U.S. patent application number 11/655678 was filed with the patent office on 2010-10-28 for staphylococcus epidermidis nucleic acids and proteins.
This patent application is currently assigned to SmithKline Beecham Corporation. Invention is credited to William John Kimmerly.
Application Number | 20100272743 11/655678 |
Document ID | / |
Family ID | 31890842 |
Filed Date | 2010-10-28 |
United States Patent
Application |
20100272743 |
Kind Code |
A1 |
Kimmerly; William John |
October 28, 2010 |
Staphylococcus epidermidis nucleic acids and proteins
Abstract
S epidermidis polypeptides and DNA (RNA) encoding such
polypeptides and a procedure for producing such polypeptides by
recombinant techniques is disclosed. Also disclosed are methods for
utilizing such polypeptides and DNA (RNA) for the treatment of
infection, particularly infections arising from S epidermidis.
Antagonists against the function of such polypeptides and their use
as therapeutics to treat infection are also disclosed. Also
disclosed are diagnostic assays for detecting diseases related to
the presence of S epidermidis nucleic acid sequences and the
polypeptides in a host. Also disclosed are diagnostic assays for
detecting polynucleotides and polypeptides related to S
epidermidis
Inventors: |
Kimmerly; William John;
(Eucinitas, CA) |
Correspondence
Address: |
GLAXOSMITHKLINE;GLOBAL PATENTS
FIVE MOORE DR., PO BOX 13398, MAIL STOP: C.2111F
RESEARCH TRIANGLE PARK
NC
27709-3398
US
|
Assignee: |
SmithKline Beecham
Corporation
Philadelphia
PA
|
Family ID: |
31890842 |
Appl. No.: |
11/655678 |
Filed: |
January 19, 2007 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10793626 |
Mar 4, 2004 |
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11655678 |
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09710279 |
Nov 9, 2000 |
6703492 |
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10793626 |
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60164258 |
Nov 9, 1999 |
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Current U.S.
Class: |
424/190.1 ;
530/350; 536/23.1 |
Current CPC
Class: |
C07K 14/31 20130101;
A61P 37/04 20180101 |
Class at
Publication: |
424/190.1 ;
536/23.1; 530/350 |
International
Class: |
A61K 39/085 20060101
A61K039/085; C07H 21/04 20060101 C07H021/04; C07H 21/02 20060101
C07H021/02; C07K 14/00 20060101 C07K014/00; A61P 37/04 20060101
A61P037/04 |
Claims
1-29. (canceled)
30. An isolated polynucleotide comprising a member selected from
the group consisting of: (a) a polynucleotide encoding a
polypeptide having at least a 70% identity to a polypeptide set
forth in the Sequence Listing as SEQ. ID. NO 2494; (b) a
polynucleotide which is complementary to the polynucleotide of (a);
and (c) a polynucleotide comprising at least 15 sequential bases of
the polynucleotide of (a) or (b).
31. The polynucleotide of claim 30 wherein the polypeptide has at
least 80% identity to the polypeptide set forth in the Sequence
Listing.
32. The polypeptide of claim 31 wherein the polypeptide has at
least 90% identity to the polypeptide set forth in the Sequence
Listing.
33. The polynucleotide of claim 30 wherein the polynucleotide is
DNA.
34. The polynucleotide of claim 30 wherein the polynucleotide is
RNA.
35. The polynucleotide of claim 33 wherein the polynucleotide has
at least 80% identity to the polynucleotide set forth in the
Sequence Listing as SEQ. ID. No. 2494.
36. A polypeptide comprising an amino acid sequence which is at
least 70% identical to a polypeptide set forth in the Sequence
Listing as SEQ. ID. No 2494.
37. The polypeptide of claim 36 further comprising an amino acid
sequence which is at least 80% identical to a polypeptide set forth
in the Sequence Listing.
38. The polypeptide of claim 37 further comprising an amino acid
sequence which is at least 90% identical to a polypeptide set forth
in the Sequence Listing.
39. The polypeptide of claim 38 further comprising an amino acid
sequence as set forth in the Sequence Listing as SEQ. ID. No.
2494.
40. A method for inducing an immunological response in a mammal
which comprises administering to the mammal the polypeptide of
claim 36, or a fragment or variant thereof.
41. An immunological composition comprising a DNA capable of
expressing a polypeptide of claim 36 which, when introduced into a
mammal, induces an immunological response in the mammal, and a
pharmaceutically acceptable carrier.
42. An immunological composition comprising a polypeptide of claim
36 which, when introduced into a mammal, induces an immunological
response in the mammal, and a pharmaceutically acceptable carrier.
Description
CROSS REFERENCE TO RELATED APPLICATIONS
[0001] This application is a continuation of U.S. application Ser.
No. 10/793,626 filed Mar. 4, 2004 which is a divisional of U.S.
application Ser. No. 09/710,279 filed Nov. 9, 2000 which claims
priority from Provisional Application No. 60/164,258 filed Nov. 9,
1999.
FIELD OF THE INVENTION
[0002] The present invention provides nucleic acids, and peptides,
polypeptides and proteins encoded by the nucleic acids, isolated
from Staphylococcus epidermidis.
BACKGROUND OF THE INVENTION
[0003] Staphylococcus epidermidis is a gram-positive bacteria
present in the normal flora of humans, and is typically present on
the skin. It is catalase positive, and grows aerobically. It is
implicated in various human conditions and diseases, including
subacute bacterial endocarditis (Baddour L M et al., Production of
experimental endocarditis by coagulase-negative staphylococci:
variability in species virulence, J. Infect. Dis. 150: 721-727,
1984; Karchmer A W, Archer G L, Dismukes W E, Staphylococcus
epidermidis causing prosthetic valve endocarditis: microbiologic
and clinical observations as guides to therapy, Ann Intern Med.
1983; 98:447-455.) and septicemia (Christensen G D et al.,
Nosocomial septicemia due to multiply antibiotic-resistant
Staphylococcus epidermidis, Ann. Intern. Med. 96: 1-10, 1982). S.
epidermidis is estimated to be responsible for about 12% of all
hospital patient infections. Because of the organism's peculiar
ability to colonize polymer and metallic surfaces, there is a
correlation of infection with the insertion of intravenous lines or
catheters or implantation of prosthetic devices. Treatment can be
difficult since different isolates of S. epidermidis show a broad
spectrum of antibiotic resistance. The organism also produces a
polysaccharide biofilm which helps to protect the bacteria from the
human immune system (Tojo M et al., Isolation and characterization
of a capsular polysaccharide adhesin from Staphylococcus
epidermidis, J. Infect. Dis. 157: 713-722, 1988).
