phe Tyr Asn Tyr Lys Glu 340 345 350Val Ile His Thr Lys Leu Tyr Lys Asp Asn Leu Phe Asp Val Lys Ala 355 360 365Lys Glu Pro Tyr Asn Val Thr Ile Thr Ser Asp Lys Tyr Ile Pro Asn 370 375 380Thr Asp Leu Lys Arg Gly Gln Ala Asp Leu Phe Val Ala Glu Gly Ser385 390 395 400Ile Lys Asp Leu Val Lys His Lys Lys His Gly Lys Ala Ile Ile Gly 405 410 415Thr Lys Lys His His Val Asn Ile Lys Leu Arg Lys Asp Ile Asn Lys 420 425 430Ile Tyr Phe Met Thr Asp Val Asp Leu Gly Gly Pro Thr Phe Val Leu 435 440 445Asn Asp Lys Asp Tyr Gln Glu Ile Arg Lys Tyr Thr Lys Ala Lys His 450

455 460Ile Val Ser Gln Phe Gly Phe Asp Leu Lys His Lys Lys Asp Ala Leu465 470 475 480Ala Leu Glu Lys Ala Lys Asn Lys Val Asp Lys Ser Ile Glu Thr Arg 485 490 495Ser Glu Ala Ile Ser Ser Ile Ser Ser Leu Thr Gly Ile Leu Leu Phe 500 505 510Val Thr Ser Phe Leu Gly Ile Thr Phe Leu Ile Ala Val Cys Cys Ile 515 520 525Ile Tyr Ile Lys Gln Ile Asp Glu Thr Glu Asp Glu Leu Glu Asn Tyr 530 535 540Ser Ile Leu Arg Lys Leu Gly Phe Thr Gln Lys Asp Met Ala Arg Gly545 550 555 560Leu Lys Phe Lys Ile Met Phe Asn Phe Gly Leu Pro Leu Val Ile Ala 565 570 575Leu Ser His Ala Tyr Phe Thr Ser Leu Ala Tyr Met Lys Leu Met Gly 580 585 590Thr Thr Asn Gln Ile Pro Val Phe Ile Val Met Gly Leu Tyr Ile Cys 595 600 605Met Tyr Ala Val Phe Ala Val Thr Ala Tyr Asn His Ser Lys Arg Thr 610 615 620Ile Arg His Ser Ile625110599PRTStaphylococcus aureus 110Met Tyr Phe Ser Phe Val Ala Leu Lys Tyr Ala His Lys Leu Asn Met1 5 10 15Thr Glu Ser Tyr Pro Ile Ile Lys Glu Gly Ser Gln Val Gly Ser Tyr 20 25 30Phe Leu Phe Phe Ile Ile Ile Ala Phe Leu Leu Tyr Ala Asn Val Leu 35 40 45Phe Ile Lys Arg Arg Ser Tyr Glu Leu Ala Leu Tyr Gln Thr Leu Gly 50 55 60Leu Ser Lys Phe Asn Ile Ile Tyr Ile Leu Met Leu Glu Gln Leu Leu65 70 75 80Ile Phe Ile Ile Thr Ala Ile Leu Gly Ile Ile Ile Gly Ile Phe Gly 85 90 95Ser Lys Leu Leu Leu Met Ile Val Phe Thr Leu Leu Gly Ile Lys Glu 100 105 110Lys Val Pro Ile Ile Phe Ser Leu Arg Ala Val Phe Glu Thr Leu Met 115 120 125Leu Ile Gly Val Ala Tyr Phe Leu Thr Ser Ala Gln Asn Phe Ile Leu 130 135 140Val Phe Lys Gln Ser Ile Ser Gln Met Ser Lys Asn Asn Gln Val Lys145 150 155 160Glu Thr Asn His Asn Lys Ile Thr Phe Glu Glu Val Val Leu Gly Ile 165 170 175Leu Gly Ile Val Leu Ile Thr Thr Gly Tyr Tyr Leu Ser Leu Asn Ile 180 185 190Val Gln Tyr Tyr Asp Ser Ile Gly Thr Leu Met Phe Ile Leu Leu Ser 195 200 205Thr Val Ile Gly Ala Tyr Leu Phe Phe Lys Ser Ser Val Ser Leu Val 210 215 220Phe Lys Met Val Lys Lys Phe Arg Lys Gly Val Ile Ser Val Asn Asp225 230 235 240Val Met Phe Ser Ser Ser Ile Met Tyr Arg Ile Lys Lys Asn Ala Phe 245 250 255Ser Leu Thr Val Met Ala Ile Ile Ser Ala Ile Thr Val Ser Val Leu 260 265 270Cys Phe Ala Ala Ile Ser Arg Ala Ser Leu Ser Ser Glu Ile Lys Tyr 275 280 285Thr Ala Pro His Asp Val Thr Ile Lys Asp Gln Gln Lys Ala Asn Gln 290 295 300Leu Ala Ser Glu Leu Asn Asn Gln Lys Ile Pro His Phe Tyr Asn Tyr305 310 315 320Lys Glu Val Ile His Thr Lys Leu Tyr Lys Asp Asn Leu Phe Asp Val 325 330 335Lys Ala Lys Glu Pro Tyr Asn Val Thr Ile Thr Ser Asp Lys Tyr Ile 340 345 350Pro Asn Thr Asp Leu Lys Arg Gly Gln Ala Asp Leu Phe Val Ala Glu 355 360 365Gly Ser Ile Lys Asp Leu Val Lys His Lys Lys His Gly Lys Ala Ile 370 375 380Ile Gly Thr Lys Lys His His Val Asn Ile Lys Leu Arg Lys Asp Ile385 390 395 400Asn Lys Ile Tyr Phe Met Thr Asp Val Asp Leu Gly Gly Pro Thr Phe 405 410 415Val Leu Asn Asp Lys Asp Tyr Gln Glu Ile Arg Lys Tyr Thr Lys Ala 420 425 430Lys His Ile Val Ser Gln Phe Gly Phe Asp Leu Lys His Lys Lys Asp 435 440 445Ala Leu Ala Leu Glu Lys Ala Lys Asn Lys Val Asp Lys Ser Ile Glu 450 455 460Thr Arg Ser Glu Ala Ile Ser Ser Ile Ser Ser Leu Thr Gly Ile Leu465 470 475 480Leu Phe Val Thr Ser Phe Leu Gly Ile Thr Phe Leu Ile Ala Val Cys 485 490 495Cys Ile Ile Tyr Ile Lys Gln Ile Asp Glu Thr Glu Asp Glu Leu Glu 500 505 510Asn Tyr Ser Ile Leu Arg Lys Leu Gly Phe Thr Gln Lys Asp Met Ala 515 520 525Arg Gly Leu Lys Phe Lys Ile Met Phe Asn Phe Gly Leu Pro Leu Val 530 535 540Ile Ala Leu Ser His Ala Tyr Phe Thr Ser Leu Ala Tyr Met Lys Leu545 550 555 560Met Gly Thr Thr Asn Gln Ile Pro Val Phe Ile Val Met Gly Leu Tyr 565 570 575Ile Cys Met Tyr Ala Val Phe Ala Val Thr Ala Tyr Asn His Ser Lys 580 585 590Arg Thr Ile Arg His Ser Ile 595111584PRTStaphylococcus aureus 111Met Thr Glu Ser Tyr Pro Ile Ile Lys Glu Gly Ser Gln Val Gly Ser1 5 10 15Tyr Phe Leu Phe Phe Ile Ile Ile Ala Phe Leu Leu Tyr Ala Asn Val 20 25 30Leu Phe Ile Lys Arg Arg Ser Tyr Glu Leu Ala Leu Tyr Gln Thr Leu 35 40 45Gly Leu Ser Lys Phe Asn Ile Ile Tyr Ile Leu Met Leu Glu Gln Leu 50 55 60Leu Ile Phe Ile Ile Thr Ala Ile Leu Gly Ile Ile Ile Gly Ile Phe65 70 75 80Gly Ser Lys Leu Leu Leu Met Ile Val Phe Thr Leu Leu Gly Ile Lys 85 90 95Glu Lys Val Pro Ile Ile Phe Ser Leu Arg Ala Val Phe Glu Thr Leu 100 105 110Met Leu Ile Gly Val Ala Tyr Phe Leu Thr Ser Ala Gln Asn Phe Ile 115 120 125Leu Val Phe Lys Gln Ser Ile Ser Gln Met Ser Lys Asn Asn Gln Val 130 135 140Lys Glu Thr Asn His Asn Lys Ile Thr Phe Glu Glu Val Val Leu Gly145 150 155 160Ile Leu Gly Ile Val Leu Ile Thr Thr Gly Tyr Tyr Leu Ser Leu Asn 165 170 175Ile Val Gln Tyr Tyr Asp Ser Ile Gly Thr Leu Met Phe Ile Leu Leu 180 185 190Ser Thr Val Ile Gly Ala Tyr Leu Phe Phe Lys Ser Ser Val Ser Leu 195 200 205Val Phe Lys Met Val Lys Lys Phe Arg Lys Gly Val Ile Ser Val Asn 210 215 220Asp Val Met Phe Ser Ser Ser Ile Met Tyr Arg Ile Lys Lys Asn Ala225 230 235 240Phe Ser Leu Thr Val Met Ala Ile Ile Ser Ala Ile Thr Val Ser Val 245 250 255Leu Cys Phe Ala Ala Ile Ser Arg Ala Ser Leu Ser Ser Glu Ile Lys 260 265 270Tyr Thr Ala Pro His Asp Val Thr Ile Lys Asp Gln Gln Lys Ala Asn 275 280 285Gln Leu Ala Ser Glu Leu Asn Asn Gln Lys Ile Pro His Phe Tyr Asn 290 295 300Tyr Lys Glu Val Ile His Thr Lys Leu Tyr Lys Asp Asn Leu Phe Asp305 310 315 320Val Lys Ala Lys Glu Pro Tyr Asn Val Thr Ile Thr Ser Asp Lys Tyr 325 330 335Ile Pro Asn Thr Asp Leu Lys Arg Gly Gln Ala Asp Leu Phe Val Ala 340 345 350Glu Gly Ser Ile Lys Asp Leu Val Lys His Lys Lys His Gly Lys Ala 355 360 365Ile Ile Gly Thr Lys Lys His His Val Asn Ile Lys Leu Arg Lys Asp 370 375 380Ile Asn Lys Ile Tyr Phe Met Thr Asp Val Asp Leu Gly Gly Pro Thr385 390 395 400Phe Val Leu Asn Asp Lys Asp Tyr Gln Glu Ile Arg Lys Tyr Thr Lys 405 410 415Ala Lys His Ile Val Ser Gln Phe Gly Phe Asp Leu Lys His Lys Lys 420 425 430Asp Ala Leu Ala Leu Glu Lys Ala Lys Asn Lys Val Asp Lys Ser Ile 435 440 445Glu Thr Arg Ser Glu Ala Ile Ser Ser Ile Ser Ser Leu Thr Gly Ile 450 455 460Leu Leu Phe Val Thr Ser Phe Leu Gly Ile Thr Phe Leu Ile Ala Val465 470 475 480Cys Cys Ile Ile Tyr Ile Lys Gln Ile Asp Glu Thr Glu Asp Glu Leu 485 490 495Glu Asn Tyr Ser Ile Leu Arg Lys Leu Gly Phe Thr Gln Lys Asp Met 500 505 510Ala Arg Gly Leu Lys Phe Lys Ile Met Phe Asn Phe Gly Leu Pro Leu 515 520 525Val Ile Ala Leu Ser His Ala Tyr Phe Thr Ser Leu Ala Tyr Met Lys 530 535 540Leu Met Gly Thr Thr Asn Gln Ile Pro Val Phe Ile Val Met Gly Leu545 550 555 560Tyr Ile Cys Met Tyr Ala Val Phe Ala Val Thr Ala Tyr Asn His Ser 565 570 575Lys Arg Thr Ile Arg His Ser Ile 580112584PRTStaphylococcus aureus 112Met Thr Glu Ser Tyr Pro Ile Ile Lys Glu Gly Ser Gln Val Gly Ser1 5 10 15Tyr Phe Leu Phe Phe Ile Ile Ile Ala Phe Leu Leu Tyr Ala Asn Val 20 25 30Leu Phe Ile Lys Arg Arg Ser Tyr Glu Leu Ala Leu Tyr Gln Thr Leu 35 40 45Gly Leu Ser Lys Phe Asn Ile Ile Tyr Ile Leu Met Leu Glu Gln Leu 50 55 60Leu Ile Phe Ile Ile Thr Ala Ile Leu Gly Ile Ile Ile Gly Ile Phe65 70 75 80Gly Ser Lys Leu Leu Leu Met Ile Val Phe Thr Leu Leu Gly Ile Lys 85 90 95Glu Lys Val Pro Ile Ile Phe Ser Leu Arg Ala Val Phe Glu Thr Leu 100 105 110Met Leu Ile Gly Val Ala Tyr Phe Leu Thr Ser Ala Gln Asn Phe Ile 115 120 125Leu Val Phe Lys Gln Ser Ile Ser Gln Met Ser Lys Asn Asn Gln Val 130 135 140Lys Glu Thr Asn His Asn Lys Ile Thr Phe Glu Glu Val Val Leu Gly145 150 155 160Ile Leu Gly Ile Val Leu Ile Ile Thr Gly Tyr Tyr Leu Ser Leu Asn 165 170 175Ile Val Gln Tyr Tyr Asp Ser Ile Gly Ile Leu Met Phe Ile Leu Leu 180 185 190Ser Thr Val Ile Gly Ala Tyr Leu Phe Phe Lys Ser Ser Val Ser Leu 195 200 205Val Phe Lys Met Val Lys Lys Phe Arg Lys Gly Val Ile Ser Val Asn 210 215 220Asp Val Met Phe Ser Ser Ser Ile Met Tyr Arg Ile Lys Lys Asn Ala225 230 235 240Phe Ser Leu Thr Val Met Ala Ile Ile Ser Ala Ile Thr Val Ser Val 245 250 255Leu Cys Phe Ala Ala Ile Ser Arg Ala Ser Leu Ser Ser Glu Ile Lys 260 265 270Tyr Thr Ala Pro His Asp Val Thr Ile Lys Asp Gln Gln Lys Ala Asn 275 280 285Gln Leu Ala Ser Glu Leu Asn Asn Gln Lys Ile Pro His Phe Tyr Asn 290 295 300Tyr Lys Glu Val Ile His Thr Lys Leu Tyr Lys Asp Asn Leu Phe Asp305 310 315 320Val Lys Ala Lys Glu Pro Tyr Asn Val Thr Ile Thr Ser Asp Lys Tyr 325 330 335Ile Pro Asn Thr Asp Leu Lys Arg Gly Gln Ala Asp Leu Phe Val Ala 340 345 350Glu Gly Ser Ile Lys Asp Leu Val Lys His Lys Lys His Gly Lys Ala 355 360 365Ile Ile Gly Thr Lys Lys His His Val Asn Ile Lys Leu Arg Lys Asp 370 375 380Ile Asn Lys Ile Tyr Phe Met Thr Asp Val Asp Leu Gly Gly Pro Thr385 390 395 400Phe Val Leu Asn Asp Lys Asp Tyr Gln Glu Ile Arg Lys Tyr Thr Lys 405 410 415Ala Lys His Ile Val Ser Gln Phe Gly Phe Asp Leu Lys His Lys Lys 420 425 430Asp Ala Leu Ala Leu Glu Lys Val Lys Asn Lys Val Asp Lys Ser Ile 435 440 445Lys Thr Arg Ser Glu Ala Ile Ser Ser Ile Ser Ser Leu Thr Gly Ile 450 455 460Leu Leu Phe Val Thr Ser Phe Leu Gly Ile Thr Phe Leu Ile Ala Val465 470 475 480Cys Cys Ile Ile Tyr Ile Lys Gln Ile Asp Glu Thr Glu Asp Glu Leu 485 490 495Glu Asn Tyr Ser Ile Leu Arg Lys Leu Gly Phe Thr Gln Lys Asp Met 500 505 510Ala Arg Gly Leu Lys Phe Lys Ile Met Phe Asn Phe Gly Leu Pro Leu 515 520 525Val Ile Ala Leu Ser His Ala Tyr Phe Thr Ser Leu Ala Tyr Met Lys 530 535 540Leu Met Gly Thr Thr Asn Gln Ile Pro Val Phe Ile Val Met Gly Leu545 550 555 560Tyr Ile Cys Met Tyr Ala Val Phe Ala Val Thr Ala Tyr Asn His Ser 565 570 575Lys Arg Thr Ile Arg His Ser Ile 580113584PRTStaphylococcus aureus 113Met Thr Glu Ser Tyr Pro Ile Ile Lys Glu Gly Ser Gln Val Gly Ser1 5 10 15Tyr Phe Leu Phe Phe Ile Ile Ile Ala Phe Leu Leu Tyr Ala Asn Val 20 25 30Leu Phe Ile Lys Arg Arg Ser Tyr Glu Leu Ala Leu Tyr Gln Thr Leu 35 40 45Gly Leu Ser Lys Phe Asn Ile Ile Tyr Ile Leu Met Leu Glu Gln Leu 50 55 60Leu Ile Phe Ile Ile Thr Ala Ile Leu Gly Ile Ile Ile Gly Ile Phe65 70 75 80Gly Ser Lys Leu Leu Leu Met Ile Val Phe Thr Leu Leu Gly Ile Lys 85 90 95Glu Lys Val Pro Ile Ile Phe Ser Leu Arg Ala Val Phe Glu Thr Leu 100 105 110Met Leu Ile Gly Val Ala Tyr Phe Leu Thr Ser Ala Gln Asn Phe Ile 115 120 125Leu Val Phe Lys Gln Ser Ile Ser Gln Met Ser Lys Asn Asn Gln Val 130 135 140Lys Glu Thr Asn His Asn Lys Ile Thr Phe Glu Glu Val Val Leu Gly145 150 155 160Ile Leu Gly Ile Val Leu Ile Ile Thr Gly Tyr Tyr Leu Ser Leu Asn 165 170 175Ile Val Gln Tyr Tyr Asp Ser Ile Gly Ile Leu Met Phe Ile Leu Leu 180 185 190Ser Thr Val Ile Gly Ala Tyr Leu Phe Phe Lys Ser Ser Val Ser Leu 195 200 205Val Phe Lys Met Val Lys Lys Phe Arg Lys Gly Val Ile Ser Val Asn 210 215 220Asp Val Met Phe Ser Ser Ser Ile Met Tyr Arg Ile Lys Lys Asn Ala225 230 235 240Phe Ser Leu Thr Val Met Ala Ile Ile Ser Ala Ile Thr Val Ser Val 245 250 255Leu Cys Phe Ala Ala Ile Ser Arg Ala Ser Leu Ser Ser Glu Ile Lys 260 265 270Tyr Thr Ala Pro His Asp Val Thr Ile Lys Asp Gln Gln Lys Ala Asn 275 280 285Gln Leu Ala Ser Glu Leu Asn Asn Gln Lys Ile Pro His Phe Tyr Asn 290 295 300Tyr Lys Glu Val Ile His Thr Lys Leu Tyr Lys Asp Asn Leu Phe Asp305 310 315 320Val Lys Ala Lys Glu Pro Tyr Asn Val Thr Ile Thr Ser Asp Lys Tyr 325 330 335Ile Pro Asn Thr Asp Leu Lys Arg Gly Gln Ala Asp Leu Phe Val Ala 340 345 350Glu Gly Ser Ile Lys Asp Leu Val Lys His Lys Lys His Gly Lys Ala 355 360 365Ile Ile Gly Thr Lys Lys His His Val Asn Ile Lys Leu Arg Lys Asp 370 375 380Ile Asn Lys Ile Tyr Phe Met Thr Asp Val Asp Leu Gly Gly Pro Thr385 390 395 400Phe Val Leu Asn Asp Lys Asp Tyr Gln Glu Ile Arg Lys Tyr Thr Lys 405 410 415Ala Lys His Ile Val Ser Gln Phe Gly Phe Asp Leu Lys His Lys Lys 420 425 430Asp Ala Leu Ala Leu Glu Lys Val Lys Asn Lys Val Asp Lys Ser Ile 435 440 445Lys Thr Arg Ser Glu Ala Ile Ser Ser Ile Ser Ser Leu Thr Gly Ile 450 455 460Leu Leu Phe Val Thr Ser Phe Leu Gly Ile Thr Phe Leu Ile Ala Val465 470 475 480Cys Cys Ile Ile Tyr Ile Lys Gln Ile Asp Glu Thr Glu Asp Glu Leu 485 490 495Glu Asn Tyr Ser Ile Leu Arg Lys Leu Gly Phe Thr Gln Lys Asp Met 500 505 510Ala Arg Gly Leu Lys Phe Lys Ile Met Phe Asn Phe Gly Leu Pro Leu 515 520 525Val

