U.S. patent application number 11/208208 was filed with the patent office on 2007-03-08 for nucleic acid and amino acid sequences relating to staphylococcus epidermidis for diagnostics and therapeutics.
Invention is credited to David Bush, Lynn A. Doucette-Stamm.
Application Number | 20070053936 11/208208 |
Document ID | / |
Family ID | 27368911 |
Filed Date | 2007-03-08 |
United States Patent
Application |
20070053936 |
Kind Code |
A1 |
Doucette-Stamm; Lynn A. ; et
al. |
March 8, 2007 |
Nucleic acid and amino acid sequences relating to Staphylococcus
epidermidis for diagnostics and therapeutics
Abstract
The invention provides isolated polypeptide and nucleic acid
sequences derived from Staphylococcus epidermidis that are useful
in diagnosis and therapy of pathological conditions; antibodies
against the polypeptides; and methods for the production of the
polypeptides. The invention also provides methods for the
detection, prevention and treatment of pathological conditions
resulting from bacterial infection.
Inventors: |
Doucette-Stamm; Lynn A.;
(Framingham, MA) ; Bush; David; (Somerville,
MA) |
Correspondence
Address: |
DRINKER BIDDLE & REATH (DC)
1500 K STREET, N.W.
SUITE 1100
WASHINGTON
DC
20005-1209
US
|
Family ID: |
27368911 |
Appl. No.: |
11/208208 |
Filed: |
August 22, 2005 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10902441 |
Jul 30, 2004 |
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11208208 |
Aug 22, 2005 |
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10092411 |
Mar 7, 2002 |
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10902441 |
Jul 30, 2004 |
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09134001 |
Aug 13, 1998 |
6380370 |
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10092411 |
Mar 7, 2002 |
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60055779 |
Aug 14, 1997 |
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60064964 |
Nov 8, 1997 |
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Current U.S.
Class: |
424/243.1 ;
435/252.3; 435/471; 435/69.3; 530/350; 536/23.7 |
Current CPC
Class: |
C07K 14/31 20130101;
Y10S 435/882 20130101; Y10S 435/884 20130101; A61P 31/04 20180101;
C12Q 1/689 20130101 |
Class at
Publication: |
424/243.1 ;
536/023.7; 530/350; 435/471; 435/069.3; 435/252.3 |
International
Class: |
A61K 39/085 20060101
A61K039/085; C07H 21/04 20060101 C07H021/04; C12N 15/74 20060101
C12N015/74; C12N 1/21 20060101 C12N001/21; C07K 14/31 20060101
C07K014/31 |
Claims
1. An isolated nucleic acid comprising a nucleotide sequence
encoding a Staphylococcus epidermidis polypeptide of SEQ ID NO:
5314 or 5547.
2. A recombinant expression vector comprising the nucleic acid of
claim 1 operably linked to a transcription regulatory element.
3. A cell comprising a recombinant expression vector of claim
2.
4. (canceled)
5. An isolated nucleic acid comprising a nucleotide sequence
encoding a S. epidermidis polypeptide or a fragment thereof, said
nucleic acid selected from the group consisting of SEQ ID NO: 2477
and SEQ ID NO: 2710.
6. A recombinant expression vector comprising the nucleic acid of
claim 5 operably linked to a transcription regulatory element.
7. A cell comprising a recombinant expression vector of claim
6.
8-9. (canceled)
10. An isolated nucleic acid comprising a nucleotide sequence of at
least 8 nucleotides in length, wherein the sequence is hybridizable
to a nucleic acid having a nucleotide sequence selected from the
group consisting of SEQ ID NOS: 2477 and 2710.
11-28. (canceled)
29. The isolated nucleic acid of claim 1, wherein the nucleic acid
comprises of SEQ ID NO: 2477.
30. The isolated nucleic acid of claim 1, wherein said nucleic acid
consists of SEQ ID NO: 2477.
31. The isolated nucleic acid of claim 1, wherein said nucleic acid
comprises SEQ ID NO: 2710.
32. The isolated nucleic acid of claim 1, wherein said nucleic acid
consists of SEQ ID NO: 2710.
33. A nucleic acid which has at least 90% homology to SEQ ID NO:
2477 or 2710.
34. The nucleic acid of claim 5, wherein the nucleic acid is DNA or
RNA.
35. A nucleic acid, wherein the nucleic acid is DNA, RNA,
single-stranded antisense, or an RNA/DNA duplex comprising SEQ ID
NO: 2477, a transcription product of SEQ ID NO: 2477, or an
antisense strand of SEQ ID NO: 2477.
36. A nucleic acid, wherein the nucleic acid is DNA, RNA,
single-stranded antisense, or an RNA/DNA duplex comprising SEQ ID
NO: 2710, a transcription product of SEQ ID NO: 2710, or an
antisense strand of SEQ ID NO: 2710.
Description
[0001] This application is a divisional of application Ser. No.
09/134,001, filed on Aug. 13, 1998, now U.S. Pat. No. 6,380,370,
issued Apr. 30, 2002, which claims priority of U.S. Provisional
Application 60/055,779, filed Aug. 14, 1997 and U.S. Provisional
Application 60/064,964, filed Nov. 8, 1997, all of which is hereby
incorporated herein by reference in their entirety.
FIELD OF THE INVENTION
[0002] The invention relates to isolated nucleic acids and
polypeptides derived from Staphylococcus epidermidis that are
useful as molecular targets for diagnostics, prophylaxis and
treatment of pathological conditions, as well as materials and
methods for the diagnosis, prevention, and amelioration of
pathological conditions resulting from bacterial infection.
BACKGROUND OF THE INVENTION
[0003] Staphylococcus epidermidis (S. epidermidis) is a species of
staphylococcal bacteria that are Gram-positive, nonmotile,
nonpigmented and coagulase-negative cocci, which are mainly found
on the skin and mucous membrane of warm-blooded animals. Their
large numbers and ubiquitous distribution result in frequent
contamination of specimens collected from or through the skin,
making these organisms amongst the most frequently isolated in the
clinical laboratory. In the past, S. epidermidis was rarely the
cause of significant infections, but with the increasing use of
implanted catheters and prosthetic devices, it has emerged as an
important agent of hospital-acquired infections and has been
recognized as a true pathogen (Lowy and Hammer, 1983, Ann Inten
Med, 99: 834-9; Blum and Rodvold, 1987, Clin Pharm, 6: 464-75;
Hamory, Parisi et al., 1987, Am J Infect Control, 15:59-74). S.
epidermidis is a major cause of infection of indwelling foreign
devices such as, orthopedic devices, intravenous catheters,
prosthetic heart valves, central nervous system shunts, and
peritoneal dialysis catheters (Blum and Rodvold, 1987, Clin Pharm,
6: 464-75; Archer, 1988, J Antimicrob Chemother, 21 Suppl C: 133-8)
(Lowy and Hammer, 1983, Ann Intern Med, 9: 834-9; Hamory, Parisi et
al.,
[0004] Staphylococcus 1987, Am J Infect Control, 15: 59-74). In
addition S. epidermidis is a common cause of postoperative wound
infections, bacteremia of immunosuppressed patients, intensive-care
unit patients and premature newborns (MacLowry, 1983, Am J Med, 75:
2-6)(Eykyn, 1988, Lancet, 1: 100-4). According to a national survey
(Centers for Disease Control, 1981:7) S. epidermidis caused 8.9% of
primary nosocomial bacteremias.
[0005] Treatment of S. epidermidis infections remains difficult
because of the occult nature, association with foreign bodies, and
frequent resistance to antimicrobial agents. Ordinarily, S.
epidermidis is an organism with low virulence, however breaks in
host defense caused by surgery, catheter placement, prosthesis
insertion or immuno-suppression is prerequisite for infection. The
presence of foreign bodies itself facilitates infection by
protecting the organism from elimination by host defenses or
antimicrobial therapy (Lowy and Hammer, 1983, Ann Intern Med, 99:
834-9). Furthermore, S. epidermidis due to its ability to produce
extracellular polysaccharide material or slime, may be uniquely
adapted to adhere to smooth surfaces such as plastics or metal.
Slime producing strains of S. epidermidis appear to be more
pathogenic than non-slime producing strains (Christensen, Simpson
et al., 1983, Infect Immun, 40: 407-10; Peters and Pulverer, 1984,
J Antimicrob Chemother, 14 Suppl D: 67-71; Gallimore, Gagnon et
al., 1991, J Infect Dis, 164: 1220-3). This property and many
factors are involved in the pathogenesis of device associated
infections. Despite the increased recognition as a pathogen, S.
epidermidis infections are difficult to diagnose. Differentiating
clinically important from clinically unimportant bacterial isolates
of S. epidermidis is difficult because of the high rate of
contamination.
[0006] Although laboratory isolates of S. epidermidis have
generally been susceptible to semisynthetic penicillins
(methicillin, nafcillin, oxacillin), cephalosporins,
amino-glycosides, vancomycin and rafampin, recent clinical isolates
have had an increased resistance. Recent reports (Karchmer, 1985,
Am J Med, 78: 116-27; Karchmer, 1991, J Hosp Infect, 18 Suppl A:
355-66) show that 83% of S. epidermidis isolates from patients with
prosthetic valve endocarditis are methicillin resistant and 32% are
gentamicin resistant as well. Multi-drug resistant staphylococci
have emerged in the midst of high level use of penicillin and
aminoglycosides (Centers for Disease Control and Prevention, 1993
MMWR 42:597; and S. Handwerger et al., 1993, Clin Infect Dis
16:750).
[0007] The use of antibiotics for therapeutics and prophylactic
purposes, promotes the selection of resistant organisms and the
spread of antibiotic resistance genes among bacteria. Previous
studies have shown that virtually all staphylococci carry some
antibiotic resistance genes on naturally occurring extrachromosomal
mobile genetic elements, such as the plasmids. Survey and analysis
of plasmids in clinical isolates of S. epidermidis have shown that
more that 80% of isolates carry plasmids and in several cases more
than one plasmid (Archer et al., 1982, Infect Immun, 35:627-632;
Kloos et al., 1981, Can J Microbiol, 27:271-278; Moller, 1988, J
Hosp Infect 12:19-27). Though the most important forms of
resistance has been the inactivation of antibiotics, particularly
penicillins and cephalosporins, recent clinical isolates have
resistance to one or more of the following antibiotics,
methicillin, tetracycline, erythromycin, gentamycin, kanamycin and
chloramphenicol. In fact due to the wide spread occurrence of
plasmids and their involvement in antibiotic resistance, plasmid
profiling has been used as an epidemiological reagent to study
nosocomial infections. This invention relates to isolated nucleic
acids and polypeptides derived from S. epidermidis plasmids that
are useful as molecular targets for diagnosis, prophylaxis and
treatment of pathological conditions, as well as materials and
methods for the diagnosis, prevention, and amelioration of
pathological conditions resulting from bacterial infection.
[0008] These concerns point to the need for diagnostic tools and
therapeutics aimed at proper identification of strain and
eradication of virulence. The design of vaccines that will limit
the spread of infection and halt transfer of resistance factors is
very desirable.
SUMMARY OF THE INVENTION
[0009] The present invention fulfills the need for diagnostic tools
and therapeutics by providing bacterial-specific compositions and
methods for detecting, treating, and preventing bacterial
infection, in particular S. epidermidis infection.
[0010] The present invention encompasses isolated nucleic acids and
polypeptides derived from S. epidermidis that are useful as
reagents for diagnosis of bacterial disease, components of
effective antibacterial vaccines, and/or as targets for
antibacterial drugs including anti-S. epidermidis drugs. They can
also be used to detect the presence of S. epidermidis and other
Staphylococcus species in a sample; and in screening compounds for
the ability to interfere with the S. epidermidis life cycle or to
inhibit S. epidermidis infection. They also has use as biocontrol
agents for plants.
[0011] More specifically, this invention features compositions of
nucleic acids corresponding to entire coding sequences of S.
epidermidis proteins, including surface or secreted proteins or
parts thereof, nucleic acids capable of binding mRNA from S.
epidermidis proteins to block protein translation, and methods for
producing S. epidermidis proteins or parts thereof using peptide
synthesis and recombinant DNA techniques. This invention also
features antibodies and nucleic acids useful as probes to detect S.
epidermidis infection. In addition, vaccine compositions and
methods for the protection or treatment of infection by S.
epidermidis are within the scope of this invention.
[0012] The nucleotide sequences provided in SEQ ID NO: 1-SEQ ID NO:
2837, a fragment thereof, or a nucleotide sequence at least 99.5%
identical to SEQ ID NO: 1-SEQ ID NO: 2837 may be "provided" in a
variety of medias to facilitate use thereof. As used herein,
"provided" refers to a manufacture, other than an isolated nucleic
acid molecule, which contains a nucleotide sequence of the present
invention, i.e., the nucleotide sequence provided in SEQ ID NO:
1-SEQ ID NO: 2837, a fragment thereof, or a nucleotide sequence at
least 99.5% identical to a sequence contained within SEQ ID NO:
1-SEQ ID NO: 2837. Uses for and methods for providing nucleotide
sequences in a variety of media is well known in the art (see e.g.,
EPO Publication No. EP 0 756 006).
[0013] In one application of this embodiment, a nucleotide sequence
of the present invention can be recorded on computer readable
media. As used herein, "computer readable media" refers to any
media which can be read and accessed directly by a computer. Such
media include, but are not limited to: magnetic storage media, such
as floppy discs, hard disc storage media, and magnetic tape;
optical storage media such as CD-ROM; electrical storage media such
as RAM and ROM; and hybrids of these categories such as
magnetic/optical storage media. A person skilled in the art can
readily appreciate how any of the presently known computer readable
media can be used to create a manufacture comprising computer
readable media having recorded thereon a nucleotide sequence of the
present invention.
[0014] As used herein, "recorded" refers to a process for storing
information on computer readable media. A person skilled in the art
can readily adopt any of the presently known methods for recording
information on computer readable media to generate manufactures
comprising the nucleotide sequence information of the present
invention.
[0015] A variety of data storage structures are available to a
person skilled in the art for creating a computer readable media
having recorded thereon a nucleotide sequence of the present
invention. The choice of the data storage structure will generally
be based on the means chosen to access the stored information. In
addition, a variety of data processor programs and formats can be
used to store the nucleotide sequence information of the present
invention on computer readable media. The sequence information can
be represented in a word processing text file, formatted in
commercially-available software such as WordPerfect and Microsoft
Word, or represented in the form of an ASCII file, stored in a
database application, such as DB2, Sybase, Oracle, or the like. A
person skilled in the art can readily adapt any number of data
processor structuring formats (e.g. text file or database) in order
to obtain computer readable media having recorded thereon the
nucleotide sequence information of the present invention.
[0016] By providing the nucleotide sequence of SEQ ID NO: 1-SEQ ID
NO: 2837, a fragment thereof, or a nucleotide sequence at least
99.5% identical to SEQ ID NO: 1-SEQ ID NO: 2837 in computer
readable form, a person skilled in the art can routinely access the
coding sequence information for a variety of purposes. Computer
software is publicly available which allows a person skilled in the
art to access sequence information provided in a computer readable
media. Examples of such computer software include programs of the
"Staden Package", "DNA Star", "MacVector", GCG "Wisconsin Package"
(Genetics Computer Group, Madison, Wis.) and "NCBI Toolbox"
(National Center For Biotechnology Information).
[0017] Computer algorithms enable the identification of S.
epidermidis open reading frames (ORFs) within SEQ ID NO: 1-SEQ ID
NO: 2837 which contain homology to ORFs or proteins from other
organisms. Examples of such similarity-search algorithms include
the BLAST [Altschul et al., J. Mol. Biol. 215:403-410 (1990)] and
Smith-Waterman [Smith and Waterman (1981) Advances in Applied
Mathematics, 2:482-489] search algorithms. These algorithms are
utilized on computer systems as exemplified below. The ORFs so
identified represent protein encoding fragments within the S.
epidermidis genome and are useful in producing commercially
important proteins such as enzymes used in fermentation reactions
and in the production of commercially useful metabolites.
[0018] The present invention further provides systems, particularly
computer-based systems, which contain the sequence information
described herein. Such systems are designed to identify
commercially important fragments of the S. epidermidis genome. As
used herein, "a computer-based system" refers to the hardware
means, software means, and data storage means used to analyze the
nucleotide sequence information of the present invention. The
minimum hardware means of the computer-based systems of the present
invention comprises a central processing unit (CPU), input means,
output means, and data storage means. A person skilled in the
artcan readily appreciate that any one of the currently available
computer-based systems is suitable for use in the present
invention. The computer-based systems of the present invention
comprise a data storage means having stored therein a nucleotide
sequence of the present invention and the necessary hardware means
and software means for supporting and implementing a search means.
As used herein, "data storage means" refers to memory which can
store nucleotide sequence information of the present invention, or
a memory access means which can access manufactures having recorded
thereon the nucleotide sequence information of the present
invention.
[0019] As used herein, "search means" refers to one or more
programs which are implemented on the computer-based system to
compare a target sequence or target structural motif with the
sequence information stored within the data storage means. Search
means are used to identify fragments or regions of the S.
epidermidis genome which are similar to, or "match", a particular
target sequence or target motif. A variety of known algorithms are
known in the art and have been disclosed publicly, and a variety of
commercially available software for conducting homology-based
similarity searches are available and can be used in the
computer-based systems of the present invention. Examples of such
software includes, but is not limited to, FASTA (GCG Wisconsin
Package), Bic_SW (Compugen Bioccelerator), BLASTN2, BLASTP2,
BLASTX2 (NCBI) and Motifs (GCG). A person skilled in the art can
readily recognize that any one of the available algorithms or
implementing software packages for conducting homology searches can
be adapted for use in the present computer-based systems.
[0020] As used herein, a "target sequence" can be any DNA or amino
acid sequence of six or more nucleotides or two or more amino
acids. A person skilled in the art can readily recognize that the
longer a target sequence is, the less likely a target sequence will
be present as a random occurrence in the database. The most
preferred sequence length of a target sequence is from about 10 to
100 amino acids or from about 30 to 300 nucleotide residues.
However, it is well recognized that many genes are longer than 500
amino acids, or 1.5 kb in length, and that commercially important
fragments of the S. epidermidis genome, such as sequence fragments
involved in gene expression and protein processing, will often be
shorter than 30 nucleotides.
[0021] As used herein, "a target structural motif," or "target
motif," refers to any rationally selected sequence or combination
of sequences in which the sequence(s) are chosen based on a
specific functional domain or three-dimensional configuration which
is formed upon the folding of the target polypeptide. There are a
variety of target motifs known in the art. Protein target motifs
include, but are not limited to, enzymatic active sites,
membrane-spanning regions, and signal sequences. Nucleic acid
target motifs include, but are not limited to, promoter sequences,
hairpin structures and inducible expression elements (protein
binding sequences).
[0022] A variety of structural formats for the input and output
means an be used to input and output the information in the
computer-based systems of the present invention. A preferred format
for an output means ranks fragments of the S. epidermidis genome
possessing varying degrees of homology to the target sequence or
target motif. Such presentation provides a person skilled in the
art with a ranking of sequences which contain various amounts of
the target sequence or target motif and identifies the degree of
homology contained in the identified fragment.
[0023] A variety of comparing means can be used to compare a target
sequence or target motif with the data storage means to identify
sequence fragments of the S. epidermidis genome. In the present
examples, implementing software which implement the BLASTP2 and
bic_SW algorithms (Altschul et al., J. Mol. Biol. 215:403-410
(1990); Compugen Biocellerator) was used to identify open reading
frames within the S. epidermidis genome.
[0024] A person skilled in the art can readily recognize that any
one of the publicly available homology search programs can be used
as the search means for the computer-based systems of the present
invention.
[0025] The invention features S. epidermidis polypeptides,
preferably a substantially pure preparation of a S. epidermidis
polypeptide, or a recombinant S. epidermidis polypeptide. In
preferred embodiments: the polypeptide has biological activity; the
polypeptide has an amino acid sequence at least 60%, 70%, 80%, 90%,
95%, 98%, or 99% identical to an amino acid sequence of the
invention contained in the Sequence Listing, preferably it has
about 65% sequence identity with an amino acid sequence of the
invention contained in the Sequence Listing, and most preferably it
has about 92% to about 99% sequence identity with an amino acid
sequence of the invention contained in the Sequence Listing; the
polypeptide has an amino acid sequence essentially the same as an
amino acid sequence of the invention contained in the Sequence
Listing; the polypeptide is at least 5, 10, 20, 50, 100, or 150
amino acid residues in length; the polypeptide includes at least 5,
preferably at least 10, more preferably at least 20, more
preferably at least 50, 100, or 150 contiguous amino acid residues
of the invention contained in the Sequence Listing. In yet another
preferred embodiment, the amino acid sequence which differs in
sequence identity by about 7% to about 8% from the S. epidermidis
amino acid sequences of the invention contained in the Sequence
Listing is also encompassed by the invention.