[0004] The present invention advantageously provides isolated
nucleic acids and their encoded peptides, polypeptides and proteins
from the genome of S. epidermidis, as well as the genomic map of S.
epidermidis. Thus, the present invention fulfils a widely-felt need
for S. epidermidis diagnostics, antigens, and products useful in
procedures for preparing antibodies and for identifying compounds
effective against S. epidermidis infection. Selected nucleic acids
and/or polypeptides of the present invention can be advantageously
utilized as targets in screenings assays for antibiotics, as
diagnostics of infections, and as means to identify S epidermidis
in any given sample and distinguish it from other bacteria.
SUMMARY OF THE INVENTION
[0005] The present invention provides an isolated polynucleotide
comprising a member selected from the group consisting of:
[0006] (a) a polynucleotide encoding a polypeptide having at least
a 70% identity to a polypeptide set forth in the Sequence
Listing;
[0007] (b) a polynucleotide which is complementary to the
polynucleotide of (a); and
[0008] (c) a polynucleotide comprising at least 15 sequential bases
of the polynucleotide of (a) or (b). The present invention further
provides polypeptides encoded by these polynucleotides and methods
of using the polynucleotides and polypeptides.
DETAILED DESCRIPTION OF THE INVENTION
Glossary
[0009] The following illustrative explanations are provided to
facilitate understanding of certain terms used frequently herein,
particularly in the Examples. The explanations are provided as a
convenience and are not limitative of the invention.
[0010] BINDING MOLECULE refers to a molecule or ion which binds or
interacts specifically with polypeptides or polynucleotides of the
present invention, including, for example enzyme substrates, cell
membrane components and classical receptors. Binding between
polypeptides (or polynucleotides) of the invention and such
molecules may be exclusive to polypeptides of the invention, which
is preferred, or it may be highly specific for polypeptides of the
invention, which is also preferred, or it may be highly specific to
a group of proteins that includes polypeptides of the invention,
which is preferred, or it may be specific to several groups of
proteins at least one of which includes a polypeptide of the
invention. Binding molecules also include antibodies and
antibody-derived reagents that bind specifically to polypeptides of
the invention.
[0011] GENETIC ELEMENT generally means a polynucleotide comprising
a region that encodes a polypeptide or a polynucleotide region that
regulates replication, transcription or translation or other
processes important to expression of the polypeptide in a host
cell, or a polynucleotide comprising both a region that encodes a
polypeptide and a region operably linked thereto that regulates
expression. Genetic elements may be comprised within a vector that
replicates as an episomal element; that is, as a molecule
physically independent of the host cell genome. They may be
comprised within plasmids. Genetic elements also may be comprised
within a host cell genome; not in their natural state but, rather,
following manipulation such as isolation, cloning and introduction
into a host cell in the form of purified DNA or in a vector, among
others.
[0012] HOST CELL is a cell which has been transformed or
transfected, or is capable of transformation or transfection by an
exogenous polynucleotide sequence.
[0013] IDENTITY, as known in the art, is the relationship between
two or more polypeptide sequences or two or more polynucleotide
sequences, as determined by comparing the sequences. In the art,
identity also means the degree of sequence relatedness between
polypeptide or polynucleotide sequences, as the case may be, as
determined by the match between strings of such sequences. Identity
can be readily calculated (Computational Molecular Biology, Lesk,
A. M., ed., Oxford University Press, New York, 1988; Biocomputing:
Informatics and Genome Projects, Smith, D. W., ed., Academic Press,
New York, 1993; Computer Analysis of Sequence Data, Part I,
Griffin, A. M., and Griffin, H. G., eds., Humana Press, New Jersey,
1994; Sequence Analysis in Molecular Biology, von Heinje, G.,
Academic Press, 1987; and Sequence Analysis Primer, Gribskov, M.
and Devereux, J., eds., M Stockton Press, New York, 1991). While
there exist a number of methods to measure identity between two
polynucleotide or two polypeptide sequences, the term is well known
to skilled artisans (Sequence Analysis in Molecular Biology, von
Heinje, G., Academic Press, 1987; Sequence Analysis Primer,
Gribskov, M. and Devereux, J., eds., M Stockton Press, New York,
1991; and Carillo, H., and Lipman, D., SIAM J. Applied Math., 48:
1073 (1988)). Methods commonly employed to determine identity
between sequences include, but are not limited to those disclosed
in Carillo, H., and Lipman, D., SIAM J. Applied Math., 48:1073
(1988). Preferred methods to determine identity are designed to
give the largest match between the sequences tested. Methods to
determine identity are codified in computer programs. Preferred
computer program methods to determine identity between two
sequences include, but are not limited to, GCG program package
(Devereux, J., et al., Nucleic Acids Research 12(1): 387 (1984)),
BLASTP, BLASTN, and FASTA (Atschul, S. F. et al., J. Molec. Biol.
215: 403 (1990)).
[0014] ISOLATED means separated "by the hand of man" from its
natural state; i.e., that, if it occurs in nature, it has been
changed or removed from its original environment, or both. For
example, a naturally occurring polynucleotide or a polypeptide
naturally present in a living organism in its natural state is not
"isolated," but the same polynucleotide or polypeptide separated
from the coexisting materials of its natural state is "isolated",
as the term is employed herein. As part of or following isolation,
such polynucleotides can be joined to other polynucleotides, such
as DNAs, for mutagenesis, to form fusion proteins, and for
propagation or expression in a host, for instance. The isolated
polynucleotides, alone or joined to other polynucleotides such as
vectors, can be introduced into host cells, in culture or in whole
organisms. Introduced into host cells in culture or in whole
organisms, such DNAs still would be isolated, as the term is used
herein, because they would not be in their naturally occurring form
or environment. Similarly, the polynucleotides and polypeptides may
occur in a composition, such as a media formulations, solutions for
introduction of polynucleotides or polypeptides, for example, into
cells, compositions or solutions for chemical or enzymatic
reactions, for instance, which are not naturally occurring
compositions, and, therein remain isolated polynucleotides or
polypeptides within the meaning of that term as it is employed
herein.