Ile Val Leu Ser His Ala Tyr Phe Thr Ser Leu Ala Tyr Met Lys 530 535 540Leu Met Gly Thr Thr Asn Gln Ile Pro Val Phe Ile Val Met Gly Leu545 550 555 560Tyr Ile Cys Met Tyr Ala Val Phe Ala Val Thr Ala Tyr Asn His Ser 565 570 575Lys Arg Thr Ile Arg His Ser Ile 580114584PRTStaphylococcus aureus 114Met Thr Glu Ser Tyr Pro Ile Ile Lys Glu Gly Ser Gln Val Gly Ser1 5 10 15Tyr Phe Leu Phe Phe Ile Ile Ile Ala Phe Leu Leu Tyr Ala Asn Val 20 25 30Leu Phe Ile Lys Arg Arg Ser Tyr Glu Leu Ala Leu Tyr Gln Thr Leu 35 40 45Gly Leu Ser Lys Phe Asn Ile Ile Tyr Ile Leu Met Leu Glu Gln Leu 50 55 60Leu Ile Phe Ile Ile Thr Ala Ile Leu Gly Ile Ile Ile Gly Ile Phe65 70 75 80Gly Ser Lys Leu Leu Leu Met Ile Val Phe Thr Leu Leu Gly Ile Lys 85 90 95Glu Lys Val Pro Ile Ile Phe Ser Leu Arg Ala Val Phe Glu Thr Leu 100 105 110Met Leu Ile Gly Val Ala Tyr Phe Leu Thr Ser Ala Gln Asn Phe Ile 115 120 125Leu Val Phe Lys Gln Ser Ile Ser Gln Met Ser Lys Asn Asn Gln Val 130 135 140Lys Glu Thr Asn His Asn Lys Ile Thr Phe Glu Glu Val Val Leu Gly145 150 155 160Ile Leu Gly Ile Val Leu Ile Thr Thr Gly Tyr Tyr Leu Ser Leu Asn 165 170 175Ile Val Gln Tyr Tyr Asp Ser Ile Gly Ile Leu Met Phe Ile Leu Leu 180 185 190Ser Thr Val Ile Gly Ala Tyr Leu Phe Phe Lys Ser Ser Val Ser Leu 195 200 205Val Phe Lys Met Val Lys Lys Phe Arg Lys Gly Val Ile Ser Val Asn 210 215 220Asp Val Met Phe Ser Ser Ser Ile Met Tyr Arg Ile Lys Lys Asn Ala225 230 235 240Phe Ser Leu Thr Val Met Ala Ile Ile Ser Ala Ile Thr Val Ser Val 245 250 255Leu Cys Phe Ala Ala Ile Ser Arg Ala Ser Leu Ser Ser Glu Ile Lys 260 265 270Tyr Thr Ala Pro His Asp Val Thr Ile Lys Asp Gln Gln Lys Ala Asn 275 280 285Gln Leu Ala Ser Glu Leu Asn Asn Gln Lys Ile Pro His Phe Tyr Asn 290 295 300Tyr Lys Glu Val Ile His Thr Lys Leu Tyr Lys Asp Asn Leu Phe Asp305 310 315 320Val Lys Ala Lys Glu Pro Tyr Asn Val Thr Ile Thr Ser Asp Lys Tyr 325 330 335Ile Pro Asn Thr Asp Leu Lys Arg Gly Gln Ala Asp Leu Phe Val Ala 340 345 350Glu Gly Ser Ile Lys Asp Leu Val Lys His Lys Lys His Gly Lys Ala 355 360 365Ile Ile Gly Thr Lys Lys His His Val Asn Ile Lys Leu Arg Lys Asp 370 375 380Ile Asn Lys Ile Tyr Phe Met Thr Asp Val Asp Leu Gly Gly Pro Thr385 390 395 400Phe Val Leu Asn Asp Lys Asp Tyr Gln Glu Ile Arg Lys Tyr Thr Lys 405 410 415Ala Lys His Ile Val Ser Gln Phe Gly Phe Asp Leu Lys His Lys Lys 420 425 430Asp Ala Leu Ala Leu Glu Lys Val Lys Asn Lys Val Asp Lys Ser Ile 435 440 445Lys Thr Arg Ser Glu Ala Ile Ser Ser Ile Ser Ser Leu Thr Gly Ile 450 455 460Leu Leu Phe Val Thr Ser Phe Leu Gly Ile Thr Phe Leu Ile Ala Val465 470 475 480Cys Cys Ile Ile Tyr Ile Lys Gln Ile Asp Glu Thr Glu Asp Glu Leu 485 490 495Glu Asn Tyr Ser Ile Leu Arg Lys Leu Gly Phe Thr Gln Lys Asp Met 500 505 510Ala Arg Gly Leu Lys Phe Lys Ile Met Phe Asn Phe Gly Leu Pro Leu 515 520 525Val Ile Ala Leu Ser His Ala Tyr Phe Thr Ser Leu Ala Tyr Met Lys 530 535 540Leu Met Gly Thr Thr Asn Gln Ile Pro Val Phe Ile Val Met Gly Leu545 550 555 560Tyr Ile Cys Met Tyr Ala Val Phe Ala Val Thr Ala Tyr Asn His Ser 565 570 575Lys Arg Thr Ile Arg His Ser Ile 580115629PRTStaphylococcus aureus 115Met Thr Phe Asn Glu Ile Ile Phe Lys Asn Phe Arg Gln Asn Leu Ser1 5 10 15His Tyr Ala Ile Tyr Leu Phe Ser Leu Ile Thr Ser Val Val Leu Tyr 20 25 30Phe Ser Phe Val Ala Leu Lys Tyr Ala His Lys Leu Asn Met Thr Glu 35 40 45Ser Tyr Pro Ile Ile Lys Glu Gly Ser Gln Val Gly Ser Tyr Phe Leu 50 55 60Phe Phe Ile Ile Ile Ala Phe Leu Leu Tyr Ala Asn Val Leu Phe Ile65 70 75 80Lys Arg Arg Ser Tyr Glu Leu Ala Leu Tyr Gln Thr Leu Gly Leu Ser 85 90 95Lys Phe Asn Ile Ile Tyr Ile Leu Met Leu Glu Gln Leu Leu Ile Phe 100 105 110Ile Ile Thr Ala Ile Leu Gly Ile Ile Ile Gly Ile Phe Gly Ser Lys 115 120 125Leu Leu Leu Met Ile Val Phe Thr Leu Leu Gly Ile Lys Glu Lys Val 130 135 140Pro Ile Ile Phe Ser Leu Arg Ala Val Phe Glu Thr Leu Met Leu Ile145 150 155 160Gly Val Ala Tyr Phe Leu Thr Ser Ala Gln Asn Phe Ile Leu Val Phe 165 170 175Lys Gln Ser Ile Ser Gln Met Ser Lys Asn Asn Gln Val Lys Glu Thr 180 185 190Asn His Asn Lys Ile Thr Phe Glu Glu Val Val Leu Gly Ile Leu Gly 195 200 205Ile Val Leu Ile Thr Thr Gly Tyr Tyr Leu Ser Leu Asn Ile Val Gln 210 215 220Tyr Tyr Asp Ser Ile Gly Ile Leu Met Phe Ile Leu Leu Ser Thr Val225 230 235 240Ile Gly Ala Tyr Leu Phe Phe Lys Ser Ser Val Ser Leu Val Phe Lys 245 250 255Met Val Lys Lys Phe Arg Lys Gly Val Ile Ser Val Asn Asp Val Met 260 265 270Phe Ser Ser Ser Ile Met Tyr Arg Ile Lys Lys Asn Ala Phe Ser Leu 275 280 285Thr Val Met Ala Ile Ile Ser Ala Ile Thr Val Ser Val Leu Cys Phe 290 295 300Ala Ala Ile Ser Arg Ala Ser Leu Ser Ser Glu Ile Lys Tyr Thr Ala305 310 315 320Pro His Asp Val Thr Ile Lys Asp Gln Gln Lys Ala Asn Gln Leu Ala 325 330 335Ser Glu Leu Asn Asn Gln Lys Ile Pro His Phe Tyr Asn Tyr Lys Glu 340 345 350Val Ile His Thr Lys Leu Tyr Lys Asp Asn Leu Phe Asp Val Lys Ala 355 360 365Lys Glu Pro Tyr Asn Val Thr Ile Thr Ser Asp Lys Tyr Ile Pro Asn 370 375 380Thr Asp Leu Lys Arg Gly Gln Ala Asp Leu Phe Val Ala Glu Gly Ser385 390 395 400Ile Lys Asp Leu Val Lys His Lys Lys His Gly Lys Ala Ile Ile Gly 405 410 415Thr Lys Lys His His Val Asn Ile Lys Leu Arg Lys Asp Ile Asn Lys 420 425 430Ile Tyr Phe Met Thr Asp Val Asp Leu Gly Gly Pro Thr Phe Val Leu 435 440 445Asn Asp Lys Asp Tyr Gln Glu Ile Arg Lys Tyr Thr Lys Ala Lys His 450 455 460Ile Val Ser Gln Phe Gly Phe Asp Leu Lys His Lys Lys Asp Ala Leu465 470 475 480Ala Leu Glu Lys Val Lys Asn Lys Val Asp Lys Ser Ile Lys Thr Arg 485 490 495Ser Glu Ala Ile Ser Ser Ile Ser Ser Leu Thr Gly Ile Leu Leu Phe 500 505 510Val Thr Ser Phe Leu Gly Ile Thr Phe Leu Ile Ala Val Cys Cys Ile 515 520 525Ile Tyr Ile Lys Gln Ile Asp Glu Thr Glu Asp Glu Leu Glu Asn Tyr 530 535 540Ser Ile Leu Arg Lys Leu Gly Phe Thr Gln Lys Asp Met Ala Arg Gly545 550 555 560Leu Lys Phe Lys Ile Met Phe Asn Phe Gly Leu Pro Leu Val Ile Ala 565 570 575Leu Ser His Ala Tyr Phe Thr Ser Leu Ala Tyr Met Lys Leu Met Gly 580 585 590Thr Thr Asn Gln Ile Pro Val Phe Ile Val Met Gly Leu Tyr Ile Cys 595 600 605Met Tyr Ala Val Phe Ala Val Thr Ala Tyr Asn His Ser Lys Arg Thr 610 615 620Ile Arg His Ser Ile625116629PRTStaphylococcus aureus 116Met Thr Phe Asn Glu Ile Ile Phe Lys Asn Phe Arg Gln Asn Leu Ser1 5 10 15His Tyr Ala Ile Tyr Leu Phe Ser Leu Ile Thr Ser Val Val Leu Tyr 20 25 30Phe Ser Phe Val Ala Leu Lys Tyr Ala His Lys Leu Asn Met Thr Glu 35 40 45Ser Tyr Pro Ile Ile Lys Glu Gly Ser Gln Val Gly Ser Tyr Phe Leu 50 55 60Phe Phe Ile Ile Ile Ala Phe Leu Leu Tyr Ala Asn Val Leu Phe Ile65 70 75 80Lys Arg Arg Ser Tyr Glu Leu Ala Leu Tyr Gln Thr Leu Gly Leu Ser 85 90 95Lys Phe Asn Ile Ile Tyr Ile Leu Met Leu Glu Gln Leu Leu Ile Phe 100 105 110Ile Ile Thr Ala Ile Leu Gly Ile Ile Ile Gly Ile Phe Gly Ser Lys 115 120 125Leu Leu Leu Met Ile Val Phe Thr Leu Leu Gly Ile Lys Glu Lys Val 130 135 140Pro Ile Ile Phe Ser Leu Arg Ala Val Phe Glu Thr Leu Met Leu Ile145 150 155 160Gly Val Ala Tyr Phe Leu Thr Ser Ala Gln Asn Phe Ile Leu Val Phe 165 170 175Lys Gln Ser Ile Ser Gln Met Ser Lys Asn Asn Gln Val Lys Glu Thr 180 185 190Asn His Asn Lys Ile Thr Phe Glu Glu Val Val Leu Gly Ile Leu Gly 195 200 205Ile Val Leu Ile Thr Thr Gly Tyr Tyr Leu Ser Leu Asn Ile Val Gln 210 215 220Tyr Tyr Asp Ser Ile Gly Ile Leu Met Phe Ile Leu Leu Ser Thr Val225 230 235 240Ile Gly Ala Tyr Leu Phe Phe Lys Ser Ser Val Ser Leu Val Phe Lys 245 250 255Met Val Lys Lys Phe Arg Lys Gly Val Ile Ser Val Asn Asp Val Met 260 265 270Phe Ser Ser Ser Ile Met Tyr Arg Ile Lys Lys Asn Ala Phe Ser Leu 275 280 285Thr Val Met Ala Ile Ile Ser Ala Ile Thr Val Ser Val Leu Cys Phe 290 295 300Ala Ala Ile Ser Arg Ala Ser Leu Ser Ser Glu Ile Lys Tyr Thr Ala305 310 315 320Pro His Asp Val Thr Ile Lys Asp Gln Gln Lys Ala Asn Gln Leu Ala 325 330 335Ser Glu Leu Asn Asn Gln Lys Ile Pro His Phe Tyr Asn Tyr Lys Glu 340 345 350Val Ile His Thr Lys Leu Tyr Lys Asp Asn Leu Phe Asp Val Lys Ala 355 360 365Lys Glu Pro Tyr Asn Val Thr Ile Thr Ser Asp Lys Tyr Ile Pro Asn 370 375 380Thr Asp Leu Lys Arg Gly Gln Ala Asp Leu Phe Val Ala Glu Gly Ser385 390 395 400Ile Lys Asp Leu Val Lys His Lys Lys His Gly Lys Ala Ile Ile Gly 405 410 415Thr Lys Lys His His Val Asn Ile Lys Leu Arg Lys Asp Ile Asn Lys 420 425 430Ile Tyr Phe Met Thr Asp Val Asp Leu Gly Gly Pro Thr Phe Val Leu 435 440 445Asn Asp Lys Asp Tyr Gln Glu Ile Arg Lys Tyr Thr Lys Ala Lys His 450 455 460Ile Val Ser Gln Phe Gly Phe Asp Leu Lys His Lys Lys Asp Ala Leu465 470 475 480Ala Leu Glu Lys Val Lys Asn Lys Val Asp Lys Ser Ile Lys Thr Arg 485 490 495Ser Glu Ala Ile Ser Ser Ile Ser Ser Leu Thr Gly Ile Leu Leu Phe 500 505 510Val Thr Ser Phe Leu Gly Ile Thr Phe Leu Ile Ala Val Cys Cys Ile 515 520 525Ile Tyr Ile Lys Gln Ile Asp Glu Thr Glu Asp Glu Leu Glu Asn Tyr 530 535 540Ser Ile Leu Arg Lys Leu Gly Phe Thr Gln Lys Asp Met Ala Arg Gly545 550 555 560Leu Lys Phe Lys Ile Met Phe Asn Phe Gly Leu Pro Leu Val Ile Ala 565 570 575Leu Ser His Ala Tyr Phe Thr Ser Leu Ala Tyr Met Lys Leu Met Gly 580 585 590Thr Thr Asn Gln Ile Pro Val Phe Ile Val Met Gly Leu Tyr Ile Cys 595 600 605Met Tyr Ala Val Phe Ala Val Thr Ala Tyr Asn His Ser Lys Arg Thr 610 615 620Ile Arg His Ser Ile625117629PRTStaphylococcus aureus 117Met Thr Phe Asn Glu Ile Ile Phe Lys Asn Phe Arg Gln Asn Leu Ser1 5 10 15His Tyr Ala Ile Tyr Leu Phe Ser Leu Ile Thr Ser Val Val Leu Tyr 20 25 30Phe Ser Phe Val Ala Leu Lys Tyr Ala His Lys Leu Asn Met Thr Glu 35 40 45Ser Tyr Pro Ile Ile Lys Glu Gly Ser Gln Val Gly Ser Tyr Phe Leu 50 55 60Phe Phe Ile Ile Ile Ala Phe Leu Leu Tyr Ala Asn Val Leu Phe Ile65 70 75 80Lys Arg Arg Ser Tyr Glu Leu Ala Leu Tyr Gln Thr Leu Gly Leu Ser 85 90 95Lys Phe Asn Ile Ile Tyr Ile Leu Met Leu Glu Gln Leu Leu Ile Phe 100 105 110Ile Ile Thr Ala Ile Leu Gly Ile Ile Ile Gly Ile Phe Gly Ser Lys 115 120 125Leu Leu Leu Met Ile Val Phe Thr Leu Leu Gly Ile Lys Glu Lys Val 130 135 140Pro Ile Ile Phe Ser Leu Arg Ala Val Phe Glu Thr Leu Met Leu Ile145 150 155 160Gly Val Ala Tyr Phe Leu Thr Ser Ala Gln Asn Phe Ile Leu Val Phe 165 170 175Lys Gln Ser Ile Ser Gln Met Ser Lys Asn Asn Gln Val Lys Glu Thr 180 185 190Asn His Asn Lys Ile Thr Phe Glu Glu Val Val Leu Gly Ile Leu Gly 195 200 205Ile Val Leu Ile Thr Thr Gly Tyr Tyr Leu Ser Leu Asn Ile Val Gln 210 215 220Tyr Tyr Asp Ser Ile Gly Ile Leu Met Phe Ile Leu Leu Ser Thr Val225 230 235 240Ile Gly Ala Tyr Leu Phe Phe Lys Ser Ser Val Ser Leu Val Phe Lys 245 250 255Met Val Lys Lys Phe Arg Lys Gly Val Ile Ser Val Asn Asp Val Met 260 265 270Phe Ser Ser Ser Ile Met Tyr Arg Ile Lys Lys Asn Ala Phe Ser Leu 275 280 285Thr Val Met Ala Ile Ile Ser Ala Ile Thr Val Ser Val Leu Cys Phe 290 295 300Ala Ala Ile Ser Arg Ala Ser Leu Ser Ser Glu Ile Lys Tyr Thr Ala305 310 315 320Pro His Asp Val Thr Ile Lys Asp Gln Gln Lys Ala Asn Gln Leu Ala 325 330 335Ser Glu Leu Asn Asn Gln Lys Ile Pro His Phe Tyr Asn Tyr Lys Glu 340 345 350Val Ile His Thr Lys Leu Tyr Lys Asp Asn Leu Phe Asp Val Lys Ala 355 360 365Lys Glu Pro Tyr Asn Val Thr Ile Thr Ser Asp Lys Tyr Ile Pro Asn 370 375 380Thr Asp Leu Lys Arg Gly Gln Ala Asp Leu Phe Val Ala Glu Gly Ser385 390 395 400Ile Lys Asp Leu Val Lys His Lys Lys His Gly Lys Ala Val Ile Gly 405 410 415Thr Lys Lys His His Val Asn Ile Lys Leu Arg Lys Asp Ile Asn Lys 420 425 430Ile Tyr Phe Met Thr Asp Val Asp Leu Gly Gly Pro Thr Phe Val Leu 435 440 445Asn Asp Lys Asp Tyr Gln Glu Ile Arg Lys Tyr Thr Lys Ala Lys His 450 455 460Ile Val Ser Gln Phe Gly Phe Asp Leu Lys His Lys Lys Asp Ala Leu465 470 475 480Ala Leu Glu Lys Val Lys Asn Lys Val Asp Lys Ser Ile Lys Thr Arg 485 490 495Ser Glu Ala Ile Ser Ser Ile Ser Ser Leu Thr Gly Ile Leu Leu Phe 500 505 510Val Thr Ser Phe Leu Gly Ile Thr Phe Leu Ile Ala Val Cys Cys Ile 515 520 525Ile Tyr Ile Lys Gln Ile Asp Glu Thr Glu Asp Glu Leu Glu Asn Tyr 530 535 540Ser Ile Leu Arg Lys Leu Gly Phe Thr Gln Lys Asp Met Ala Arg Gly545 550 555 560Leu Lys Phe Lys Ile Met Phe Asn Phe Gly Leu Pro Leu Val Ile