[0026] In preferred embodiments: the S. epidermidis polypeptide is
encoded by a nucleic acid of the invention contained in the
Sequence Listing, or by a nucleic acid having at least 60%, 70%,
80%, 90%, 95%, 98%, or 99% homology with a nucleic acid of the
invention contained in the Sequence Listing.
[0027] In a preferred embodiment, the subject S. epidermidis
polypeptide differs in amino acid sequence at 1, 2, 3, 5, 10 or
more residues from a sequence of the invention contained in the
Sequence Listing. The differences, however, are such that the S.
epidermidis polypeptide exhibits a S. epidermidis biological
activity, e.g., the S. epidermidis polypeptide retains a biological
activity of a naturally occurring S. epidermidis enzyme.
[0028] In preferred embodiments, the polypeptide includes all or a
fragment of an amino acid sequence of the invention contained in
the Sequence Listing; fused, in reading frame, to additional amino
acid residues, preferably to residues encoded by genomic DNA 5' or
3' to the genomic DNA which encodes a sequence of the invention
contained in the Sequence Listing.
[0029] In yet other preferred embodiments, the S. epidermidis
polypeptide is a recombinant fusion protein having a first S.
epidermidis polypeptide portion and a second polypeptide portion,
e.g., a second polypeptide portion having an amino acid sequence
unrelated to S. epidermidis. The second polypeptide portion can be,
e.g., any of glutathione-5-transferase, a DNA binding domain, or a
polymerase activating domain. In preferred embodiment the fusion
protein can be used in a two-hybrid assay.
[0030] Polypeptides of the invention include those which arise as a
result of alternative transcription events, alternative RNA
splicing events, and alternative translational and postranslational
events.
[0031] In a preferred embodiment, the encoded S. epidermidis
polypeptide differs (e.g., by amino acid substitution, addition or
deletion of at least one amino acid residue) in amino acid sequence
at 1, 2, 3, 5, 10 or more residues, from a sequence of the
invention contained in the Sequence Listing. The differences,
however, are such that: the S. epidermidis encoded polypeptide
exhibits a S. epidermidis biological activity, e.g., the encoded S.
epidermidis enzyme retains a biological activity of a naturally
occurring S. epidermidis.
[0032] In preferred embodiments, the encoded polypeptide includes
all or a fragment of an amino acid sequence of the invention
contained in the Sequence Listing; fused, in reading frame, to
additional amino acid residues, preferably to residues encoded by
genomic DNA 5' or 3' to the genomic DNA which encodes a sequence of
the invention contained in the Sequence Listing.
[0033] The S. epidermidis strain, from which the nucleotide
sequences have been sequenced, was deposited on Jul. 10, 1997 in
the American Type Culture Collection (ATCC #55998) as strain
18972.
[0034] Included in the invention are: allelic variations; natural
mutants; induced mutants; proteins encoded by DNA that hybridize
under high or low stringency conditions to a nucleic acid which
encodes a polypeptide of the invention contained in the Sequence
Listing (for definitions of high and low stringency see Current
Protocols in Molecular Biology, John Wiley & Sons, New York,
1989, 6.3.1-6.3.6, hereby incorporated by reference); and,
polypeptides specifically bound by antisera to S. epidermidis
polypeptides, especially by antisera to an active site or binding
domain of S. epidermidis polypeptide. The invention also includes
fragments, preferably biologically active fragments. These and
other polypeptides are also referred to herein as S. epidermidis
polypeptide analogs or variants.
[0035] The invention further provides nucleic acids, e.g., RNA or
DNA, encoding a polypeptide of the invention. This includes double
stranded nucleic acids as well as coding and antisense single
strands.
[0036] In preferred embodiments, the subject S. epidermidis nucleic
acid will include a transcriptional regulatory sequence, e.g. at
least one of a transcriptional promoter or transcriptional enhancer
sequence, operably linked to the S. epidermidis gene sequence,
e.g., to render the S. epidermidis gene sequence suitable for
expression in a recombinant host cell.
[0037] In yet a further preferred embodiment, the nucleic acid
which encodes a S. epidermidis polypeptide of the invention,
hybridizes under stringent conditions to a nucleic acid probe
corresponding to at least 8 consecutive nucleotides of the
invention contained in the Sequence Listing; more preferably to at
least 12 consecutive nucleotides of the invention contained in the
Sequence Listing; more preferably to at least 20 consecutive
nucleotides of the invention contained in the Sequence Listing;
more preferably to at least 40 consecutive nucleotides of the
invention contained in the Sequence Listing.
[0038] In another aspect, the invention provides a substantially
pure nucleic acid having a nucleotide sequence which encodes a S.
epidermidis polypeptide. In preferred embodiments: the encoded
polypeptide has biological activity; the encoded polypeptide has an
amino acid sequence at least 60%; 70%, 80%, 90%) 95%, 98%, or 99%
homologous to an amino acid sequence of the invention contained in
the Sequence Listing; the encoded polypeptide has an amino acid
sequence essentially the same as an amino acid sequence of the
invention contained in the Sequence Listing; the encoded
polypeptide is at least 5, 10, 20, 50, 100, or 150 amino acids in
length; the encoded polypeptide comprises at least 5, preferably at
least 10, more preferably at least 20, more preferably at least 50,
100, or 150 contiguous amino acids of the invention contained in
the Sequence Listing.
[0039] In another aspect, the invention encompasses: a vector
including a nucleic acid which encodes a S. epidermidis polypeptide
or a S. epidermidis polypeptide variant as described herein; a host
cell transfected with the vector; and a method of producing a
recombinant S. epidermidis polypeptide or S. epidermidis
polypeptide variant; including culturing the cell, e.g., in a cell
culture medium, and isolating a S. epidermidis or S. epidermidis
polypeptide variant, e.g., from the cell or from the cell culture
medium.
[0040] One embodiment of the invention is directed to substantially
isolated nucleic acids. Nucleic acids of the invention include
sequences comprising at least about 8 nucleotides in length, more
preferably at least about 12 nucleotides in length, even more
preferably at least about 15-20 nucleotides in length, that
correspond to a subsequence of any one of SEQ ID NO: 1-SEQ ID NO:
2837 or complements thereof. Alternatively, the nucleic acids
comprise sequences contained within any ORF (open reading frame),
including a complete protein-coding sequence, of which any of SEQ
ID NO: 1-SEQ ID NO: 2837 forms a part. The invention encompasses
sequence-conservative variants and function-conservative variants
of these sequences. The nucleic acids may be DNA, RNA, DNA/RNA
duplexes, protein-nucleic acid (PNA), or derivatives thereof.
[0041] In another aspect, the invention features, a purified
recombinant nucleic acid having at least 50%, 60%, 70%, 80%, 90%,
95%, 98%, or 99% homology with a sequence of the invention
contained in the Sequence Listing
[0042] The invention also encompasses recombinant DNA (including
DNA cloning and expression vectors) comprising these S.
epidermidis-derived sequences; host cells comprising such DNA,
including fungal, bacterial, yeast, plant, insect, and mammalian
host cells; and methods for producing expression products
comprising RNA and polypeptides encoded by the S. epidermidis
sequences. These methods are carried out by incubating a host cell
comprising a S. epidermidis-derived nucleic acid sequence under
conditions in which the sequence is expressed. The host cell may be
native or recombinant. The polypeptides can be obtained by (a)
harvesting the incubated cells to produce a cell fraction and a
medium fraction; and (b) recovering the S. epidermidis polypeptide
from the cell fraction, the medium fraction, or both. The
polypeptides can also be made by in vitro translation.
[0043] In another aspect, the invention features nucleic acids
capable of binding mRNA of S. epidermidis. Such nucleic acid is
capable of acting as antisense nucleic acid to control the
translation of mRNA of S. epidermidis. A further aspect features a
nucleic acid which is capable of binding specifically to a S.
epidermidis nucleic acid. These nucleic acids are also referred to
herein as complements and have utility as probes and as capture
reagents.
[0044] In another aspect, the invention features an expression
system comprising an open reading frame corresponding to S.
epidermidis nucleic acid. The nucleic acid further comprises a
control sequence compatible with an intended host. The expression
system is useful for making polypeptides corresponding to S.
epidermidis nucleic acid.
[0045] In another aspect, the invention encompasses: a vector
including a nucleic acid which encodes a S. epidermidis polypeptide
or a S. epidermidis polypeptide variant as described herein; a host
cell transfected with the vector; and a method of producing a
recombinant S. epidermidis polypeptide or S. epidermidis
polypeptide variant; including culturing the cell, e.g., in a cell
culture medium, and isolating the S. epidermidis or S. epidermidis
polypeptide variant, e.g., from the cell or from the cell culture
medium.
[0046] In yet another embodiment of the invention encompasses
reagents for detecting bacterial infection, including S.
epidermidis infection, which comprise at least one S.
epidermidis-derived nucleic acid defined by any one of SEQ ID NO:
1-SEQ ID NO: 2837, or sequence-conservative or
function-conservative variants thereof. Alternatively, the
diagnostic reagents comprise polypeptide sequences that are
contained within any open reading frames (ORFs), including complete
protein-coding sequences, contained within any of SEQ ID NO: 1-SEQ
ID NO: 2837, or polypeptide sequences contained within any of SEQ
ID NO: 2838-SEQ ID NO: 5674, or polypeptides of which any of the
above sequences forms a part, or antibodies directed against any of
the above peptide sequences or function-conservative variants
and/or fragments thereof.
[0047] The invention further provides antibodies, preferably
monoclonal antibodies, which specifically bind to the polypeptides
of the invention. Methods are also provided for producing
antibodies in a host animal. The methods of the invention comprise
immunizing an animal with at least one S. epidermidis-derived
immunogenic component, wherein the immunogenic component comprises
one or more of the polypeptides encoded by any one of SEQ ID NO:
1-SEQ ID NO: 2837 or sequence-conservative or function-conservative
variants thereof; or polypeptides that are contained within any
ORFs, including complete protein-coding sequences, of which any of
SEQ ID NO: 1-SEQ ID NO: 2837 forms a part; or polypeptide sequences
contained within any of SEQ ID NO: 2838-SEQ ID NO: 5674, or
polypeptides of which any of SEQ ID NO: 2838-SEQ ID NO: 5674 forms
a part. Host animals include any warm blooded animal, including
without limitation mammals and birds. Such antibodies have utility
as reagents for immunoassays to evaluate the abundance and
distribution of S. epidermidis-specific antigens.
[0048] In yet another aspect, the invention provides diagnostic
methods for detecting S. epidermidis antigenic components or
anti-S. epidermidis antibodies in a sample. S. epidermidis
antigenic components are detected by a process comprising: (i)
contacting a sample suspected to contain a bacterial antigenic
component with a bacterial-specific antibody, under conditions in
which a stable antigen-antibody complex can form between the
antibody and bacterial antigenic components in the sample; and (ii)
detecting any antigen-antibody complex formed in step (i), wherein
detection of an antigen-antibody complex indicates the presence of
at least one bacterial antigenic component in the sample. In
different embodiments of this method, the antibodies used are
directed against a sequence encoded by any of SEQ ID NO: 1-SEQ ID
NO: 2837 or sequence-conservative or function-conservative variants
thereof, or against a polypeptide sequence contained in any of SEQ
ID NO: 2838-SEQ ID NO: 5674 or function-conservative variants
thereof.
[0049] In yet another aspect, the invention provides a method for
detecting antibacterial-specific antibodies in a sample, which
comprises: (i) contacting a sample suspected to contain
antibacterial-specific antibodies with a S. epidermidis antigenic
component, under conditions in which a stable antigen-antibody
complex can form between the S. epidermidis antigenic component and
antibacterial antibodies in the sample; and (ii) detecting any
antigen-antibody complex formed in step (i), wherein detection of
an antigen-antibody complex indicates the presence of antibacterial
antibodies in the sample. In different embodiments of this method,
the antigenic component is encoded by a sequence contained in any
of SEQ ID NO: 1-SEQ ID NO: 2837 or sequence-conservative and
function-conservative variants thereof, or is a polypeptide
sequence contained in any of SEQ ID NO: 2838-SEQ ID NO: 5674 or
function-conservative variants thereof.
[0050] In another aspect, the invention features a method of
generating vaccines for immunizing an individual against S.
epidermidis. The method includes: immunizing a subject with a S.
epidermidis polypeptide, e.g., a surface or secreted polypeptide,
or a combination of such peptides or active portion(s) thereof, and
a pharmaceutically acceptable carrier. Such vaccines have
therapeutic and prophylactic utilities.
[0051] In another aspect, the invention features a method of
evaluating a compound, e.g. a polypeptide, e.g., a fragment of a
host cell polypeptide, for the ability to bind a S. epidermidis
polypeptide. The method includes: contacting the Staphylococcus
compound with a S. epidermidis polypeptide and determining if the
compound binds or otherwise interacts with a S. epidermidis
polypeptide. Compounds which bind S. epidermidis are candidates as
activators or inhibitors of the bacterial life cycle. These assays
can be performed in vitro or in vivo.
[0052] In another aspect, the invention features a method of
evaluating a compound, e.g. a polypeptide, e.g., a fragment of a
host cell polypeptide, for the ability to bind a S. epidermidis
nucleic acid, e.g., DNA or RNA. The method includes: contacting the
Staphylococcus compound with a S. epidermidis nucleic acid and
determining if the compound binds or otherwise interacts with a S.
epidermidis polypeptide. Compounds which bind S. epidermidis are
candidates as activators or inhibitors of the bacterial life cycle.
These assays can be performed in vitro or in vivo.
[0053] A particularly preferred embodiment of the invention is
directed to a method of screening test compounds for anti-bacterial
activity, which method comprises: selecting as a target a bacterial
specific sequence, which sequence is essential to the viability of
a bacterial species; contacting a test compound with said target
sequence; and selecting those test compounds which bind to said
target sequence as potential anti-bacterial candidates. In one
embodiment, the target sequence selected is specific to a single
species, or even a single strain, i.e., the S. epidermidis 18972.
In a second embodiment, the target sequence is common to at least
two species of bacteria. In a third embodiment, the target sequence
is common to a family of bacteria. The target sequence may be a
nucleic acid sequence or a polypeptide sequence. Methods employing
sequences common to more than one species of microorganism may be
used to screen candidates for broad spectrum anti-bacterial
activity.
[0054] The invention also provides methods for preventing or
treating disease caused by certain bacteria, including S.
epidermidis, which are carried out by administering to an animal in
need of such treatment, in particular a warm-blooded vertebrate,
including but not limited to birds and mammals, a compound that
specifically inhibits or interferes with the function of a
bacterial polypeptide or nucleic acid. In a particularly preferred
embodiment, the mammal to be treated is human.
DETAILED DESCRIPTION OF THE INVENTION
[0055] The sequences of the present invention include the specific
nucleic acid and amino acid sequences set forth in the Sequence
Listing that forms a part of the present specification, and which
are designated SEQ ID NO: 1-SEQ ID NO: 5674. Use of the terms "SEQ
ID NO: 1-SEQ ID NO: 2837," "SEQ ID NO: 2838-SEQ ID NO: 5674," and
"the sequences depicted in Table 2", etc., is intended, for
convenience, to refer to each individual SEQ ID. NO individually,
and is not intended to refer to the genus of these sequences. In
other words, it is a shorthand for listing all of these sequences
individually. The invention encompasses each sequence individually,
as well as any combination thereof.
Definitions
[0056] "Nucleic acid" or "polynucleotide" as used herein refers to
purine- and pyrimidine-containing polymers of any length, either
polyribonucleotides or polydeoxyribonucleotides or mixed
polyribo-polydeoxyribo nucleotides. This includes single- and
double-stranded molecules, i.e., DNA-DNA, DNA-RNA and RNA-RNA
hybrids, as well as "protein nucleic acids" (PNA) formed by
conjugating bases to an amino acid backbone. This also includes
nucleic acids containing modified bases.
[0057] A nucleic acid or polypeptide sequence that is "derived
from" a designated sequence refers to a sequence that corresponds
to a region of the designated sequence. For nucleic acid sequences,
this encompasses sequences that are homologous or complementary to
the sequence, as well as "sequence-conservative variants" and
"function-conservative variants." For polypeptide sequences, this
encompasses "function-conservative variants." Sequence-conservative
variants are those in which a change of one or more nucleotides in
a given codon position results in no alteration in the amino acid
encoded at that position. Function-conservative variants are those
in which a given amino acid residue in a polypeptide has been
changed without altering the overall conformation and function of
the native polypeptide, including, but not limited to, replacement
of an amino acid with one having similar physico-chemical
properties (such as, for example, acidic, basic, hydrophobic, and
the like): "Function-conservative" variants also include any
polypeptides that have the ability to elicit antibodies specific to
a designated polypeptide.
[0058] An "S. epidermidis-derived" nucleic acid or polypeptide
sequence may or may not be present in other bacterial species, and
may or may not be present in all S. epidermidis strains. This term
is intended to refer to the source from which the sequence was
originally isolated. Thus, a S. epidermidis-derived polypeptide, as
used herein, may be used, e.g., as a target to screen for a broad
spectrum antibacterial agent, to search for homologous proteins in
other species of bacteria or in eukaryotic organisms such as fungi
and humans, etc.
[0059] A purified or isolated polypeptide or a substantially pure
preparation of a polypeptide are used interchangeably herein and,
as used herein, mean a polypeptide that has been separated from
other proteins, lipids, and nucleic acids with which it naturally
occurs. Preferably, the polypeptide is also separated from
substances, e.g., antibodies or gel matrix, e.g., polyacrylamide,
which are used to purify it. Preferably, the polypeptide
constitutes at least 10, 20, 50 70, 80 or 95% dry weight of the
purified preparation. Preferably, the preparation contains:
sufficient polypeptide to allow protein sequencing; at least 1, 10,
or 100 mg of the polypeptide.
[0060] A purified preparation of cells refers to, in the case of
plant or animal cells, an in vitro preparation of cells and not an
entire intact plant or animal. In the case of cultured cells or
microbial cells, it consists of a preparation of at least 10% and
more preferably 50% of the subject cells.
[0061] A purified or isolated or a substantially pure nucleic acid,
e.g., a substantially pure DNA, (are terms used interchangeably
herein) is a nucleic acid which is one or both of the following:
not immediately contiguous with both of the coding sequences with
which it is immediately contiguous (i.e., one at the 5' end and one
at the 3' end) in the naturally-occurring genome of the organism
from which the nucleic acid is derived; or which is substantially
free of a nucleic acid with which it occurs in the organism from
which the nucleic acid is derived. The term includes, for example,
a recombinant DNA which is incorporated into a vector, e.g., into
an autonomously replicating plasmid or virus, or into the genomic
DNA of a prokaryote or eukaryote, or which exists as a separate
molecule (e.g., a cDNA or a genomic DNA fragment produced by PCR or
restriction endonuclease treatment) independent of other DNA
sequences. Substantially pure DNA also includes a recombinant DNA
which is part of a hybrid gene encoding additional S. epidermidis
DNA sequence.
[0062] A "contig" as used herein is a nucleic acid representing a
continuous stretch of genomic sequence of an organism.
[0063] An "open reading frame", also referred to herein as ORF, is
a region of nucleic acid which encodes a polypeptide. This region
may represent a portion of a coding sequence or a total sequence
and can be determined from a stop to stop codon or from a start to
stop codon.
[0064] As used herein, a "coding sequence" is a nucleic acid which
is transcribed into messenger RNA and/or translated into a
polypeptide when placed under the control of appropriate regulatory
sequences. The boundaries of the coding sequence are determined by
a translation start codon at the five prime terminus and a
translation stop code at the three prime terminus. A coding
sequence can include but is not limited to messenger. RNA,
synthetic DNA, and recombinant nucleic acid sequences.
[0065] A "complement" of a nucleic acid as used herein refers to an
anti-parallel or antisense sequence that participates in
Watson-Crick base-pairing with the original sequence.
[0066] A "gene product" is a protein or structural RNA which is
specifically encoded by a gene.
[0067] As used herein, the term "probe" refers to a nucleic acid,
peptide or other chemical entity which specifically binds to a
molecule of interest. Probes are often associated with or capable
of associating with a label. A label is a chemical moiety capable
of detection. Typical labels comprise dyes, radioisotopes,
luminescent and chemiluminescent moieties, fluorophores, enzymes,
precipitating agents, amplification sequences, and the like.
Similarly, a nucleic acid, peptide or other chemical entity which
specifically binds to a molecule of interest and immobilizes such
molecule is referred herein as a "capture ligand". Capture ligands
are typically associated with or capable of associating with a
support such as nitro-cellulose, glass, nylon membranes, beads,
particles and the like. The specificity of hybridization is
dependent on conditions such as the base pair composition of the
nucleotides, and the temperature and salt concentration of the
reaction. These conditions are readily discernable to one of
ordinary skill in the art using routine experimentation.