[0015] POLYNUCLEOTIDE(S) generally refers to any polyribonucleotide
or polydeoxyribonucleotide, which may be unmodified RNA or DNA or
modified RNA or DNA. Thus, for instance, polynucleotides as used
herein refers to, among others, single- and double-stranded DNA,
DNA that is a mixture of single- and double-stranded regions or
single-, double- and triple-stranded regions, single- and
double-stranded RNA, and RNA that is mixture of single- and
double-stranded regions, hybrid molecules comprising DNA and RNA
that may be single-stranded or, more typically, double-stranded, or
triple-stranded, or a mixture of single- and double-stranded
regions. In addition, polynucleotide as used herein refers to
triple-stranded regions comprising RNA or DNA or both RNA and DNA.
The strands in such regions may be from the same molecule or from
different molecules. The regions may include all of one or more of
the molecules, but more typically involve only a region of some of
the molecules. One of the molecules of a triple-helical region
often is an oligonucleotide. As used herein, the term
polynucleotide includes DNAs or RNAs as described above that
contain one or more modified bases. Thus, DNAs or RNAs with
backbones modified for stability or for other reasons are
"polynucleotides" as that term is intended herein. Moreover, DNAs
or RNAs comprising unusual bases, such as inosine, or modified
bases, such as tritylated bases, to name just two examples, are
polynucleotides as the term is used herein. It will be appreciated
that a great variety of modifications have been made to DNA and RNA
that serve many useful purposes known to those of skill in the art.
The term polynucleotide as it is employed herein embraces such
chemically, enzymatically or metabolically modified forms of
polynucleotides, as well as the chemical forms of DNA and RNA
characteristic of viruses and cells, including simple and complex
cells, inter alia. The term polynucleotide also embraces short
polynucleotides often referred to as oligonucleotide(s).
"Polynucleotide" and "nucleic acid" are often used interchangeably
herein.
[0016] POLYPEPTIDES, as used herein, includes all polypeptides as
described below. The basic structure of polypeptides is well known
and has been described in innumerable textbooks and other
publications in the art. In this context, the term is used herein
to refer to any peptide or protein comprising two or more amino
acids joined to each other in a linear chain by peptide bonds. As
used herein, unless otherwise indicated, the term refers to both
short chains, which also commonly are referred to in the art as
peptides, oligopeptides and oligomers, for example, and to longer
chains, which generally are referred to in the art as proteins, of
which there are many types. It will be appreciated that
polypeptides often contain amino acids other than the 20 amino
acids commonly referred to as the 20 naturally occurring amino
acids, and that many amino acids, including the terminal amino
acids, may be modified in a given polypeptide, either by natural
processes, such as processing and other post-translational
modifications, but also by chemical modification techniques which
are well known to the art. Even the common modifications that occur
naturally in polypeptides are too numerous to list exhaustively
here, but they are well described in basic texts and in more
detailed monographs, as well as in a voluminous research
literature, and they are well known to those of skill in the art.
Among the known modifications which may be present in polypeptides
of the present are, to name an illustrative few, acetylation,
acylation, ADP-ribosylation, amidation, covalent attachment of
flavin, covalent attachment of a heme moiety, covalent attachment
of a nucleotide or nucleotide derivative, covalent attachment of a
lipid or lipid derivative, covalent attachment of
phosphotidylinositol, cross-linking, cyclization, disulfide bond
formation, demethylation, formation of covalent cross-links,
formation of cystine, formation of pyroglutamate, formylation,
gamma-carboxylation, glycosylation, GPI anchor formation,
hydroxylation, iodination, methylation, myristoylation, oxidation,
proteolytic processing, phosphorylation, prenylation, racemization,
selenoylation, sulfation, transfer-RNA mediated addition of amino
acids to proteins such as arginylation, and ubiquitination. Such
modifications are well known to those of skill and have been
described in great detail in the scientific literature. Several
particularly common modifications, glycosylation, lipid attachment,
sulfation, gamma-carboxylation of glutamic acid residues,
hydroxylation and ADP-ribosylation, for instance, are described in
most basic texts, such as, for instance PROTEINS--STRUCTURE AND
MOLECULAR PROPERTIES, 2nd Ed., T. E. Creighton, W. H. Freeman and
Company, New York (1993). Many detailed reviews are available on
this subject, such as, for example, those provided by Wold, F.,
Posttranslational Protein Modifications: Perspectives and
Prospects, pgs. 1-12 in POSTTRANSLATIONAL COVALENT MODIFICATION OF
PROTEINS, B. C. Johnson, Ed., Academic Press, New York (1983);
Seifter et al., Meth. Enzymol. 182:626-646 (1990) and Rattan et
al., Protein Synthesis: Posttranslational Modifications and Aging,
Ann. N.Y. Acad. Sci. 663: 48-62 (1992). It will be appreciated, as
is well known and as noted above, that polypeptides are not always
entirely linear. For instance, polypeptides may be generally as a
result of posttranslational events, including natural processing
event and events brought about by human manipulation which do not
occur naturally. Circular, branched and branched circular
polypeptides may be synthesized by non-translation natural process
and by entirely synthetic methods, as well. Modifications can occur
anywhere in a polypeptide, including the peptide backbone, the
amino acid side-chains and the amino or carboxyl termini. In fact,
blockage of the amino or carboxyl group in a polypeptide, or both,
by a covalent modification, is common in naturally occurring and
synthetic polypeptides and such modifications may be present in
polypeptides of the present invention, as well. For instance, the
amino terminal residue of polypeptides made in E. coli or other
cells, prior to proteolytic processing, almost invariably will be
N-formylmethionine. During post-translational modification of the
peptide, a methionine residue at the NH.sub.2-terminus may be
deleted. Accordingly, this invention contemplates the use of both
the methionine-containing and the methionineless amino terminal
variants of the protein of the invention. The modifications that
occur in a polypeptide often will be a function of how it is made.
For polypeptides made by expressing a cloned gene in a host, for
instance, the nature and extent of the modifications in large part
will be determined by the host cell posttranslational modification
capacity and the modification signals present in the polypeptide
amino acid sequence. For instance, as is well known, glycosylation
often does not occur in bacterial hosts such as, for example, E.
coli. Accordingly, when glycosylation is desired, a polypeptide
should be expressed in a glycosylating host, generally a eukaryotic
cell. Insect cell often carry out the same posttranslational
glycosylations as mammalian cells and, for this reason, insect cell
expression systems have been developed to express efficiently
mammalian proteins having native patterns of glycosylation, inter
alia. Similar considerations apply to other modifications. It will
be appreciated that the same type of modification may be present in
the same or varying degree at several sites in a given polypeptide.