Ala 565 570 575Leu Ser His Ala Tyr Phe Thr Ser Leu Ala Tyr Met Lys Leu Met Gly 580 585 590Thr Thr Asn Gln Ile Pro Val Phe Ile Val Met Gly Leu Tyr Ile Cys 595 600 605Met Tyr Ala Val Phe Ala Val Thr Ala Tyr Asn His Ser Lys Arg Thr 610 615 620Ile Arg His Ser Ile625118629PRTStaphylococcus aureus 118Met Thr Phe Asn Glu Ile Ile Phe Lys Asn Phe Arg Gln Asn Leu Ser1 5 10 15His Tyr Ala Ile Tyr Leu Phe Ser Leu Ile Thr Ser Val Val Leu Tyr 20 25 30Phe Ser Phe Val Ala Leu Lys Tyr Ala His Lys Leu Asn Met Thr Glu 35 40 45Ser Tyr Pro Ile Ile Lys Glu Gly Ser Gln Val Gly Ser Tyr Phe Leu 50 55 60Phe Phe Ile Ile Ile Ala Phe Leu Leu Tyr Ala Asn Val Leu Phe Ile65 70 75 80Lys Arg Arg Ser Tyr Glu Leu Ala Leu Tyr Gln Thr Leu Gly Leu Ser 85 90 95Lys Phe Asn Ile Ile Tyr Ile Leu Met Leu Glu Gln Leu Leu Ile Phe 100 105 110Ile Ile Thr Ala Ile Leu Gly Ile Ile Ile Gly Ile Phe Gly Ser Lys 115 120 125Leu Leu Leu Met Ile Val Phe Thr Leu Leu Gly Ile Lys Glu Lys Val 130 135 140Pro Ile Ile Phe Ser Leu Arg Ala Val Phe Glu Thr Leu Met Leu Ile145 150 155 160Gly Val Ala Tyr Phe Leu Thr Ser Ala Gln Asn Phe Ile Leu Val Phe 165 170 175Lys Gln Ser Ile Ser Gln Met Ser Lys Asn Asn Gln Val Lys Glu Thr 180 185 190Asn His Asn Lys Ile Thr Phe Glu Glu Val Val Leu Gly Ile Leu Gly 195 200 205Ile Val Leu Ile Thr Thr Gly Tyr Tyr Leu Ser Leu Asn Ile Val Gln 210 215 220Tyr Tyr Asp Ser Ile Gly Ile Leu Met Phe Ile Leu Leu Ser Thr Val225 230 235 240Ile Gly Ala Tyr Leu Phe Phe Lys Ser Ser Val Ser Leu Val Phe Lys 245 250 255Met Val Lys Lys Phe Arg Lys Gly Val Ile Ser Val Asn Asp Val Met 260 265 270Phe Ser Ser Ser Ile Met Tyr Arg Ile Lys Lys Asn Ala Phe Ser Leu 275 280 285Thr Val Met Ala Ile Ile Ser Ala Ile Thr Val Ser Val Leu Cys Phe 290 295 300Ala Ala Ile Ser Arg Ala Ser Leu Ser Ser Glu Ile Lys Tyr Thr Ala305 310 315 320Pro His Asp Val Thr Ile Lys Asp Gln Gln Lys Ala Asn Gln Leu Ala 325 330 335Ser Glu Leu Asn Asn Gln Lys Ile Pro His Phe Tyr Asn Tyr Lys Glu 340 345 350Val Ile His Thr Lys Leu Tyr Lys Asp Asn Leu Phe Asp Val Lys Ala 355 360 365Lys Glu Pro Tyr Asn Val Thr Ile Thr Ser Asp Lys Tyr Ile Pro Asn 370 375 380Thr Asp Leu Lys Arg Gly Gln Ala Asp Leu Phe Val Ala Glu Gly Ser385 390 395 400Ile Lys Asp Leu Val Lys His Lys Lys His Gly Lys Ala Val Ile Gly 405 410 415Thr Lys Lys His His Val Asn Ile Lys Leu Arg Lys Asp Ile Asn Lys 420 425 430Ile Tyr Phe Met Thr Asp Val Asp Leu Gly Gly Pro Thr Phe Val Leu 435 440 445Asn Asp Lys Asp Tyr Gln Glu Ile Arg Lys Tyr Thr Lys Ala Lys His 450 455 460Ile Val Ser Gln Phe Gly Phe Asp Leu Lys His Lys Lys Asp Ala Leu465 470 475 480Ala Leu Glu Lys Val Lys Asn Lys Val Asp Lys Ser Ile Lys Thr Arg 485 490 495Ser Glu Ala Ile Ser Ser Ile Ser Ser Leu Thr Gly Ile Leu Leu Phe 500 505 510Val Thr Ser Phe Leu Gly Ile Thr Phe Leu Ile Ala Val Cys Cys Ile 515 520 525Ile Tyr Ile Lys Gln Ile Asp Glu Thr Glu Asp Glu Leu Glu Asn Tyr 530 535 540Ser Ile Leu Arg Lys Leu Gly Phe Thr Gln Lys Asp Met Ala Arg Gly545 550 555 560Leu Lys Phe Lys Ile Met Phe Asn Phe Gly Leu Pro Leu Val Ile Ala 565 570 575Leu Ser His Ala Tyr Phe Thr Ser Leu Ala Tyr Met Lys Leu Met Gly 580 585 590Thr Thr Asn Gln Ile Pro Val Phe Ile Val Met Gly Leu Tyr Ile Cys 595 600 605Met Tyr Ala Val Phe Ala Val Thr Ala Tyr Asn His Ser Lys Arg Thr 610 615 620Ile Arg His Ser Ile625119629PRTStaphylococcus aureus 119Met Thr Phe Asn Glu Ile Ile Phe Lys Asn Phe Arg Gln Asn Leu Ser1 5 10 15His Tyr Ala Ile Tyr Leu Phe Ser Leu Ile Thr Ser Val Val Leu Tyr 20 25 30Phe Ser Phe Val Ala Leu Lys Tyr Ala His Lys Leu Asn Met Thr Glu 35 40 45Ser Tyr Pro Ile Ile Lys Glu Gly Ser Gln Val Gly Ser Tyr Phe Leu 50 55 60Phe Phe Ile Ile Ile Ala Phe Leu Leu Tyr Ala Asn Val Leu Phe Ile65 70 75 80Lys Arg Arg Ser Tyr Glu Leu Ala Leu Tyr Gln Thr Leu Gly Leu Ser 85 90 95Lys Phe Asn Ile Ile Tyr Ile Leu Met Leu Glu Gln Leu Leu Ile Phe 100 105 110Ile Ile Thr Ala Ile Leu Gly Ile Ile Ile Gly Ile Phe Gly Ser Lys 115 120 125Leu Leu Leu Met Ile Val Phe Thr Leu Leu Gly Ile Lys Glu Lys Val 130 135 140Pro Ile Ile Phe Ser Leu Arg Ala Val Phe Glu Thr Leu Met Leu Ile145 150 155 160Gly Val Ala Tyr Phe Leu Thr Ser Ala Gln Asn Phe Ile Leu Val Phe 165 170 175Lys Gln Ser Ile Ser Gln Met Ser Lys Asn Asn Gln Val Lys Glu Thr 180 185 190Asn His Asn Lys Ile Thr Phe Glu Glu Val Val Leu Gly Ile Leu Gly 195 200 205Ile Val Leu Ile Thr Thr Gly Tyr Tyr Leu Ser Leu Asn Ile Val Gln 210 215 220Tyr Tyr Asp Ser Ile Gly Thr Leu Met Phe Ile Leu Leu Ser Thr Val225 230 235 240Ile Gly Ala Tyr Leu Phe Phe Lys Ser Ser Val Ser Leu Val Phe Lys 245 250 255Met Val Lys Lys Phe Arg Lys Gly Val Ile Ser Val Asn Asp Val Met 260 265 270Phe Ser Ser Ser Ile Met Tyr Arg Ile Lys Lys Asn Ala Phe Ser Leu 275 280 285Thr Val Met Ala Ile Ile Ser Ala Ile Thr Val Ser Val Leu Cys Phe 290 295 300Ala Ala Ile Ser Arg Ala Ser Leu Ser Ser Glu Ile Lys Tyr Thr Ala305 310 315 320Pro His Asp Val Thr Ile Lys Asp Gln Gln Lys Ala Asn Gln Leu Ala 325 330 335Ser Glu Leu Asn Asn Gln Lys Ile Pro His Phe Tyr Asn Tyr Lys Glu 340 345 350Val Ile His Thr Lys Leu Tyr Lys Asp Asn Leu Phe Asp Val Lys Ser 355 360 365Lys Gln Pro Tyr Asn Val Thr Ile Thr Ser Asp Lys Tyr Ile Pro Ser 370 375 380Thr Asp Leu Lys Arg Gly Gln Ala Asp Leu Phe Val Ala Glu Gly Ser385 390 395 400Ile Lys Asp Leu Val Lys His Lys Lys His Gly Lys Ala Val Ile Gly 405 410 415Thr Lys Lys His His Val Asn Ile Lys Leu Arg Lys Asp Ile Asn Lys 420 425 430Ile Tyr Phe Met Thr Asp Val Asp Leu Gly Gly Pro Thr Phe Val Leu 435 440 445Asn Asp Lys Asp Tyr Gln Glu Ile Arg Lys Tyr Thr Lys Ala Lys His 450 455 460Ile Val Ser Gln Phe Gly Phe Asp Leu Lys His Lys Lys Asp Ala Leu465 470 475 480Ala Leu Glu Lys Ala Lys Asn Lys Val Asp Lys Ser Ile Glu Thr Arg 485 490 495Ser Glu Ala Ile Ser Ser Ile Ser Ser Leu Thr Gly Ile Leu Leu Phe 500 505 510Val Thr Ser Phe Leu Gly Ile Thr Phe Leu Ile Ala Val Cys Cys Ile 515 520 525Ile Tyr Ile Lys Gln Ile Asp Glu Thr Glu Asp Glu Leu Glu Asn Tyr 530 535 540Ser Ile Leu Arg Lys Leu Gly Phe Thr Gln Lys Asp Met Ala Arg Gly545 550 555 560Leu Lys Phe Lys Ile Met Phe Asn Phe Gly Leu Pro Leu Val Ile Ala 565 570 575Leu Ser His Ala Tyr Phe Thr Ser Leu Ala Tyr Met Lys Leu Met Gly 580 585 590Thr Thr Asn Gln Ile Pro Val Phe Ile Val Met Gly Leu Tyr Ile Cys 595 600 605Met Tyr Ala Val Phe Ala Val Thr Ala Tyr Asn His Ser Lys Arg Thr 610 615 620Ile Arg His Ser Ile625120629PRTArtificial sequencesynthetic sequencemisc_feature(1)..(45)Xaa can be any naturally occurring amino acidmisc_feature(213)..(213)Xaa can be any naturally occurring amino acidmisc_feature(231)..(231)Xaa can be any naturally occurring amino acidmisc_feature(368)..(368)Xaa can be any naturally occurring amino acidmisc_feature(370)..(370)Xaa can be any naturally occurring amino acidmisc_feature(384)..(384)Xaa can be any naturally occurring amino acidmisc_feature(414)..(414)Xaa can be any naturally occurring amino acidmisc_feature(486)..(486)Xaa can be any naturally occurring amino acidmisc_feature(494)..(494)Xaa can be any naturally occurring amino acidmisc_feature(576)..(576)Xaa can be any naturally occurring amino acid 120Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa1 5 10 15Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 20 25 30Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Met Thr Glu 35 40 45Ser Tyr Pro Ile Ile Lys Glu Gly Ser Gln Val Gly Ser Tyr Phe Leu 50 55 60Phe Phe Ile Ile Ile Ala Phe Leu Leu Tyr Ala Asn Val Leu Phe Ile65 70 75 80Lys Arg Arg Ser Tyr Glu Leu Ala Leu Tyr Gln Thr Leu Gly Leu Ser 85 90 95Lys Phe Asn Ile Ile Tyr Ile Leu Met Leu Glu Gln Leu Leu Ile Phe 100 105 110Ile Ile Thr Ala Ile Leu Gly Ile Ile Ile Gly Ile Phe Gly Ser Lys 115 120 125Leu Leu Leu Met Ile Val Phe Thr Leu Leu Gly Ile Lys Glu Lys Val 130 135 140Pro Ile Ile Phe Ser Leu Arg Ala Val Phe Glu Thr Leu Met Leu Ile145 150 155 160Gly Val Ala Tyr Phe Leu Thr Ser Ala Gln Asn Phe Ile Leu Val Phe 165 170 175Lys Gln Ser Ile Ser Gln Met Ser Lys Asn Asn Gln Val Lys Glu Thr 180 185 190Asn His Asn Lys Ile Thr Phe Glu Glu Val Val Leu Gly Ile Leu Gly 195 200 205Ile Val Leu Ile Xaa Thr Gly Tyr Tyr Leu Ser Leu Asn Ile Val Gln 210 215 220Tyr Tyr Asp Ser Ile Gly Xaa Leu Met Phe Ile Leu Leu Ser Thr Val225 230 235 240Ile Gly Ala Tyr Leu Phe Phe Lys Ser Ser Val Ser Leu Val Phe Lys 245 250 255Met Val Lys Lys Phe Arg Lys Gly Val Ile Ser Val Asn Asp Val Met 260 265 270Phe Ser Ser Ser Ile Met Tyr Arg Ile Lys Lys Asn Ala Phe Ser Leu 275 280 285Thr Val Met Ala Ile Ile Ser Ala Ile Thr Val Ser Val Leu Cys Phe 290 295 300Ala Ala Ile Ser Arg Ala Ser Leu Ser Ser Glu Ile Lys Tyr Thr Ala305 310 315 320Pro His Asp Val Thr Ile Lys Asp Gln Gln Lys Ala Asn Gln Leu Ala 325 330 335Ser Glu Leu Asn Asn Gln Lys Ile Pro His Phe Tyr Asn Tyr Lys Glu 340 345 350Val Ile His Thr Lys Leu Tyr Lys Asp Asn Leu Phe Asp Val Lys Xaa 355 360 365Lys Xaa Pro Tyr Asn Val Thr Ile Thr Ser Asp Lys Tyr Ile Pro Xaa 370 375 380Thr Asp Leu Lys Arg Gly Gln Ala Asp Leu Phe Val Ala Glu Gly Ser385 390 395 400Ile Lys Asp Leu Val Lys His Lys Lys His Gly Lys Ala Xaa Ile Gly 405 410 415Thr Lys Lys His His Val Asn Ile Lys Leu Arg Lys Asp Ile Asn Lys 420 425 430Ile Tyr Phe Met Thr Asp Val Asp Leu Gly Gly Pro Thr Phe Val Leu 435 440 445Asn Asp Lys Asp Tyr Gln Glu Ile Arg Lys Tyr Thr Lys Ala Lys His 450 455 460Ile Val Ser Gln Phe Gly Phe Asp Leu Lys His Lys Lys Asp Ala Leu465 470 475 480Ala Leu Glu Lys Ala Xaa Asn Lys Val Asp Lys Ser Ile Xaa Thr Arg 485 490 495Ser Glu Ala Ile Ser Ser Ile Ser Ser Leu Thr Gly Ile Leu Leu Phe 500 505 510Val Thr Ser Phe Leu Gly Ile Thr Phe Leu Ile Ala Val Cys Cys Ile 515 520 525Ile Tyr Ile Lys Gln Ile Asp Glu Thr Glu Asp Glu Leu Glu Asn Tyr 530 535 540Ser Ile Leu Arg Lys Leu Gly Phe Thr Gln Lys Asp Met Ala Arg Gly545 550 555 560Leu Lys Phe Lys Ile Met Phe Asn Phe Gly Leu Pro Leu Val Ile Xaa 565 570 575Leu Ser His Ala Tyr Phe Thr Ser Leu Ala Tyr Met Lys Leu Met Gly 580 585 590Thr Thr Asn Gln Ile Pro Val Phe Ile Val Met Gly Leu Tyr Ile Cys 595 600 605Met Tyr Ala Val Phe Ala Val Thr Ala Tyr Asn His Ser Lys Arg Thr 610 615 620Ile Arg His Ser Ile62512155PRTStaphylococcus aureus 121Met Leu Glu Ser Arg Glu Gln Leu Ser Val Glu Glu Tyr Glu Thr Phe1 5 10 15Phe Asn Arg Phe Asp Asn Gln Glu Phe Asp Phe Glu Arg Glu Leu Thr 20 25 30Gln Asp Pro Tyr Ser Lys Val Tyr Leu Tyr Ser Ile Glu Asp His Ile 35 40 45Arg Thr Tyr Lys Ile Glu Lys 50 5512255PRTStaphylococcus aureus 122Met Leu Glu Ser Arg Glu Gln Leu Ser Val Glu Glu Tyr Glu Thr Phe1 5 10 15Phe Asn Arg Phe Asp Asn Gln Glu Phe Asp Phe Glu Arg Glu Leu Thr 20 25 30Gln Asp Pro Tyr Ser Lys Val Tyr Leu Tyr Ser Ile Glu Asp His Ile 35 40 45Arg Thr Tyr Lys Ile Glu Lys 50 5512388PRTStaphylococcus aureus 123Cys Leu Phe Ser Tyr Gly Ser Gly Ala Val Gly Glu Ile Phe Ser Gly1 5 10 15Ser Ile Val Lys Gly Tyr Asp Lys Ala Leu Asp Lys Glu Lys His Leu 20 25 30Asn Met Leu Glu Ser Arg Glu Gln Leu Ser Val Glu Glu Tyr Glu Thr 35 40 45Phe Phe Asn Arg Phe Asp Asn Gln Glu Phe Asp Phe Glu Arg Glu Leu 50 55 60Thr Gln Asp Pro Tyr Ser Lys Val Tyr Leu Tyr Ser Ile Glu Asp His65 70 75 80Ile Arg Thr Tyr Lys Ile Glu Lys 8512488PRTStaphylococcus aureus 124Cys Leu Phe Ser Tyr Gly Ser Gly Ala Val Gly Glu Ile Phe Ser Gly1 5 10 15Ser Ile Val Lys Gly Tyr Asp Lys Ala Leu Asp Lys Glu Lys His Leu 20 25 30Asn Met Leu Glu Ser Arg Glu Gln Leu Ser Val Glu Glu Tyr Glu Thr 35 40 45Phe Phe Asn Arg Phe Asp Asn Gln Glu Phe Asp Phe Glu Arg Glu Leu 50 55 60Thr Gln Asp Pro Tyr Ser Lys Val Tyr Leu Tyr Ser Ile Glu Asp His65 70 75 80Ile Arg Thr Tyr Lys Ile Glu Lys 8512588PRTStaphylococcus aureus 125Cys Leu Phe Ser Tyr Gly Ser Gly Ala Val Gly Glu Ile Phe Ser Gly1 5 10 15Ser Ile Val Lys Gly Tyr Asp Lys Ala Leu Asp Lys Glu Lys His Leu 20 25 30Asn Met Leu Glu Ser Arg Glu Gln Leu Ser Val Glu Glu Tyr Glu Thr 35 40 45Phe Phe Asn Arg Phe Asp Asn Gln Glu Phe Asp Phe Glu Arg Glu Leu 50 55 60Thr Gln Asp Pro Tyr Ser Lys Val Tyr Leu Tyr Ser Ile Glu Asp His65 70 75 80Ile Arg Thr Tyr Lys Ile Glu Lys 8512678PRTStaphylococcus aureus 126Gly Glu Phe Tyr Ser Ala Thr Leu Val Glu Gly Tyr Lys Asp His Leu1 5 10 15Asp Gln Ala Ala His Lys Ala Leu Leu Asn Asn Arg