[0068] "Homologous" refers to the sequence similarity or sequence
identity between two polypeptides or between two nucleic acid
molecules. When a position in both of the two compared sequences is
occupied by the same base or amino acid monomer subunit, e.g., if a
position in each of two DNA molecules is occupied by adenine, then
the molecules are homologous at that position. The percent of
homology between two sequences is a function of the number of
matching or homologous positions shared by the two sequences
divided by the number of positions compared.times.100. For example,
if 6 of 10 of the positions in two sequences are matched or
homologous then the two sequences are 60% homologous. By way of
example, the DNA sequences ATTGCC and TATGGC share 50% homology.
Generally, a comparison is made when two sequences are aligned to
give maximum homology.
[0069] Nucleic acids are hybridizable to each other when at least
one strand of a nucleic acid can anneal to the other nucleic acid
under defined stringency conditions. Stringency of hybridization is
determined by: (a) the temperature at which hybridization and/or
washing is performed; and (b) the ionic strength and polarity of
the hybridization and washing solutions. Hybridization requires
that the two nucleic acids contain complementary sequences;
depending on the stringency of hybridization, however, mismatches
may be tolerated. Typically, hybridization of two sequences at high
stringency (such as, for example, in a solution of 0.5.times.SSC,
at 65.degree. C.) requires that the sequences be essentially
completely homologous. Conditions of intermediate stringency (such
as, for example, 2.times.SSC at 65.degree. C.) and low stringency
(such as, for example 2.times.SSC at 55.degree. C.), require
correspondingly less overall complementarity between the
hybridizing sequences. (1.times.SSC is 0.15 M NaCl, 0.015 M Na
citrate).
[0070] The terms peptides, proteins, and polypeptides are used
interchangeably herein.
[0071] As used herein, the term "surface protein" refers to all
surface accessible proteins, e.g. inner and outer membrane
proteins, proteins adhering to the cell wall, and secreted
proteins.
[0072] A polypeptide has S. epidermidis biological activity if it
has one, two and preferably more of the following properties: (1)
if when expressed in the course of a S. epidermidis infection, it
can promote, or mediate the attachment of S. epidermidis to a cell;
(2) it has an enzymatic activity, structural or regulatory function
characteristic of a S. epidermidis protein; (3) or the gene which
encodes it can rescue a lethal mutation in a S. epidermidis gene. A
polypeptide has biological activity if it is an antagonist,
agonist, or super-agonist of a polypeptide having one of the
above-listed properties.
[0073] A biologically active fragment or analog is one having an in
vivo or in vitro activity which is characteristic of the S.
epidermidis polypeptides of the invention contained in the Sequence
Listing, or of other naturally occurring S. epidermidis
polypeptides, e.g., one or more of the biological activities
described herein. Especially preferred are fragments which exist in
vivo, e.g., fragments which arise from post transcriptional
processing or which arise from translation of alternatively spliced
RNA's. Fragments include those expressed in native or endogenous
cells as well as those made in expression systems, e.g., in CHO
(Chinese Hamster Ovary) cells. Because peptides such as S.
epidermidis polypeptides often exhibit a range of physiological
properties and because such properties may be attributable to
different portions of the molecule, a useful S. epidermidis
fragment or S. epidermidis analog is one which exhibits a
biological activity in any biological assay for S. epidermidis
activity. Most preferably the fragment or analog possesses 10%,
preferably 40%, more preferably 60%, 70%, 80% or 90% or greater of
the activity of S. epidermidis, in any in vivo or in vitro
assay.
[0074] Analogs can differ from naturally occurring S. epidermidis
polypeptides in amino acid sequence or in ways that do not involve
sequence, or both. Non-sequence modifications include changes in
acetylation, methylation, phosphorylation, carboxylation, or
glycosylation. Preferred analogs include S. epidermidis
polypeptides (or biologically active fragments thereof) whose
sequences differ from the wild-type sequence by one or more
conservative amino acid substitutions or by one or more
non-conservative amino acid substitutions, deletions, or insertions
which do not substantially diminish the biological activity of the
S. epidermidis polypeptide. Conservative substitutions typically
include the substitution of one amino acid for another with similar
characteristics, e.g., substitutions within the following groups:
valine, glycine; glycine, alanine; valine, isoleucine, leucine;
aspartic acid, glutamic acid; asparagine, glutamine; serine,
threonine; lysine, arginine; and phenylalanine, tyrosine. Other
conservative substitutions can be made in view of the table below.
TABLE-US-00001 TABLE 1 CONSERVATIVE AMINO ACID REPLACEMENTS For
Amino Acid Code Replace with any of Alanine A D-Ala, Gly, beta-Ala,
L-Cys, D-Cys Arginine R D-Arg, Lys, D-Lys, homo-Arg, D-homo-Arg,
Met, Ile, D-Met, D-Ile, Orn, D-Orn Asparagine N D-Asn, Asp, D-Asp,
Glu, D-Glu, Gln, D-Gln Aspartic Acid D D-Asp, D-Asn, Asn, Glu,
D-Glu, Gln, D-Gln Cysteine C D-Cys, S-Me-Cys, Met, D-Met, Thr,
D-Thr Glutamine Q D-Gln, Asn, D-Asn, Glu, D-Glu, Asp, D-Asp
Glutamic Acid E D-Glu, D-Asp, Asp, Asn, D-Asn, Gln, D-Gln Glycine G
Ala, D-Ala, Pro, D-Pro, .beta.-Ala, Acp Isoleucine I D-Ile, Val,
D-Val, Leu, D-Leu, Met, D-Met Leucine L D-Leu, Val, D-Val, Leu,
D-Leu, Met, D-Met Lysine K D-Lys, Arg, D-Arg, homo-Arg, D-homo-Arg,
Met, D-Met, Ile, D-Ile, Orn, D-Orn Methionine M D-Met, S-Me-Cys,
Ile, D-Ile, Leu, D-Leu, Val, D-Val Phenylalanine F D-Phe, Tyr,
D-Thr, L-Dopa, His, D-His, Trp, D-Trp, Trans-3,4, or
5-phenylproline, cis-3,4, or 5-phenylproline Proline P D-Pro,
L-I-thioazolidine-4-carboxylic acid, D-or
L-1-oxazolidine-4-carboxylic acid Serine S D-Ser, Thr, D-Thr,
allo-Thr, Met, D-Met, Met(O), D-Met(O), L-Cys, D-Cys Threonine T
D-Thr, Ser, D-Ser, allo-Thr, Met, D-Met, Met(O), D-Met(O), Val,
D-Val Tyrosine Y D-Tyr, Phe, D-Phe, L-Dopa, His, D-His Valine V
D-Val, Leu, D-Leu, Ile, D-Ile, Met, D-Met
[0075] Other analogs within the invention are those with
modifications which increase peptide stability; such analogs may
contain, for example, one or more non-peptide bonds (which replace
the peptide bonds) in the peptide sequence. Also included are:
analogs that include residues other than naturally occurring
L-amino acids, e.g., D-amino acids or non-naturally occurring or
synthetic amino acids, e.g., .beta. or .gamma. amino acids; and
cyclic analogs.
[0076] As used herein, the term "fragment", as applied to a S.
epidermidis analog, will ordinarily be at least about 20 residues,
more typically at least about 40 residues, preferably at least
about 60 residues in length. Fragments of S. epidermidis
polypeptides can be generated by methods known to those skilled in
the art. The ability of a Staphylococcus fragment to exhibit a
biological activity of S. epidermidis polypeptide can be assessed
by methods known to those skilled in the art as described herein.
Also included are S. epidermidis polypeptides containing residues
that are not required for biological activity of the peptide or
that result from alternative mRNA splicing or alternative protein
processing events.
[0077] An "immunogenic component" as used herein is a moiety, such
as a S. epidermidis polypeptide, analog or fragment thereof, that
is capable of eliciting a humoral and/or cellular immune response
in a host animal.
[0078] An "antigenic component" as used herein is a moiety, such as
a S. epidermidis polypeptide, analog or fragment thereof, that is
capable of binding to a specific antibody with sufficiently high
affinity to form a detectable antigen-antibody complex.
[0079] The term "antibody" as used herein is intended to include
fragments thereof which are specifically reactive with S.
epidermidis polypeptides.
[0080] As used herein, the term "cell-specific promoter" means a
DNA sequence that serves as a promoter, i.e., regulates expression
of a selected DNA sequence operably linked to the promoter, and
which effects expression of the selected DNA sequence in specific
cells of a tissue. The term also covers so-called "leaky"
promoters, which regulate expression of a selected DNA primarily in
one tissue, but cause expression in other tissues as well.
[0081] Misexpression, as used herein, refers to a non-wild type
pattern of gene expression. It includes: expression at non-wild
type levels, i.e., over or under expression; a pattern of
expression that differs from wild type in terms of the time or
stage at which the gene is expressed, e.g., increased or decreased
expression (as compared with wild type) at a predetermined
developmental period or stage; a pattern of expression that differs
from wild type in terms of increased expression (as compared with
wild type) in a predetermined cell type or tissue type; a pattern
of expression that differs from wild type in terms of the splicing
size, amino acid sequence, post-translational modification, or
biological activity of the expressed polypeptide; a pattern of
expression that differs from wild type in terms of the effect of an
environmental stimulus or extracellular stimulus on expression of
the gene, e.g., a pattern of increased or decreased expression (as
compared with wild type) in the presence of an increase or decrease
in the strength of the stimulus.
[0082] As used herein, "host cells" and other such terms denoting
microorganisms or higher eukaryotic cell lines cultured as
unicellular entities refers to cells which can become or have been
used as recipients for a recombinant vector or other transfer DNA,
and include the progeny of the original cell which has been
transfected. It is understood by individuals skilled in the art
that the progeny of a single parental cell may not necessarily be
completely identical in genomic or total DNA compliment to the
original parent, due to accident or deliberate mutation.
[0083] As used herein, the term "control sequence" refers to a
nucleic acid having a base sequence which is recognized by the host
organism to effect the expression of encoded sequences to which
they are ligated. The nature of such control sequences differs
depending upon the host organism; in prokaryotes, such control
sequences generally include a promoter, ribosomal binding site,
terminators, and in some cases operators; in eukaryotes, generally
such control sequences include promoters, terminators and in some
instances, enhancers. The term control sequence is intended to
include at a minimum, all components whose presence is necessary
for expression, and may also include additional components whose
presence is advantageous, for example, leader sequences.
[0084] As used herein, the term "operably linked" refers to
sequences joined or ligated to function in their intended manner.
For example, a control sequence is operably linked to coding
sequence by ligation in such a way that expression of the coding
sequence is achieved under conditions compatible with the control
sequence and host cell.
[0085] The "metabolism" of a substance, as used herein, means any
aspect of the expression, function, action, or regulation of the
substance. The metabolism of a substance includes modifications,
e.g., covalent or non-covalent modifications of the substance. The
metabolism of a substance includes modifications, e.g., covalent or
non-covalent modification, the substance induces in other
substances. The metabolism of a substance also includes changes in
the distribution of the substance. The metabolism of a substance
includes changes the substance induces in the distribution of other
substances.
[0086] A "sample" as used herein refers to a biological sample,
such as, for example, tissue or fluid isolated from an individual
(including without limitation plasma, serum, cerebrospinal fluid,
lymph, tears, saliva and tissue sections) or from in vitro cell
culture constituents, as well as samples from the environment.
[0087] Technical and scientific terms used herein have the meanings
commonly understood by one of ordinary skill in the art to which
the present invention pertains, unless otherwise defined. Reference
is made herein to various methodologies known to those of skill in
the art. Publications and other materials setting forth such known
methodologies to which reference is made are incorporated herein by
reference in their entireties as though set forth in full. The
practice of the invention will employ, unless otherwise indicated,
conventional techniques of chemistry, molecular biology,
microbiology, recombinant DNA, and immunology, which are within the
skill of the art. Such techniques are explained fully in the
literature. See e.g., Sambrook, Fritsch, and Maniatis, Molecular
Cloning; Laboratory Manual 2nd ed. (1989); DNA Cloning, Volumes I
and II (D. N Glover ed. 1985); Oligonucleotide Synthesis (M. J.
Gait ed, 1984); Nucleic Acid Hybridization (B. D. Hames & S. J.
Higgins eds. 1984); the series, Methods in Enzymology (Academic
Press, Inc.), particularly Vol. 154 and Vol. 155 (Wu and Grossman,
eds.); PCR-A Practical Approach (McPherson, Quirke, and Taylor,
eds., 1991); Immunology, 2d Edition, 1989, Roitt et al., C. V.
Mosby Company, and New York; Advanced Immunology, 2d Edition, 1991,
Male et al., Grower Medical Publishing, New York; DNA Cloning: A
Practical Approach, Volumes I and II, 1985 (D. N. Glover ed.);
Oligonucleotide Synthesis, 1984, (M. L. Gait ed); Transcription and
Translation, 1984 (Hames and Higgins eds.); Animal Cell Culture,
1986 (R. I. Freshney ed.); Immobilized Cells and Enzymes, 1986 (IRL
Press); Perbal, 1984, A Practical Guide to Molecular Cloning; and
Gene Transfer Vectors for Mammalian Cells, 1987 (J. H. Miller and
M. P. Calos eds., Cold Spring Harbor Laboratory);
[0088] Any suitable materials and/or methods known to those of
skill can be utilized in carrying out the present invention:
however preferred materials and/or methods are described.
Materials, reagents and the like to which reference is made in the
following description and examples are obtainable from commercial
sources, unless otherwise noted.
S. epidermidis Genomic Sequence
[0089] This invention provides nucleotide sequences of the genome
of S. epidermidis which thus comprises a DNA sequence library of S.
epidermidis genomic DNA. The detailed description that follows
provides nucleotide sequences of S. epidermidis, and also describes
how the sequences were obtained and how ORFs and protein-coding
sequences were identified. Also described are methods of using the
disclosed S. epidermidis sequences in methods including diagnostic
and therapeutic applications. Furthermore, the library can be used
as a database for identification and comparison of medically
important sequences in this and other strains of S.
epidermidis.
[0090] To determine the genomic sequence of S. epidermidis, DNA
from strain 18972 of S. epidermidis was isolated after Zymolyase
digestion, sodium dodecyl sulfate lysis, potassium acetate
precipitation, phenol:chloroform extraction and ethanol
precipitation (Soll, D. R., T. Srikantha and S. R. Lockhart:
Characterizing Developmentally Regulated Genes in S. epidermidis.
In Microbial Genome Methods. K. W. Adolph, editor. CRC Press. New
York. p 17-37). DNA was sheared hydrodynamically using an HPLC
(Oefner, et. al., 1996) to an insert size of 2000-3000 bp. After
size fractionation by gel electrophoresis the fragments were
blunt-ended, ligated to adapter oligonucleotides and cloned into
the pGTC (Thomann) vector to construct a "shotgun" subclone
library
[0091] DNA sequencing was achieved using established ABI sequencing
methods on ABI377 automated DNA sequencers. The cloning and
sequencing procedures are described in more detail in the
Exemplification.
[0092] Individual sequence reads were assembled using PHRAP (P.
Green, Abstracts of DOE Human Genome Program Contractor-Grantee
Workshop V, Jan. 1996, p. 157). The average contig length was about
3-4 kb.
[0093] All subsequent steps were based on sequencing by ABI377
automated DNA sequencing methods. The cloning and sequencing
procedures are described in more detail in the Exemplification.
[0094] A variety of approaches are used to order the contigs so as
to obtain a continuous sequence representing the entire S.
epidermidis genome. Synthetic oligonucleotides are designed that
are complementary to sequences at the end of each contig. These
oligonucleotides may be hybridized to libraries of S. epidermidis
genomic DNA in, for example, lambda phage vectors or plasmid
vectors to identify clones that contain sequences corresponding to
the junctional regions between individual contigs. Such clones are
then used to isolate template DNA and the same oligonucleotides are
used as primers in polymerase chain reaction (PCR) to amplify
junctional fragments, the nucleotide sequence of which is then
determined.
[0095] The S. epidermidis sequences were analyzed for the presence
of open reading frames (ORFs) comprising at least 180 nucleotides.
As a result of the analysis of ORFs based on stop-to-stop codon
reads, it should be understood that these ORFs may not correspond
to the ORF of a naturally-occurring S. epidermidis polypeptide.
These ORFs may contain start codons which indicate the initiation
of protein synthesis of a naturally-occurring S. epidermidis
polypeptide. Such start codons within the ORFs provided herein were
identified by those of ordinary skill in the relevant art, and the
resulting ORF and the encoded S. epidermidis polypeptide is within
the scope of this invention. For example, within the ORFs a codon
such as AUG or GUG (encoding methionine or valine) which is part of
the initiation signal for protein synthesis were identified and the
portion of an ORF to corresponding to a naturally-occurring S.
epidermidis polypeptide was recognized. The predicted coding
regions were defined by evaluating the coding potential of such
sequences with the program GENEMARK.TM. (Borodovsky and McIninch,
1993, Comp. 17:123).
[0096] Each predicted ORF amino acid sequence was compared with all
sequences found in current GENBANK, SWISS-PROT, and PIR databases
using the BLAST algorithm. BLAST identifies local alignments
occurring by chance between the ORF sequence and the sequence in
the databank (Altschal et al., 1990, L Mol. Biol. 215:403-410).
Homologous ORFs (probabilities less than 10.sup.-5 by chance) and
ORF's that are probably non-homologous (probabilities greater than
10.sup.-5 by chance) but have good codon usage were identified.
Both homologous, sequences and non-homologous sequences with good
codon usage, are likely to encode proteins and are encompassed by
the invention.
S. epidermidis Nucleic Acids
[0097] The present invention provides a library of S.
epidermidis-derived nucleic acid sequences. The libraries provide
probes, primers, and markers which are used as markers in
epidemiological studies. The present invention also provides a
library of S. epidermidis-derived nucleic acid sequences which
comprise or encode targets for therapeutic drugs.
[0098] The nucleic acids of this invention may be obtained directly
from the DNA of the above referenced S. epidermidis strain by using
the polymerase chain reaction (PCR). See "PCR, A Practical
Approach" (McPherson, Quirke, and Taylor, eds., IRL Press, Oxford,
UK, 1991) for details about the PCR. High fidelity PCR is used to
ensure a faithful DNA copy prior to expression. In addition, the
authenticity of amplified products is verified by conventional
sequencing methods. Clones carrying the desired sequences described
in this invention may also be obtained by screening the libraries
by means of the PCR or by hybridization of synthetic
oligonucleotide probes to filter lifts of the library colonies or
plaques as known in the art (see, e.g., Sambrook et al., Molecular
Cloning, A Laboratory Manual 2nd edition, 1989, Cold Spring Harbor
Press, NY).
[0099] It is also possible to obtain nucleic acids encoding S.
epidermidis polypeptides from a cDNA library in accordance with
protocols herein described. A cDNA encoding a S. epidermidis
polypeptide can be obtained by isolating total mRNA from an
appropriate strain. Double stranded cDNAs can then be prepared from
the total mRNA. Subsequently, the cDNAs can be inserted into a
suitable plasmid or viral (e.g., bacteriophage) vector using any
one of a number of known techniques. Genes encoding S. epidermidis
polypeptides can also be cloned using established polymerase chain
reaction techniques in accordance with the nucleotide sequence
information provided by the invention. The nucleic acids of the
invention can be DNA or RNA. Preferred nucleic acids of the
invention are contained in the Sequence Listing.
[0100] The nucleic acids of the invention can also be chemically
synthesized using standard techniques. Various methods of
chemically synthesizing polydeoxynucleotides are known, including
solid-phase synthesis which, like peptide synthesis, has been fully
automated in commercially available DNA synthesizers (See e.g.,
Itakura et al. U.S. Pat. No. 4,598,049; Caruthers et al. U.S. Pat.
No. 4,458,066; and Itakura U.S. Pat. Nos. 4,401,796 and 4,373,071,
incorporated by reference herein).
[0101] In another example, DNA can be chemically synthesized using,
e.g., the phosphoramidite solid support method of Matteucci et al.,
1981, J. Am. Chem. Soc. 103:3185, the method of Yoo et al., 1989,
J. Biol. Chem. 764:17078, or other well known methods. This can be
done by sequentially linking a series of oligonucleotide cassettes
comprising pairs of synthetic oligonucleotides, as described
below.
[0102] Nucleic acids isolated or synthesized in accordance with
features of the present invention are useful, by way of example,
without limitation, as probes, primers, capture ligands, antisense
genes and for developing expression systems for the synthesis of
proteins and peptides corresponding to such sequences. As probes,
primers, capture ligands and antisense agents, the nucleic acid
normally consists of all or part (approximately twenty or more
nucleotides for specificity as well as the ability to form stable
hybridization products) of the nucleic acids of the invention
contained in the Sequence Listing. These uses are described in
further detail below.