Also, a given polypeptide may contain many types of modifications.
In general, as used herein, the term polypeptide encompasses all
such modifications, particularly those that are present in
polypeptides synthesized recombinantly by expressing a
polynucleotide in a host cell.
[0017] VARIANT(S) of polynucleotides or polypeptides, as the term
is used herein, are polynucleotides or polypeptides that differ
from a reference polynucleotide or polypeptide, respectively.
Variants in this sense are described below and elsewhere in the
present disclosure in greater detail. (1) A polynucleotide that
differs in nucleotide sequence from another, reference
polynucleotide. Generally, differences are limited so that the
nucleotide sequences of the reference and the variant are closely
similar overall and, in many regions, identical. As noted below,
changes in the nucleotide sequence of the variant may be silent.
That is, they may not alter the amino acids encoded by the
polynucleotide. Where alterations are limited to silent changes of
this type a variant will encode a polypeptide with the same amino
acid sequence as the reference. Also as noted below, changes in the
nucleotide sequence of the variant may alter the amino acid
sequence of a polypeptide encoded by the reference polynucleotide.
Such nucleotide changes may result in amino acid substitutions,
additions, deletions, fusions and truncations in the polypeptide
encoded by the reference sequence, as discussed below. (2) A
polypeptide that differs in amino acid sequence from another,
reference polypeptide. Generally, differences are limited so that
the sequences of the reference and the variant are closely similar
overall and, in many regions, identical. A variant and reference
polypeptide may differ in amino acid sequence by one or more
substitutions, additions, deletions, fusions and truncations, which
may be present in any combination.
[0018] Techniques are available to evaluate temporal gene
expression in bacteria, particularly as it applies to viability
under laboratory and host infection conditions. A number of methods
can be used to identify genes which are essential to survival per
se, or essential to the establishment/maintenance of an infection.
Identification of expression of a sequence by one of these methods
yields additional information about its function and permits the
selection of such sequence for further development as a screening
target. Briefly, these approaches include:
1) Signature Tagged Mutagenesis (STM)
[0019] This technique is described by Hensel et al., Science 269:
400-403 (1995), the contents of which is incorporated by reference
for background purposes. Signature tagged mutagenesis identifies
genes necessary for the establishment/maintenance of infection in a
given infection model.
[0020] The basis of the technique is the random mutagenesis of
target organism by various means (e.g., transposons) such that
unique DNA sequence tags are inserted in close proximity to the
site of mutation. The tags from a mixed population of bacterial
mutants and bacteria recovered from an infected host are detected
by amplification, radiolabeling and hybridization analysis. Mutants
attenuated in virulence are revealed by absence of the tag from the
pool of bacteria recovered from infected hosts.
[0021] In Streptococcus pneumoniae, because the transposon system
is less well developed, a more efficient way of creating the tagged
mutants is to use the insertion-duplication mutagenesis technique
as described by Morrison et al., J. Bacteria 159:870 (1984) the
contents of which is incorporated by reference for background
purposes.
2) In Vivo Expression Technology (IVET)
[0022] This technique is described by Camilli et al., Proc. Nat'l.
Acad. Sci. USA. 91:2634-2638 (1994), the contents of which is
incorporated by reference for background purposes. IVET identifies
genes up-regulated during infection when compared to laboratory
cultivation, implying an important role in infection. Sequences
identified by this technique are implied to have a significant role
in infection establishment/maintenance.
[0023] In this technique random chromosomal fragments of target
organism are cloned upstream of a promoter-less reporter gene in a
plasmid vector. The pool is introduced into a host and at various
times after infection bacteria may be recovered and assessed for
the presence of reporter gene expression. The chromosomal fragment
carried upstream of an expressed reporter gene should carry a
promoter or portion of a gene normally upregulated during
infection. Sequencing upstream of the reporter gene allows
identification of the up regulated gene.
3) Differential Display
[0024] This technique is described by Chuang et al., J. Bacteriol.
175:2026-2036 (1993), the contents of which is incorporated by
reference for background purposes. This method identifies those
genes which are expressed in an organism by identifying mRNA
present using randomly-primed RT-PCR. By comparing pre-infection
and post infection profiles, genes up and down regulated during
infection can be identified and the RT-PCR product sequenced and
matched to library sequences.
4) Generation of Conditional Lethal Mutants by Transposon
Mutagenesis.
[0025] This technique, described by de Lorenzo, V. et al., Gene
123:17-24 (1993); Neuwald, A. F. et al., Gene 125: 69-73 (1993);
and Takiff, H. E. et al., J. Bacteriol. 174:1544-1553 (1992), the
contents of which is incorporated by reference for background
purposes, identifies genes whose expression are essential for cell
viability.
[0026] In this technique transposons carrying controllable
promoters, which provide transcription outward from the transposon
in one or both directions, are generated. Random insertion of these
transposons into target organisms and subsequent isolation of
insertion mutants in the presence of inducer of promoter activity
ensures that insertions which separate promoter from coding region
of a gene whose expression is essential for cell viability will be
recovered. Subsequent replica plating in the absence of inducer
identifies such insertions, since they fail to survive. Sequencing
of the flanking regions of the transposon allows identification of
site of insertion and identification of the gene disrupted. Close
monitoring of the changes in cellular processes/morphology during
growth in the absence of inducer yields information on likely
function of the gene. Such monitoring could include flow cytometry
(cell division, lysis, redox potential, DNA replication),
incorporation of radiochemically labeled precursors into DNA, RNA,
protein, lipid, peptidoglycan, monitoring reporter enzyme gene
fusions which respond to known cellular stresses.
5) Generation of Conditional Lethal Mutants by Chemical
Mutagenesis.
[0027] This technique is described by Beckwith, J., Methods in
Enzymology 204: 3-18 (1991), the contents of which are incorporated
herein by reference for background purposes. In this technique
random chemical mutagenesis of target organism, growth at
temperature other than physiological temperature (permissive
temperature) and subsequent replica plating and growth at different
temperature (e.g. 42.degree. C. to identify ts, 25.degree. C. to
identify cs) are used to identify those isolates which now fail to
grow (conditional mutants). As above close monitoring of the
changes upon growth at the non-permissive temperature yields
information on the function of the mutated gene. Complementation of
conditional lethal mutation by library from target organism and
sequencing of complementing gene allows matching with library
sequences.