Thr Glu Val Ser 20 25 30Val Asp Ala Tyr Glu Thr Phe Phe Lys Arg Phe Asp Asp Val Asp Phe 35 40 45Asp Glu Gln Gln Asp Ala Val His Glu Asp Arg Arg Ile Phe Tyr Leu 50 55 60Ser Asn Ile Glu Asn Asn Val Arg Glu Tyr His Arg Pro Glu65 70 7512788PRTStaphylococcus aureus 127Gly Leu Phe Ser Tyr Gly Ser Gly Ser Val Gly Glu Phe Tyr Ser Ala1 5 10 15Thr Leu Val Glu Gly Tyr Lys Asp His Leu Asp Gln Ala Ala His Lys 20 25 30Ala Leu Leu Asn Asn Arg Thr Glu Val Ser Val Asp Ala Tyr Glu Thr 35 40 45Phe Phe Lys Arg Phe Asp Asp Val Asp Phe Asp Glu Gln Gln Asp Ala 50 55 60Val His Glu Asp Arg Arg Ile Phe Tyr Leu Ser Asn Ile Glu Asn Asn65 70 75 80Val Arg Glu Tyr His Arg Pro Glu 8512878PRTStaphylococcus aureus 128Gly Glu Phe Tyr Ser Ala Thr Leu Val Glu Gly Tyr Lys Asp His Leu1 5 10 15Asp Gln Ala Ala His Lys Ala Leu Leu Asn Asn Arg Thr Glu Val Ser 20 25 30Val Asp Ala Tyr Glu Thr Phe Phe Lys Arg Phe Asp Asp Val Asp Phe 35 40 45Asp Glu Glu Gln Asp Ala Val His Glu Asp Arg His Ile Phe Tyr Leu 50 55 60Ser Asn Ile Glu Asn Asn Val Arg Glu Tyr His Arg Pro Glu65 70 7512988PRTStaphylococcus aureus 129Gly Leu Phe Ser Tyr Gly Ser Gly Ser Val Gly Glu Phe Tyr Ser Ala1 5 10 15Thr Leu Val Glu Gly Tyr Lys Asp His Leu Asp Gln Ala Ala His Lys 20 25 30Ala Leu Leu Asn Asn Arg Thr Glu Val Ser Val Asp Ala Tyr Glu Thr 35 40 45Phe Phe Lys Arg Phe Asp Asp Val Glu Phe Asp Glu Glu Gln Asp Ala 50 55 60Val His Glu Asp Arg His Ile Phe Tyr Leu Ser Asn Ile Glu Asn Asn65 70 75 80Val Arg Glu Tyr His Arg Pro Glu 8513088PRTStaphylococcus aureus 130Gly Leu Phe Ser Tyr Gly Ser Gly Ser Val Gly Glu Phe Tyr Ser Ala1 5 10 15Thr Leu Val Glu Gly Tyr Lys Asp His Leu Asp Gln Ala Ala His Lys 20 25 30Ala Leu Leu Asn Asn Arg Thr Glu Val Ser Val Asp Ala Tyr Glu Thr 35 40 45Phe Phe Lys Arg Phe Asp Asp Val Glu Phe Asp Glu Glu Gln Asp Ala 50 55 60Val His Glu Asp Arg His Ile Phe Tyr Leu Ser Asn Ile Glu Asn Asn65 70 75 80Val Arg Glu Tyr His Arg Pro Glu 8513188PRTArtificial sequencesynthetic sequencemisc_feature(1)..(34)Xaa can be any naturally occurring amino acidmisc_feature(36)..(37)Xaa can be any naturally occurring amino acidmisc_feature(39)..(41)Xaa can be any naturally occurring amino acidmisc_feature(44)..(45)Xaa can be any naturally occurring amino acidmisc_feature(51)..(51)Xaa can be any naturally occurring amino acidmisc_feature(55)..(57)Xaa can be any naturally occurring amino acidmisc_feature(60)..(72)Xaa can be any naturally occurring amino acidmisc_feature(75)..(76)Xaa can be any naturally occurring amino acidmisc_feature(79)..(81)Xaa can be any naturally occurring amino acidmisc_feature(83)..(83)Xaa can be any naturally occurring amino acidmisc_feature(85)..(88)Xaa can be any naturally occurring amino acid 131Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa1 5 10 15Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 20 25 30Xaa Xaa Leu Xaa Xaa Arg Xaa Xaa Xaa Ser Val Xaa Xaa Tyr Glu Thr 35 40 45Phe Phe Xaa Arg Phe Asp Xaa Xaa Xaa Phe Asp Xaa Xaa Xaa Xaa Xaa 50 55 60Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Tyr Leu Xaa Xaa Ile Glu Xaa Xaa65 70 75 80Xaa Arg Xaa Tyr Xaa Xaa Xaa Xaa 8513214PRTStaphylococcus aureus 132Glu Ser Arg Glu Gln Leu Ser Val Glu Glu Tyr Glu Thr Phe1 5 1013314PRTStaphylococcus aureus 133Asn Asn Arg Thr Glu Val Ser Val Asp Ala Tyr Glu Thr Phe1 5 1013414PRTStaphylococcus aureus 134Asn Gln Glu Phe Asp Phe Glu Arg Glu Leu Thr Gln Asp Pro1 5 1013514PRTStaphylococcus aureus 135Asp Val Asp Phe Asp Glu Gln Gln Asp Ala Val His Glu Asp1 5 1013614PRTStaphylococcus aureus 136Asp Val Asp Phe Asp Glu Glu Gln Asp Ala Val His Glu Asp1 5 1013714PRTStaphylococcus aureus 137Asp Val Glu Phe Asp Glu Glu Gln Asp Ala Val His Glu Asp1 5 1013814PRTArtificial sequencesynthetic sequencemisc_feature(1)..(2)Xaa can be any naturally occurring amino acidmisc_feature(4)..(6)Xaa can be any naturally occurring amino acidmisc_feature(9)..(10)Xaa can be any naturally occurring amino acid 138Xaa Xaa Arg Xaa Xaa Xaa Ser Val Xaa Xaa Tyr Glu Thr Phe1 5 1013914PRTArtificial sequencesynthetic sequencemisc_feature(1)..(3)Xaa can be any naturally occurring amino acidmisc_feature(6)..(14)Xaa can be any naturally occurring amino acid 139Xaa Xaa Xaa Phe Asp Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa1 5 10140720DNAStaphylococcus aureus 140ttattgttca atgtgctttt gaataaaatc tttgatttga ggcgtaaagt atacggcacc 60attatattca gaaccaattt ttccaatacc gccatacacc acacctatga cctcattgtt 120agaatttaga actggtgatc ctgaattccc gggttcaatg tatgcatcaa aatttaaaat 180attgtctttg attcttttta tagttcctgt agattcaaac tgtttaaaac tattttgagc 240aggtaatggg taaccaataa ctttaatttt gtcatcaact ttagcatctt tcgcaaaatt 300taatgcttgg acattttcat taaaattaaa gccttttggt ccacgttcaa ctgcttgttc 360ttcaatattc atgacagaga tgtcttcatc acccggataa tcagaaatgc ttttaatttt 420atatatacca ccatttcctt tgtcaccgtc tggatgggca gtaattctat cgccaacttt 480ataatctttt gatacatgtt tattggtgat aattgtattt tttccaatta caaaacctgt 540cgcatcttta aatgaaacga cgccattata tggaaaaata tttgtatctt taacttgcgt 600aacattcttc tctgcatttg ctatttgttg tgtctcttca acgactgcag catttattcc 660agttactgag gttagaatgg ctaatgctgc catgctttta atgactatat ttttattcat 720141720DNAStaphylococcus aureus 141ttattgttca atgtgctttt gaataaaatc tttgatttga ggcgtaaagt atacggcacc 60attatattca gaaccaattt ttccaatacc gccatacacc acacctatga cctcattgtt 120agaatttaga actggtgatc ctgaattccc gggttcaatg tatgcatcaa aatttaaaat 180attgtctttg attcttttta tagttcctgt agattcaaac tgtttaaaac tattttgagc 240aggtaatggg taaccaataa ctttaatttt gtcatcaact ttagcatctt tcgcaaaatt 300taatgcttgg acattttcat taaaattaaa gccttttggt ccacgttcaa ctgcttgttc 360ttcaatattc atgacagaga tgtcttcatc acccggataa tcagaaatgc ttttaatttt 420atatatacca ccatttcctt tgtcaccgtc tggatgggca gtaattctat cgccaacttt 480ataatctttt gatacatgtt tattggtgat aattgtattt tttccaatta caaaacctgt 540cgcatcttta aatgaaacga cgccattata tggaaaaata tttgtatctt taacttgcgt 600aacattcttc tctgcatttg ctatttgttg tgtctcttca acgactgcag catttattcc 660agttgctgag gttagaatgg ctaatgctgc catgctttta atgactatat ttttattcat 720142740DNAStaphylococcus aureus 142ttattgttca atgtgctttt gaataaaatc tttgatttga ggcgtaaagt atacggcacc 60attatattca gaaccaattt ttccaatacc gccatacacc acacctatga cctcattgtt 120agaatttaga actggtgatc ctgaattccc gggttcaatg tatgcatcaa aatttaaaat 180attgtctttg attcttttta tagttcctgt agattcaaac tgtttaaaac tattttgagc 240aggtaatggg taaccaataa ctttaatttt gtcatcaact ttagcatctt tcgcaaaatt 300gaatgcttgg acattttcat taaaattaaa gccttttggt ccacgttcga ctgcttgttc 360ttcaatattc atgacagaga tgtcttcatc acccggataa tcagaaatgc ttttaatttt 420atatatacca ccatttcctt tgtcaccgtt tggatgggca gtaattctat cgccaacttt 480ataatctttt gatacatgtt tattggtgat aattgtattt tttccaatta caaaacctgt 540cgcatcttta aatgaaacga cgccattata tggaaaaata tttgtatctt taacttgcgt 600aacattcttc tctgcatttg ctatttgttg tgtctcttca acgactgcag catttattcc 660agttactgag gttagaatgg ctaatgctgc catgctttta atgactatat ttttattcat 720aaaagcacat tgaacaataa 740143720DNAStaphylococcus aureus 143ttattgttca atgtgctttt gaataaaatc tttgatttga ggcgtaaagt atacggcacc 60attatattca gaaccaattt ttccaatacc gccatacacc acacctatga cctcattgtt 120agaatttaga actggtgatc ctgaattccc gggttcaatg tatgcatcaa aatttaaaat 180attgtctttg attcttttta tagttcctgt agattcaaac tgtttaaaac tattttgagc 240aggtaatggg taaccaataa ctttaatttt gtcatcaact ttagcatctt tcgcaaaatt 300gaatgcttgg acattttcat taaaattaaa gccttttggt ccacgttcga ctgcttgttc 360ttcaatattc atgacagaga tgtcttcatc acccggataa tcagaaatgc ttttaatttt 420atatatacca ccatttcctt tgtcaccgtt tggatgggca gtaattctat cgccaacttt 480ataatctttt gatacatgtt tattggtgat aattgtattt tttccaatta caaaacctgt 540cgcatcttta aatgaaacga cgccattata tggaaaaata tttgtatctt taacttgcgt 600aacattcttc tctgcatttg ctatttgttg tgtctcttca acgactgcag catttattcc 660agttactgag gttagaatgg ctaatgctgc catgctttta atgactatat ttttattcat 720144720DNAStaphylococcus aureus 144ttattgttca atgtgctttt gaataaaatc tttgatttga ggcgtaaagt atacggcacc 60attatattca gaaccaattt ttccaatacc gccatacacc acacctatga cctcattgtt 120agaatttaga actggtgatc ctgaattccc gggttcaatg tatgcatcaa aatttaaaat 180attgtctttg attcttttta tagttcctgt agattcaaac tgtttaaaac tattttgagc 240aggtaatggg taaccaataa ctttaatttt gtcatcaact ttagcatctt tcgcaaaatt 300taatgcttgg acattttcat taaaattaaa gccttttggt ccacgttcga ctgcttgttc 360ttcaatattc atgacagaga tgtcttcatc acccggataa tcagaaatgc ttttaatttt 420atatatacca ccatttcctt tgtcaccgtt tggatgggca gtaattctat cgccaacttt 480ataatctttt gatacatgtt tattggtgat aattgtattt tttccaatta caaaacctgt 540cgcatcttta aatgaaacga cgccattata tggaaaaata tttgtatctt taacttgcgt 600aacattcttc tctgcatttg ctatttgttg tgtctcttca acgactgcag catttattcc 660agttactgag gttagaatgg ctaatgctgc catgctttta atgactatat ttttattcat 720145720DNAStaphylococcus aureus 145ttattgttca atgtgctttt gaataaaatc tttgatttga ggcgtaaagt atacggcacc 60attatattca gaaccaattt ttccaatacc gccatacacc acacctatga cctcattgtt 120agaatttaga actggtgatc ctgaattccc gggttcaatg tatgcatcaa aatttaaaat 180attgtctttg attcttttta tagttcctgt agattcaaac tgtttaaaac tattttgagc 240aggtaatggg taaccaataa ctttaatttt gtcatcaact ttagcatctt tcgcaaaatt 300gaatgcttgg acattttcat taaaattaaa gccttttggt ccacgttcga ctgcttgttc 360ttcaatattc atgacagaga tgtcttcatc acccggataa tcagaaatgc ttttaatttt 420atatatacca ccatttcctt tgtcaccgtt tggatgggca gtaattctat cgccaacttt 480ataatctttt gatacatgtt tattggtgat aattgtattt tttccaatta caaaacctgt 540cgcatcttta aatgaaacga cgccattata tggaaaaata tttgtatctt taacttgcgt 600aacattcttc tctgcatttg ctatttgttg tgtctcttca acgactgcag catttattcc 660agttactgag gttagaatgg ctaatgctgc catgctttta atgactatat ttttattcat 720146720DNAStaphylococcus aureus 146ttattgttca atgtgctttt gaataaaatc tttgatttga ggcgtaaagt atacggcacc 60attatattca gaaccaattt ttccaatacc gccatacacc acacctatga cctcattgtt 120agaatttaga actggtgatc ctgaattccc gggttcaatg tatgcatcaa aatttaaaat 180attgtctttg attcttttta tagttcctgt agattcaaac tgtttaaaac tattttgagc 240aggtaatggg taaccaataa ctttaatttt gtcatcaact ttagcatctt tcgcaaaatt 300gaatgcttgg acattttcat taaaattaaa gccttttggt ccacgttcaa ctgcttgttc 360ttcaatattc atgacagaga tgtcttcatc acccggataa tcagaaatgc ttttaatttt 420atatatacca ccatttcctt tgtcaccgtt tggatgggca gtaattctat cgccaacttt 480ataatctttt gatacatgtt tattggtgat aattgtattt tttccaatta caaaacctgt 540cgcatcctta aatgaaacga cgccattata tggaaaatta tttgtatctt taacttgcgt 600aacattcttc tctgcatttg ctatttgttg tgtctcttca acgactgcag catttattcc 660agttactgag gttagaatgg ctaatgctgc catgctttta atgactatat ttttattcat 720147720DNAStaphylococcus aureus 147ttattgttca atgtgctttt gaataaaatc tttgatttga ggcgtaaagt atacggcacc 60attatattca gaaccaattt ttccaatacc gccatacacc acacctatga cctcattgtt 120agaatttaga actggtgatc ctgaattccc gggttcaatg tatgcatcaa aatttaaaat 180attgtctttg attcttttta tagttcctgt agattcaaac tgtttaaaac tattttgagc 240aggtaatggg taaccaataa ctttaatttt gtcatcaact ttagcatctt tcgcaaaatt 300gaatgcttgg acattttcat taaaattaaa gccttttggt ccacgttcaa ctgcttgttc 360ttcaatattc atgacagaga tgtcttcatc acccggataa tcagaaatgc ttttaatttt 420atatatacca ccatttcctt tgtcaccgtt tggatgggca gtaattctat cgccaacttt 480ataatctttt gatacatgtt tattggtgat aattgtattt tttccaatta caaaacctgt 540cgcatcctta aatgaaacga cgccattata tggaaaatta tttgtatctt taacttgcgt 600aacattcttc tctgcatttg ctatttgttg tgtctcttca acgactgcag catttattcc 660agttactgag gttagaatgg ctaatgctgc catgctttta atgactatat ttttattcat 720148720DNAStaphylococcus aureus 148ttattgttca atgtgctttt gaataaaatc tttgatttga ggcgtaaagt atacggcacc 60attatattca gaaccaattt ttccaatacc gccatacacc acacctatga cctcattgtt 120agaatttaga actggtgatc ctgaattccc gggttcaatg tatgcatcaa aatttaaaat 180attgtctttg attcttttta tagttcctgt agattcaaac tgtttaaaac tattttgagc 240aggtaatggg taaccaataa ctttaatttt gtcatcaact ttagcatctt tcgcaaaatt 300gaatgcttgg acattttcat taaaattaaa gccttttggt ccacgttcaa ctgcttgttc 360ttcaatattc atgacagaga tgtcttcatc acccggataa tcagaaatgc ttttaatttt 420atatatacca ccatttcctt tgtcaccgtt tggatgggca gtaattctat cgccaacttt 480ataatctttt gatacatgtt tattggtgat aattgtattt tttccaatta caaaacctgt 540cgcatcctta aatgaaacga cgccattata tggaaaatta tttgtatctt taacttgcgt 600aacattcttc tctgcatttg ctatttgttg tgtctcttca acgactgcag catttattcc 660agttactgag gttagaatgg ctaatgctgc catgctttta atgactatat ttttattcat 720149720DNAStaphylococcus aureus 149ttattgttca atgtgctttt gaataaaatc tttgatttga ggcgtaaagt atacggcacc 60attatattca gaaccaattt ttccaatacc gccatacacc acacctatga cctcattgtt 120agaatttaga actggtgatc ctgaattccc gggttcaatg tatgcatcaa aatttaaaat 180attgtctttg attcttttta tagttcctgt agattcaaac tgtttaaaac tattttgagc 240aggtaatggg taaccaataa ctttaatttt gtcatcaact ttagcatctt tcgcaaaatt 300gaatgcttgg acattttcat taaaattaaa gccttttggt ccacgttcaa ctgcttgttc 360ttcaatattc atgacagaga tgtcttcatc acccggataa tcagaaatgc ttttaatttt 420atatatacca ccatttcctt tgtcaccgtt tggatgggca gtaattctat cgccaacttt 480ataatctttt gatacatgtt tattggtgat aattgtattt tttccaatta caaaacctgt 540cgcatcctta aatgaaacga cgccattata tggaaaatta tttgtatctt taacttgcgt 600aacattcttc tctgcatttg ctatttgttg tgtctcttca acgactgcag catttattcc 660agttactgag gttagaatgg ctaatgctgc catgctttta atgactatat ttttattcat 720150720DNAStaphylococcus aureus 150ttattgttca atgtgctttt gaataaaatc tttgatttga ggcgtaaagt atacggcacc 60attatattca gaaccaattt ttccaatacc gccatacacc acacctatga cctcattgtt 120agaatttaga actggtgatc ctgaattccc gggttcaatg tatgcatcaa aatttaaaat 180attgtctttg attcttttta tagttcctgt agattcaaac tgtttaaaac tattttgagc 240aggtaatggg taaccaataa ctttaatttt gtcatcaact ttagcatctt tcgcaaaatt 300gaatgcttgg acattttcat taaaattaaa gccttttggt ccacgttcaa ctgcttgttc 360ttcaatattc atgacagaga tgtcttcatc acccggataa tcagaaatgc ttttaatttt 420atatatacca ccatttcctt tgtcaccgtt tggatgggca gtaattctat cgccaacttt 480ataatctttt gatacatgtt tattggtgat aattgtattt tttccaatta caaaacctgt 540cgcatcctta aatgaaacga cgccattata tggaaaatta tttgtatctt taacttgcgt 600aacattcttc tctgcatttg ctatttgttg tgtctcttca acgactgcag catttattcc 660agttactgag gttagaatgg ctaatgctgc catgctttta atgactatat ttttattcat 720151720DNAStaphylococcus aureus 151ttattgttca atgtgctttt gaataaattc tttgatttga ggcgtaaaat aaactgcacc 60attgtattct gatccgattt ttccaatacc tccatacaca actcctacga cttcattatt 120taaatttaaa actggggatc ctgaatttcc aggctcgata tatgcatcaa aatttaaatt 180attatcttta atacttttta cagttccagt tgattcaaat tgtttgaatg tattttgagc 240tggtaaaggg tatccaataa ctttaatttt gtcgtcaact ttagcatctt tcgcaaaatt 300gaatgcttgg acattttcat taaaattata accatttgct ccacgttcaa cagcattttc 360ttcaacgttc attactgata tatcctcatt acctggataa tcagaaatat ttttaatttt 420ataaattcca ccgttgcctt tgtcaccatt tgggtgggca gtaattctat cgccaacttt 480atagtccttt gatacatgtt tattggtgat aattgtattt ttttcaattg caaaacctgt 540cgcatcttta aatgaaacga cgccattata tggaaaaaca tttgtatctt taacttgcgt 600aacattcttc tctgcatttg ctatttgttg tgtctcatca acgactgcag catttattcc 660agttactgag gttagaatgg ctaatgctgc catgctttta atgactatat ttttattcat 720152239PRTStaphylococcus aureus 152Met Asn Lys Asn Ile Val Ile Lys Ser Met Ala Ala Leu Ala Ile Leu1 5 10 15Thr Ser Val Thr Gly Ile Asn Ala Ala Val Val Glu Glu Thr Gln Gln 20 25 30Ile Ala Asn Ala Glu Lys Asn Val Thr Gln Val Lys Asp Thr Asn Ile 35 40 45Phe Pro Tyr Asn Gly Val Val Ser Phe Lys Asp Ala Thr Gly Phe Val 50 55 60Ile Gly Lys Asn Thr Ile Ile Thr Asn Lys His Val Ser Lys Asp Tyr65 70 75 80Lys Val Gly Asp Arg Ile Thr Ala His Pro Asp Gly Asp Lys Gly Asn 85 90 95Gly Gly Ile Tyr Lys Ile Lys Ser Ile Ser Asp Tyr Pro Gly Asp Glu 100 105 110Asp Ile Ser Val Met Asn Ile Glu Glu Gln Ala Val Glu Arg Gly Pro 115 120 125Lys Gly Phe Asn Phe Asn Glu Asn Val Gln Ala Leu Asn Phe Ala Lys 130 135 140Asp Ala Lys Val Asp Asp Lys Ile Lys Val Ile Gly Tyr Pro Leu Pro145 150 155 160Ala Gln Asn Ser Phe Lys Gln Phe Glu Ser Thr Gly Thr Ile Lys Arg 165 170