[0103] Probes
[0104] A nucleic acid isolated or synthesized in accordance with
the sequence of the invention contained in the Sequence Listing can
be used as a probe to specifically detect S. epidermidis. With the
sequence information set forth in the present application,
sequences of twenty or more nucleotides are identified which
provide the desired inclusively and exclusivity with respect to S.
epidermidis, and extraneous nucleic acids likely to be encountered
during hybridization conditions. More preferably, the sequence will
comprise at least twenty to thirty nucleotides to convey stability
to the hybridization product formed between the probe and the
intended target molecules.
[0105] Sequences larger than 1000 nucleotides in length are
difficult to synthesize but can be generated by recombinant DNA
techniques. Individuals skilled in the art will readily recognize
that the nucleic acids, for use as probes, can be provided with a
label to facilitate detection of a hybridization product.
[0106] Nucleic acid isolated and synthesized in accordance with the
sequence of the invention contained in the Sequence Listing can
also be useful as probes to detect homologous regions (especially
homologous genes) of other Staphylococcus species using appropriate
stringency hybridization conditions as described herein.
[0107] Capture Ligand
[0108] For use as a capture ligand, the nucleic acid selected in
the manner described above with respect to probes, can be readily
associated with a support. The manner in which nucleic acid is
associated with supports is well known. Nucleic acid having twenty
or more nucleotides in a sequence of the invention contained in the
Sequence Listing have utility to separate S. epidermidis nucleic
acid from one strain from the nucleic acid of other another strain
as well as from other organisms. Nucleic acid having twenty or more
nucleotides in a sequence of the invention contained in the
Sequence Listing can also have utility to separate other
Staphylococcus species from each other and from other organisms.
Preferably, the sequence will comprise at least twenty nucleotides
to convey stability to the hybridization product formed between the
probe and the intended target molecules. Sequences larger than 1000
nucleotides in length are difficult to synthesize but can be
generated by recombinant DNA techniques.
[0109] Primers
[0110] Nucleic acid isolated or synthesized in accordance with the
sequences described herein have utility as primers for the
amplification of S. epidermidis nucleic acid. These nucleic acids
may also have utility as primers for the amplification of nucleic
acids in other Staphylococcus species. With respect to polymerase
chain reaction (PCR) techniques, nucleic acid sequences of
.gtoreq.10.sup.-15 nucleotides of the invention contained in the
Sequence Listing have utility in conjunction with suitable enzymes
and reagents to create copies of S. epidermidis nucleic acid. More
preferably, the sequence will comprise twenty or more nucleotides
to convey stability to the hybridization product formed between the
primer and the intended target molecules. Binding conditions of
primers greater than 100 nucleotides are more difficult to control
to obtain specificity. High fidelity PCR can be used to ensure a
faithful DNA copy prior to expression. In addition, amplified
products can be checked by conventional sequencing methods.
[0111] The copies can be used in diagnostic assays to detect
specific sequences, including genes from S. epidermidis and/or
other Staphylococcus species. The copies can also be incorporated
into cloning and expression vectors to generate polypeptides
corresponding to the nucleic acid synthesized by PCR, as is
described in greater detail herein.
[0112] The nucleic acids of the present invention find use as
templates for the recombinant production of S. epidermidis-derived
peptides or polypeptides
[0113] Antisense
[0114] Nucleic acid or nucleic acid-hybridizing derivatives
isolated or synthesized in accordance with the sequences described
herein have utility as antisense agents to prevent the expression
of S. epidermidis genes. These sequences also have utility as
antisense agents to prevent expression of genes of other
Staphylococcus species.
[0115] In one embodiment, nucleic acid or derivatives corresponding
to S. epidermidis nucleic acids is loaded into a suitable carrier
such as a liposome or bacteriophage for introduction into bacterial
cells. For example, a nucleic acid having twenty or more
nucleotides is capable of binding to bacteria nucleic acid or
bacteria messenger RNA. Preferably, the antisense nucleic acid is
comprised of 20 or more nucleotides to provide necessary stability
of a hybridization product of non-naturally occurring nucleic acid
and bacterial nucleic acid and/or bacterial messenger RNA. Nucleic
acid having a sequence greater than 1000 nucleotides in length is
difficult to synthesize but can be generated by recombinant DNA
techniques. Methods for loading antisense nucleic acid in liposomes
is known in the art as exemplified by U.S. Pat. No. 4,241,046
issued Dec. 23, 1980 to Papahadjopoulos et al.
[0116] The present invention encompasses isolated polypeptides and
nucleic acids derived from S. epidermidis that are useful as
reagents for diagnosis of bacterial infection, components of
effective anti-bacterial vaccines, and/or as targets for
anti-bacterial drugs, including anti-S. epidermidis drugs.
Expression of S. epidermidis Nucleic Acids
[0117] Table 2, which is appended herewith and which forms part of
the present specification, provides a list of open reading frames
(ORFs) in both strands and a putative identification of the
particular function of a polypeptide which is encoded by each ORF,
based on the homology match (determined by the BLAST algorithm) of
the predicted polypeptide with known proteins encoded by ORFs in
other organisms. An ORF is a region of nucleic acid which encodes a
polypeptide. This region may represent a portion of a coding
sequence or a total sequence and was determined from stop to stop
codons. The first column contains a designation for the contig from
which each ORF was identified (numbered arbitrarily). Each contig
represents a continuous stretch of the genomic sequence of the
organism. The second column lists the ORF designation. The third
and fourth columns list the SEQ ID numbers for the nucleic acid and
amino acid sequences corresponding to each ORF, respectively. The
fifth and sixth columns list the length of the nucleic acid and the
length of the amino acid, respectively. The nucleotide sequence
corresponding to each ORF designation begins at the first
nucleotide immediately following a stop codon and ends at the
nucleotide immediately preceding the next downstream stop codon in
the same reading frame. It will be recognized by one skilled in the
art that the natural translation initiation sites will correspond
to ATG, GTG, or TTG codons located within the ORFs. The natural
initiation sites depend not only on the sequence of a start codon
but also on the context of the DNA sequence adjacent to the start
codon. Usually, a recognizable ribosome binding site is found
within 20 nucleotides upstream from the initiation codon. In some
cases where genes are translationally coupled and coordinately
expressed together in "operons", ribosome binding sites are not
present, but the initiation codon of a downstream gene may occur
very close to, or overlap, the stop codon of the an upstream gene
in the same operon. The correct start codons can be generally
identified without undue experimentation because only a few codons
need be tested. It is recognized that the translational machinery
in bacteria initiates all polypeptide chains with the amino acid
methionine, regardless of the sequence of the start codon. In some
cases, polypeptides are post-translationally modified, resulting in
an N-terminal amino acid other than methionine in vivo. The seventh
and eighth columns provide metrics for assessing the likelihood of
the homology match (determined by the BLASTP2 algorithm), as is
known in the art, to the genes indicated in the eleventh column
when the designated ORF was compared against a non-redundant
comprehensive protein database. Specifically, the seventh column
represents the "Blast Score" for the match (a higher score is a
better match), and the eighth column represents the "P-value" for
the match (the probability that such a match can have occurred by
chance; the lower the value, the more likely the match is valid).
If a BLASTP2 score of less than 46 was obtained, no value is
reported in the table the "P-value". Column nine, Subject
Taxonomy," provides the name of the organism that was identified as
having the closest homology match. The tenth column, "Subject
Name," provides where available, either a public database accession
number or our own sequence name. The eleventh column provides,
where available, the Swissprot accession number (SP), the locus
name (LN), the Organism (OR), Source of variant (SR), E.C. number
(EC), the gene name (GN), the product name (PN), the Function
Description (FN), Left End (LE), Right End (RE), Coding Direction
(DI), and the description (DE) or notes (NT) for each ORF.
Information that is not preceded by a code designation in the
eleventh column represents a description of the ORF. This
information allows one of ordinary skill in the art to determine a
potential use for each identified coding sequence and, as a result,
allows use of the polypeptides of the present invention for
commercial and industrial purposes.
[0118] Using the information provided in SEQ ID NO: 1-SEQ ID NO:
2837 and in Table 2 together with routine cloning and sequencing
methods, one of ordinary skill in the art will be able to clone and
sequence all the nucleic acid fragments of interest including open
reading frames (ORFs) encoding a large variety proteins of S.
epidermidis.
[0119] Nucleic acid isolated or synthesized in accordance with the
sequences described herein have utility to generate polypeptides.
The nucleic acid of the invention exemplified in SEQ ID NO: 1-SEQ
ID NO: 2837 and in Table 2 or fragments of said nucleic acid
encoding active portions of S. epidermidis polypeptides can be
cloned into suitable vectors or used to isolate nucleic acid. The
isolated nucleic acid is combined with suitable DNA linkers and
cloned into a suitable vector.
[0120] The function of a specific gene or operon can be ascertained
by expression in a bacterial strain under conditions where the
activity of the gene product(s) specified by the gene or operon in
question can be specifically measured. Alternatively, a gene
product may be produced in large quantities in an expressing strain
for use as an antigen, an industrial reagent, for structural
studies, etc. This expression can be accomplished in a mutant
strain which lacks the activity of the gene to be tested, or in a
strain that does not produce the same gene product(s). This
includes, but is not limited to, Eucaryotic species such as the
yeast Saccharomyces cerevisiae, Methanobacterium strains or other
Archaea, and Eubacteria such as E. coli, B. Subtilis, S. Aureus, S.
Pneumonia or Pseudomonas putida. In some cases the expression host
will utilize the natural S. epidermidis promoter whereas in others,
it will be necessary to drive the gene with a promoter sequence
derived from the expressing organism (e.g., an E. coli
beta-galactosidase promoter for expression in E. coli).
[0121] To express a gene product using the natural S. epidermidis
promoter, a procedure such as the following can be used. A
restriction fragment containing the gene of interest, together with
its associated natural promoter element and regulatory sequences
(identified using the DNA sequence data) is cloned into an
appropriate recombinant plasmid containing an origin of replication
that functions in the host organism and an appropriate selectable
marker. This can be accomplished by a number of procedures known to
those skilled in the art. It is most preferably done by cutting the
plasmid and the fragment to be cloned with the same restriction
enzyme to produce compatible ends that can be ligated to join the
two pieces together. The recombinant plasmid is introduced into the
host organism by, for example, electroporation and cells containing
the recombinant plasmid are identified by selection for the marker
on the plasmid. Expression of the desired gene product is detected
using an assay specific for that gene product.
[0122] In the case of a gene that requires a different promoter,
the body of the gene (coding sequence) is specifically excised and
cloned into an appropriate expression plasmid. This subcloning can
be done by several methods, but is most easily accomplished by PCR
amplification of a specific fragment and ligation into an
expression plasmid after treating the PCR product with a
restriction enzyme or exonuclease to create suitable ends for
cloning.
[0123] A suitable host cell for expression of a gene can be any
procaryotic or eucaryotic cell. Suitable methods for transforming
host cells can be found in Sambrook et al. (Molecular Cloning: A
Laboratory Manual, 2nd Edition, Cold Spring Harbor Laboratory press
(1989)), and other laboratory textbooks.
[0124] For example, a host cell transfected with a nucleic acid
vector directing expression of a nucleotide sequence encoding a S.
epidermidis polypeptide can be cultured under appropriate
conditions to allow expression of the polypeptide to occur.
Suitable media for cell culture are well known in the art.
Polypeptides of the invention can be isolated from cell culture
medium, host cells, or both using techniques known in the art for
purifying proteins including ion-exchange chromatography, gel
filtration chromatography, ultrafiltration, electrophoresis, and
immunoaffinity purification with antibodies specific for such
polypeptides. Additionally, in many situations, polypeptides can be
produced by chemical cleavage of a native protein (e.g., tryptic
digestion) and the cleavage products can then be purified by
standard techniques.
[0125] In the case of membrane bound proteins, these can be
isolated from a host cell by contacting a membrane-associated
protein fraction with a detergent forming a solubilized complex,
where the membrane-associated protein is no longer entirely
embedded in the membrane fraction and is solubilized at least to an
extent which allows it to be chromatographically isolated from the
membrane fraction. Chromatographic techniques which can be used in
the final purification step are known in the art and include
hydrophobic interaction, lectin affinity, ion exchange, dye
affinity and immunoaffinity.
[0126] One strategy to maximize recombinant S. epidermidis peptide
expression in E. coli is to express the protein in a host bacteria
with an impaired capacity to proteolytically cleave the recombinant
protein (Gottesman, S., Gene Expression Technology: Methods in
Enzymology 185, Academic Press, San Diego, Calif. (1990) 1119-128).
Another strategy would be to alter the nucleic acid encoding a S.
epidermidis peptide to be inserted into an expression vector so
that the individual codons for each amino acid would be those
preferentially utilized in highly expressed E. coli proteins (Wada
et al., (1992) Nuc. Acids Res. 20:2111-2118). Such alteration of
nucleic acids of the invention can be carried out by standard DNA
synthesis techniques.
[0127] The nucleic acids of the invention can also be chemically
synthesized using standard techniques. Various methods of
chemically synthesizing polydeoxynucleotides are known, including
solid-phase synthesis which, like peptide synthesis, has been fully
automated in commercially available DNA synthesizers (See, e.g.,
Itakura et al. U.S. Pat. No. 4,598,049; Caruthers et al. U.S. Pat.
No. 4,458,066; and Itakura U.S. Pat. Nos. 4,401,796 and 4,373,071,
incorporated by reference herein).
[0128] The present invention provides a library of S.
epidermidis-derived nucleic acid sequences. The libraries provide
probes, primers, and markers which can be used as markers in
epidemiological studies. The present invention also provides a
library of S. epidermidis-derived nucleic acid sequences which
comprise or encode targets for therapeutic drugs.
[0129] Nucleic acids comprising any of the sequences disclosed
herein or sub-sequences thereof can be prepared by standard methods
using the nucleic acid sequence information provided in SEQ ID NO:
1-SEQ ID NO: 2837. For example, DNA can be chemically synthesized
using, e.g., the phosphoramidite solid support method of Matteucci
et al., 1981, J. Am. Chem. Soc. 103:3185, the method of Yoo et al.,
1989, J. Biol. Chem. 764:17078, or other well known methods. This
can be done by sequentially linking a series of oligonucleotide
cassettes comprising pairs of synthetic oligonucleotides, as
described below.
[0130] Of course, due to the degeneracy of the genetic code, many
different nucleotide sequences can encode polypeptides having the
amino acid sequences defined by SEQ ID NO: 2838-SEQ ID NO: 5674 or
sub-sequences thereof. The codons can be selected for optimal
expression in prokaryotic or eukaryotic systems. Such degenerate
variants are also encompassed by this invention.
[0131] Insertion of nucleic acids (typically DNAs) encoding the
polypeptides of the invention into a vector is easily accomplished
when the termini of both the DNAs and the vector comprise
compatible restriction sites. If this cannot be done, it may be
necessary to modify the termini of the DNAs and/or vector by
digesting back single-stranded DNA overhangs generated by
restriction endonuclease cleavage to produce blunt ends, or to
achieve the same result by filling in the single-stranded termini
with an appropriate DNA polymerase.
[0132] Alternatively, any site desired may be produced, e.g., by
ligating nucleotide sequences (linkers) onto the termini. Such
linkers may comprise specific oligonucleotide sequences that define
desired restriction sites. Restriction sites can also be generated
by the use of the polymerase chain reaction (PCR). See, e.g., Saiki
et al., 1988, Science 239:48. The cleaved vector and the DNA
fragments may also be modified if required by homopolymeric
tailing.
[0133] The nucleic acids of the invention may be isolated directly
from cells. Alternatively, the polymerase chain reaction (PCR)
method can be used to produce the nucleic acids of the invention,
using either chemically synthesized strands or genomic material as
templates. Primers used for PCR can be synthesized using the
sequence information provided herein and can further be designed to
introduce appropriate new restriction sites, if desirable, to
facilitate incorporation into a given vector for recombinant
expression.
[0134] The nucleic acids of the present invention may be flanked by
natural S. epidermidis regulatory sequences, or may be associated
with heterologous sequences, including promoters, enhancers,
response elements, signal sequences, polyadenylation sequences,
introns, 5'- and 3'-noncoding regions, and the like. The nucleic
acids may also be modified by many means known in the art.
Non-limiting examples of such modifications include methylation,
"caps", substitution of one or more of the naturally occurring
nucleotides with an analog, internucleotide modifications such as,
for example, those with uncharged linkages (e.g., methyl
phosphonates, phosphotriesters, phosphoroamidates, carbamates,
etc.) and with charged linkages (e.g., phosphorothioates,
phosphorodithioates, etc.). Nucleic acids may contain one or more
additional covalently linked moieties, such as, for example,
proteins (e.g., nucleases, toxins, antibodies, signal peptides,
poly-L-lysine, etc.), intercalators (e.g., acridine, psoralen,
etc.), chelators (e.g., metals, radioactive metals, iron, oxidative
metals, etc.), and alkylators. PNAs are also included. The nucleic
acid may be derivatized by formation of a methyl or ethyl
phosphotriester or an alkyl phosphoramidate linkage. Furthermore,
the nucleic acid sequences of the present invention may also be
modified with a label capable of providing a detectable signal,
either directly or indirectly. Exemplary labels include
radioisotopes, fluorescent molecules, biotin, and the like.
[0135] The invention also provides nucleic acid vectors comprising
the disclosed S. epidermidis-derived sequences or derivatives or
fragments thereof. A large number of vectors, including plasmid and
bacterial vectors, have been described for replication and/or
expression in a variety of eukaryotic and prokaryotic hosts, and
may be used for cloning or protein expression.
[0136] The encoded S. epidermidis polypeptides may be expressed by
using many known vectors, such as pUC plasmids, pET plasmids
(Novagen, Inc., Madison, Wis.), or pRSET or pREP (Invitrogen, San
Diego, Calif.), and many appropriate host cells, using methods
disclosed or cited herein or otherwise known to those skilled in
the relevant art. The particular choice of vector/host is not
critical to the practice of the invention.
[0137] Recombinant cloning vectors will often include one or more
replication systems for cloning or expression, one or more markers
for selection in the host, e.g. antibiotic resistance, and one or
more expression cassettes. The inserted S. epidermidis coding
sequences may be synthesized by standard methods, isolated from
natural sources, or prepared as hybrids, etc. Ligation of the S.
epidermidis coding sequences to transcriptional regulatory elements
and/or to other amino acid coding sequences may be achieved by
known methods. Suitable host cells may be
transformed/transfected/infected as appropriate by any suitable
method including electroporation, CaCl.sub.2 mediated DNA uptake,
bacterial infection, microinjection, microprojectile, or other
established methods.
[0138] Appropriate host cells include bacteria, archebacteria,
fungi, especially yeast, and plant and animal cells, especially
mammalian cells. Of particular interest are S. epidermidis, E.
coli, B. Subtilis, Saccharomyces cerevisiae, Saccharomyces
carlsbergensis, Schizosaccharomyces pombi. SF9 cells, C129 cells,
293 cells, Neurospora, and CHO cells, COS cells, HeLa cells, and
immortalized mammalian myeloid and lymphoid cell lines. Preferred
replication systems include M13, ColE1, SV40, baculovirus, lambda,
adenovirus, and the like. A large number of transcription
initiation and termination regulatory regions have been isolated
and shown to be effective in the transcription and translation of
heterologous proteins in the various hosts. Examples of these
regions, methods of isolation, manner of manipulation, etc. are
known in the art. Under appropriate expression conditions, host
cells can be used as a source of recombinantly produced S.
epidermidis-derived peptides and polypeptides.
[0139] Advantageously, vectors may also include a transcription
regulatory element (i.e., a promoter) operably linked to the S.
epidermidis portion. The promoter may optionally contain operator
portions and/or ribosome binding sites. Non-limiting examples of
bacterial promoters compatible with E. coli include: b-lactamase
(penicillinase) promoter; lactose promoter; tryptophan (trp)
promoter; araBAD (arabinose) operon promoter; lambda-derived
P.sub.1 promoter and N gene ribosome binding site; and the hybrid
tac promoter derived from sequences of the trp and lac UV5
promoters. Non-limiting examples of yeast promoters include
3-phosphoglycerate kinase promoter, glyceraldehyde-3-phosphate
dehydrogenase (GAPDH) promoter, galactokinase (GAL1) promoter,
galactoepimerase promoter, and alcohol dehydrogenase (ADH)
promoter. Suitable promoters for mammalian cells include without
limitation viral promoters such as that from Simian Virus 40
(SV40), Rous sarcoma virus (RSV), adenovirus (ADV), and bovine
papilloma virus (BPV). Mammalian cells may also require terminator
sequences, polyA addition sequences and enhancer sequences to
increase expression. Sequences which cause amplification of the
gene may also be desirable. Furthermore, sequences that facilitate
secretion of the recombinant product from cells, including, but not
limited to, bacteria, yeast, and animal cells, such as secretory
signal sequences and/or prohormone pro region sequences, may also
be included. These sequences are well described in the art.