[0028] Each of these techniques may have advantages or
disadvantages depending on the particular application. The skilled
artisan would choose the approach that is the most relevant with
the particular end use in mind. For example, some genes might be
recognised as essential for infection but in reality are only
necessary for the initiation of infection and so their products
would represent relatively unattractive targets for antibacterials
developed to cure established and chronic infections.
6) RT-PCR
[0029] Bacterial messenger RNA, preferably that of S. epidermidis,
is isolated from bacterial infected tissue e.g. 48 hour murine lung
infections, and the amount of each mRNA species assessed by reverse
transcription of the RNA sample primed with random hexanucleotides
followed by PCR with gene specific primer pairs. The determination
of the presence and amount of a particular mRNA species by
quantification of the resultant PCR product provides information on
the bacterial genes which are transcribed in the infected tissue.
Analysis of gene transcription can be carried out at different
times of infection to gain a detailed knowledge of gene regulation
in bacterial pathogenesis allowing for a clearer understanding of
which gene products represent targets for screens for novel
antibacterials. Because of the gene specific nature of the PCR
primers employed it should be understood that the bacterial mRNA
preparation need not be free of mammalian RNA. This allows the
investigator to carry out a simple and quick RNA preparation from
infected tissue to obtain bacterial mRNA species which are very
short lived in the bacterium (in the order of 2 minute halflives).
Optimally the bacterial mRNA is prepared from infected murine lung
tissue by mechanical disruption in the presence of TRIzole
(GIBCO-BRL) for very short periods of time, subsequent processing
according to the manufacturers of TRIzole reagent and DNAase
treatment to remove contaminating DNA. Preferably the process is
optimized by finding those conditions which give a maximum amount
of bacterial 16S ribosomal RNA, preferably that of S. epidermidis,
as detected by probing Northerns with a suitably labeled sequence
specific oligonucleotide probe. Typically a 5' dye labelled primer
is used in each PCR primer pair in a PCR reaction which is
terminated optimally between 8 and 25 cycles. The PCR products are
separated on 6% polyacrylamide gels with detection and
quantification using GeneScanner (manufactured by ABI).
[0030] Use of the of these technologies when applied to the
sequences of the present invention enables identification of
bacterial proteins expressed during infection, inhibitors of which
would have utility in anti-bacterial therapy.
Polynucleotides
[0031] The present invention relates to novel polynucleotides and
novel polypeptides of S. epidermidis, among other things, as
described below. The invention particularly relates to the
nucleotide sequences set forth in the Sequence Listing SEQ ID NOs:
1-3334, typically as odd numbered ID numbers, and the corresponding
deduced amino acid sequences also set forth in the Sequence Listing
SEQ ID NOs:1-3334, typically as even numbered ID numbers. SEQ ID
NOs 1-3334 refer to open reading frames (ORFs). The invention also
relates to consensus polynucleotide sequences from which the ORFs
were extracted. These genomic sequences include the ORFs,
intergenic regions and ribosomal RNA genes. Such genomic
polynucleotides are set forth as SEQ ID Nos 3335-4464. It will be
noted that minor errors in sequencing can occur which do not depart
from the spirit of the invention; S. epidermidis polynucleotides
and polypeptides having any corrected sequences are thus
encompassed by this invention.
[0032] Using the information provided herein and known, standard
methods, such as those for cloning and sequencing and those for
synthesizing polynucleotides and polypeptides (see, e.g., Sambrook
et al., Molecular Cloning: A Laboratory Manual, 2d Ed., Cold Spring
Harbor Laboratory Press, Cold Spring Harbor, N.Y. (1989)), one can
generate numerous unique fragments, both longer and shorter than
the polynucleotides and polypeptides set forth in the Sequence
Listing, of the S. epidermidis genome and the S. epidermidis coding
regions, which are encompassed by the present invention. To be
unique, a fragment must be of sufficient size to distinguish it
from other known nucleic acid sequences, most readily determined by
comparing any selected S. epidermidis fragment to the nucleotide
sequences in computer databases such as GenBank. Such comparative
searches are standard in the art. Many unique fragments will be S.
epidermidis-specific. Typically, a unique fragment useful as a
primer or probe will be at least about 20 to about 25 nucleotides
in length, depending upon the specific nucleotide content of the
sequence. Additionally, fragments can be, for example, at least
about 30, 40, 50, 60, 75, 80, 90, 100, 150, 200, 250, 300, 400, 500
or more nucleotides in length. The nucleic acid fragment can be
single, double or triple stranded, depending upon the purpose for
which it is intended.
[0033] Additionally, as discussed above and below, modifications
can be made to the S. epidermidis polynucleotides and polypeptides
that are encompassed by the present invention. For example,
nucleotide substitutions can be made which do not affect the
polypeptide encoded by the nucleic acid, and thus any
polynucleotide which encodes the polypeptides of this invention is
within the present invention. Additionally, certain amino acid
substitutions (and corresponding nucleotide substitutions to encode
them) can be made which are known in the art to be neutral
(Robinson W. E. Jr. and Mitchell, W. m., AIDS 4: S141-S162 (1990).
Such variations may arise naturally as allelic variations (e.g.,
due to genetic polymorphism) or may be produced by human
intervention (e.g., by mutagenesis of cloned DNA sequences), such
as induced point, deletion, insertion and substitution mutations.
Minor changes in amino acid sequence are generally preferred, such
as conservative amino acid replacements, small internal deletions
or insertions, and additions or deletions at the ends of the
molecules. Substitutions may be designed based on, for example, the
model of Dayhoff, et al. (in Atlas of Protein Sequence and
Structure 1978, Nat'l Biomed. Res. Found., Washington D.C.). These
modifications can result in changes in the amino acid sequence,
provide silent mutations, modify a restriction site, or provide
other specific mutations. Likewise, such amino acid changes result
in a different nucleic acid encoding the polypeptides and proteins.
Thus, alternative polynucleotides, which are within the parameters
of the present invention, are contemplated by such
modifications.
[0034] Furthermore, the polynucleotide sequences set forth as SEQ
ID Nos: 1-3334 in the Sequence Listing are open reading frames
(ORFs), i.e., coding regions of S. epidermidis. The polypeptide
encoded by each open reading frame can be deduced, and the
molecular weight of the polypeptide thus calculated using amino
acid residue molecular weight values well known in the art. Any
selected coding region can be functionally linked, using standard
techniques such as standard subcloning techniques, to any desired
regulatory sequence, whether a S. epidermidis regulatory sequence
or a heterologous regulatory sequence, or to a heterologous coding
sequence to create a fusion protein, as further described
herein.