175Ile Lys Asp Asn Ile Leu Asn Phe Asp Ala Tyr Ile Glu Pro Gly Asn 180 185 190Ser Gly Ser Pro Val Leu Asn Ser Asn Asn Glu Val Ile Gly Val Val 195 200 205Tyr Gly Gly Ile Gly Lys Ile Gly Ser Glu Tyr Asn Gly Ala Val Tyr 210 215 220Phe Thr Pro Gln Ile Lys Asp Phe Ile Gln Lys His Ile Glu Gln225 230 235153239PRTStaphylococcus aureus 153Met Asn Lys Asn Ile Val Ile Lys Ser Met Ala Ala Leu Ala Ile Leu1 5 10 15Thr Ser Ala Thr Gly Ile Asn Ala Ala Val Val Glu Glu Thr Gln Gln 20 25 30Ile Ala Asn Ala Glu Lys Asn Val Thr Gln Val Lys Asp Thr Asn Ile 35 40 45Phe Pro Tyr Asn Gly Val Val Ser Phe Lys Asp Ala Thr Gly Phe Val 50 55 60Ile Gly Lys Asn Thr Ile Ile Thr Asn Lys His Val Ser Lys Asp Tyr65 70 75 80Lys Val Gly Asp Arg Ile Thr Ala His Pro Asp Gly Asp Lys Gly Asn 85 90 95Gly Gly Ile Tyr Lys Ile Lys Ser Ile Ser Asp Tyr Pro Gly Asp Glu 100 105 110Asp Ile Ser Val Met Asn Ile Glu Glu Gln Ala Val Glu Arg Gly Pro 115 120 125Lys Gly Phe Asn Phe Asn Glu Asn Val Gln Ala Leu Asn Phe Ala Lys 130 135 140Asp Ala Lys Val Asp Asp Lys Ile Lys Val Ile Gly Tyr Pro Leu Pro145 150 155 160Ala Gln Asn Ser Phe Lys Gln Phe Glu Ser Thr Gly Thr Ile Lys Arg 165 170 175Ile Lys Asp Asn Ile Leu Asn Phe Asp Ala Tyr Ile Glu Pro Gly Asn 180 185 190Ser Gly Ser Pro Val Leu Asn Ser Asn Asn Glu Val Ile Gly Val Val 195 200 205Tyr Gly Gly Ile Gly Lys Ile Gly Ser Glu Tyr Asn Gly Ala Val Tyr 210 215 220Phe Thr Pro Gln Ile Lys Asp Phe Ile Gln Lys His Ile Glu Gln225 230 235154239PRTStaphylococcus aureus 154Met Asn Lys Asn Ile Val Ile Lys Ser Met Ala Ala Leu Ala Ile Leu1 5 10 15Thr Ser Val Thr Gly Ile Asn Ala Ala Val Val Glu Glu Thr Gln Gln 20 25 30Ile Ala Asn Ala Glu Lys Asn Val Thr Gln Val Lys Asp Thr Asn Ile 35 40 45Phe Pro Tyr Asn Gly Val Val Ser Phe Lys Asp Ala Thr Gly Phe Val 50 55 60Ile Gly Lys Asn Thr Ile Ile Thr Asn Lys His Val Ser Lys Asp Tyr65 70 75 80Lys Val Gly Asp Arg Ile Thr Ala His Pro Asn Gly Asp Lys Gly Asn 85 90 95Gly Gly Ile Tyr Lys Ile Lys Ser Ile Ser Asp Tyr Pro Gly Asp Glu 100 105 110Asp Ile Ser Val Met Asn Ile Glu Glu Gln Ala Val Glu Arg Gly Pro 115 120 125Lys Gly Phe Asn Phe Asn Glu Asn Val Gln Ala Phe Asn Phe Ala Lys 130 135 140Asp Ala Lys Val Asp Asp Lys Ile Lys Val Ile Gly Tyr Pro Leu Pro145 150 155 160Ala Gln Asn Ser Phe Lys Gln Phe Glu Ser Thr Gly Thr Ile Lys Arg 165 170 175Ile Lys Asp Asn Ile Leu Asn Phe Asp Ala Tyr Ile Glu Pro Gly Asn 180 185 190Ser Gly Ser Pro Val Leu Asn Ser Asn Asn Glu Val Ile Gly Val Val 195 200 205Tyr Gly Gly Ile Gly Lys Ile Gly Ser Glu Tyr Asn Gly Ala Val Tyr 210 215 220Phe Thr Pro Gln Ile Lys Asp Phe Ile Gln Lys His Ile Glu Gln225 230 235155239PRTStaphylococcus aureus 155Met Asn Lys Asn Ile Val Ile Lys Ser Met Ala Ala Leu Ala Ile Leu1 5 10 15Thr Ser Val Thr Gly Ile Asn Ala Ala Val Val Glu Glu Thr Gln Gln 20 25 30Ile Ala Asn Ala Glu Lys Asn Val Thr Gln Val Lys Asp Thr Asn Ile 35 40 45Phe Pro Tyr Asn Gly Val Val Ser Phe Lys Asp Ala Thr Gly Phe Val 50 55 60Ile Gly Lys Asn Thr Ile Ile Thr Asn Lys His Val Ser Lys Asp Tyr65 70 75 80Lys Val Gly Asp Arg Ile Thr Ala His Pro Asn Gly Asp Lys Gly Asn 85 90 95Gly Gly Ile Tyr Lys Ile Lys Ser Ile Ser Asp Tyr Pro Gly Asp Glu 100 105 110Asp Ile Ser Val Met Asn Ile Glu Glu Gln Ala Val Glu Arg Gly Pro 115 120 125Lys Gly Phe Asn Phe Asn Glu Asn Val Gln Ala Phe Asn Phe Ala Lys 130 135 140Asp Ala Lys Val Asp Asp Lys Ile Lys Val Ile Gly Tyr Pro Leu Pro145 150 155 160Ala Gln Asn Ser Phe Lys Gln Phe Glu Ser Thr Gly Thr Ile Lys Arg 165 170 175Ile Lys Asp Asn Ile Leu Asn Phe Asp Ala Tyr Ile Glu Pro Gly Asn 180 185 190Ser Gly Ser Pro Val Leu Asn Ser Asn Asn Glu Val Ile Gly Val Val 195 200 205Tyr Gly Gly Ile Gly Lys Ile Gly Ser Glu Tyr Asn Gly Ala Val Tyr 210 215 220Phe Thr Pro Gln Ile Lys Asp Phe Ile Gln Lys His Ile Glu Gln225 230 235156239PRTStaphylococcus aureus 156Met Asn Lys Asn Ile Val Ile Lys Ser Met Ala Ala Leu Ala Ile Leu1 5 10 15Thr Ser Val Thr Gly Ile Asn Ala Ala Val Val Glu Glu Thr Gln Gln 20 25 30Ile Ala Asn Ala Glu Lys Asn Val Thr Gln Val Lys Asp Thr Asn Ile 35 40 45Phe Pro Tyr Asn Gly Val Val Ser Phe Lys Asp Ala Thr Gly Phe Val 50 55 60Ile Gly Lys Asn Thr Ile Ile Thr Asn Lys His Val Ser Lys Asp Tyr65 70 75 80Lys Val Gly Asp Arg Ile Thr Ala His Pro Asn Gly Asp Lys Gly Asn 85 90 95Gly Gly Ile Tyr Lys Ile Lys Ser Ile Ser Asp Tyr Pro Gly Asp Glu 100 105 110Asp Ile Ser Val Met Asn Ile Glu Glu Gln Ala Val Glu Arg Gly Pro 115 120 125Lys Gly Phe Asn Phe Asn Glu Asn Val Gln Ala Phe Asn Phe Ala Lys 130 135 140Asp Ala Lys Val Asp Asp Lys Ile Lys Val Ile Gly Tyr Pro Leu Pro145 150 155 160Ala Gln Asn Ser Phe Lys Gln Phe Glu Ser Thr Gly Thr Ile Lys Arg 165 170 175Ile Lys Asp Asn Ile Leu Asn Phe Asp Ala Tyr Ile Glu Pro Gly Asn 180 185 190Ser Gly Ser Pro Val Leu Asn Ser Asn Asn Glu Val Ile Gly Val Val 195 200 205Tyr Gly Gly Ile Gly Lys Ile Gly Ser Glu Tyr Asn Gly Ala Val Tyr 210 215 220Phe Thr Pro Gln Ile Lys Asp Phe Ile Gln Lys His Ile Glu Gln225 230 235157239PRTStaphylococcus aureus 157Met Asn Lys Asn Ile Val Ile Lys Ser Met Ala Ala Leu Ala Ile Leu1 5 10 15Thr Ser Val Thr Gly Ile Asn Ala Ala Val Val Glu Glu Thr Gln Gln 20 25 30Ile Ala Asn Ala Glu Lys Asn Val Thr Gln Val Lys Asp Thr Asn Ile 35 40 45Phe Pro Tyr Asn Gly Val Val Ser Phe Lys Asp Ala Thr Gly Phe Val 50 55 60Ile Gly Lys Asn Thr Ile Ile Thr Asn Lys His Val Ser Lys Asp Tyr65 70 75 80Lys Val Gly Asp Arg Ile Thr Ala His Pro Asn Gly Asp Lys Gly Asn 85 90 95Gly Gly Ile Tyr Lys Ile Lys Ser Ile Ser Asp Tyr Pro Gly Asp Glu 100 105 110Asp Ile Ser Val Met Asn Ile Glu Glu Gln Ala Val Glu Arg Gly Pro 115 120 125Lys Gly Phe Asn Phe Asn Glu Asn Val Gln Ala Phe Asn Phe Ala Lys 130 135 140Asp Ala Lys Val Asp Asp Lys Ile Lys Val Ile Gly Tyr Pro Leu Pro145 150 155 160Ala Gln Asn Ser Phe Lys Gln Phe Glu Ser Thr Gly Thr Ile Lys Arg 165 170 175Ile Lys Asp Asn Ile Leu Asn Phe Asp Ala Tyr Ile Glu Pro Gly Asn 180 185 190Ser Gly Ser Pro Val Leu Asn Ser Asn Asn Glu Val Ile Gly Val Val 195 200 205Tyr Gly Gly Ile Gly Lys Ile Gly Ser Glu Tyr Asn Gly Ala Val Tyr 210 215 220Phe Thr Pro Gln Ile Lys Asp Phe Ile Gln Lys His Ile Glu Gln225 230 235158239PRTStaphylococcus aureus 158Met Asn Lys Asn Ile Val Ile Lys Ser Met Ala Ala Leu Ala Ile Leu1 5 10 15Thr Ser Val Thr Gly Ile Asn Ala Ala Val Val Glu Glu Thr Gln Gln 20 25 30Ile Ala Asn Ala Glu Lys Asn Val Thr Gln Val Lys Asp Thr Asn Asn 35 40 45Phe Pro Tyr Asn Gly Val Val Ser Phe Lys Asp Ala Thr Gly Phe Val 50 55 60Ile Gly Lys Asn Thr Ile Ile Thr Asn Lys His Val Ser Lys Asp Tyr65 70 75 80Lys Val Gly Asp Arg Ile Thr Ala His Pro Asn Gly Asp Lys Gly Asn 85 90 95Gly Gly Ile Tyr Lys Ile Lys Ser Ile Ser Asp Tyr Pro Gly Asp Glu 100 105 110Asp Ile Ser Val Met Asn Ile Glu Glu Gln Ala Val Glu Arg Gly Pro 115 120 125Lys Gly Phe Asn Phe Asn Glu Asn Val Gln Ala Phe Asn Phe Ala Lys 130 135 140Asp Ala Lys Val Asp Asp Lys Ile Lys Val Ile Gly Tyr Pro Leu Pro145 150 155 160Ala Gln Asn Ser Phe Lys Gln Phe Glu Ser Thr Gly Thr Ile Lys Arg 165 170 175Ile Lys Asp Asn Ile Leu Asn Phe Asp Ala Tyr Ile Glu Pro Gly Asn 180 185 190Ser Gly Ser Pro Val Leu Asn Ser Asn Asn Glu Val Ile Gly Val Val 195 200 205Tyr Gly Gly Ile Gly Lys Ile Gly Ser Glu Tyr Asn Gly Ala Val Tyr 210 215 220Phe Thr Pro Gln Ile Lys Asp Phe Ile Gln Lys His Ile Glu Gln225 230 235159239PRTStaphylococcus aureus 159Met Asn Lys Asn Ile Val Ile Lys Ser Met Ala Ala Leu Ala Ile Leu1 5 10 15Thr Ser Val Thr Gly Ile Asn Ala Ala Val Val Glu Glu Thr Gln Gln 20 25 30Ile Ala Asn Ala Glu Lys Asn Val Thr Gln Val Lys Asp Thr Asn Asn 35 40 45Phe Pro Tyr Asn Gly Val Val Ser Phe Lys Asp Ala Thr Gly Phe Val 50 55 60Ile Gly Lys Asn Thr Ile Ile Thr Asn Lys His Val Ser Lys Asp Tyr65 70 75 80Lys Val Gly Asp Arg Ile Thr Ala His Pro Asn Gly Asp Lys Gly Asn 85 90 95Gly Gly Ile Tyr Lys Ile Lys Ser Ile Ser Asp Tyr Pro Gly Asp Glu 100 105 110Asp Ile Ser Val Met Asn Ile Glu Glu Gln Ala Val Glu Arg Gly Pro 115 120 125Lys Gly Phe Asn Phe Asn Glu Asn Val Gln Ala Phe Asn Phe Ala Lys 130 135 140Asp Ala Lys Val Asp Asp Lys Ile Lys Val Ile Gly Tyr Pro Leu Pro145 150 155 160Ala Gln Asn Ser Phe Lys Gln Phe Glu Ser Thr Gly Thr Ile Lys Arg 165 170 175Ile Lys Asp Asn Ile Leu Asn Phe Asp Ala Tyr Ile Glu Pro Gly Asn 180 185 190Ser Gly Ser Pro Val Leu Asn Ser Asn Asn Glu Val Ile Gly Val Val 195 200 205Tyr Gly Gly Ile Gly Lys Ile Gly Ser Glu Tyr Asn Gly Ala Val Tyr 210 215 220Phe Thr Pro Gln Ile Lys Asp Phe Ile Gln Lys His Ile Glu Gln225 230 235160239PRTStaphylococcus aureus 160Met Asn Lys Asn Ile Val Ile Lys Ser Met Ala Ala Leu Ala Ile Leu1 5 10 15Thr Ser Val Thr Gly Ile Asn Ala Ala Val Val Glu Glu Thr Gln Gln 20 25 30Ile Ala Asn Ala Glu Lys Asn Val Thr Gln Val Lys Asp Thr Asn Asn 35 40 45Phe Pro Tyr Asn Gly Val Val Ser Phe Lys Asp Ala Thr Gly Phe Val 50 55 60Ile Gly Lys Asn Thr Ile Ile Thr Asn Lys His Val Ser Lys Asp Tyr65 70 75 80Lys Val Gly Asp Arg Ile Thr Ala His Pro Asn Gly Asp Lys Gly Asn 85 90 95Gly Gly Ile Tyr Lys Ile Lys Ser Ile Ser Asp Tyr Pro Gly Asp Glu 100 105 110Asp Ile Ser Val Met Asn Ile Glu Glu Gln Ala Val Glu Arg Gly Pro 115 120 125Lys Gly Phe Asn Phe Asn Glu Asn Val Gln Ala Phe Asn Phe Ala Lys 130 135 140Asp Ala Lys Val Asp Asp Lys Ile Lys Val Ile Gly Tyr Pro Leu Pro145 150 155 160Ala Gln Asn Ser Phe Lys Gln Phe Glu Ser Thr Gly Thr Ile Lys Arg 165 170 175Ile Lys Asp Asn Ile Leu Asn Phe Asp Ala Tyr Ile Glu Pro Gly Asn 180 185 190Ser Gly Ser Pro Val Leu Asn Ser Asn Asn Glu Val Ile Gly Val Val 195 200 205Tyr Gly Gly Ile Gly Lys Ile Gly Ser Glu Tyr Asn Gly Ala Val Tyr 210 215 220Phe Thr Pro Gln Ile Lys Asp Phe Ile Gln Lys His Ile Glu Gln225 230 235161239PRTStaphylococcus aureus 161Met Asn Lys Asn Ile Val Ile Lys Ser Met Ala Ala Leu Ala Ile Leu1 5 10 15Thr Ser Val Thr Gly Ile Asn Ala Ala Val Val Glu Glu Thr Gln Gln 20 25 30Ile Ala Asn Ala Glu Lys Asn Val Thr Gln Val Lys Asp Thr Asn Asn 35 40 45Phe Pro Tyr Asn Gly Val Val Ser Phe Lys Asp Ala Thr Gly Phe Val 50 55 60Ile Gly Lys Asn Thr Ile Ile Thr Asn Lys His Val Ser Lys Asp Tyr65 70 75 80Lys Val Gly Asp Arg Ile Thr Ala His Pro Asn Gly Asp Lys Gly Asn 85 90 95Gly Gly Ile Tyr Lys Ile Lys Ser Ile Ser Asp Tyr Pro Gly Asp Glu 100 105 110Asp Ile Ser Val Met Asn Ile Glu Glu Gln Ala Val Glu Arg Gly Pro 115 120 125Lys Gly Phe Asn Phe Asn Glu Asn Val Gln Ala Phe Asn Phe Ala Lys 130 135 140Asp Ala Lys Val Asp Asp Lys Ile Lys Val Ile Gly Tyr Pro Leu Pro145 150 155 160Ala Gln Asn Ser Phe Lys Gln Phe Glu Ser Thr Gly Thr Ile Lys Arg 165 170 175Ile Lys Asp Asn Ile Leu Asn Phe Asp Ala Tyr Ile Glu Pro Gly Asn 180 185 190Ser Gly Ser Pro Val Leu Asn Ser Asn Asn Glu Val Ile Gly Val Val 195 200 205Tyr Gly Gly Ile Gly Lys Ile Gly Ser Glu Tyr Asn Gly Ala Val Tyr 210 215 220Phe Thr Pro Gln Ile Lys Asp Phe Ile Gln Lys His Ile Glu Gln225 230 235162239PRTStaphylococcus aureus 162Met Asn Lys Asn Ile Val Ile Lys Ser Met Ala Ala Leu Ala Ile Leu1 5 10 15Thr Ser Val Thr Gly Ile Asn Ala Ala Val Val Glu Glu Thr Gln Gln 20 25 30Ile Ala Asn Ala Glu Lys Asn Val Thr Gln Val Lys Asp Thr Asn Asn 35 40 45Phe Pro Tyr Asn Gly Val Val Ser Phe Lys Asp Ala Thr Gly Phe Val 50 55 60Ile Gly Lys Asn Thr Ile Ile Thr Asn Lys His Val Ser Lys Asp Tyr65 70 75 80Lys Val Gly Asp Arg Ile Thr Ala His Pro Asn Gly Asp Lys Gly Asn 85 90 95Gly Gly Ile Tyr Lys Ile Lys Ser Ile Ser Asp Tyr Pro Gly Asp Glu 100 105 110Asp Ile Ser Val Met Asn Ile Glu Glu Gln Ala Val Glu Arg Gly Pro 115 120 125Lys Gly Phe Asn Phe Asn Glu Asn Val Gln Ala Phe Asn Phe Ala Lys 130 135 140Asp Ala Lys Val Asp Asp Lys Ile Lys Val Ile Gly Tyr Pro Leu Pro145 150 155 160Ala Gln Asn Ser Phe Lys Gln Phe Glu Ser Thr Gly Thr Ile Lys Arg 165 170 175Ile Lys Asp Asn Ile Leu Asn Phe Asp Ala Tyr Ile Glu Pro Gly Asn 180 185 190Ser Gly Ser Pro Val Leu Asn Ser Asn Asn Glu Val Ile Gly Val Val 195 200 205Tyr Gly Gly Ile Gly Lys Ile Gly Ser Glu Tyr Asn Gly Ala Val Tyr 210 215 220Phe Thr Pro Gln Ile Lys Asp Phe Ile Gln Lys His Ile Glu Gln225 230