[0140] Nucleic acids encoding wild-type or variant S.
epidermidis-derived polypeptides may also be introduced into cells
by recombination events. For example, such a sequence can be
introduced into a cell, and thereby effect homologous recombination
at the site of an endogenous gene or a sequence with substantial
identity to the gene. Other recombination-based methods such as
nonhomologous recombinations or deletion of endogenous genes by
homologous recombination may also be used.
[0141] The nucleic acids of the present invention find use as
templates for the recombinant production of S. epidermidis-derived
peptides or polypeptides.
Identification and Use of S. epidermidis Nucleic Acid Sequences
[0142] The disclosed S. epidermidis polypeptide and nucleic acid
sequences, or other sequences that are contained within ORFs,
including complete protein-coding sequences, of which any of the
disclosed S. epidermidis-specific sequences forms a part, are
useful as target components for diagnosis and/or treatment of S.
epidermidis-caused infection
[0143] It will be understood that the sequence of an entire
protein-coding sequence of which each disclosed nucleic acid
sequence forms a part can be isolated and identified based on each
disclosed sequence. This can be achieved, for example, by using an
isolated nucleic acid encoding the disclosed sequence, or fragments
thereof, to prime a sequencing reaction with genomic S. epidermidis
DNA as template; this is followed by sequencing the amplified
product. The isolated nucleic acid encoding the disclosed sequence,
or fragments thereof, can also be hybridized to S. epidermidis
genomic libraries to identify clones containing additional complete
segments of the protein-coding sequence of which the shorter
sequence forms a part. Then, the entire protein-coding sequence, or
fragments thereof, or nucleic acids encoding all or part of the
sequence, or sequence-conservative or function-conservative
variants thereof, may be employed in practicing the present
invention.
[0144] Preferred sequences are those that are useful in diagnostic
and/or therapeutic applications. Diagnostic applications include
without limitation nucleic-acid-based and antibody-based methods
for detecting bacterial infection. Therapeutic applications include
without limitation vaccines, passive immunotherapy, and drug
treatments directed against gene products that are both unique to
bacteria and essential for growth and/or replication of
bacteria.
Identification of Nucleic Acids Encoding Vaccine Components and
Targets for Agents Effective Against S. epidermidis
[0145] The disclosed S. epidermidis genome sequence includes
segments that direct the synthesis of ribonucleic acids and
polypeptides, as well as origins of replication, promoters, other
types of regulatory sequences, and intergenic nucleic acids. The
invention encompasses nucleic acids encoding immunogenic components
of vaccines and targets for agents effective against S.
epidermidis. Identification of said immunogenic components involved
in the determination of the function of the disclosed sequences,
which can be achieved using a variety of approaches. Non-limiting
examples of these approaches are described briefly below.
Homology to Known Sequences:
[0146] Computer-assisted comparison of the disclosed S. epidermidis
sequences with previously reported sequences present in publicly
available databases is useful for identifying functional S.
epidermidis nucleic acid and polypeptide sequences. It will be
understood that protein-coding sequences, for example, may be
compared as a whole, and that a high degree of sequence homology
between two proteins (such as, for example, >80-90%) at the
amino acid level indicates that the two proteins also possess some
degree of functional homology, such as, for example, among enzymes
involved in metabolism, DNA synthesis, or cell wall synthesis, and
proteins involved in transport, cell division, etc. In addition,
many structural features of particular protein classes have been
identified and correlate with specific consensus sequences, such
as, for example, binding domains for nucleotides, DNA, metal ions,
and other small molecules; sites for covalent modifications such as
phosphorylation, acylation, and the like; sites of protein:protein
interactions, etc. These consensus sequences may be quite short and
thus may represent only a fraction of the entire protein-coding
sequence. Identification of such a feature in a S. epidermidis
sequence is therefore useful in determining the function of the
encoded protein and identifying useful targets of antibacterial
drugs.
[0147] Of particular relevance to the present invention are
structural features that are common to secretory, transmembrane,
and surface proteins, including secretion signal peptides and
hydrophobic transmembrane domains. S. epidermidis proteins
identified as containing putative signal sequences and/or
transmembrane domains are useful as immunogenic components of
vaccines.
[0148] Targets for therapeutic drugs according to the invention
include, but are not limited to, polypeptides, of the invention,
whether unique to S. epidermidis or not, that are essential for
growth and/or viability of S. epidermidis under at least one growth
condition. Polypeptides essential for growth and/or viability can
be determined by examining the effect of deleting and/or disrupting
the genes, i.e., by so-called gene "knockout". Alternatively,
genetic footprinting can be used (Smith et al., 1995, Proc. Natl.
Acad. Sci. USA 92:5479-6433; Published International Application WO
94/26933; U.S. Pat. No. 5,612,180). Still other methods for
assessing essentiality includes the ability to isolate conditional
lethal mutations in the specific gene (e.g., temperature sensitive
mutations). Other useful targets for therapeutic drugs, which
include polypeptides that are not essential for growth or viability
per se but lead to loss of viability of the cell, can be used to
target therapeutic agents to cells.
Strain-Specific Sequences:
[0149] Because of the evolutionary relationship between different
S. epidermidis strains, it is believed that the presently disclosed
S. epidermidis sequences are useful for identifying, and/or
discriminating between, previously known and new S. epidermidis
strains. It is believed that other S. epidermidis strains will
exhibit at least 70% sequence homology with the presently disclosed
sequence. Systematic and routine analyses of DNA sequences derived
from samples containing S. epidermidis strains, and comparison with
the present sequence allows for the identification of sequences
that can be used to discriminate between strains, as well as those
that are common to all S. epidermidis strains. In one embodiment,
the invention provides nucleic acids, including probes, and peptide
and polypeptide sequences that discriminate between different
strains of S. epidermidis. Strain-specific components can also be
identified functionally by their ability to elicit or react with
antibodies that selectively recognize one or more S. epidermidis
strains.
[0150] In another embodiment, the invention provides nucleic acids,
including probes, and peptide and polypeptide sequences that are
common to all S. epidermidis strains but are not found in other
bacterial species.
S. epidermidis Polypeptides
[0151] This invention encompasses isolated S. epidermidis
polypeptides encoded by the disclosed S. epidermidis genomic
sequences, including the polypeptides of the invention contained in
the Sequence Listing. Polypeptides of the invention are preferably
at least 5 amino acid residues in length. Using the DNA sequence
information provided herein, the amino acid sequences of the
polypeptides encompassed by the invention can be deduced using
methods well-known in the art. It will be understood that the
sequence of an entire nucleic acid encoding a S. epidermidis
polypeptide can be isolated and identified based on an ORF that
encodes only a fragment of the cognate protein-coding region. This
can be achieved, for example, by using the isolated nucleic acid
encoding the ORF, or fragments thereof, to prime a polymerase chain
reaction with genomic S. epidermidis DNA as template; this is
followed by sequencing the amplified product.
[0152] The polypeptides of the present invention, including
function-conservative variants of the disclosed ORFs, may be
isolated from wild-type or mutant S. epidermidis cells, or from
heterologous organisms or cells (including, but not limited to,
bacteria, fungi, insect, plant, and mammalian cells) including S.
epidermidis into which a S. epidermidis-derived protein-coding
sequence has been introduced and expressed. Furthermore, the
polypeptides may be part of recombinant fusion proteins.
[0153] S. epidermidis polypeptides of the invention can be
chemically synthesized using commercially automated procedures such
as those referenced herein, including, without limitation,
exclusive solid phase synthesis, partial solid phase methods,
fragment condensation or classical solution synthesis. The
polypeptides are preferably prepared by solid phase peptide
synthesis as described by Merrifield, 1963, J. Am. Chem. Soc.
85:2149. The synthesis is carried out with amino acids that are
protected at the alpha-amino terminus. Trifunctional amino acids
with labile side-chains are also protected with suitable groups to
prevent undesired chemical reactions from occurring during the
assembly of the polypeptides. The alpha-amino protecting group is
selectively removed to allow subsequent reaction to take place at
the amino-terminus. The conditions for the removal of the
alpha-amino protecting group do not remove the side-chain
protecting groups.
[0154] Methods for polypeptide purification are well-known in the
art, including, without limitation, preparative disc-gel
electrophoresis, isoelectric focusing, HPLC, reversed-phase HPLC,
gel filtration, ion exchange and partition chromatography, and
countercurrent distribution. For some purposes, it is preferable to
produce the polypeptide in a recombinant system in which the S.
epidermidis protein contains an additional sequence tag that
facilitates purification, such as, but not limited to, a
polyhistidine sequence. The polypeptide can then be purified from a
crude lysate of the host cell by chromatography on an appropriate
solid-phase matrix. Alternatively, antibodies produced against a S.
epidermidis protein or against peptides derived therefrom can be
used as purification reagents. Other purification methods are
possible.
[0155] The present invention also encompasses derivatives and
homologues of S. epidermidis-encoded polypeptides. For some
purposes, nucleic acid sequences encoding the peptides may be
altered by substitutions, additions, or deletions that provide for
functionally equivalent molecules, i.e., function-conservative
variants. For example, one or more amino acid residues within the
sequence can be substituted by another amino acid of similar
properties, such as, for example, positively charged amino acids
(arginine, lysine, and histidine); negatively charged amino acids
(aspartate and glutamate); polar neutral amino acids; and non-polar
amino acids.
[0156] The isolated polypeptides may be modified by, for example,
phosphorylation, sulfation, acylation, or other protein
modifications. They may also be modified with a label capable of
providing a detectable signal, either directly or indirectly,
including, but not limited to, radioisotopes and fluorescent
compounds.
[0157] To identify S. epidermidis-derived polypeptides for use in
the present invention, essentially the complete genomic sequence of
a Staphyolococcus epidermidis isolate was analyzed. While, in very
rare instances, a nucleic acid sequencing error may be revealed,
resolving a rare sequencing error is well within the art, and such
an occurrence will not prevent one skilled in the art from
practicing the invention.
[0158] Also encompassed are any S. epidermidis polypeptide
sequences that are contained within the open reading frames (ORFs),
including complete protein-coding sequences, of which any of SEQ ID
NO: 2838-SEQ ID NQ: 5674 forms a part. Table 2, which is appended
herewith and which forms part of the present specification,
provides a putative identification of the particular function of a
polypeptide which is encoded by each ORF, based on the homology
match (determined by the BLAST algorithm) of the predicted
polypeptide with known proteins encoded by ORFs in other organisms.
As a result, one skilled in the art can use the polypeptides of the
present invention for commercial and industrial purposes consistent
with the type of putative identification of the polypeptide.
[0159] The present invention provides a library of S.
epidermidis-derived polypeptide sequences, and a corresponding
library of nucleic acid sequences encoding the polypeptides,
wherein the polypeptides themselves, or polypeptides contained
within ORFs of which they form a part, comprise sequences that are
contemplated for use as components of vaccines. Non-limiting
examples of such sequences are listed by SEQ ID NO in Table 2,
which is appended herewith and which forms part of the present
specification.
[0160] The present invention also provides a library of S.
epidermidis-derived polypeptide sequences, and a corresponding
library of nucleic acid sequences encoding the polypeptides,
wherein the polypeptides themselves, or polypeptides contained
within ORFs of which they form a part, comprise sequences lacking
homology to any known prokaryotic or eukaryotic sequences. Such
libraries provide probes, primers, and markers which can be used to
diagnose S. epidermidis infection, including use as markers in
epidemiological studies. Non-limiting examples of such sequences
are listed by SEQ ID NO in Table 2, which is appended The present
invention also provides a library of S. epidermidis-derived
polypeptide sequences, and a corresponding library of nucleic acid
sequences encoding the polypeptides, wherein the polypeptides
themselves, or polypeptides contained within ORFs of which they
form a part, comprise targets for therapeutic drugs.
SPECIFIC EXAMPLE
Determination of Staphylococcus Protein Antigens for Antibody and
Vaccine Development
[0161] The selection of Staphylococcus protein antigens for vaccine
development can be derived from the nucleic acids encoding S.
epidermidis polypeptides. First, the ORF's can be analyzed for
homology to other known exported or membrane proteins and analyzed
using the discriminant analysis described by Klein, et al. (Klein,
P., Kanehsia, M., and DeLisi, C. (1985) Biochimica et Biophysica
Acta 815, 468-476) for predicting exported and membrane
proteins.
[0162] Homology searches can be performed using the BLAST algorithm
contained in the Wisconsin Sequence Analysis Package (Genetics
Computer Group, University Research Park, 575 Science Drive,
Madison, Wis. 53711) to compare each predicted ORF amino acid
sequence with all sequences found in the current GenBank,
SWISS-PROT and PIR databases. BLAST searches for local alignments
between the ORF and the databank sequences and reports a
probability score which indicates the probability of finding this
sequence by chance in the database. ORF's with significant homology
(e.g. probabilities lower than 1.times.10.sup.-6 that the homology
is only due to random chance) to membrane or exported proteins
represent protein antigens for vaccine development. Possible
functions can be provided to S. epidermidis genes based on sequence
homology to genes cloned in other organisms.
[0163] Discriminant analysis (Klein, et al. supra) can be used to
examine the ORF amino acid sequences. This algorithm uses the
intrinsic information contained in the ORF amino acid sequence and
compares it to information derived from the properties of known
membrane and exported proteins. This comparison predicts which
proteins will be exported, membrane associated or cytoplasmic. ORF
amino acid sequences identified as exported or membrane associated
by this algorithm are likely protein antigens for vaccine
development.
Production of Fragments and Analogs of S. epidermidis Nucleic Acids
and Polypeptides
[0164] Based on the discovery of the S. epidermidis gene products
of the invention provided in the Sequence Listing, one skilled in
the art can alter the disclosed structure of S. epidermidis genes,
e.g., by producing fragments or analogs, and test the newly
produced structures for activity. Examples of techniques known to
those skilled in the relevant art which allow the production and
testing of fragments and analogs are discussed below. These, or
analogous methods can be used to make and screen libraries of
polypeptides, e.g., libraries of random peptides or libraries of
fragments or analogs of cellular proteins for the ability to bind
S. epidermidis polypeptides. Such screens are useful for the
identification of inhibitors of S. epidermidis.
[0165] Generation of Fragments
[0166] Fragments of a protein can be produced in several ways,
e.g., recombinantly, by proteolytic digestion, of by chemical
synthesis. Internal or terminal fragments of a polypeptide can be
generated by removing one or more nucleotides from one end (for a
terminal fragment) or both ends (for an internal fragment) of a
nucleic acid which encodes the polypeptide. Expression of the
mutagenized DNA produces polypeptide fragments. Digestion with
"end-nibbling" endonucleases can thus generate DNAs which encode an
array of fragments. DNAs which encode fragments of a protein can
also be generated by random shearing, restriction digestion or a
combination of the above-discussed methods.
[0167] Fragments can also be chemically synthesized using
techniques known in the art such as conventional Merrifield solid
phase f-Moc or t-Boc chemistry. For example, peptides of the
present invention may be arbitrarily divided into fragments of
desired length with no overlap of the fragments, or divided into
overlapping fragments of a desired length.
Alteration of Nucleic Acids and Polypeptides: Random Methods
[0168] Amino acid sequence variants of a protein can be prepared by
random mutagenesis of DNA which encodes a protein or a particular
domain or region of a protein. Useful methods include PCR
mutagenesis and saturation mutagenesis. A library of random amino
acid sequence variants can also be generated by the synthesis of a
set of degenerate oligonucleotide sequences. (Methods for screening
proteins in a library of variants are elsewhere herein).
[0169] PCR Mutagenesis
[0170] In PCR mutagenesis, reduced Taq polymerase fidelity is used
to introduce random mutations into a cloned fragment of DNA (Leunig
et al., 1989, Technique 1:11-15). The DNA region to be mutagenized
is amplified using the polymerase chain reaction (PCR) under
conditions that reduce the fidelity of DNA synthesis by Taq DNA
polymerase, e.g., by using a dGTP/dATP ratio of five and adding
Mn.sup.2+ to the PCR reaction. The pool of amplified DNA fragments
are inserted into appropriate cloning vectors to provide random
mutant libraries.
[0171] Saturation Mutagenesis
[0172] Saturation mutagenesis allows for the rapid introduction of
a large number of single base substitutions into cloned DNA
fragments (Mayers et al., 1985, Science 229:242). This technique
includes generation of mutations, e.g., by chemical treatment or
irradiation of single-stranded DNA in vitro, and synthesis of a
complimentary DNA strand. The mutation frequency can be modulated
by modulating the severity of the treatment, and essentially all
possible base substitutions can be obtained. Because this procedure
does not involve a genetic selection for mutant fragments both
neutral substitutions, as well as those that alter function, are
obtained. The distribution of point mutations is not biased toward
conserved sequence elements.
[0173] Degenerate Oligonucleotides
[0174] A library of homologs can also be generated from a set of
degenerate oligonucleotide sequences. Chemical synthesis of a
degenerate sequences can be carried out in an automatic DNA
synthesizer, and the synthetic genes then ligated into an
appropriate expression vector. The synthesis of degenerate
oligonucleotides is known in the art (see for example, Narang, S A
(1983) Tetrahedron 39:3; Itakura et al. (1981) Recombinant DNA,
Proc 3rd Cleveland Sympos. Macromolecules, ed. A G Walton,
Amsterdam: Elsevier pp 273-289; Itakura et al. (1984) Annu. Rev.
Biochem. 53:323; Itakura et al. (1984) Science 198:1056; Ike et al.
(1983) Nucleic Acid Res. 11:477. Such techniques have been employed
in the directed evolution of other proteins (see, for example,
Scott et al. (1990) Science 249:386-390; Roberts et al. (1992) PNAS
89:2429-2433; Devlin et al. (1990) Science 249: 404-406; Cwirla et
al. (1990) PNAS 87: 6378-6382; as well as U.S. Pat. Nos. 5,223,409,
5,198,346, and 5,096,815).
Alteration of Nucleic Acids and Polypeptides: Methods for Directed
Mutagenesis
[0175] Non-random or directed, mutagenesis techniques can be used
to provide specific sequences or mutations in specific regions.
These techniques can be used to create variants which include,
e.g., deletions, insertions, or substitutions, of residues of the
known amino acid sequence of a protein. The sites for mutation can
be modified individually or in series, e.g., by (1) substituting
first with conserved amino acids and then with more radical choices
depending upon results achieved, (2) deleting the target residue,
or (3) inserting residues of the same or a different class adjacent
to the located site, or combinations of options 1-3.
[0176] Alanine Scanning Mutagenesis
[0177] Alanine scanning mutagenesis is a useful method for
identification of certain residues or regions of the desired
protein that are preferred locations or domains for mutagenesis,
Cunningham and Wells (Science 244:1081-1085, 1989). In alanine
scanning, a residue or group of target residues are identified
(e.g., charged residues such as Arg, Asp, His, Lys, and Glu) and
replaced by a neutral or negatively charged amino acid (most
preferably alanine or polyalanine). Replacement of an amino acid
can affect the interaction of the amino acids with the surrounding
aqueous environment in or outside the cell. Those domains
demonstrating functional sensitivity to the substitutions are then
refined by introducing further or other variants at or for the
sites of substitution. Thus, while the site for introducing an
amino acid sequence variation is predetermined, the nature of the
mutation per se need not be predetermined. For example, to optimize
the performance of a mutation at a given site, alanine scanning or
random mutagenesis may be conducted at the target codon or region
and the expressed desired protein subunit variants are screened for
the optimal combination of desired activity.
[0178] Oligonucleotide-Mediated Mutagenesis
[0179] Oligonucleotide-mediated mutagenesis is a useful method for
preparing substitution, deletion, and insertion variants of DNA,
see, e.g., Adelman et al., (DNA 2:183, 1983). Briefly, the desired
DNA is altered by hybridizing an oligonucleotide encoding a
mutation to a DNA template, where the template is the
single-stranded form of a plasmid or bacteriophage containing the
unaltered or native DNA sequence of the desired protein. After
hybridization, a DNA polymerase is used to synthesize an entire
second complementary strand of the template that will thus
incorporate the oligonucleotide primer, and will code for the
selected alteration in the desired protein DNA. Generally,
oligonucleotides of at least 25 nucleotides in length are used. An
optimal oligonucleotide will have 12 to 15 nucleotides that are
completely complementary to the template on either side of the
nucleotide(s) coding for the mutation. This ensures that the
oligonucleotide will hybridize properly to the single-stranded DNA
template molecule. The oligonucleotides are readily synthesized
using techniques known in the art such as that described by Crea et
al. (Proc. Natl. Acad. Sci. USA, 75: 5765[1978]).