[0035] Polynucleotides of the present invention may be in the form
of RNA, such as mRNA or cRNA, or in the form of DNA, including, for
instance, cDNA and genomic DNA obtained by cloning or produced by
chemical synthetic techniques or by a combination thereof. The DNA
may be triple-stranded, double-stranded or single-stranded.
Single-stranded DNA may be the coding strand, also known as the
sense strand, or it may be the non-coding strand, also referred to
as the anti-sense strand.
[0036] The coding sequence which encodes a S. epidermidis
polypeptide of this invention may be identical to the coding
sequence of a polynucleotide set forth in the sequence listing. It
also may be a polynucleotide with a different sequence which, as a
result of the redundancy (degeneracy) of the genetic code, encodes
a S. epidermidis polypeptide set forth in the sequence listing.
[0037] Polynucleotides of the present invention which encode a S.
epidermidis polypeptide set forth in the sequence listing may
include, but are not limited to, the coding sequence for a mature
polypeptide, by itself; the coding sequence for a mature
polypeptide and additional coding sequences, such as those encoding
a leader or secretory sequence, such as a pre-, or pro- or
prepro-protein sequence; the coding sequence of a mature
polypeptide, with or without the aforementioned additional coding
sequences, together with additional, non-coding sequences,
including for example, but not limited to non-coding 5' and 3'
sequences, such as the transcribed, non-translated sequences that
play a role in transcription (including termination signals, for
example), ribosome binding, mRNA stability elements, and additional
coding sequence which encode additional amino acids, such as those
which provide additional functionalities. Thus, for instance, a
polypeptide may be fused to a marker sequence, such as a peptide,
which facilitates purification of the fused polypeptide. In certain
embodiments of this aspect of the invention, the marker sequence is
a hexa-histidine peptide, such as the tag provided in the pQE
vector (Qiagen, Inc.), among others, many of which are commercially
available. As described in Gentz et al., Proc. Natl. Acad. Sci.,
USA 86: 821-824 (1989), for instance, hexa-histidine provides for
convenient purification of the fusion protein. The HA tag may also
be used to create fusion proteins and corresponds to an epitope
derived of influenza hemagglutinin protein, which has been
described by Wilson et al., Cell 37: 767 (1984), for instance.
Polynucleotides of the invention also include, but are not limited
to, polynucleotides comprising a structural gene and its naturally
associated genetic elements.
[0038] In accordance with the foregoing, the term "polynucleotide
encoding a polypeptide" as used herein encompasses polynucleotides
which include a sequence encoding a polypeptide of the present
invention, particularly a polypeptide having a S. epidermidis amino
acid sequence set forth in the Sequence Listing. The term
encompasses polynucleotides that include a single continuous region
or discontinuous regions encoding the polypeptide (for example,
interrupted by integrated phage or insertion sequence or editing)
together with additional regions, that also may contain coding
and/or non-coding sequences.
[0039] The present invention further relates to variants of the
herein above described polynucleotides which encode for fragments,
analogs and derivatives of the polypeptide having a deduced S.
epidermidis amino acid sequence set forth in the Sequence Listing.
A variant of the polynucleotide may be a naturally occurring
variant such as a naturally occurring allelic variant, or it may be
a variant that is not known to occur naturally. Such non-naturally
occurring variants of the polynucleotide may be made by mutagenesis
techniques, including those applied to polynucleotides, cells or
organisms.
[0040] Among variants in this regard are variants that differ from
the aforementioned polynucleotides by nucleotide substitutions,
deletions or additions. The substitutions, deletions or additions
may involve one or more nucleotides. The variants may be altered in
coding or non-coding regions or both. Alterations in the coding
regions may produce conservative or non-conservative amino acid
substitutions, deletions or additions. Preferred are
polynucleotides encoding a variant, analog, derivative or fragment,
or a variant, analogue or derivative of a fragment, which have a S.
epidermidis sequence as set forth in the Sequence Listing, in which
several, a few, 5 to 10, 1 to 5, 1 to 3, 2, 1 or no amino acid(s)
is substituted, deleted or added, in any combination. Especially
preferred among these are silent substitutions, additions and
deletions, which do not alter the properties and activities of the
S. epidermidis polypeptides set forth in the Sequence Listing. Also
especially preferred in this regard are conservative
substitutions.
[0041] Further preferred embodiments of the invention are
polynucleotides that are at least 70% identical over their entire
length to a polynucleotide encoding a polypeptide having an amino
acid sequence set forth in the Sequence Listing, and
polynucleotides which are complementary to such polynucleotides.
Alternatively, most highly preferred are polynucleotides that
comprise a region that is at least 80% or at least 85% identical
over their entire length to a polynucleotide encoding a S.
epidermidis polypeptide set forth in the Sequence Listing,
including complementary polynucleotides. In this regard,
polynucleotides at least 90%, 91%, 92%, 93%, 94%, 95%, or 96%
identical over their entire length to the same are particularly
preferred, and among these particularly preferred polypeptides,
those with at least 95% are especially preferred. Furthermore,
those with at least 97% are highly preferred among those with at
least 95%, and among these, those with at least 98% and at least
99% are particularly highly preferred, with at least 99% or 99.5%
being the more preferred.
[0042] Preferred embodiments in this respect, moreover, are
polynucleotides which encode polypeptides which retain
substantially the same biological function or activity as the
mature polypeptide encoded by the DNA set forth in the Sequence
Listing.
[0043] The present invention further relates to polynucleotides
that hybridize to the herein above-described sequences. In this
regard, the present invention especially relates to polynucleotides
which hybridize under stringent conditions to the herein
above-described polynucleotides. Stringent conditions are typically
selective conditions. As herein used, the term "stringent
conditions" means hybridization will occur only if there is at
least 95% and preferably at least 97% identity between the
sequences. For a specific sequence, stringent conditions can be
determined empirically according to the nucleotide content, as is
known in the art. For example, a typical example of stringent
conditions is hybridization of a 48mer having 55% GC content at
42.degree. C. in 50% formamide and 750 mM NaCl followed by washing
at 55.degree. C. in 15 mM NaCl and 0.1% SDS.