235163239PRTStaphylococcus aureus 163Met Asn Lys Asn Ile Val Ile Lys Ser Met Ala Ala Leu Ala Ile Leu1 5 10 15Thr Ser Val Thr Gly Ile Asn Ala Ala Val Val Asp Glu Thr Gln Gln 20 25 30Ile Ala Asn Ala Glu Lys Asn Val Thr Gln Val Lys Asp Thr Asn Val 35 40 45Phe Pro Tyr Asn Gly Val Val Ser Phe Lys Asp Ala Thr Gly Phe Ala 50 55 60Ile Glu Lys Asn Thr Ile Ile Thr Asn Lys His Val Ser Lys Asp Tyr65 70 75 80Lys Val Gly Asp Arg Ile Thr Ala His Pro Asn Gly Asp Lys Gly Asn 85 90 95Gly Gly Ile Tyr Lys Ile Lys Asn Ile Ser Asp Tyr Pro Gly Asn Glu 100 105 110Asp Ile Ser Val Met Asn Val Glu Glu Asn Ala Val Glu Arg Gly Ala 115 120 125Asn Gly Tyr Asn Phe Asn Glu Asn Val Gln Ala Phe Asn Phe Ala Lys 130 135 140Asp Ala Lys Val Asp Asp Lys Ile Lys Val Ile Gly Tyr Pro Leu Pro145 150 155 160Ala Gln Asn Thr Phe Lys Gln Phe Glu Ser Thr Gly Thr Val Lys Ser 165 170 175Ile Lys Asp Asn Asn Leu Asn Phe Asp Ala Tyr Ile Glu Pro Gly Asn 180 185 190Ser Gly Ser Pro Val Leu Asn Leu Asn Asn Glu Val Val Gly Val Val 195 200 205Tyr Gly Gly Ile Gly Lys Ile Gly Ser Glu Tyr Asn Gly Ala Val Tyr 210 215 220Phe Thr Pro Gln Ile Lys Glu Phe Ile Gln Lys His Ile Glu Gln225 230 235164239PRTArtificial sequencesynthetic sequencemisc_feature(19)..(19)Xaa can be any naturally occurring amino acidmisc_feature(28)..(28)Xaa can be any naturally occurring amino acidmisc_feature(48)..(48)Xaa can be any naturally occurring amino acidmisc_feature(64)..(64)Xaa can be any naturally occurring amino acidmisc_feature(66)..(66)Xaa can be any naturally occurring amino acidmisc_feature(91)..(91)Xaa can be any naturally occurring amino acidmisc_feature(104)..(104)Xaa can be any naturally occurring amino acidmisc_feature(111)..(111)Xaa can be any naturally occurring amino acidmisc_feature(119)..(119)Xaa can be any naturally occurring amino acidmisc_feature(122)..(122)Xaa can be any naturally occurring amino acidmisc_feature(128)..(129)Xaa can be any naturally occurring amino acidmisc_feature(131)..(131)Xaa can be any naturally occurring amino acidmisc_feature(140)..(140)Xaa can be any naturally occurring amino acidmisc_feature(164)..(164)Xaa can be any naturally occurring amino acidmisc_feature(174)..(174)Xaa can be any naturally occurring amino acidmisc_feature(176)..(176)Xaa can be any naturally occurring amino acidmisc_feature(181)..(181)Xaa can be any naturally occurring amino acidmisc_feature(200)..(200)Xaa can be any naturally occurring amino acidmisc_feature(205)..(205)Xaa can be any naturally occurring amino acidmisc_feature(231)..(231)Xaa can be any naturally occurring amino acid 164Met Asn Lys Asn Ile Val Ile Lys Ser Met Ala Ala Leu Ala Ile Leu1 5 10 15Thr Ser Xaa Thr Gly Ile Asn Ala Ala Val Val Xaa Glu Thr Gln Gln 20 25 30Ile Ala Asn Ala Glu Lys Asn Val Thr Gln Val Lys Asp Thr Asn Xaa 35 40 45Phe Pro Tyr Asn Gly Val Val Ser Phe Lys Asp Ala Thr Gly Phe Xaa 50 55 60Ile Xaa Lys Asn Thr Ile Ile Thr Asn Lys His Val Ser Lys Asp Tyr65 70 75 80Lys Val Gly Asp Arg Ile Thr Ala His Pro Xaa Gly Asp Lys Gly Asn 85 90 95Gly Gly Ile Tyr Lys Ile Lys Xaa Ile Ser Asp Tyr Pro Gly Xaa Glu 100 105 110Asp Ile Ser Val Met Asn Xaa Glu Glu Xaa Ala Val Glu Arg Gly Xaa 115 120 125Xaa Gly Xaa Asn Phe Asn Glu Asn Val Gln Ala Xaa Asn Phe Ala Lys 130 135 140Asp Ala Lys Val Asp Asp Lys Ile Lys Val Ile Gly Tyr Pro Leu Pro145 150 155 160Ala Gln Asn Xaa Phe Lys Gln Phe Glu Ser Thr Gly Thr Xaa Lys Xaa 165 170 175Ile Lys Asp Asn Xaa Leu Asn Phe Asp Ala Tyr Ile Glu Pro Gly Asn 180 185 190Ser Gly Ser Pro Val Leu Asn Xaa Asn Asn Glu Val Xaa Gly Val Val 195 200 205Tyr Gly Gly Ile Gly Lys Ile Gly Ser Glu Tyr Asn Gly Ala Val Tyr 210 215 220Phe Thr Pro Gln Ile Lys Xaa Phe Ile Gln Lys His Ile Glu Gln225 230 23516514PRTStaphylococcus aureus 165Pro Tyr Asn Gly Val Val Ser Phe Lys Asp Ala Thr Gly Phe1 5 1016614PRTStaphylococcus aureus 166Ala His Pro Asp Gly Asp Lys Gly Asn Gly Gly Ile Tyr Lys1 5 1016714PRTStaphylococcus aureus 167Ala His Pro Asn Gly Asp Lys Gly Asn Gly Gly Ile Tyr Lys1 5 1016814PRTartificial sequencesynthetic sequencemisc_feature(4)..(4)Xaa can be any naturally occurring amino acid 168Ala His Pro Xaa Gly Asp Lys Gly Asn Gly Gly Ile Tyr Lys1 5 1016914PRTStaphylococcus aureus 169Ser Ile Ser Asp Tyr Pro Gly Asp Glu Asp Ile Ser Val Met1 5 1017014PRTStaphylococcus aureus 170Asn Ile Ser Asp Tyr Pro Gly Asn Glu Asp Ile Ser Val Met1 5 1017114PRTartificial sequencesynthetic sequencemisc_feature(1)..(1)Xaa can be any naturally occurring amino acidmisc_feature(8)..(8)Xaa can be any naturally occurring amino acid 171Xaa Ile Ser Asp Tyr Pro Gly Xaa Glu Asp Ile Ser Val Met1 5 1017214PRTStaphylococcus aureus 172Arg Gly Pro Lys Gly Phe Asn Phe Asn Glu Asn Val Gln Ala1 5 1017314PRTStaphylococcus aureus 173Arg Gly Ala Asn Gly Tyr Asn Phe Asn Glu Asn Val Gln Ala1 5 1017414PRTartificial sequencesynthetic sequencemisc_feature(3)..(4)Xaa can be any naturally occurring amino acidmisc_feature(6)..(6)Xaa can be any naturally occurring amino acid 174Arg Gly Xaa Xaa Gly Xaa Asn Phe Asn Glu Asn Val Gln Ala1 5 1017514PRTStaphylococcus aureus 175Gln Phe Glu Ser Thr Gly Thr Ile Lys Arg Ile Lys Asp Asn1 5 1017614PRTStaphylococcus aureus 176Gln Phe Glu Ser Thr Gly Thr Val Lys Ser Ile Lys Asp Asn1 5 1017714PRTartificial sequencesynthetic sequencemisc_feature(8)..(8)Xaa can be any naturally occurring amino acidmisc_feature(10)..(10)Xaa can be any naturally occurring amino acid 177Gln Phe Glu Ser Thr Gly Thr Xaa Lys Xaa Ile Lys Asp Asn1 5 1017814PRTStaphylococcus aureus 178Gly Asn Ser Gly Ser Pro Val Leu Asn Ser Asn Asn Glu Val1 5 1017914PRTStaphylococcus aureus 179Gly Asn Ser Gly Ser Pro Val Leu Asn Leu Asn Asn Glu Val1 5 1018014PRTartificial sequencesynthetic sequencemisc_feature(10)..(10)Xaa can be any naturally occurring amino acid 180Gly Asn Ser Gly Ser Pro Val Leu Asn Xaa Asn Asn Glu Val1 5 10181975DNAStaphylococcus aureus 181atgaaatttg atagacatag aagattgaga tcatcagcga caatgagaga tatggttaga 60gagaatcatg taagaaaaga agatttaata tatccaattt ttgtagttga aaaagacgat 120gtgaaaaaag aaattaagtc attgccaggt gtataccaaa tcagtttgaa tttacttgaa 180agtgaattaa aagaagctta tgacttaggc atacgtgcca ttatgttttt cggtgttcca 240aactcaaaag atgatatagg tactggtgca tacattcacg atggtgttat tcaacaggca 300acacgtattg ctaaaaaaat gtatgatgac ttattaattg ttgcagacac ttgtttatgt 360gaatatactg atcatggtca ttgtggcgtg attgatgacc atacacatga cgttgacaat 420gataaatcat tgccactact tgttaaaaca gcaatttctc aagtggaagc tggtgctgat 480attattgcgc caagtaatat gatggatggt tttgttgctg aaattcgtcg tggattagat 540gaagccggct attacaatat tcctataatg agttatggtg tcaagtatgc atcaagtttc 600tttggacctt ttagagatgc agcagattca gcgccatcat ttggggatag aaaaacgtat 660cagatggacc ctgctaaccg tttggaagca cttcgtgaat tagaaagtga tcttaaagaa 720gggtgcgaca tgatgattgt taaacctgct ctaagttatt tagatatagt tcgagatgtt 780aaaaatcata cgaatgttcc agttgttgca tataatgtga gtggagaata tagtatgact 840aaagcagcgg cacaaaatgg ttggatagat gaagaacgtg tcgttatgga acaaatggtt 900tcaatgaaac gtgcaggtgc tgatatgatt attacgtatt ttgcaaagga catttgtcgc 960tatttagata aataa 975182324PRTStaphylococcus aureus 182Met Lys Phe Asp Arg His Arg Arg Leu Arg Ser Ser Ala Thr Met Arg1 5 10 15Asp Met Val Arg Glu Asn His Val Arg Lys Glu Asp Leu Ile Tyr Pro 20 25 30Ile Phe Val Val Glu Lys Asp Asp Val Lys Lys Glu Ile Lys Ser Leu 35 40 45Pro Gly Val Tyr Gln Ile Ser Leu Asn Leu Leu Glu Ser Glu Leu Lys 50 55 60Glu Ala Tyr Asp Leu Gly Ile Arg Ala Ile Met Phe Phe Gly Val Pro65 70 75 80Asn Ser Lys Asp Asp Ile Gly Thr Gly Ala Tyr Ile His Asp Gly Val 85 90 95Ile Gln Gln Ala Thr Arg Ile Ala Lys Lys Met Tyr Asp Asp Leu Leu 100 105 110Ile Val Ala Asp Thr Cys Leu Cys Glu Tyr Thr Asp His Gly His Cys 115 120 125Gly Val Ile Asp Asp His Thr His Asp Val Asp Asn Asp Lys Ser Leu 130 135 140Pro Leu Leu Val Lys Thr Ala Ile Ser Gln Val Glu Ala Gly Ala Asp145 150 155 160Ile Ile Ala Pro Ser Asn Met Met Asp Gly Phe Val Ala Glu Ile Arg 165 170 175Arg Gly Leu Asp Glu Ala Gly Tyr Tyr Asn Ile Pro Ile Met Ser Tyr 180 185 190Gly Val Lys Tyr Ala Ser Ser Phe Phe Gly Pro Phe Arg Asp Ala Ala 195 200 205Asp Ser Ala Pro Ser Phe Gly Asp Arg Lys Thr Tyr Gln Met Asp Pro 210 215 220Ala Asn Arg Leu Glu Ala Leu Arg Glu Leu Glu Ser Asp Leu Lys Glu225 230 235 240Gly Cys Asp Met Met Ile Val Lys Pro Ala Leu Ser Tyr Leu Asp Ile 245 250 255Val Arg Asp Val Lys Asn His Thr Asn Val Pro Val Val Ala Tyr Asn 260 265 270Val Ser Gly Glu Tyr Ser Met Thr Lys Ala Ala Ala Gln Asn Gly Trp 275 280 285Ile Asp Glu Glu Arg Val Val Met Glu Gln Met Val Ser Met Lys Arg 290 295 300Ala Gly Ala Asp Met Ile Ile Thr Tyr Phe Ala Lys Asp Ile Cys Arg305 310 315 320Tyr Leu Asp Lys1832802DNAStaphylococcus aureus 183atgaatatga agaaaaaaga aaaacacgca attcggaaaa aatcgattgg cgtggcttca 60gtgcttgtag gtacgttaat cggttttgga ctactcagca gtaaagaagc agatgcaagt 120gaaaatagtg ttacgcaatc tgatagcgca agtaacgaaa gcaaaagtaa tgattcaagt 180agcgttagtg ctgcacctaa aacagacgac acaaacgtga gtgatactaa aacatcgtca 240aacactaata atggcgaaac gagtgtggcg caaaatccag cacaacagga aacgacacaa 300tcatcatcaa caaatgcaac tacggaagaa acgccggtaa ctggtgaagc tactactacg 360acaacgaatc aagctaatac accggcaaca actcaatcaa gcaatacaaa tgcggaggaa 420ttagtgaatc aaacaagtaa tgaaacgact tttaatgata ctaatacagt atcatctgta 480aattcacctc aaaattctac aaatgcggaa aatgtttcaa caacgcaaga tacttcaact 540gaagcaacac cttcaaacaa tgaatcagct ccacagagta cagatgcaag taataaagat 600gtagttaatc aagcggttaa tacaagtgcg cctagaatga gagcatttag tttagcggca 660gtagctgcag atgcaccggc agctggcaca gatattacga atcagttgac gaatgtgaca 720gttggtattg actctggtac gactgtgtat ccgcaccaag caggttatgt caaactgaat 780tatggttttt cagtgcctaa ttctgctgtt aaaggtgaca cattcaaaat aactgtacct 840aaagaattaa acttaaatgg tgtaacttca actgctaaag tgccaccaat tatggctgga 900gatcaagtat tggcaaatgg tgtaatcgat agtgatggta atgttattta tacatttaca 960gactatgtaa atactaaaga tgatgtaaaa gcaactttga ccatgcccgc ttatattgac 1020cctgaaaatg ttaaaaagac aggtaatgtg acattggcta ctggcatagg tagtacaaca 1080gcaaacaaaa cagtattagt agattatgaa aaatatggta agttttataa cttatctatt 1140aaaggtacaa ttgaccaaat cgataaaaca aataatacgt atcgtcagac aatttatgtc 1200aatccaagtg gagataacgt tattgcgccg gttttaacag gtaatttaaa accaaatacg 1260gatagtaatg cattaataga tcagcaaaat acaagtatta aagtatataa agtagataat 1320gcagctgatt tatctgaaag ttactttgtg aatccagaaa actttgagga tgtcactaat 1380agtgtgaata ttacattccc aaatccaaat caatataaag tagagtttaa tacgcctgat 1440gatcaaatta caacaccgta tatagtagtt gttaatggtc atattgatcc gaatagcaaa 1500ggtgatttag ctttacgttc aactttatat gggtataact cgaatataat ttggcgctct 1560atgtcatggg acaacgaagt agcatttaat aacggatcag gttctggtga cggtatcgat 1620aaaccagttg ttcctgaaca acctgatgag cctggtgaaa ttgaaccaat tccagaggat 1680tcagattctg acccaggttc agattctggc agcgattcta attcagatag cggttcagat 1740tcgggtagtg attctacatc agatagtggt tcagattcag cgagtgattc agattcagca 1800agtgattcag actcagcgag tgattcagat tcagcaagcg attccgactc agcgagcgat 1860tccgactcag acaatgactc ggattcagat agcgattctg actcagacag tgactcagat 1920tccgacagtg actcagattc agatagcgat tctgactcag acagtgactc agattcagat 1980agcgattcag attcagatag cgattcagat tccgacagtg attccgactc agacagcgat 2040tctgactccg acagtgattc cgactcagac agcgattcag attccgacag tgattccgac 2100tcagatagcg attccgactc agatagcgac tcagattcag acagcgattc agattcagac 2160agcgattcag attcagatag cgattcagat tccgacagtg actcagattc cgacagtgac 2220tcggattcag atagcgattc agattccgac agtgactcag attccgacag tgactcagac 2280tcagacagtg attcggattc agcgagtgat tcggattcag atagtgattc cgactccgac 2340agtgactcgg attcagatag cgactcagac tcggatagcg actcggattc agatagcgat 2400tcggactcag atagcgattc agaatcagac agcgattcag aatcagacag cgattcagat 2460tcagacagcg actcagacag tgactcagat tcagatagtg actcggattc agcgagtgat 2520tcagactcag gtagtgactc cgattcatca agtgattccg actcagaaag tgattcaaat 2580agcgattccg agtcaggttc taacaataat gtagttccgc ctaattcacc taaaaatggt 2640actaatgctt ctaataaaaa tgaggctaaa gatagtaaag aaccattacc agatacaggt 2700tctgaagatg aagcaaatac gtcactaatt tggggattat tagcatcaat aggttcatta 2760ctacttttca gaagaaaaaa agaaaataaa gataagaaat aa 2802184933PRTStaphylococcus aureus 184Met Asn Met Lys Lys Lys Glu Lys His Ala Ile Arg Lys Lys Ser Ile1 5 10 15Gly Val Ala Ser Val Leu Val Gly Thr Leu Ile Gly Phe Gly Leu Leu 20 25 30Ser Ser Lys Glu Ala Asp Ala Ser Glu Asn Ser Val Thr Gln Ser Asp 35 40 45Ser Ala Ser Asn Glu Ser Lys Ser Asn Asp Ser Ser Ser Val Ser Ala 50 55 60Ala Pro Lys Thr Asp Asp Thr Asn Val Ser Asp Thr Lys Thr Ser Ser65 70 75 80Asn Thr Asn Asn Gly Glu Thr Ser Val Ala Gln Asn Pro Ala Gln Gln 85 90 95Glu Thr Thr Gln Ser Ser Ser Thr Asn Ala Thr Thr Glu Glu Thr Pro 100 105 110Val Thr Gly Glu Ala Thr Thr Thr Thr Thr Asn Gln Ala Asn Thr Pro 115 120 125Ala Thr Thr Gln Ser Ser Asn Thr Asn Ala Glu Glu Leu Val Asn Gln 130 135 140Thr Ser Asn Glu Thr Thr Phe Asn Asp Thr Asn Thr Val Ser Ser Val145 150 155 160Asn Ser Pro Gln Asn Ser Thr Asn Ala Glu Asn Val Ser Thr Thr Gln 165 170 175Asp Thr Ser Thr Glu Ala Thr Pro Ser Asn Asn Glu Ser Ala Pro Gln 180 185 190Ser Thr Asp Ala Ser Asn Lys Asp Val Val Asn Gln Ala Val Asn Thr 195 200 205Ser Ala Pro Arg Met Arg Ala Phe Ser Leu Ala Ala Val Ala Ala Asp 210 215 220Ala Pro Ala Ala Gly Thr Asp Ile Thr Asn Gln Leu Thr Asn Val Thr225 230 235 240Val Gly Ile Asp Ser Gly Thr Thr Val Tyr Pro His Gln Ala Gly Tyr 245 250 255Val Lys Leu Asn Tyr Gly Phe Ser Val Pro Asn Ser Ala Val Lys Gly 260 265 270Asp Thr Phe Lys Ile Thr Val Pro Lys Glu Leu Asn Leu Asn Gly Val 275 280 285Thr Ser Thr Ala Lys Val Pro Pro Ile Met Ala Gly Asp Gln Val Leu 290 295 300Ala Asn Gly Val Ile Asp Ser Asp Gly Asn Val Ile Tyr Thr Phe Thr305 310 315 320Asp Tyr Val Asn Thr Lys Asp Asp Val Lys Ala Thr Leu Thr Met Pro 325 330 335Ala Tyr Ile Asp Pro Glu Asn Val Lys Lys Thr Gly Asn Val Thr Leu 340 345 350Ala Thr Gly Ile Gly Ser Thr Thr Ala Asn Lys Thr Val Leu Val Asp 355 360 365Tyr Glu Lys Tyr Gly Lys Phe Tyr Asn Leu Ser Ile Lys Gly Thr Ile 370 375 380Asp Gln Ile Asp Lys Thr Asn Asn Thr Tyr Arg Gln Thr Ile Tyr Val385 390 395 400Asn Pro Ser Gly Asp Asn Val Ile Ala Pro Val Leu Thr Gly Asn Leu 405 410 415Lys Pro Asn Thr Asp Ser Asn Ala Leu Ile Asp Gln Gln Asn Thr Ser 420