[0180] Cassette Mutagenesis
[0181] Another method for preparing variants, cassette mutagenesis,
is based on the technique described by Wells et al. (Gene,
34:315[1985]). The starting material is a plasmid (or other vector)
which includes the protein subunit DNA to be mutated. The codon(s)
in the protein subunit DNA to be mutated are identified. There must
be a unique restriction endonuclease site on each side of the
identified mutation site(s). If no such restriction sites exist,
they may be generated using the above-described
oligonucleotide-mediated mutagenesis method to introduce them at
appropriate locations in the desired protein subunit DNA. After the
restriction sites have been introduced into the plasmid, the
plasmid is cut at these sites to linearize it. A double-stranded
oligonucleotide encoding the sequence of the DNA between the
restriction sites but containing the desired mutation(s) is
synthesized using standard procedures. The two strands are
synthesized separately and then hybridized together using standard
techniques. This double-stranded oligonucleotide is referred to as
the cassette. This cassette is designed to have 3' and 5' ends that
are comparable with the ends of the linearized plasmid, such that
it can be directly ligated to the plasmid. This plasmid now
contains the mutated desired protein subunit DNA sequence.
[0182] Combinatorial Mutagenesis
[0183] Combinatorial mutagenesis can also be used to generate
mutants (Ladner et al., WO 88/06630). In this method, the amino
acid sequences for a group of homologs or other related proteins
are aligned, preferably to promote the highest homology possible.
All of the amino acids which appear at a given position of the
aligned sequences can be selected to create a degenerate set of
combinatorial sequences. The variegated library of variants is
generated by combinatorial mutagenesis at the nucleic acid level,
and is encoded by a variegated gene library. For example, a mixture
of synthetic oligonucleotides can be enzymatically ligated into
gene sequences such that the degenerate set of potential sequences
are expressible as individual peptides, or alternatively, as a set
of larger fusion proteins containing the set of degenerate
sequences.
Other Modifications of S. epidermidis Nucleic Acids and
Polypeptides
[0184] It is possible to modify the structure of a S. epidermidis
polypeptide for such purposes as increasing solubility, enhancing
stability (e.g., shelf life ex vivo and resistance to proteolytic
degradation in vivo). A modified S. epidermidis protein or peptide
can be produced in which the amino acid sequence has been altered,
such as by amino acid substitution, deletion, or addition as
described herein.
[0185] An S. epidermidis peptide can also be modified by
substitution of cysteine residues preferably with alanine, serine,
threonine, leucine or glutamic acid residues to minimize
dimerization via disulfide linkages. In addition, amino acid side
chains of fragments of the protein of the invention can be
chemically modified. Another modification is cyclization of the
peptide.
[0186] In order to enhance stability and/or reactivity, a S.
epidermidis polypeptide can be modified to incorporate one or more
polymorphisms in the amino acid sequence of the protein resulting
from any natural allelic variation. Additionally, D-amino acids,
non-natural amino acids, or non-amino acid analogs can be
substituted or added to produce a modified protein within the scope
of this invention. Furthermore, an S. epidermidis polypeptide can
be modified using polyethylene glycol (PEG) according to the method
of A. Sehon and co-workers (Wie et al., supra) to produce a protein
conjugated with PEG. In addition, PEG can be added during chemical
synthesis of the protein. Other modifications of S. epidermidis
proteins include reduction/alkylation (Tarr, Methods of Protein
Microcharacterization, J. E. Silver ed., Humana Press, Clifton N.J.
155-194 (1986)); acylation (Tarr, supra); chemical coupling to an
appropriate carrier (Mishell and Shiigi, eds, Selected Methods in
Cellular Immunology, WH Freeman, San Francisco, Calif. (1980), U.S.
Pat. No. 4,939,239; or mild formalin treatment (Marsh, (1971) Int.
Arch. of Allergy and Appl. Immunol., 41: 199-215).
[0187] To facilitate purification and potentially increase
solubility of a S. epidermidis protein or peptide, it is possible
to add an amino acid fusion moiety to the peptide backbone. For
example, hexa-histidine can be added to the protein for
purification by immobilized metal ion affinity chromatography
(Hochuli, E. et al., (1988) Bio/Technology, 6: 1321-1325). In
addition, to facilitate isolation of peptides free of irrelevant
sequences, specific endoprotease cleavage sites can be introduced
between the sequences of the fusion moiety and the peptide.
[0188] To potentially aid proper antigen processing of epitopes
within an S. epidermidis polypeptide, canonical protease sensitive
sites can be engineered between regions, each comprising at least
one epitope via recombinant or synthetic methods. For example,
charged amino acid pairs, such as KK or RR, can be introduced
between regions within a protein or fragment during recombinant
construction thereof. The resulting peptide can be rendered
sensitive to cleavage by cathepsin and/or other trypsin-like
enzymes which would generate portions of the protein containing one
or more epitopes. In addition, such charged amino acid residues can
result in an increase in the solubility of the peptide.
Primary Methods for Screening Polypeptides and Analogs
[0189] Various techniques are known in the art for screening
generated mutant gene products. Techniques for screening large gene
libraries often include cloning the gene library into replicable
expression vectors, transforming appropriate cells with the
resulting library of vectors, and expressing the genes under
conditions in which detection of a desired activity, e.g., in this
case, binding to S. epidermidis polypeptide or an interacting
protein, facilitates relatively easy isolation of the vector
encoding the gene whose product was detected. Each of the
techniques described below is amenable to high through-put analysis
for screening large numbers of sequences created, e.g., by random
mutagenesis techniques.
[0190] Two Hybrid Systems
[0191] Two hybrid assays such as the system described below (as
with the other screening methods described herein), can be used to
identify polypeptides, e.g., fragments or analogs of a
naturally-occurring S. epidermidis polypeptide, e.g., of cellular
proteins, or of randomly generated polypeptides which bind to an S.
epidermidis protein. (The S. epidermidis domain is used as the bait
protein and the library of variants are expressed as prey fusion
proteins.) In an analogous fashion, a two hybrid assay (as with the
other screening methods described herein), can be used to find
polypeptides which bind a S. epidermidis polypeptide.
[0192] Display Libraries
[0193] In one approach to screening assays, the Staphylococcus
peptides are displayed on the surface of a cell or viral particle,
and the ability of particular cells or viral particles to bind an
appropriate receptor protein via the displayed product is detected
in a "panning assay". For example, the gene library can be cloned
into the gene for a surface membrane protein of a bacterial cell,
and the resulting fusion protein detected by panning (Ladner et
al., WO 88/06630; Fuchs et al. (1991) Bio/Technology 9:1370-1371;
and Goward et al. (1992) TIBS 18:136-140). In a similar fashion, a
detectably labeled ligand can be used to score for potentially
functional peptide homologs. Fluorescently labeled ligands, e.g.,
receptors, can be used to detect homologs which retain
ligand-binding activity. The use of fluorescently labeled ligands,
allows cells to be visually inspected and separated under a
fluorescence microscope, or, where the morphology of the cell
permits, to be separated by a fluorescence-activated cell
sorter.
[0194] A gene library can be expressed as a fusion protein on the
surface of a viral particle. For instance, in the filamentous phage
system, foreign peptide sequences can be expressed on the surface
of infectious phage, thereby conferring two significant benefits.
First, since these phage can be applied to affinity matrices at
concentrations well over 10.sup.13 phage per milliliter, a large
number of phage can be screened at one time. Second, since each
infectious phage displays a gene product on its surface, if a
particular phage is recovered from an affinity matrix in low yield,
the phage can be amplified by another round of infection. The group
of almost identical E. coli filamentous phages, M13, fd., and fl,
are most often used in phage display libraries. Either of the phage
gIII or gVIII coat proteins can be used to generate fusion proteins
without disrupting the ultimate packaging of the viral particle.
Foreign epitopes can be expressed at the NH.sub.2-terminal end of
pIII and phage bearing such epitopes recovered from a large excess
of phage lacking this epitope (Ladner et al. PCT publication WO
90/02909; Garrard et al., PCT publication WO 92/09690; Marks et al.
(1992) J. Biol. Chem. 267:16007-16010; Griffiths et al. (1993) EMBO
J 12:725-734; Clackson et al. (1991) Nature 352:624-628; and Barbas
et al. (1992) PNAS 89:4457-4461).
[0195] A common approach uses the maltose receptor of E. coli (the
outer membrane protein, LamB) as a peptide fusion partner (Charbit
et al. (1986) EMBO 5, 3029-3037). Oligonucleotides have been
inserted into plasmids encoding the LamB gene to produce peptides
fused into one of the extracellular loops of the protein. These
peptides are available for binding to ligands, e.g., to antibodies,
and can elicit an immune response when the cells are administered
to animals. Other cell surface proteins, e.g., OmpA (Schorr et al.
(1991) Vaccines 91, pp. 387-392), PhoE (Agterberg, et al. (1990)
Gene 88, 37-45), and PAL (Fuchs et al. (1991) Bio/Tech 9,
1369-1372), as well as large bacterial surface structures have
served as vehicles for peptide display. Peptides can be fused to
pilin, a protein which polymerizes to form the pilus-a conduit for
interbacterial exchange of genetic information (Thiry et al. (1989)
Appl. Environ. Microbiol: 55, 984-993). Because of its role in
interacting with other cells, the pilus provides a useful support
for the presentation of peptides to the extracellular environment.
Another large surface structure used for peptide display is the
bacterial motive organ, the flagellum. Fusion of peptides to the
subunit protein flagellin offers a dense array of many peptide
copies on the host cells (Kuwajima et al. (1988) Bio/Tech. 6,
1080-1083). Surface proteins of other bacterial species have also
served as peptide fusion partners. Examples include the
Staphylococcus protein A and the outer membrane IgA protease of
Neisseria (Hansson et al. (1992) J. Bacteriol. 174, 4239-4245 and
Klauser et al. (1990) EMBO J. 9, 1991-1999).
[0196] In the filamentous phage systems and the LamB system
described above, the physical link between the peptide and its
encoding DNA occurs by the containment of the DNA within a particle
(cell or phage) that carries the peptide on its surface. Capturing
the peptide captures the particle and the DNA within. An
alternative scheme uses the DNA-binding protein LacI to form a link
between peptide and DNA (Cull et al. (1992) PNAS USA 89:1865-1869).
This system uses a plasmid containing the LacI gene with an
oligonucleotide cloning site at its 3'-end. Under the controlled
induction by arabinose, a LacI-peptide fusion protein is produced.
This fusion retains the natural ability of LacI to bind to a short
DNA sequence known as LacO operator (LacO). By installing two
copies of LacO on the expression plasmid, the LacI-peptide fusion
binds tightly to the plasmid that encoded it. Because the plasmids
in each cell contain only a single oligonucleotide sequence and
each cell expresses only a single peptide sequence, the peptides
become specifically and stablely associated with the DNA sequence
that directed its synthesis. The cells of the library are gently
lysed and the peptide-DNA complexes are exposed to a matrix of
immobilized receptor to recover the complexes containing active
peptides. The associated plasmid DNA is then reintroduced into
cells for amplification and DNA sequencing to determine the
identity of the peptide ligands. As a demonstration of the
practical utility of the method, a large random library of
dodecapeptides was made and selected on a monoclonal antibody
raised against the opioid peptide dynorphin B. A cohort of peptides
was recovered, all related by a consensus sequence corresponding to
a six-residue portion of dynorphin B. (Cull et al. (1992) Proc.
Natl. Acad. Sci. U.S.A. 89-1869)
[0197] This scheme, sometimes referred to as peptides-on-plasmids,
differs in two important ways from the phage display methods.
First, the peptides are attached to the C-terminus of the fusion
protein, resulting in the display of the library members as
peptides having free carboxy termini. Both of the filamentous phage
coat proteins, pIII and pVIII, are anchored to the phage through
their C-termini, and the guest peptides are placed into the
outward-extending N-terminal domains. In some designs, the
phage-displayed peptides are presented right at the amino terminus
of the fusion protein. (Cwirla, et al. (1990) Proc. Natl. Acad.
Sci. USA. 87, 6378-6382) A second difference is the set of
biological biases affecting the population of peptides actually
present in the libraries. The LacI fusion molecules are confined to
the cytoplasm of the host cells. The phage coat fusions are exposed
briefly to the cytoplasm during translation but are rapidly
secreted through the inner membrane into the periplasmic
compartment, remaining anchored in the membrane by their C-terminal
hydrophobic domains, with the N-termini, containing the peptides,
protruding into the periplasm while awaiting assembly into phage
particles. The peptides in the LacI and phage libraries may differ
significantly as a result of their exposure to different
proteolytic activities. The phage coat proteins require transport
across the inner membrane and signal peptidase processing as a
prelude to incorporation into phage. Certain peptides exert a
deleterious effect on these processes and are underrepresented in
the libraries (Gallop et al. (1994) J. Med. Chem. 37(9):1233-1251).
These particular biases are not a factor in the LacI display
system.
[0198] The number of small peptides available in recombinant random
libraries is enormous. Libraries of 10.sup.7-10.sup.9 independent
clones are routinely prepared. Libraries as large as 10.sup.11
recombinants have been created, but this size approaches the
practical limit for clone libraries. This limitation in library
size occurs at the step of transforming the DNA containing
randomized segments into the host bacterial cells. To circumvent
this limitation, an in vitro system based on the display of nascent
peptides in polysome complexes has recently been developed. This
display library method has the potential of producing libraries 3-6
orders of magnitude larger than the currently available
phage/phagemid or plasmid libraries. Furthermore, the construction
of the libraries, expression of the peptides, and screening, is
done in an entirely cell-free format.
[0199] In one application of this method (Gallop et al. (1994) J.
Med. Chem. 37(9):1233-1251), a molecular DNA library encoding
10.sup.12 decapeptides was constructed and the library expressed in
an E. coli S30 in vitro coupled transcription/translation system.
Conditions were chosen to stall the ribosomes on the mRNA, causing
the accumulation of a substantial proportion of the RNA in
polysomes and yielding complexes containing nascent peptides still
linked to their encoding RNA. The polysomes are sufficiently robust
to be affinity purified on immobilized receptors in much the same
way as the more conventional recombinant peptide display libraries
are screened. RNA from the bound complexes is recovered, converted
to cDNA, and amplified by PCR to produce a template for the next
round of synthesis and screening. The polysome display method can
be coupled to the phage display system. Following several rounds of
screening, cDNA from the enriched pool of polysomes was cloned into
a phagemid vector. This vector serves as both a peptide expression
vector, displaying peptides fused to the coat proteins, and as a
DNA sequencing vector for peptide identification. By expressing the
polysome-derived peptides on phage, one can either continue the
affinity selection procedure in this format or assay the peptides
on individual clones for binding activity in a phage ELISA, or for
binding specificity in a completion phage ELISA (Barret, et al.
(1992) Anal. Biochem 204,357-364). To identify the sequences of the
active peptides one sequences the DNA produced by the phagemid
host.
Secondary Screening of Polypeptides and Analogs
[0200] The high through-put assays described above can be followed
by secondary screens in order to identify further biological
activities which will, e.g., allow one skilled in the art to
differentiate agonists from antagonists. The type of a secondary
screen used will depend on the desired activity that needs to be
tested. For example, an assay can be developed in which the ability
to inhibit an interaction between a protein of interest and its
respective ligand can be used to identify antagonists from a group
of peptide fragments isolated though one of the primary screens
described above.
[0201] Therefore, methods for generating fragments and analogs and
testing them for activity are known in the art. Once the core
sequence of interest is identified, it is routine for one skilled
in the art to obtain analogs and fragments.
Peptide Mimetics of S. epidermidis Polypeptides
[0202] The invention also provides for reduction of the protein
binding domains of the subject S. epidermidis polypeptides to
generate mimetics, e.g. peptide or non-peptide agents. The peptide
mimetics are able to disrupt binding of a polypeptide to its
counter ligand, e.g., in the case of a S. epidermidis polypeptide
binding to a naturally occurring ligand. The critical residues of a
subject S. epidermidis polypeptide which are involved in molecular
recognition of a polypeptide can be determined and used to generate
S. epidermidis-derived peptidomimetics which competitively or
noncompetitively inhibit binding of the S. epidermidis polypeptide
with an interacting polypeptide (see, for example, European patent
applications EP412,762A and EP-B31,080A).
[0203] For example, scanning mutagenesis can be used to map the
amino acid residues of a particular S. epidermidis polypeptide
involved in binding an interacting polypeptide, peptidomimetic
compounds (e.g. diazepine or isoquinoline derivatives) can be
generated which mimic those residues in binding to an interacting
polypeptide, and which therefore can inhibit binding of a S.
epidermidis polypeptide to an interacting polypeptide and thereby
interfere with the function of S. epidermidis polypeptide. For
instance, non-hydrolyzable peptide analogs of such residues can be
generated using benzodiazepine (e.g., see Freidinger et al. in
Peptides: Chemistry and Biology, G. R Marshall ed., ESCOM
Publisher: Leiden, Netherlands, 1988), azepine (e.g., see Huffman
et al. in Peptides: Chemistry and Biology, G. R. Marshall ed.,
ESCOM Publisher: Leiden, Netherlands, 1988), substituted gama
lactam rings (Garvey et al. in Peptides: Chemistry and Biology, G.
R. Marshall ed., ESCOM Publisher: Leiden, Netherlands, 1988),
keto-methylene pseudopeptides (Ewenson et al. (1986) J Med Chem
29:295; and Ewenson et al. in Peptides: Structure and Function
(Proceedings of the 9th American Peptide Symposium) Pierce Chemical
Co. Rockland, Ill., 1985), b-turn dipeptide cores (Nagai et al.
(1985) Tetrahedron Lett 26:647; and Sato et al. (1986) J Chem Soc
Perkin Trans 1:1231), and b-aminoalcohols (Gordon et al. (1985)
Biochem Biophys Res Commun 126:419; and et al. (1986) Biochem
Biophys Res Commun 134:71).
Vaccine Formulations for S. epidermidis Nucleic Acids and
Polypeptides
[0204] This invention also features vaccine compositions for
protection against infection by S. epidermidis or for treatment of
S. epidermidis infection, a gram-positive spiral bacterium. In one
embodiment, the vaccine compositions contain one or more
immunogenic components such as a surface protein from S.
epidermidis, or portion thereof, and a pharmaceutically acceptable
carrier. Nucleic acids within the scope of the invention are
exemplified by the nucleic acids of the invention contained in the
Sequence Listing which encode S. epidermidis surface proteins. Any
nucleic acid encoding an immunogenic S. epidermidis protein, or
portion thereof, which is capable of expression in a cell, can be
used in the present invention. These vaccines have therapeutic and
prophylactic utilities.
[0205] One aspect of the invention provides a vaccine composition
for protection against infection by S. epidermidis which contains
at least one immunogenic fragment of an S. epidermidis protein and
a pharmaceutically acceptable carrier. Preferred fragments include
peptides of at least about 10 amino acid residues in length,
preferably about 10-20 amino acid residues in length, and more
preferably about 12-16 amino acid residues in length.
[0206] Immunogenic components of the invention can be obtained, for
example, by screening polypeptides recombinantly produced from the
corresponding fragment of the nucleic acid encoding the full-length
S. epidermidis protein. In addition, fragments can be chemically
synthesized using techniques known in the art such as conventional
Merrifield solid phase f-Moc or t-Boc chemistry.
[0207] In one embodiment, immunogenic components are identified by
the ability of the peptide to stimulate T cells. Peptides which
stimulate T cells, as determined by, for example, T cell
proliferation or cytokine secretion are defined herein as
comprising at least one T cell epitope. T cell epitopes are
believed to be involved in initiation and perpetuation of the
immune response to the protein allergen which is responsible for
the clinical symptoms of allergy. These T cell epitopes are thought
to trigger early events at the level of the T helper cell by
binding to an appropriate HLA molecule on the surface of an antigen
presenting cell, thereby stimulating the T cell subpopulation with
the relevant T cell receptor for the epitope. These events lead to
T cell proliferation, lymphokine secretion, local inflammatory
reactions, recruitment of additional immune cells to the site of
antigen/T cell interaction, and activation of the B cell cascade,
leading to the production of antibodies. A T cell epitope is the
basic element, or smallest unit of recognition by a T cell
receptor, where the epitope comprises amino acids essential to
receptor recognition (e.g., approximately 6 or 7 amino acid
residues). Amino acid sequences which mimic those of the T cell
epitopes are within the scope of this invention.