[0044] As discussed additionally herein regarding polynucleotide
assays of the invention, for instance, polynucleotides of the
invention as discussed above, may be used as a hybridization probe
for RNA, cDNA and genomic DNA to isolate full-length cDNAs and
genomic clones encoding polypeptides of the present invention and
to isolate cDNA and genomic clones of other genes that have a high
sequence similarity to the polynucleotides of the present
invention. Such probes generally will comprise at least 15 bases.
Preferably, such probes will have at least 20, at least 25 or at
least 30 bases, and may have at least 50 bases. Particularly
preferred probes will have at least 30 bases, and will have 50
bases or less, such as 30, 35, 40, 45, or 50 bases.
[0045] For example, the coding region of the polynucleotide of the
present invention may be isolated by screening using the known DNA
sequence to synthesize an oligonucleotide probe. A labeled
oligonucleotide having a sequence complementary to that of a gene
of the present invention is then used to screen a library of cDNA,
genomic DNA or mRNA to determine to which members of the library
the probe hybridizes.
[0046] The polynucleotides and polypeptides of the present
invention may be employed as reagents and materials for development
of treatments of and diagnostics for disease, particularly human
disease, as further discussed herein relating to polynucleotide
assays, inter alia.
[0047] The polynucleotides of the present invention that are
oligonucleotides can be used in the processes herein as described,
but preferably for PCR, to determine whether or not the S.
epidermidis genes identified herein in whole or in part are present
and/or transcribed in infected tissue such as blood. It is
recognized that such sequences will also have utility in diagnosis
of the stage of infection and type of infection the pathogen has
attained.
[0048] The polynucleotides may encode a polypeptide which is the
mature protein plus additional amino or carboxyl-terminal amino
acids, or amino acids interior to the mature polypeptide (when the
mature form has more than one polypeptide chain, for instance).
Such sequences may play a role in processing of a protein from
precursor to a mature form, may allow protein transport, may
lengthen or shorten protein half-life or may facilitate
manipulation of a protein for assay or production, among other
things. As generally is the case in vivo, the additional amino
acids may be processed away from the mature protein by cellular
enzymes.
[0049] A precursor protein, having the mature form of the
polypeptide fused to one or more prosequences may be an inactive
form of the polypeptide. When prosequences are removed such
inactive precursors generally are activated. Some or all of the
prosequences may be removed before activation. Generally, such
precursors are called proproteins.
[0050] The present invention additionally contemplates
polynucleotides functionally encoding fusion polypeptides wherein
the fusion polypeptide comprises a fragment of a S. epidermidis
polypeptide and one or more polypeptide(s) derived from another S.
epidermidis polypeptide or from another organism or a synthetic
polyamino acid sequence. Such polynucleotides may or may not encode
amino acid sequences to facilitate cleavage of the S. epidermidis
polypeptide from the other polypeptide(s) under appropriate
conditions.
[0051] In sum, a polynucleotide of the present invention may encode
a mature protein, a mature protein plus a leader sequence (which
may be referred to as a preprotein), a precursor of a mature
protein having one or more prosequences which are not the leader
sequences of a preprotein, or a preproprotein, which is a precursor
to a proprotein, having a leader sequence and one or more
prosequences, which generally are removed during processing steps
that produce active and mature forms of the polypeptide.
Polypeptides
[0052] The present invention further relates to peptides,
polypeptides and proteins (collectively referred to as
"polypeptides") of S. epidermidis. The amino acid sequence of these
polypeptides is set forth in the Sequence Listing.
[0053] The invention also relates to fragments, analogs and
derivatives of these polypeptides. The terms "fragment,"
"derivative" and "analog" when referring to a polypeptide whose
amino acid sequence is set forth in the Sequence Listing, means a
polypeptide which retains essentially the same biological function
or activity as such polypeptide. Thus, an analog includes a
proprotein which can be activated by cleavage of the proprotein
portion to produce an active mature polypeptide.
[0054] The fragment, derivative or analog of the polypeptide of the
present invention may be (i) one in which one or more of the amino
acid residues are substituted with a conserved or non-conserved
amino acid residue (preferably a conserved amino acid residue) and
such substituted amino acid residue may or may not be one encoded
by the genetic code, or (ii) one in which one or more of the amino
acid residues includes a substituent group, or (iii) one in which
the mature polypeptide is fused with another compound, such as a
compound to increase the half-life of the polypeptide (for example,
polyethylene glycol), or (iv) one in which the additional amino
acids are fused to the mature polypeptide, such as a leader or
secretory sequence or a sequence which is employed for purification
of the mature polypeptide or a proprotein sequence. Such fragments,
derivatives and analogs are deemed to be within the scope of those
skilled in the art from the teachings herein.
[0055] Among the particularly preferred embodiments of the
invention in this regard are polypeptides set forth in the Sequence
Listing, variants, analogs, derivatives and fragments thereof, and
variants, analogs and derivatives of the fragments. Additionally,
fusion polypeptides comprising such polypeptides, variants,
analogs, derivatives and fragments thereof, and variants, analogs
and derivatives of the fragments, in addition to a heterologous
polypeptide, are contemplated by the present invention. Such fusion
polypeptides and proteins, as well as polynucleotides encoding
them, can readily be made using standard techniques, including
standard recombinant techniques for producing and expressing a
recombinant polynucleic acid encoding a fusion protein.
[0056] Among preferred variants are those that vary from a
reference by conservative amino acid substitutions. Such
substitutions are those that substitute a given amino acid in a
polypeptide by another amino acid of like characteristics.
Typically seen as conservative substitutions are the replacements,
one for another, among the aliphatic amino acids Ala, Val, Leu and
Ile; interchange of the hydroxyl residues Ser and Thr, exchange of
the acidic residues Asp and Glu, substitution between the amide
residues Asn and Gln, exchange of the basic residues Lys and Arg
and replacements among the aromatic residues Phe, Tyr.
[0057] Further particularly preferred in this regard are variants,
analogs, derivatives and fragments, and variants, analogs and
derivatives of the fragments, having the amino acid sequence of any
polypeptide set forth in the Sequence Listing, in which several, a
few, 5 to 10, 1 to 5, 1 to 3, 2, 1 or no amino acid residues are
substituted, deleted or added, in any combination. Especially
preferred among these are silent substitutions, additions and
deletions, which do not alter the properties and activities of the
polypeptide of the present invention. Also especially preferred in
this regard are conservative substitutions. Most highly preferred
are polypeptides having an amino acid sequence set forth in the
Sequence Listing without substitutions.