425 430Ile Lys Val Tyr Lys Val Asp Asn Ala Ala Asp Leu Ser Glu Ser Tyr 435 440 445Phe Val Asn Pro Glu Asn Phe Glu Asp Val Thr Asn Ser Val Asn Ile 450 455 460Thr Phe Pro Asn Pro Asn Gln Tyr Lys Val Glu Phe Asn Thr Pro Asp465 470 475 480Asp Gln Ile Thr Thr Pro Tyr Ile Val Val Val Asn Gly His Ile Asp 485 490 495Pro Asn Ser Lys Gly Asp Leu Ala Leu Arg Ser Thr Leu Tyr Gly Tyr 500 505 510Asn Ser Asn Ile Ile Trp Arg Ser Met Ser Trp Asp Asn Glu Val Ala 515 520 525Phe Asn Asn Gly Ser Gly Ser Gly Asp Gly Ile Asp Lys Pro Val Val 530 535 540Pro Glu Gln Pro Asp Glu Pro Gly Glu Ile Glu Pro Ile Pro Glu Asp545 550 555 560Ser Asp Ser Asp Pro Gly Ser Asp Ser Gly Ser Asp Ser Asn Ser Asp 565 570 575Ser Gly Ser Asp Ser Gly Ser Asp Ser Thr Ser Asp Ser Gly Ser Asp 580 585 590Ser Ala Ser Asp Ser Asp Ser Ala Ser Asp Ser Asp Ser Ala Ser Asp 595 600 605Ser Asp Ser Ala Ser Asp Ser Asp Ser Ala Ser Asp Ser Asp Ser Asp 610 615 620Asn Asp Ser Asp Ser Asp Ser Asp Ser Asp Ser Asp Ser Asp Ser Asp625 630 635 640Ser Asp Ser Asp Ser Asp Ser Asp Ser Asp Ser Asp Ser Asp Ser Asp 645 650 655Ser Asp Ser Asp Ser Asp Ser Asp Ser Asp Ser Asp Ser Asp Ser Asp 660 665 670Ser Asp Ser Asp Ser Asp Ser Asp Ser Asp Ser Asp Ser Asp Ser Asp 675 680 685Ser Asp Ser Asp Ser Asp Ser Asp Ser Asp Ser Asp Ser Asp Ser Asp 690 695 700Ser Asp Ser Asp Ser Asp Ser Asp Ser Asp Ser Asp Ser Asp Ser Asp705 710 715 720Ser Asp Ser Asp Ser Asp Ser Asp Ser Asp Ser Asp Ser Asp Ser Asp 725 730 735Ser Asp Ser Asp Ser Asp Ser Asp Ser Asp Ser Asp Ser Asp Ser Asp 740 745 750Ser Asp Ser Asp Ser Asp Ser Asp Ser Asp Ser Asp Ser Asp Ser Ala 755 760 765Ser Asp Ser Asp Ser Asp Ser Asp Ser Asp Ser Asp Ser Asp Ser Asp 770 775 780Ser Asp Ser Asp Ser Asp Ser Asp Ser Asp Ser Asp Ser Asp Ser Asp785 790 795 800Ser Asp Ser Asp Ser Asp Ser Glu Ser Asp Ser Asp Ser Glu Ser Asp 805 810 815Ser Asp Ser Asp Ser Asp Ser Asp Ser Asp Ser Asp Ser Asp Ser Asp 820 825 830Ser Asp Ser Asp Ser Ala Ser Asp Ser Asp Ser Gly Ser Asp Ser Asp 835 840 845Ser Ser Ser Asp Ser Asp Ser Glu Ser Asp Ser Asn Ser Asp Ser Glu 850 855 860Ser Gly Ser Asn Asn Asn Val Val Pro Pro Asn Ser Pro Lys Asn Gly865 870 875 880Thr Asn Ala Ser Asn Lys Asn Glu Ala Lys Asp Ser Lys Glu Pro Leu 885 890 895Pro Asp Thr Gly Ser Glu Asp Glu Ala Asn Thr Ser Leu Ile Trp Gly 900 905 910Leu Leu Ala Ser Ile Gly Ser Leu Leu Leu Phe Arg Arg Lys Lys Glu 915 920 925Asn Lys Asp Lys Lys 930185219PRTStaphylococcus aureus 185Met Ile Glu Ile Asn Asn Leu Ser Lys Arg Tyr Arg Asn Lys Gln Ile1 5 10 15Phe Asn His Leu Thr Met Ser Phe Asp Ser Asn Arg Leu Thr Val Leu 20 25 30Leu Gly Asp Asn Gly Ala Gly Lys Ser Thr Leu Leu Arg Met Ile Ala 35 40 45Gly Ile Glu Lys Ala Asn Asp Gly Thr Ile Asn Tyr Phe Gly Glu Lys 50 55 60Trp Asn Gln Arg Gln Ile Gln Asn His Ile Gly Tyr Val Pro Gln Asp65 70 75 80Ile Ala Leu Phe Glu His Met Thr Val Ala Glu Asn Ile Lys Phe Phe 85 90 95Lys Ser Leu Cys Lys Asn Pro Ile Asn Asp Thr Thr Ile Asn Glu Tyr 100 105 110Leu Gln Gln Leu Asn Phe Asp Asp Thr Ser Ala Lys Val Ser Thr Leu 115 120 125Ser Gly Gly Asn Lys Arg Lys Ile Asn Ile Leu Val Gly Leu Leu Gly 130 135 140Gln Pro Arg Ile Leu Ile Leu Asp Glu Pro Thr Val Gly Ile Asp Leu145 150 155 160Lys Ser Arg His Asp Ile His Gln Leu Leu Asn Ile Met Lys Ser Lys 165 170 175Cys Leu Ile Ile Leu Thr Thr His His Leu Asp Glu Val Glu Ala Leu 180 185 190Ala Asp Asp Ile Lys Leu Ile Gly Gln Asp Pro Phe Tyr Gln His Val 195 200 205Leu Glu Asp Lys Gln Trp Thr Tyr Thr Tyr Tyr 210 215186253PRTStaphylococcus aureus 186Met Ala Ile Leu Glu Val Lys Gln Leu Thr Lys Ile Tyr Gly Thr Lys1 5 10 15Lys Met Ala Gln Glu Val Leu Arg Asp Ile Asn Met Ser Ile Glu Glu 20 25 30Gly Glu Phe Ile Ala Ile Met Gly Pro Ser Gly Ser Gly Lys Thr Thr 35 40 45Leu Leu Asn Val Leu Ser Ser Ile Asp Tyr Ile Ser Gln Gly Ser Ile 50 55 60Thr Leu Lys Gly Lys Lys Leu Glu Lys Leu Ser Asn Lys Glu Leu Ser65 70 75 80Asp Ile Arg Lys His Asp Ile Gly Phe Ile Phe Gln Glu Tyr Asn Leu 85 90 95Leu His Thr Leu Thr Val Lys Glu Asn Ile Met Leu Pro Leu Thr Val 100 105 110Gln Lys Leu Asp Lys Glu His Met Leu Asn Arg Tyr Glu Lys Val Ala 115 120 125Glu Ala Leu Asn Ile Leu Asp Ile Ser Asp Lys Tyr Pro Ser Glu Leu 130 135 140Ser Gly Gly Gln Arg Gln Arg Thr Ser Ala Ala Arg Ala Phe Ile Thr145 150 155 160Leu Pro Ser Ile Ile Phe Ala Asp Glu Pro Thr Gly Ala Leu Asp Ser 165 170 175Lys Ser Thr Gln Asp Leu Leu Lys Arg Leu Thr Arg Met Asn Glu Ala 180 185 190Phe Lys Ser Thr Ile Ile Met Val Thr His Asp Pro Val Ala Ala Ser 195 200 205Tyr Ala Asn Arg Val Val Met Leu Lys Asp Gly Gln Ile Phe Thr Glu 210 215 220Leu Tyr Gln Gly Asp Asp Asp Lys His Thr Phe Phe Lys Glu Ile Ile225 230 235 240Arg Val Gln Ser Val Leu Gly Gly Val Asn Tyr Asp Leu 245 250187243PRTStaphylococcus aureus 187Met Ile Leu Ser Tyr Leu Lys Ile Glu Phe Lys Val Ile Met Arg Lys1 5 10 15Lys Thr Thr Leu Ile Leu Ser Ile Leu Phe Pro Val Ile Phe Tyr Ile 20 25 30Leu Phe Thr Ser Ile Leu Glu Leu Pro Glu Asp Val Lys Pro Lys Phe 35 40 45Tyr Lys Glu Tyr Met Tyr Ser Met Thr Val Tyr Ser Leu Leu Ser Phe 50 55 60Ser Leu Leu Thr Phe Pro Leu Asp Ile Ile Asn Glu Lys Gln Asn Glu65 70 75 80Trp Arg Gln Arg Leu Met Val Thr Pro Phe Thr Phe Thr Ser Tyr Tyr 85 90 95Ile Ser Lys Val Val Lys Thr Met Leu Gln Phe Ala Ile Ala Ile Leu 100 105 110Val Ile Phe Met Val Gly His Phe Tyr Lys Gly Val Ala Met Ser Ala 115 120 125Val Gln Trp Leu Glu Ser Gly Ile Phe Leu Trp Leu Gly Ala Ser Leu 130 135 140Leu Ile Thr Phe Gly Ile Leu Phe Ser Leu Leu Asn Asp Ile Gln Lys145 150 155 160Thr Ser Ala Leu Ala Asn Ile Val Thr Ile Gly Leu Ala Val Leu Gly 165 170 175Gly Leu Trp Phe Pro Ile Asn Thr Phe Pro Asn Trp Leu Gln His Val 180 185 190Ala His Val Leu Pro Ser Tyr His Leu Arg Lys Leu Gly Val Asp Ile 195 200 205Ala Ser Asn His His Ile Asn Leu Ile Ser Phe Ala Ile Ile Leu Leu 210 215 220Tyr Ala Leu Gly Ser Ile Ile Ala Val Tyr Cys Ile Ser His Phe Lys225 230 235 240Arg Ala Glu188276PRTStaphylococcus aureus 188Met His Lys Ile Phe Ser Lys Asn Asn Leu Ile Phe Phe Val Phe Val1 5 10 15Ala Phe Ile Phe Val Val Ile Val Leu Gln Phe Phe Val Ser Ser Glu 20 25 30Asn Ala Thr Lys Val Asn Leu Ser Gln Thr Phe Glu Pro Ile Ser Trp 35 40 45Leu His Leu Leu Gly Thr Asp Asp Tyr Gly Arg Asp Leu Phe Thr Arg 50 55 60Ile Ile Ile Gly Ala Arg Ser Thr Leu Phe Val Thr Val Leu Thr Leu65 70 75 80Ile Ala Ile Val Val Ile Gly Val Thr Leu Gly Leu Phe Ala Gly Tyr 85 90 95Lys Lys Gly Trp Ile Glu Arg Leu Val Leu Arg Phe Ile Asp Val Gly 100 105 110Leu Ser Ile Pro Glu Phe Ile Ile Met Ile Ala Leu Ala Ser Phe Phe 115 120 125Gln Pro Ser Leu Trp Asn Leu Val Ile Ser Ile Thr Leu Ile Lys Trp 130 135 140Met Asn Tyr Thr Arg Leu Thr Arg Ser Ile Val Asn Ser Glu Met Asn145 150 155 160Lys Pro Tyr Ile Lys Met Ala Gln Leu Phe His Val Pro Thr Arg Thr 165 170 175Ile Leu Ile Arg His Leu Thr Pro Lys Ile Ile Pro Ala Ile Ile Val 180 185 190Leu Met Val Val Asp Phe Gly Lys Ile Ile Leu Tyr Ile Ser Ser Leu 195 200 205Ser Phe Ile Gly Leu Gly Ala Gln Pro Pro Thr Pro Glu Trp Gly Ala 210 215 220Met Leu Gln Gln Gly Arg Asp Phe Ile Ser Ser His Pro Ile Met Leu225 230 235 240Ile Ala Pro Ala Ser Val Ile Ala Ile Thr Ile Leu Ile Phe Asn Leu 245 250 255Thr Gly Asp Ala Leu Arg Asp Arg Leu Leu Lys Gln Arg Gly Glu Tyr 260 265 270Asp Glu Ser His 275189136PRTStaphylococcus aureus 189Met Asn Thr Asn Asp Ala Ile Lys Ile Leu Lys Glu Asn Gly Leu Lys1 5 10 15Tyr Thr Asp Lys Arg Lys Asp Met Leu Asp Ile Phe Val Glu Glu Asp 20 25 30Lys Tyr Ile Asn Ala Lys Tyr Ile Gln Gln Val Met Asp Glu Asn Tyr 35 40 45Pro Gly Ile Ser Phe Asp Thr Ile Tyr Arg Asn Leu His Leu Phe Lys 50 55 60Asp Leu Gly Ile Ile Glu Asn Thr Glu Leu Asp Gly Glu Met Lys Phe65 70 75 80Arg Ile Ala Cys Thr Asn His His His His His Phe Ile Cys Glu Lys 85 90 95Cys Gly Asp Thr Lys Val Ile Asp Tyr Cys Pro Ile Asp Gln Ile Lys 100 105 110Leu Ser Leu Pro Gly Val Asn Ile His Lys His Lys Leu Glu Val Tyr 115 120 125Gly Val Cys Glu Ser Cys Gln Asp 130 135190233PRTStaphylococcus aureus 190Met Thr His Lys Tyr Ile Ser Thr Gln Met Leu Ile Ile Phe Thr Ala1 5 10 15Leu Met Ile Ile Ala Asn Phe Tyr Tyr Ile Phe Phe Glu Lys Ile Gly 20 25 30Phe Leu Leu Val Leu Leu Leu Gly Cys Val Leu Val Tyr Val Gly Tyr 35 40 45Leu Tyr Phe His Lys Ile Arg Gly Leu Leu Ala Phe Trp Ile Gly Ala 50 55 60Leu Leu Ile Ala Phe Thr Leu Leu Ser Asn Lys Tyr Thr Ile Ile Ile65 70 75 80Leu Phe Val Phe Leu Leu Leu Leu Ile Val Arg Tyr Leu Ile His Lys 85 90 95Phe Lys Pro Lys Lys Val Val Ala Thr Asp Glu Val Met Thr Ser Pro 100 105 110Ser Phe Ile Lys Gln Lys Trp Phe Gly Glu Gln Arg Thr Pro Val Tyr 115 120 125Val Tyr Lys Trp Glu Asp Val Gln Ile Gln His Gly Ile Gly Asp Leu 130 135 140His Ile Asp Leu Thr Lys Ala Ala Asn Ile Lys Glu Asn Asn Thr Ile145 150 155 160Val Val Arg His Ile Leu Gly Lys Val Gln Val Ile Leu Pro Val Asn 165 170 175Tyr Asn Ile Asn Leu His Val Ala Ala Phe Tyr Gly Ser Thr Tyr Val 180 185 190Asn Glu Lys Ser Tyr Lys Val Glu Asn Asn Asn Ile His Ile Glu Glu 195 200 205Met Met Lys Pro Asp Asn Tyr Thr Val Asn Ile Tyr Val Ser Thr Phe 210 215 220Ile Gly Asp Val Glu Val Ile Tyr Arg225 230191260PRTStaphylococcus aureus 191Met Leu Ile Gln Leu Asp Gln Ile Gly Arg Met Lys Gln Gly Lys Thr1 5 10 15Ile Leu Lys Lys Ile Ser Trp Gln Ile Ala Lys Gly Asp Lys Trp Ile 20 25 30Leu Tyr Gly Leu Asn Gly Ala Gly Lys Thr Thr Leu Leu Asn Ile Leu 35 40 45Asn Ala Tyr Glu Pro Ala Thr Ser Gly Thr Val Asn Leu Phe Gly Lys 50 55 60Met Pro Gly Lys Val Gly Tyr Ser Ala Glu Thr Val Arg Gln His Ile65 70 75 80Gly Phe Val Ser His Ser Leu Leu Glu Lys Phe Gln Glu Gly Glu Arg 85 90 95Val Ile Asp Val Val Ile Ser Gly Ala Phe Lys Ser Ile Gly Val Tyr 100 105 110Gln Asp Ile Asp Asp Glu Ile Arg Asn Glu Ala His Gln Leu Leu Lys 115 120 125Leu Val Gly Met Ser Ala Lys Ala Gln Gln Tyr Ile Gly Tyr Leu Ser 130 135 140Thr Gly Glu Lys Gln Arg Val Met Ile Ala Arg Ala Leu Met Gly Gln145 150 155 160Pro Gln Val Leu Ile Leu Asp Glu Pro Ala Ala Gly Leu Asp Phe Ile 165 170 175Ala Arg Glu Ser Leu Leu Ser Ile Leu Asp Ser Leu Ser Asp Ser Tyr 180 185 190Pro Thr Leu Ala Met Ile Tyr Val Thr His Phe Ile Glu Glu Ile Thr 195 200 205Ala Asn Phe Ser Lys Ile Leu Leu Leu Lys Asp Gly Gln Ser Ile Gln 210 215 220Gln Gly Ala Val Glu Asp Ile Leu Thr Ser Glu Asn Met Ser Arg Phe225 230 235 240Phe Gln Lys Asn Val Ala Val Gln Arg Trp Asn Asn Arg Phe Ser Met 245 250 255Ala Met Leu Glu 260192209PRTStaphylococcus aureus 192Met Lys Arg Leu Val Thr Gly Leu Leu Ala Leu Ser Leu Phe Leu Ala1 5 10 15Ala Cys Gly Gln Asp Ser Asp Gln Gln Lys Asp Gly Asn Lys Glu Lys 20 25 30Asp Asp Lys Ala Lys Thr Glu Gln Gln Asp Lys Lys Thr Asn Asp Ser 35 40 45Ser Lys Asp Lys Lys Asp Asn Lys Asp Asp Ser Lys Asp Val Asn Lys 50 55 60Asp Asn Lys Asp Asn Ser Ala Asn Asp Asn Gln Gln Gln Ser Asn Ser65 70 75 80Asn Ala Thr Asn Asn Asp Gln Asn Gln Thr Asn Asn Asn Gln Ser Ser 85 90 95Asn Asn Gln Ala Asn Asn Asn Gln Lys Ser Ser Tyr Val Ala Pro Tyr 100 105 110Tyr Gly Gln Asn Ala Ala Pro Val Ala Arg Gln Ile Tyr Pro Phe Asn 115 120 125Gly Asn Lys Asn Gln Ala Leu Gln Gln Leu Pro Asn Phe Gln Thr Ala 130 135 140Leu Asn Ala Ala Asn Asn Glu Ala Asn Lys Phe Gly Ser Asn Asn Lys145 150 155 160Val Tyr Asn Asp Tyr Ser Ile Glu Glu His Asn Gly Asn Tyr Lys Tyr 165 170 175Val Phe Ser Phe Lys Asp Pro Asn Ala Asn Gly Lys Tyr Ser Ile Val 180 185 190Thr Val Asp Tyr Thr Gly Gln Ala Met Val Thr Asp Pro Asn Tyr Gln 195 200 205Gln193439PRTStaphylococcus aureus 193Met Arg Thr Gly His Tyr Thr Pro Ile Pro Asn Glu Pro His Tyr Leu1 5 10 15Val Ile Ser His Ala Asp Lys Leu Thr Ala Thr Glu Lys Ala Lys Leu 20 25 30Arg Leu Leu Ile Ile Lys Gln Lys Leu Asp Ile Ser Leu Ala Glu Ser 35 40 45Val Val Ser Ser Pro Ile Ala Ser Glu His Val Ile Glu Gln Leu Thr 50 55 60Leu Phe Gln His Glu Arg Arg His Leu Arg Pro Lys Ile Ser Ala Thr65 70 75 80Phe Leu Ala Trp Leu Leu Ile Phe Leu Met Phe Ala Leu Pro Ile Gly 85 90 95Ile Ala Tyr Gln Phe Ser Asp Trp Phe Gln Asn Gln Tyr Val Ser Ala 100