[0208] Screening immunogenic components can be accomplished using
one or more of several different assays. For example, in vitro,
peptide T cell stimulatory activity is assayed by contacting a
peptide known or suspected of being immunogenic with an antigen
presenting cell which presents appropriate MHC molecules in a T
cell culture. Presentation of an immunogenic S. epidermidis peptide
in association with appropriate MHC molecules to T cells in
conjunction with the necessary co-stimulation has the effect of
transmitting a signal to the T cell that induces the production of
increased levels of cytokines, particularly of interleukin-2 and
interleukin-4. The culture supernatant can be obtained and assayed
for interleukin-2 or other known cytokines. For example, any one of
several conventional assays for interleukin-2 can be employed, such
as the assay described in Proc. Natl. Acad. Sci USA, 86: 1333
(1989) the pertinent portions of which are incorporated herein by
reference. A kit for an assay for the production of interferon is
also available from Genzyme Corporation (Cambridge, Mass.).
[0209] Alternatively, a common assay for T cell proliferation
entails measuring tritiated thymidine incorporation. The
proliferation of T cells can be measured in vitro by determining
the amount of .sup.3H-labeled thymidine incorporated into the
replicating DNA of cultured cells. Therefore, the rate of DNA
synthesis and, in turn, the rate of cell division can be
quantified.
[0210] Vaccine compositions of the invention containing immunogenic
components (e.g., S. epidermidis polypeptide or fragment thereof or
nucleic acid encoding an S. epidermidis polypeptide or fragment
thereof) preferably include a pharmaceutically acceptable carrier.
The term "pharmaceutically acceptable carrier" refers to a carrier
that does not cause an allergic reaction or other untoward effect
in patients to whom it is administered. Suitable pharmaceutically
acceptable carriers include, for example, one or more of water,
saline, phosphate buffered saline, dextrose, glycerol, ethanol and
the like, as well as combinations thereof. Pharmaceutically
acceptable carriers may further comprise minor amounts of auxiliary
substances such as wetting or emulsifying agents, preservatives or
buffers, which enhance the shelf life or effectiveness of the
antibody. For vaccines of the invention containing S. epidermidis
polypeptides, the polypeptide is co-administered with a suitable
adjuvant.
[0211] It will be apparent to those of skill in the art that the
therapeutically effective amount of DNA or protein of this
invention will depend, inter alia, upon the administration
schedule, the unit dose of antibody administered, whether the
protein or DNA is administered in combination with other
therapeutic agents, the immune status and health of the patient,
and the therapeutic activity of the particular protein or DNA.
[0212] Vaccine compositions are conventionally administered
parenterally, e.g., by injection, either subcutaneously or
intramuscularly. Methods for intramuscular immunization are
described by Wolff et al. (1990) Science 247: 1465-1468 and by
Sedegah et al. (1994) Immunology 91: 9866-9870. Other modes of
administration include oral and pulmonary formulations,
suppositories, and transdermal applications. Oral immunization is
preferred over parenteral methods for inducing protection against
infection by S. epidermidis. Cain et. al. (1993) Vaccine 11:
637-642. Oral formulations include such normally employed
excipients as, for example, pharmaceutical grades of mannitol,
lactose, starch, magnesium stearate, sodium saccharine, cellulose,
magnesium carbonate, and the like.
[0213] The vaccine compositions of the invention can include an
adjuvant, including, but not limited to aluminum hydroxide;
N-acetyl-muramyl-L-threonyl-D-isoglutamine (thr-MDP);
N-acetyl-nor-muramyl-L-alanyl-D-isoglutamine (CGP 11637, referred
to as nor-MDP);
N-acetylmuramyl-L-alanyl-D-isoglutaminyl-L-alanine-2-(1'-2'-dip-
almitoyl-sn-glycero-3-hydroxyphos-phoryloxy)-ethylamine (CGP
19835A, referred to a MTP-PE); RIBI, which contains three
components from bacteria; monophosphoryl lipid A; trehalose
dimycoloate; cell wall skeleton (MPL+TDM+CWS) in a 2%
squalene/Tween 80 emulsion; and cholera toxin. Others which may be
used are non-toxic derivatives of cholera toxin, including its B
subunit, and/or conjugates or genetically engineered fusions of the
S. epidermidis polypeptide with cholera toxin or its B subunit,
procholeragenoid, fungal polysaccharides, including schizophyllan,
muramyl dipeptide, muramyl dipeptide derivatives, phorbol esters,
labile toxin of E. coli, non-S. epidermidis bacterial lysates,
block polymers or saponins.
[0214] Other suitable delivery methods include biodegradable
microcapsules or immuno-stimulating complexes (ISCOMs), cochleates,
or liposomes, genetically engineered attenuated live vectors such
as viruses or bacteria, and recombinant (chimeric) virus-like
particles, e.g., bluetongue. The amount of adjuvant employed will
depend on the type of adjuvant used. For example, when the mucosal
adjuvant is cholera toxin, it is suitably used in an amount of 5 mg
to 50 mg, for example 10 mg to 35 mg. When used in the form of
microcapsules, the amount used will depend on the amount employed
in the matrix of the microcapsule to achieve the desired dosage.
The determination of this amount is within the skill of a person of
ordinary skill in the art.
[0215] Carrier systems in humans may include enteric release
capsules protecting the antigen from the acidic environment of the
stomach, and including S. epidermidis polypeptide in an insoluble
form as fusion proteins. Suitable carriers for the vaccines of the
invention are enteric coated capsules and polylactide-glycolide
microspheres. Suitable diluents are 0.2 N NaHCO3 and/or saline.
[0216] Vaccines of the invention can be administered as a primary
prophylactic agent in adults or in children, as a secondary
prevention, after successful eradication of S. epidermidis in an
infected host, or as a therapeutic agent in the aim to induce an
immune response in a susceptible host to prevent infection by S.
epidermidis. The vaccines of the invention are administered in
amounts readily determined by persons of ordinary skill in the art.
Thus, for adults a suitable dosage will be in the range of 10 mg to
10 g, preferably 10 mg to 100 mg. A suitable dosage for adults will
also be in the range of 5 mg to 500 mg. Similar dosage ranges will
be applicable for children. Those skilled in the art will recognize
that the optimal dose may be more or less depending upon the
patient's body weight, disease, the route of administration, and
other factors. Those skilled in the art will also recognize that
appropriate dosage levels can be obtained based on results with
known oral vaccines such as, for example, a vaccine based on an E.
coli lysate (6 mg dose daily up to total of 540 mg) and with an
enterotoxigenic E. coli purified antigen (4 doses of 1 mg)
(Schulman et al., J. Urol. 150:917-921 (1993); Boedecker et al.,
American Gastroenterological Assoc. 999:A-222 (1993)). The number
of doses will depend upon the disease, the formulation, and
efficacy data from clinical trials. Without intending any
limitation as to the course of treatment, the treatment can be
administered over 3 to 8 doses for a primary immunization schedule
over 1 month (Boedeker, American Gastroenterological Assoc.
888:A-222 (1993)).
[0217] In a preferred embodiment, a vaccine composition of the
invention can be based on a killed whole E. coli preparation with
an immunogenic fragment of a S. epidermidis protein of the
invention expressed on its surface or it can be based on an E. coli
lysate, wherein the killed E. coli acts as a carrier or an
adjuvant.
[0218] It will be apparent to those skilled in the art that some of
the vaccine compositions of the invention are useful only for
preventing S. epidermidis infection, some are useful only for
treating S. epidermidis infection, and some are useful for both
preventing and treating S. epidermidis infection. In a preferred
embodiment, the vaccine composition of the invention provides
protection against S. epidermidis infection by stimulating humoral
and/or cell-mediated immunity against S. epidermidis. It should be
understood that amelioration of any of the symptoms of S.
epidermidis infection is a desirable clinical goal, including a
lessening of the dosage of medication used to treat S.
epidermidis-caused disease, or an increase in the production of
antibodies in the serum or mucous of patients.
Antibodies Reactive with S. epidermidis Polypeptides
[0219] The invention also includes antibodies specifically reactive
with the subject S. epidermidis polypeptide.
Anti-protein/anti-peptide antisera or monoclonal antibodies can be
made by standard protocols (See, for example, Antibodies: A
Laboratory Manual ed. by Harlow and Lane (Cold Spring Harbor Press:
1988)). A mammal such as a mouse, a hamster or rabbit can be
immunized with an immunogenic form of the peptide. Techniques for
conferring immunogenicity on a protein or peptide include
conjugation to carriers or other techniques well known in the art.
An immunogenic portion of the subject S. epidermidis polypeptide
can be administered in the presence of adjuvant. The progress of
immunization can be monitored by detection of antibody titers in
plasma or serum. Standard ELISA or other immunoassays can be used
with the immunogen as antigen to assess the levels of
antibodies.
[0220] In a preferred embodiment, the subject antibodies are
immunospecific for antigenic determinants of the S. epidermidis
polypeptides of the invention, e.g. antigenic determinants of a
polypeptide of the invention contained in the Sequence Listing, or
a closely related human or non-human mammalian homolog (e.g., 90%
homologous, more preferably at least 95% homologous). In yet a
further preferred embodiment of the invention, the anti-S.
epidermidis antibodies do not substantially cross react (i.e.,
react specifically) with a protein which is for example, less than
80% percent homologous to a sequence of the invention contained in
the Sequence Listing. By "not substantially cross react", it is
meant that the antibody has a binding affinity for a non-homologous
protein which is less than 10 percent, more preferably less than 5
percent, and even more preferably less than 1 percent, of the
binding affinity for a protein of the invention contained in the
Sequence Listing. In a most preferred embodiment, there is no
cross-reactivity between bacterial and mammalian antigens.
[0221] The term antibody as used herein is intended to include
fragments thereof which are also specifically reactive with S.
epidermidis polypeptides. Antibodies can be fragmented using
conventional techniques and the fragments screened for utility in
the same manner as described above for whole antibodies. For
example, F(ab').sub.2 fragments can be generated by treating
antibody with pepsin. The resulting F(ab').sub.2 fragment can be
treated to reduce disulfide bridges to produce Fab' fragments. The
antibody of the invention is further intended to include bispecific
and chimeric molecules having an anti-S. epidermidis portion.
[0222] Both monoclonal and polyclonal antibodies (Ab) directed
against S. epidermidis polypeptides or S. epidermidis polypeptide
variants, and antibody fragments such as Fab' and F(ab').sub.2, can
be used to block the action of S. epidermidis polypeptide and allow
the study of the role of a particular S. epidermidis polypeptide of
the invention in aberrant or unwanted intracellular signaling, as
well as the normal cellular function of the S. epidermidis and by
microinjection of anti-S. epidermidis polypeptide antibodies of the
present invention.
[0223] Antibodies which specifically bind S. epidermidis epitopes
can also be used in immunohistochemical staining of tissue samples
in order to evaluate the abundance and pattern of expression of S.
epidermidis antigens. Anti-S. epidermidis polypeptide antibodies
can be used diagnostically in immuno-precipitation and
immuno-blotting to detect and evaluate S. epidermidis levels in
tissue or bodily fluid as part of a clinical testing procedure:
Likewise, the ability to monitor S. epidermidis polypeptide levels
in an individual can allow determination of the efficacy of a given
treatment regimen for an individual afflicted with such a disorder.
The level of a S. epidermidis polypeptide can be measured in cells
found in bodily fluid, such as in urine samples or can be measured
in tissue, such as produced by gastric biopsy. Diagnostic assays
using anti-S. epidermidis antibodies can include, for example,
immunoassays designed to aid in early diagnosis of S. epidermidis
infections. The present invention can also be used as a method of
detecting antibodies contained in samples from individuals infected
by this bacterium using specific S. epidermidis antigens.
[0224] Another application of anti-S. epidermidis polypeptide
antibodies of the invention is in the immunological screening of
cDNA libraries constructed in expression vectors such as
.lamda.gt11, .lamda.gt18-23, .lamda.ZAP, and .lamda.ORF8. Messenger
libraries of this type, having coding sequences inserted in the
correct reading frame and orientation, can produce fusion proteins.
For instance, .lamda.gt11 will produce fusion proteins whose amino
termini consist of .beta.-galactosidase amino acid sequences and
whose carboxy termini consist of a foreign polypeptide. Antigenic
epitopes of a subject S. epidermidis polypeptide can then be
detected with antibodies, as, for example, reacting nitrocellulose
filters lifted from infected plates with anti-S. epidermidis
polypeptide antibodies. Phage, scored by this assay, can then be
isolated from the infected plate. Thus, the presence of S.
epidermidis gene homologs can be detected and cloned from other
species, and alternate isoforms (including splicing variants) can
be detected and cloned.
Kits Containing Nucleic Acids, Polypeptides or Antibodies of the
Invention
[0225] The nucleic acid, polypeptides and antibodies of the
invention can be combined with other reagents and articles to form
kits. Kits for diagnostic purposes typically comprise the nucleic
acid, polypeptides or antibodies in vials or other suitable
vessels. Kits typically comprise other reagents for performing
hybridization reactions, polymerase chain reactions (PCR), or for
reconstitution of lyophilized components, such as aqueous media,
salts, buffers, and the like. Kits may also comprise reagents for
sample processing such as detergents, chaotropic salts and the
like. Kits may also comprise immobilization means such as
particles, supports, wells, dipsticks and the like. Kits may also
comprise labeling means such as dyes, developing reagents,
radioisotopes, fluorescent agents, luminescent or chemiluminescent
agents, enzymes, intercalating agents and the like. With the
nucleic acid and amino acid sequence information provided herein,
individuals skilled in art can readily assemble kits to serve their
particular purpose. Kits further can include instructions for
use.
Drug Screening Assays Using S. epidermidis Polypeptides
[0226] By making available purified and recombinant S. epidermidis
polypeptides, the present invention provides assays which can be
used to screen for drugs which are either agonists or antagonists
of the normal cellular function, in this case, of the subject S.
epidermidis polypeptides, or of their role in intracellular
signaling. Such inhibitors or potentiators may be useful as new
therapeutic agents to combat S. epidermidis infections in humans. A
variety of assay formats will suffice and, in light of the present
inventions, will be comprehended by the person skilled in the
art.
[0227] In many drug screening programs which test libraries of
compounds and natural extracts, high throughput assays are
desirable in order to maximize the number of compounds surveyed in
a given period of time. Assays which are performed in cell-free
systems, such as may be derived with purified or semi-purified
proteins, are often preferred as "primary" screens in that they can
be generated to permit rapid development and relatively easy
detection of an alteration in a molecular target which is mediated
by a test compound. Moreover, the effects of cellular toxicity
and/or bioavailability of the test compound can be generally
ignored in the in vitro system, the assay instead being focused
primarily on the effect of the drug on the molecular target as may
be manifest in an alteration of binding affinity with other
proteins or change in enzymatic properties of the molecular target.
Accordingly, in an exemplary screening assay of the present
invention, the compound of interest is contacted with an isolated
and purified S. epidermidis polypeptide.
[0228] Screening assays can be constructed in vitro with a purified
S. epidermidis polypeptide or fragment thereof, such as a S.
epidermidis polypeptide having enzymatic activity, such that the
activity of the polypeptide produces a detectable reaction product.
The efficacy of the compound can be assessed by generating dose
response curves from data obtained using various concentrations of
the test compound. Moreover, a control assay can also be performed
to provide a baseline for comparison. Suitable products include
those with distinctive absorption, fluorescence, or
chemi-luminescence properties, for example, because detection may
be easily automated. A variety of synthetic or naturally occurring
compounds can be tested in the assay to identify those which
inhibit or potentiate the activity of the S. epidermidis
polypeptide. Some of these active compounds may directly, or with
chemical alterations to promote membrane permeability or
solubility, also inhibit or potentiate the same activity (e.g.,
enzymatic activity) in whole, live S. epidermidis cells.
[0229] Overexpression Assays
[0230] Overexpression assays are based on the premise that
overproduction of a protein would lead to a higher level of
resistance to compounds that selectively interfere with the
function of that protein. Overexpression assays may be used to
identify compounds that interfere with the function of virtually
any type of protein, including without limitations enzymes,
receptors, DNA- or RNA-binding proteins, or any proteins that are
directly or indirectly involved in regulating cell growth.
[0231] Typically, two bacterial strains are constructed. One
contains a single copy of the gene of interest, and a second
contains several copies of the same gene. Identification of useful
inhibitory compounds of this type of assay is based on a comparison
of the activity of a test compound in inhibiting growth and/or
viability of the two strains. The method involves constructing a
nucleic acid vector that directs high level expression of a
particular target nucleic acid. The vectors are then transformed
into host cells in single or multiple copies to produce strains
that express low to moderate and high levels of protein encoding by
the target sequence (strain A and B, respectively). Nucleic acid
comprising sequences encoding the target gene can, of course, be
directly integrated into the host cell.
[0232] Large numbers of compounds (or crude substances which may
contain active compounds) are screened for their effect on the
growth of the two strains. Agents which interfere with an unrelated
target equally inhibit the growth of both strains. Agents which
interfere with the function of the target at high concentration
should inhibit the growth of both strains. It should be possible,
however, to titrate out the inhibitory effect of the compound in
the overexpressing strain. That is, if the compound is affecting
the particular target that is being tested, it should be possible
to inhibit the growth of strain A at a concentration of the
compound that allows strain B to grow.
[0233] Alternatively, a bacterial strain is constructed that
contains the gene of interest under the control of an inducible
promoter. Identification of useful inhibitory agents using this
type of assay is based on a comparison of the activity of a test
compound in inhibiting growth and/or viability of this strain under
both inducing and non-inducing conditions. The method involves
constructing a nucleic acid vector that directs high-level
expression of a particular target nucleic acid. The vector is then
transformed into host cells that are grown under both non-inducing
and inducing conditions (conditions A and B, respectively).
[0234] Large numbers of compounds (or crude substances which may
contain active compounds) are screened for their effect on growth
under these two conditions. Agents that interfere with the function
of the target should inhibit growth under both conditions. It
should be possible, however, to titrate out the inhibitory effect
of the compound in the overexpressing strain. That is, if the
compound is affecting the particular target that is being tested,
it should be possible to inhibit growth under condition A at a
concentration that allows the strain to grow under condition B.
[0235] Ligand-Binding Assays
[0236] Many of the targets according to the invention have
functions that have not yet been identified. Ligand-binding assays
are useful to identify inhibitor compounds that interfere with the
function of a particular target, even when that function is
unknown. These assays are designed to detect binding of test
compounds to particular targets. The detection may involve direct
measurement of binding. Alternatively, indirect indications of
binding may involve stabilization of protein structure or
disruption of a biological function. Non-limiting examples of
useful ligand-binding assays are detailed below.
[0237] A useful method for the detection and isolation of binding
proteins is the Biomolecular Interaction Assay (BIAcore) system
developed by Pharmacia Biosensor and described in the
manufacturer's protocol (LKB Pharmacia, Sweden). The BIAcore system
uses an affinity purified anti-GST antibody to immobilize
OST-fusion proteins onto a sensor chip. The sensor utilizes surface
plasmon resonance which is an optical phenomenon that detects
changes in refractive indices. In accordance with the practice of
the invention, a protein of interest is coated onto a chip and test
compounds are passed over the chip. Binding is detected by a change
in the refractive index (surface plasmon resonance).
[0238] A different type of ligand-binding assay involves
scintillation proximity assays (SPA, described in U.S. Pat. No.
4,568,649).
[0239] Another type of ligand binding assay, also undergoing
development, is based on the fact that proteins containing
mitochondrial targeting signals are imported into isolated
mitochondria in vitro (Hurt et al., 1985, Embo J 4:2061-2068;
Eilers and Schatz, Nature, 1986, 322:228-231). In a mitochondrial
import assay, expression vectors are constructed in which nucleic
acids encoding particular target proteins are inserted downstream
of sequences encoding mitochondrial import signals. The chimeric
proteins are synthesized and tested for their ability to be
imported into isolated mitochondria in the absence and presence of
test compounds. A test compound that binds to the target protein
should inhibit its uptake into isolated mitochondria in vitro.
[0240] Another ligand-binding assay is the yeast two-hybrid system
(Fields and Song, 1989, Nature 340:245-246). The yeast two-hybrid
system takes advantage of the properties of the GAL4 protein of the
yeast Saccharomyces cerevisiae. The GAL4 protein is a
transcriptional activator required for the expression of genes
encoding enzymes of galactose utilization. This protein consists of
two separable and functionally essential domains: an N-terminal
domain which binds to specific DNA sequences (UAS.sub.G); and a
C-terminal domain containing acidic regions, which is necessary to
activate transcription. The native GAL4 protein, containing both
domains, is a potent activator of transcription when yeast are
grown on galactose media. The N-terminal domain binds to DNA in a
sequence-specific manner but is unable to activate transcription.