105 110Trp Ile Glu Tyr Leu Thr Gln Thr Thr Leu Leu Asn His Asp Ile Leu 115 120 125Gln His Ile Leu Phe Gly Asp Tyr Gly Val Leu Ser Leu Gly Thr Tyr 130 135 140Ser Leu Val Trp Ala Leu Pro Val Val Ile Leu Ile Ser Leu Ser Thr145 150 155 160Ala Ile Ile Asp Gln Thr Gly Leu Lys Ser Trp Met Ile Trp Ala Ile 165 170 175Glu Pro Ser Met Leu Trp Ile Gly Leu Gln Gly Asn Asp Ile Val Pro 180 185 190Leu Leu Glu Gly Phe Gly Cys Asn Ala Ala Ala Ile Ser Gln Ala Ala 195 200 205His Gln Cys His Thr Cys Thr Lys Thr Gln Cys Met Ser Leu Ile Ser 210 215 220Phe Gly Ser Ser Cys Ser Tyr Gln Ile Gly Ala Thr Leu Ser Ile Phe225 230 235 240Ser Val Ala Gly Lys Ser Trp Leu Phe Met Pro Tyr Leu Ile Leu Val 245 250 255Leu Leu Gly Gly Ile Leu His Asn Arg Ile Trp Leu Lys Lys Asn Asp 260 265 270Gln Gln Leu Ser Val Pro Leu Pro Tyr Asp Arg Gln Leu His Met Pro 275 280 285Asn Ile Arg Gln Met Leu Leu Gln Met Trp Gln Asn Ile Gln Met Phe 290 295 300Ile Val Gln Ala Leu Pro Ile Phe Ile Thr Ile Cys Leu Ile Val Ser305 310 315 320Ile Leu Ser Leu Thr Pro Ile Leu Asn Val Leu Ser Gln Ile Phe Thr 325 330 335Pro Ile Leu Ser Leu Leu Gly Ile Ser Ser Glu Leu Ser Pro Gly Ile 340 345 350Leu Phe Ser Met Ile Arg Lys Asp Gly Met Leu Leu Phe Asn Leu His 355 360 365Gln Gly Ala Leu Leu Gln Gly Met Thr Ala Thr Gln Leu Leu Leu Leu 370 375 380Val Phe Phe Ser Ser Thr Phe Thr Ala Cys Ser Val Thr Met Thr Met385 390 395 400Leu Leu Lys His Leu Gly Gly Gln Ser Ala Leu Lys Leu Ile Gly Lys 405 410 415Gln Met Val Thr Ser Leu Ser Leu Val Ile Gly Val Gly Ile Ile Val 420 425 430Lys Ile Val Met Leu Ile Ile 43519422DNAArtificial sequencesynthetic sequence 194tcgtcgttgt cgttttgtcg tt 2219513PRTArtificial sequencesynthetic sequence 195Ile Leu Pro Trp Lys Trp Pro Trp Trp Pro Trp Arg Arg1 5 1019613PRTArtificial sequencesynthetic sequence 196Lys Asp Tyr Glu Arg Lys Tyr Lys Lys His Ile Val Ser1 5 10

Claims

1. A fusion construct of formula (I): X-A-linker-B-Z (1) wherein (1) A and B are different and (a) wherein A is a polypeptide comprising a SACOL0029 polypeptide as set forth in any one of SEQ ID NOs: 5 and 121 to 131, or a SACOL0442 polypeptide as set forth in any one of SEQ ID NOs: 29 and 82 to 92, and wherein B is a SACOL0720 polypeptide as set forth in any one of SEQ ID NOs: 11 and 109 to 120, a SACOL 1867 polypeptide as set forth in any one of SEQ ID NOs: 152 to 164, or a SACOL0442 polypeptide as set forth in any one of SEO ID NOs: 29 and 82 to 92; (b) a polypeptide comprising an immunogenic fragment of at least 13 consecutive amino acids of (a); or (c) a polypeptide comprising an amino acid sequence at least 60% identical overall to the sequence of the polypeptide of any one of (a) or (b); (2) the linker is an amino acid sequence of at least one amino acid or is absent; (3) X is absent or is an amino acid sequence of at least one amino acid; and (4) Z is absent or is an amino acid sequence of at least one amino acid.

2. The construct of claim 1, wherein A is a polypeptide comprising a SACOL0029 polypeptide as set forth in any one of SEQ ID NOs: 5 and 121 to 131, or a SACOL0442 polypeptide as set forth in any one of SEQ ID NOs: 29 and 82 to 92, and B is a SACOL0720 polypeptide as set forth in any one of SEQ ID NOs: 11 and 109 to 120, or a SACOL 1867 polypeptide as set forth in any one of SEQ ID NOs: 152 to 164.

3. The construct of claim 1, wherein A is (a) a polypeptide comprising a SACOL0029 polypeptide as set forth in any one of SEQ ID NOs: 5 and 121 to 131; (b) a polypeptide comprising an immunogenic fragment of at least 13 consecutive amino acids of (a); or (c) a polypeptide comprising an amino acid sequence at least 60% identical overall to the sequence of the polypeptide of any one of (a) or (b); and B is (i) a polypeptide comprising a SACOL1867 polypeptide as set forth in any one of SEQ ID NOs: 152 to 164; (ii) a polypeptide comprising an immunogenic fragment of at least 13 consecutive amino acids of (i); or (iii) a polypeptide comprising an amino acid sequence at least 60% identical overall to the sequence of the polypeptide of any one of (i) or (ii).

4. The construct of claim 1, wherein is (a) a polypeptide comprising a SACOL0442 polypeptide as set forth in any one of SEQ ID NOs: 29 and 82 to 92; (b) a polypeptide comprising an immunogenic fragment of at least 13 consecutive amino acids of (a); or (c) a polypeptide comprising an amino acid sequence at least 60% identical overall to the sequence of the polypeptide of any one of (a) or (b); and B is (a') a polypeptide comprising a SACOL0720 polypeptide as set forth in any one of the SEQ ID NOs: 11 and 109 to 120; (b') a polypeptide comprising an immunogenic fragment of at least 13 consecutive amino acids of (a'); or (c') a polypeptide comprising an amino acid sequence at least 60% identical overall to the sequence of the polypeptide of any one of (a') or (b').

5. The construct of claim 22L wherein A is (a) a polypeptide comprising a SACOL0029 polypeptide as set forth in any one of SEO ID NOs: 5 and 121 to 131; (b) a polypeptide comprising an immunogenic fragment of at least 13 consecutive amino acids of (a); or (c) a polypeptide comprising an amino acid sequence at least 60% identical overall to the sequence of the polypeptide of any one of (a) or (b); and B is (1) a polypeptide comprising a SACOL0442 polypeptide as set forth in any one of SEO ID NOs: 29 and 82 to 92; (2) a polypeptide comprising an immunogenic fragment of at least 13 consecutive amino acids of (1) or (1); or (3) a polypeptide comprising an amino acid sequence at least 60% identical overall to the sequence of the polypeptide of any one of (1) or (2).

6. The construct of claim 1, wherein the immunogenic fragment (b) comprises an amino acid sequence selected from the group consisting of TABLE-US-00013 (SEQ ID NO: 34) KDTINGKSNKSRNW[[;]] and (SEQ ID NO: 1) KDGGKYTLESHKELQ.

7. The construct of claim 6, wherein the immunogenic fragment (c) comprises an amino acid sequence selected from the group consisting of: TABLE-US-00014 (SEQ ID NO: 30) STQNSSSVQDKQLQKVEEVPNNSEKALVKKLYDRYSKDTINGKSNKSRN WVYSERPLNENQVRIHLEGTYTVAGRVYTPKRNITLNKEWTLKELDHII RFAHISYGLYMGEHLPKGNIVINTKDGGKYTLESHKELQKDRENVKINT ADIKNVTFKLVKSVNDIEQV; (SEQ ID NO: 32) DKQLQKVEEVPNNSEKALVKKLYDRYSKDTINGKSNKSRNWVYSERPLN ENQVRIHLEGTYTVAGRVYTPKRNITLNKEWTLKELDHIIRFAHISYGL YMGEHLPKGNIVINTK; and (SEQ ID NO: 33) DKQLQKVEEVPNNSEKALVKKLYDRYSKDTINGKSNKSRNWVYSERPLN ENQVRIHLEGTYTVAGRVYTPKRNITLNKEWTLKELDHIIRFAHISYGL YMGEHLPKGNIVINTKDGGKYTLESHKELQKDRENVKINTADIKNVTFK LVKSVNDIEQV.

8. The construct of claim 1, wherein the immunogenic fragment (c) comprises an amino acid sequence selected from the group consisting of: TABLE-US-00015 (SEQ ID NO: 21) QFGFDLKHKKDALA; (SEQ ID NO: 22) TIKDQQKANQLAS; (SEQ ID NO: 23) KDINKIYFMTDVDL; and (SEQ ID NO: 24) DVDLGGPTFVLND.

9. The construct of claim 1, wherein the immunogenic fragment (c) comprises an amino acid sequence selected from the group consisting of: TABLE-US-00016 (SEQ ID NO: 12) RASLSSEIKYTAPHDVTIKDQQKANQLASELNNQKIPHFYNYKEVIHTKLYKDNLFDVKAK EPYNVTITSDKYIPNTDLKRGQADLFVAEGSIKOLVKHKKHGKAIIGTKKHHVNIKLRKDIN KIYFMTDVDLGGPTFVLNDKDYQEIRKYTKAKHIVSQFGFDLKHKKDALALEKAKNKVDKS IETRSEAISSISSLTG; (SEQ ID NO: 13) ASLSSEIKYTAPHDVTIKDQQKANQLASELNNQKIPHFYNYKEVIHTKLYKDNLFDVKAKEP YNVTITSDKYIPNTDLKRGQADLFVAEGSIKOLVKHKKHGKAIIGTKKHHVNIKLRKDINKIY FMTDVDLGGPTFVLNDKDYQEIRKYTKAKHIVSQFGFDLKHKKDALALEKAKNKVDKSIET RSEAISSISSLTG; (SEQ ID NO: 14) ASLSSEIKYTAPHDVTIKDQQKANQLASELNNQKIPHFYNYKEVIHTKLYKDNLFDVKAKEP YNVTITSDKYIPNTDLKRGQADLFVAEGSIKDLVKHKKHGKAIIGTKKHHVNIKLRKDINKIY FMTDVDLGGPTFVLNDKDYQE; (SEQ ID NO: 15) KDINKIYFMTDVDLGGPTFVLNDKDYQEIRKYTKAKHIVSQFGFDLKHKKDALA; (SEQ ID NO: 17) KDINKIYFMTDVDLGGPTFVLNDKDY; (SEQ ID NO: 16) KDINKIYFMTDVDLGGPTFVLNDKD; (SEQ ID NO: 19) KDINKIYFMTDVDLGGPTFVLND; (SEQ ID NO: 20) KHIVSQFGFDLKHKKDALA and (SEQ ID NO: 18) SQFGFDLKHKKDALA.

10. The construct of claim 1, wherein the immunogenic fragment (b) comprises an amino acid sequence selected from the group consisting of: TABLE-US-00017 (SEQ ID NO: 165) PYNGWSFKDATGF; (SEQ ID NO: 167) AHPNGDKGNGGIYK; (SEQ ID NO: 169) SISDYPGDEDISVM; (SEQ ID NO: 172) RGPKGFNFNENVQA; (SEQ ID NO: 175) QFESTGTIKRIKDN; and (SEQ ID NO: 178) GNSGSPVLNSNNEV.

11. The construct of claim 10, wherein the immunogenic fragment (c) comprises the following amino acid sequence: TABLE-US-00018 (SEQ ID NO: 39) TQVKDTNIFPYNGWSFKDATGFVIGKNTIITNKHVSKDYKVGDRITAHP NGDKGNGGIYKIKSISDYPGDEDISVMNIEEQAVERGPKGFNFNENVQA FNFAKDAKVDDKIKVIGYPLPAQNSFKQFESTGTIKRIKDNILNFDAYI EPGNSGSPVLNSNNEVIGWYGGIGKIGSEYNGAVYFTPQIKDFIQKHIE Q.

12. The construct of claim 1, wherein the linker comprises at least four identical or different amino acids selected from the group consisting of glycine, serine, alanine, aspartate, glutamate, and lysine.

13. The construct of claim 1, wherein the linker comprises (GGGGS)n (SEQ ID NO: 67), (ERKYK)n (SEQ ID NO: 61); or (EAAAK)n (SEQ ID NO: 63), wherein n=1 to 5.

14. The construct of claim 1, wherein the X comprises a polyhistidine of 6 to 10 amino acids.

15. The construct of claim 1, wherein the X is absent.

16. The construct of claim 1, wherein the Z is absent.

17. An isolated nucleic acid molecule encoding the construct defined in claim 1.

18. A vector comprising the isolated nucleic acid defined in claim 17.

19. A host cell comprising the vector defined in claim 18.

20. The cell of claim 19, which is a live attenuated form of Staphylococcus aureus.

21. The cell of claim 20, wherein the live attenuated form of Staphylococcus aureus has a stabilized small colony variant (SCV) phenotype.

22. The cell of claim 21, wherein the live attenuated form of Staphylococcus aureus having a stabilized SCV phenotype is a .DELTA.hemB.DELTA.720 S. aureus.

23. A composition comprising: the construct of claim 1.

24. (canceled)

25. The composition of claim 23, wherein the live attenuated form of Staphylococcus aureus has a stabilized small colony variant (SCV) phenotype.

26. The composition of claim 23, wherein the adjuvant comprises alum, an oil, saponin, cyclicdiguanosine-5'-monophosphate (c-di-GMP), polyphosphasine, indolicidin, pathogen-associated molecular patterns (PAMPS), liposome or a combination of at least two thereof.

27. A method for preventing and/or treating a Staphylococcal intramammary infection (IMI) in a mammal comprising administrating to the mammal an effective amount of the construct of claim 1.

28. The method of claim 27, wherein the Staphylococcal IMI is caused by one or more Staphylococcus aureus strains.

29. The method of claim 27, wherein the mammal is a cow.

30. (canceled)

31. (canceled)

32. (canceled)

33. (canceled)

34. (canceled)

35. (canceled)

36. A kit for preventing and/or treating a Staphylococcal intramammary infection (IMI) in a mammal comprising the composition of claim 23.