The C-terminal domain contains the activating regions but cannot
activate transcription because it fails to be localized to
UAS.sub.G. In the two-hybrid system, a system of two hybrid
proteins containing parts of GAL4: (1) a GAL4 DNA-binding domain
fused to a protein `X` and (2) a GAL4 activation region fused to a
protein `Y`. If X and Y can form a protein-protein complex and
reconstitute proximity of the GAL4 domains, transcription of a gene
regulated by UAS.sub.G occurs. Creation of two hybrid proteins,
each containing one of the interacting proteins X and Y, allows the
activation region of UAS.sub.G to be brought to its normal site of
action.
[0241] The binding assay described in Fodor et al., 1991, Science
251:767-773, which involves testing the binding affinity of test
compounds for a plurality of defined polymers synthesized on a
solid substrate, may also be useful.
[0242] Compounds which bind to the polypeptides of the invention
are potentially useful as antibacterial agents for use in
therapeutic compositions.
[0243] Pharmaceutical formulations suitable for antibacterial
therapy comprise the antibacterial agent in conjunction with one or
more biologically acceptable carriers. Suitable biologically
acceptable carriers include, but are not limited to,
phosphate-buffered saline, saline, deionized water, or the like.
Preferred biologically acceptable carriers are physiologically or
pharmaceutically acceptable carriers.
[0244] The antibacterial compositions include an antibacterial
effective amount of active agent. Antibacterial effective amounts
are those quantities of the antibacterial agents of the present
invention that afford prophylactic protection against bacterial
infections or which result in amelioration or cure of an existing
bacterial infection. This antibacterial effective amount will
depend upon the agent, the location and nature of the infection,
and the particular host. The amount can be determined by
experimentation known in the art, such as by establishing a matrix
of dosages and frequencies and comparing a group of experimental
units or subjects to each point in the matrix.
[0245] The antibacterial active agents or compositions can be
formed into dosage unit forms, such as for example, creams,
ointments, lotions, powders, liquids, tablets, capsules,
suppositories, sprays, aerosols or the like. If the antibacterial
composition is formulated into a dosage unit form, the dosage unit
form may contain an antibacterial effective amount of active agent.
Alternatively, the dosage unit form may include less than such an
amount if multiple dosage unit forms or multiple dosages are to be
used to administer a total dosage of the active agent. Dosage unit
forms can include, in addition, one or more excipient(s),
diluent(s), disintegrant(s), lubricant(s), plasticizer(s),
colorant(s), dosage vehicle(s), absorption enhancer(s),
stabilizer(s), bactericide(s), or the like.
[0246] For general information concerning formulations, see, e.g.,
Gilman et al. (eds.), 1990, Goodman and Gilman's: The
Pharmacological Basis of Therapeutics, 8th ed., Pergamon Press; and
Remington's Pharmaceutical Sciences, 17th ed., 1990, Mack
Publishing Co., Easton, Pa.; Avis et al. (eds.), 1993,
Pharmaceutical Dosage Forms: Parenteral Medications, Dekker, New
York; Lieberman et al (eds.), 1990, Pharmaceutical Dosage Forms:
Disperse Systems, Dekker, New York.
[0247] The antibacterial agents and compositions of the present
invention are useful for preventing or treating S. epidermidis
infections. Infection prevention methods incorporate a
prophylactically effective amount of an antibacterial agent or
composition. A prophylactically effective amount is an amount
effective to prevent S. epidermidis infection and will depend upon
the specific bacterial strain, the agent, and the host. These
amounts can be determined experimentally by methods known in the
art and as described above.
[0248] S. epidermidis infection treatment methods incorporate a
therapeutically effective amount of an antibacterial agent or
composition. A therapeutically effective amount is an amount
sufficient to ameliorate or eliminate the infection. The
prophylactically and/or therapeutically effective amounts can be
administered in one administration or over repeated
administrations. Therapeutic administration can be followed by
prophylactic administration, once the initial bacterial infection
has been resolved.
[0249] The antibacterial agents and compositions can be
administered topically or systemically. Topical application is
typically achieved by administration of creams, ointments, lotions,
or sprays as described above. Systemic administration includes both
oral and parental routes. Parental routes include, without
limitation, subcutaneous, intramuscular, intraperitoneal,
intravenous, transdermal, inhalation and intranasal
administration.
EXEMPLIFICATION
Cloning and Sequencing S. epidermidis Genomic Sequence
[0250] This invention provides nucleotide sequences of the genome
of S. epidermidis which thus comprises a DNA sequence library of S.
epidermidis genomic DNA. The detailed description that follows
provides nucleotide sequences of S. epidermidis, and also describes
how the sequences were obtained and how ORFs (Open Reading Frames)
and protein-coding sequences can be identified. Also described are
methods of using the disclosed S. epidermidis sequences in methods
including diagnostic and therapeutic applications. Furthermore, the
library can be used as a database for identification and comparison
of medically important sequences in this and other strains of S.
epidermidis as well as other species of Staphylococcus.
[0251] Chromosomal DNA from strain 19804 of S. epidermidis was
isolated after Zymolyase digestion, sodium dodecyl sulfate lysis,
potassium acetate precipitation, phenol:chloroform extraction and
ethanol precipitation (Soll, D. R., T. Srikantha and S. R.
Lockhart: Characterizing Developmentally Regulated Genes in S.
epidermidis. In Microbial Genome Methods. K. W. Adolph, editor. CRC
Press. New York. p 17-37). Genomic S. epidermidis DNA was
hydrodynamically sheared in an HPLC and then separated on a
standard 1% agarose gel. Fractions corresponding to 2500-3000 bp in
length were excised from the gel and purified by the GeneClean
procedure (Bio101, Inc.).
[0252] The purified DNA fragments were then blunt-ended using T4
DNA polymerase. The healed DNA was then ligated to unique
BstXI-linker adapters (5'-GTCTTCACCACGGGG-3' (SEQ ID NO: 5675) and
5'-GTGGTGAAGAC-3' (SEQ ID NO: 5676) in 100-1000 fold molar excess).
These linkers are complimentary to the BstXI-cut pGTC vector, while
the overhang is not self-complimentary. Therefore, the linkers will
not concatermerize nor will the cut-vector religate itself easily.
The linker-adapted inserts were separated from the unincorporated
linkers on a 1% agarose gel and purified using GeneClean. The
linker-adapted inserts were then ligated to BstXI-cut vector to
construct a "shotgun" sublclone libraries.
[0253] Only major modifications to the protocols are highlighted.
Briefly, the library was then transformed into DH5a competent cells
(Gibco/BRL, DH5a transformation protocol). It was assessed by
plating onto antibiotic plates containing ampicillin and IPTG/Xgal.
The plates were incubated overnight at 37.degree. C. Transformants
were then used for plating of clones and picking for sequencing.
The cultures were grown overnight at 37.degree. C. DNA was purified
using a silica bead DNA preparation (Engelstein, 1996) method. In
this manner, 25 .mu.g of DNA was obtained per clone.
[0254] These purified DNA samples were then sequenced using
primarily ABI dye-terminator chemistry. All subsequent steps were
based on sequencing by ABI377 automated DNA sequencing methods. The
ABI dye terminator sequence reads were run on AB1377 machines and
the data was transferred to UNIX machines following lane tracking
of the gels. Base calls and quality scores were determined using
the program PHRED (Ewing et al., 1998, Genome Res. 8: 175-185;
Ewing and Green, 1998, Genome Res. 8: 685-734). Reads were
assembled using PHRAP (P. Green, Abstracts of DOE Human Genome
Program Contractor-Grantee Workshop V, January 1996, p. 157) with
default program parameters and quality scores. The initial assembly
was done at 2.3-fold coverage and yielded 5821 contigs.
[0255] Finishing can follow the initial assembly. Missing mates
(sequences from clones that only gave reads from one end of the
Staphylococcus DNA inserted in the plasmid) can be identified and
sequenced with ABI technology to allow the identification of
additional overlapping contigs.
[0256] End-sequencing of randomly picked genomic lambda was also
performed. Sequencing on a both sides was done for all lambda
sequences. The lambda library backbone helped to verify the
integrity of the assembly and allowed closure of some of the
physical gaps. Primers for walking off the ends of contigs would be
selected using pick_primer (a GTC program) near the ends of the
clones to facilitate gap closure. These walks can be sequenced
using the selected clones and primers. These data are then
reassembled with PHRAP. Additional sequencing using PCR-generated
templates and screened and/or unscreened lambda templates can be
done in addition.
[0257] To identify S. epidermidis polypeptides the complete genomic
sequence of S. epidermidis were analyzed essentially as follows:
First, all possible stop-to-stop open reading frames (ORFs) greater
than 180 nucleotides in all six reading frames were translated into
amino acid sequences. Second, the identified ORFs were analyzed for
homology to known (archeabacter, prokaryotic and eukaryotic)
protein sequences. Third, the coding potential of non-homologous
sequences were evaluated with the program GENEMARK.TM. (Borodovsky
and McIninch, 1993, Comp. Chem. 17:123).
EE341901427USIdentification, Cloning and Expression of S.
epidermidis Nucleic Acids
[0258] Expression and purification of the S. epidermidis
polypeptides of the invention can be performed essentially as
outlined below.
[0259] To facilitate the cloning, expression and purification of
membrane and secreted proteins from S. epidermidis, a gene
expression system, such as the pET System (Novagen), for cloning
and expression of recombinant proteins in E. coli, is selected.
Also, a DNA sequence encoding a peptide tag, the His-Tag, is fused
to the 3' end of DNA sequences of interest in order to facilitate
purification of the recombinant protein products. The 3' end is
selected for fusion in order to avoid alteration of any 5' terminal
signal sequence.
PCR Amplification and Cloning of Nucleic Acids Containing ORF's
Encoding Enzymes
[0260] Nucleic acids chosen (for example, from the nucleic acids
set forth in SEQ ID NO: 1-SEQ ID NO: 2837) for cloning from the
18972 strain of S. epidermidis are prepared for amplification
cloning by polymerase chain reaction (PCR). Synthetic
oligonucleotide primers specific for the 5' and 3' ends of open
reading frames (ORFs) are designed and purchased from GibcoBRL Life
Technologies (Gaithersburg, Md., USA). All forward primers
(specific for the 5' end of the sequence) are designed to include
an NcoI cloning site at the extreme 5' terminus. These primers are
designed to permit initiation of protein translation at a
methionine residue followed by a valine residue and the coding
sequence for the remainder of the native S. epidermidis DNA
sequence. All reverse primers (specific for the 3' end of any S.
epidermidis ORF) include a EcoRI site at the extreme 5' terminus to
permit cloning of each S. epidermidis sequence into the reading
frame of the pET-28b. The pET-28b vector provides sequence encoding
an additional 20 carboxy-terminal amino acids including six
histidine residues (at the extreme C-terminus), which comprise the
His-Tag.
[0261] Genomic DNA prepared from the 18972 strain of S. epidermidis
is used as the source of template DNA for PCR amplification
reactions (Current Protocols in Molecular Biology, John Wiley and
Sons, Inc., F. Ausubel et al., eds., 1994). To amplify a DNA
sequence containing a S. epidermidis ORF, genomic DNA (50
nanograms) is introduced into a reaction vial containing 2 mM
MgCl.sub.2, 1 micromolar synthetic oligonucleotide primers (forward
and reverse primers) complementary to and flanking a defined S.
epidermidis ORF, 0.2 mM of each deoxynucleotide triphosphate; dATP,
dGTP, dCTP, dTTP and 2.5 units of heat stable DNA polymerase
(Amplitaq, Roche Molecular Systems, Inc., Branchburg, N.J., USA) in
a final volume of 100 microliters.
[0262] Upon completion of thermal cycling reactions, each sample of
amplified DNA is washed and purified using the Qiaquick Spin PCR
purification kit (Qiagen, Gaithersburg, Md., USA). All amplified
DNA samples are subjected to digestion with the restriction
endonucleases, e.g., NcoI and EcoRI (New England BioLabs, Beverly,
Mass., USA)(Current Protocols in Molecular Biology, John Wiley and
Sons, Inc., F. Ausubel et al., eds., 1994). DNA samples are then
subjected to electrophoresis on 1.0% NuSeive (FMC BioProducts,
Rockland, Me. USA) agarose gels. DNA is visualized by exposure to
ethidium bromide and long wave uv irradiation. DNA contained in
slices isolated from the agarose gel is purified using the Bio 101
GeneClean Kit protocol (Bio 101 Vista, Calif., USA).
Cloning of S. epidermidis Nucleic Acids into an Expression
Vector
[0263] The pET-28b vector is prepared for cloning by digestion with
restriction endonucleases, e.g., NcoI and EcoRI (Current Protocols
in Molecular Biology, John Wiley and Sons, Inc., F. Ausubel et al.,
eds., 1994). The pET-28a vector, which encodes a His-Tag that can
be fused to the 5' end of an inserted gene, is prepared by
digestion with appropriate restriction endonucleases.
[0264] Following digestion, DNA inserts are cloned (Current
Protocols in Molecular Biology, John Wiley and Sons, Inc., F.
Ausubel et al., eds., 1994) into the previously digested pET-28b
expression vector. Products of the ligation reaction are then used
to transform the BL21 strain of E. coli (Current Protocols in
Molecular Biology, John Wiley and Sons, Inc., F. Ausubel et al.,
eds., 1994) as described below.
Transformation of Competent Bacteria with Recombinant Plasmids
[0265] Competent bacteria, E. coli strain BL21 or E. coli strain
BL21(DE3), are transformed with recombinant pET expression plasmids
carrying the cloned S. epidermidis sequences according to standard
methods (Current Protocols in Molecular, John Wiley and Sons, Inc.,
F. Ausubel et al., eds., 1994). Briefly, 1 microliter of ligation
reaction is mixed with 50 microliters of electrocompetent cells and
subjected to a high voltage pulse, after which, samples are
incubated in 0.45 milliliters SOC medium (0.5% yeast extract, 2.0%
tryptone, 10 mM NaCl, 2.5 mM KCl, 10 nM MgCl2, 10 mM MgSO4 and 20,
mM glucose) at 37.degree. C. with shaking for 1 hour. Samples are
then spread on LB agar plates containing 25 microgram/ml kanamycin
sulfate for growth overnight.
[0266] Transformed colonies of BL21 are then picked and analyzed to
evaluate cloned inserts as described below.
Identification of Recombinant Expression Vectors with S.
epidermidis Nucleic Acids
[0267] Individual BL21 clones transformed with recombinant pET-28b
S. epidermidis ORFs are analyzed by PCR amplification of the cloned
inserts using the same forward and reverse primers, specific for
each S. epidermidis sequence, that were used in the original PCR
amplification cloning reactions. Successful amplification verifies
the integration of the S. epidermidis sequences in the expression
vector (Current Protocols in Molecular Biology, John Wiley and
Sons, Inc., F. Ausubel et al., eds., 1994).
Isolation and Preparation of Nucleic Acids from Transformants
[0268] Individual clones of recombinant pET-28b vectors carrying
properly cloned S. epidermidis ORFs are picked and incubated in 5
mls of LB broth plus 25 microgram/ml kanamycin sulfate overnight.
The following day plasmid DNA is isolated and purified using the
Qiagen plasmid purification protocol (Qiagen Inc., Chatsworth,
Calif., USA).
Expression of Recombinant S. epidermidis Sequences In E. coli
[0269] The pET vector can be propagated in any E. coli K-12 strain
e.g. HMS174, HB101, JM109, DH5, etc. for the purpose of cloning or
plasmid preparation. Hosts for expression include E. coli strains
containing a chromosomal copy of the gene for T7 RNA polymerase.
These hosts are lysogens of bacteriophage DE3, a lambda derivative
that carries the lacI gene, the lacUV5 promoter and the gene for T7
RNA polymerase. T7 RNA polymerase is induced by addition of
isopropyl-B-D-thiogalactoside (IPTG), and to the T7 RNA polymerase
transcribes any target plasmid, such as pET-28b, carrying its gene
of interest. Strains used include: BL21(DE3) (Studier, F. W.,
Rosenberg, A. H., Dunn, J. J., and Dubendorff, J. W. (1990) Meth.
Enzymol. 185, 60-89).
[0270] To express recombinant S. epidermidis sequences, 50
nanograms of plasmid DNA isolated as described above is used to
transform competent BL21(DE3) bacteria as described above (provided
by Novagen as part of the pET expression system kit). The lacZ gene
(beta-galactosidase) is expressed in the pET-System as described
for the S. epidermidis recombinant constructions. Transformed cells
are cultured in SOC medium for 1 hour, and the culture is then
plated on LB plates containing 25 micrograms/ml kanamycin sulfate.
The following day, bacterial colonies are pooled and grown in LB
medium containing kanamycin sulfate (25 micrograms/ml) to an
optical density at 600 nM of 0.5 to 1.0 O.D. units, at which point,
1 millimolar IPTG was added to the culture for 3 hours to induce
gene expression of the S. epidermidis recombinant DNA
constructions.
[0271] After induction of gene expression with IPTG, bacteria are
pelleted by centrifugation in a Sorvall RC-3B centrifuge at
3500.times.g for 15 minutes at 4.degree. C. Pellets are resuspended
in 50 milliliters of cold 10 mM Tris-HCl, pH 8.0, 0.1 M NaCl and
0.1 mM EDTA (STE buffer). Cells are then centrifuged at
2000.times.g for 20 min at 4.degree. C. Wet pellets are weighed and
frozen at -80.degree. C. until ready for protein purification.
[0272] A variety of methodologies known in the art can be utilized
to purify the isolated proteins. (Current Protocols in Protein
Science, John Wiley and Sons, Inc., J. E. Coligan et al., eds.,
1995). For example, the frozen cells may be thawed, resuspended in
buffer and ruptured by several passages through a small volume
microfluidizer (Model M-110S, Microfluidics International
Corporation, Newton, Mass.). The resultant homogenate may be
centrifuged to yield a clear supernatant (crude extract) and
following filtration the crude extract may be fractionated over
columns. Fractions may be monitored by absorbance at OD.sub.280 nm
and peak fractions may analyzed by SDS-PAGE
[0273] The concentrations of purified protein preparations may be
quantified spectrophotometrically using absorbance coefficients
calculated from amino acid content (Perkins, S. J. 1986 Eur. J.
Biochem. 157, 169-180). Protein concentrations are also measured by
the method of Bradford, M. M. (1976) Anal. Biochem. 72, 248-254,
and Lowry, O. H., Rosebrough, N., Farr, A. L. & Randall, R. J.
(1951) J. Biol. Chem. 193, pages 265-275, using bovine serum
albumin as a standard.
[0274] SDS-polyacrylamide gels of various concentrations may be
purchased from BioRad (Hercules, Calif., USA), and stained with
Coomassie blue. Molecular weight markers may include rabbit
skeletal muscle myosin (200 kDa), E. coli (-galactosidase (116
kDa), rabbit muscle phosphorylase B (97.4 kDa), bovine serum
albumin (66.2 kDa), ovalbumin (45 kDa), bovine carbonic anhydrase
(31 kDa), soybean trypsin inhibitor (21.5 kDa), egg white lysozyme
(14.4 kDa) and bovine aprotinin (6.5 kDa).
EQUIVALENTS
[0275] Those skilled in the art will recognize, or be able to
ascertain using no more than routine experimentation, many
equivalents to the specific embodiments and methods described
herein. The specific embodiments described herein are offered by
way of example only, and the invention is to limited only by the
terms of the appended claims, along with the full scope of
equivalents to which such claims are entitled. TABLE-US-00002
LENGTHY TABLE REFERENCED HERE US20070053936A1-20070308-T00001
Please refer to the end of the specification for access
instructions.
TABLE-US-00003 LENGTHY TABLE The patent application contains a
lengthy table section. A copy of the table is available in
electronic form from the USPTO web site
(http://seqdata.uspto.gov/?pageRequest=docDetail&DocID=US20070053936A1).
An electronic copy of the table will also be available from the
USPTO upon request and payment of the fee set forth in 37 CFR
1.19(b)(3).
Sequence CWU 0 SQTB SEQUENCE LISTING The patent application
contains a lengthy "Sequence Listing" section. A copy of the
"Sequence Listing" is available in electronic form from the USPTO
web site
(http://seqdata.uspto.gov/?pageRequest=docDetail&DocID=US20070053936A1).
An electronic copy of the "Sequence Listing" will also be available
from the USPTO upon request and payment of the fee set forth in 37
CFR 1.19(b)(3).
0 SQTB SEQUENCE LISTING The patent application contains a lengthy
"Sequence Listing" section. A copy of the "Sequence Listing" is
available in electronic form from the USPTO web site
(http://seqdata.uspto.gov/?pageRequest=docDetail&DocID=US20070053936A1).
An electronic copy of the "Sequence Listing" will also be available
from the USPTO upon request and payment of the fee set forth in 37
CFR 1.19(b)(3).
* * * * *
References