[0058] The polypeptides and polynucleotides of the present
invention are preferably provided in an isolated form, and
preferably are purified to homogeneity.
[0059] The polypeptides of the present invention include any
polypeptide set forth in the Sequence Listing (in particular a
mature polypeptide) as well as polypeptides which have at least 70%
identity to a polypeptide set forth in the Sequence Listing,
preferably at least 80% or 85% identity to a polypeptide set forth
in the Sequence Listing, and more preferably at least 90%
similarity (more preferably at least 90% identity) to a polypeptide
set forth in the Sequence Listing and still more preferably at
least 95%, 96%, 97%, 98%, 99%, or 99.5% similarity (still more
preferably at least 95%, 96%, 97%, 98%, 99%, or 99.5% identity) to
a polypeptide set forth in the Sequence Listing and also include
portions of such polypeptides with such portion of the polypeptide
generally containing at least 30 amino acids and more preferably at
least 50 amino acids, such as 30, 35, 40, 45 or 50 amino acids.
[0060] Fragments or portions of the polypeptides of the present
invention may be employed for producing the corresponding
full-length polypeptide by peptide synthesis; therefore, the
fragments may be employed as intermediates for producing the
full-length polypeptides. Fragments or portions of the
polynucleotides of the present invention may be used to synthesize
full-length polynucleotides of the present invention.
[0061] Fragments
[0062] Also among preferred embodiments of this aspect of the
present invention are polypeptides comprising fragments of the
polypeptide having the amino acid sequence set forth in the
Sequence Listing, and fragments of variants and derivatives of the
polypeptides set forth in the Sequence Listing.
[0063] In this regard a fragment is a polypeptide having an amino
acid sequence that entirely is the same as part but not all of the
amino acid sequence of the aforementioned S. epidermidis
polypeptides and variants or derivatives thereof.
[0064] Such fragments may be "free-standing," i.e., not part of or
fused to other amino acids or polypeptides, or they may be
comprised within a larger polypeptide of which they form a part or
region. When comprised within a larger polypeptide, the presently
discussed fragments most preferably form a single continuous
region. However, several fragments may be comprised within a single
larger polypeptide. For instance, certain preferred embodiments
relate to a fragment of a polypeptide of the present invention
comprised within a precursor polypeptide designed for expression in
a host and having heterologous pre and pro-polypeptide regions
fused to the amino terminus of the fragment and an additional
region fused to the carboxyl terminus of the fragment. Therefore,
fragments in one aspect of the meaning intended herein, refers to
the portion or portions of a fusion polypeptide or fusion protein
derived from a polypeptide of the present invention.
[0065] Representative examples of polypeptide fragments of the
invention, include, for example, in any selected polypeptide,
fragments from about amino acid number 1-20, 21-40, 41-60, 61-80,
81-100, and 101-200, 201-300, or, at the COOH-terminal end, the
C-terminal 20 amino acids, the C-terminal 30 amino acids, the
C-terminal 40 amino acids, the C-terminal 50 amino acids, and any
combination of these fragments, such as fragment from about amino
acid number 1-40, 1-60, 21-60, 41-80, 61-100, and the like.
[0066] In this context "about" herein includes the particularly
recited ranges larger or smaller by several, a few, 5, 4, 3, 2 or 1
amino acid at either extreme or at both extremes.
[0067] Preferred fragments of the invention include, for example,
truncation polypeptides including polypeptides having an amino acid
sequence set forth in the Sequence Listing, or of variants or
derivatives thereof, except for deletion of a continuous series of
residues (that is, a continuous region, part or portion) that
includes the amino terminus, or a continuous series of residues
that includes the carboxyl terminus or, as in double truncation
mutants, deletion of two continuous series of residues, one
including the amino terminus and one including the carboxyl
terminus. Fragments having the size ranges set out above also are
preferred embodiments of truncation fragments, which are especially
preferred among fragments generally. Degradation forms of the
polypeptides of the invention in a host cell are also
preferred.
[0068] Also preferred in this aspect of the invention are fragments
characterized by structural or functional attributes of the
polypeptide of the present invention. Preferred embodiments of the
invention in this regard include fragments that comprise
alpha-helix and alpha-helix forming regions, beta-sheet and
beta-sheet-forming regions, turn and turn-forming regions, coil and
coil-forming regions, hydrophilic regions, hydrophobic regions,
alpha amphipathic regions, beta amphipathic regions, flexible
regions, surface-forming regions, substrate binding region, and
high antigenic index regions of the polypeptide of the present
invention, and combinations of such fragments.
[0069] Preferred regions are those that mediate activities of the
polypeptide of the present invention. Most highly preferred in this
regard are fragments that have a chemical, biological or other
activity of the polypeptide of the present invention, including
those with a similar activity or an improved activity, or with a
decreased undesirable activity. Particularly preferred are
fragments comprising receptors or domains of enzymes that confer a
function essential for viability of S. epidermidis or the ability
to cause disease in humans. Further preferred polypeptide fragments
are those that comprise or contain antigenic or immunogenic
determinants in an animal, especially in a human.
[0070] It will be appreciated that the invention also relates to,
among others, polynucleotides encoding the aforementioned
fragments, polynucleotides that hybridize to polynucleotides
encoding the fragments, particularly those that hybridize under
stringent conditions, and polynucleotides, such as PCR primers, for
amplifying polynucleotides that encode the fragments. In these
regards, preferred polynucleotides are those that correspond to the
preferred fragments, as discussed above.
Vectors, Host Cells, Expression
[0071] The present invention also relates to vectors which comprise
a polynucleotide or polynucleotides of the present invention, host
cells which are genetically engineered with vectors of the
invention and the production of polypeptides of the invention by
recombinant techniques.
[0072] Host cells can be genetically engineered to incorporate
polynucleotides and express polypeptides of the present invention.
Introduction of a polynucleotides into the host cell can be
effected by calcium phosphate transfection, DEAE-dextran mediated
transfection, transvection, microinjection, cationic lipid-mediated
transfection, electroporation, transduction, scrape loading,
ballistic introduction, infection or other methods. Such methods
are described in many standard laboratory manuals, such as Davis et
al., BASIC METHODS IN MOLECULAR BIOLOGY, (1986) and Sambrook et
al., MOLECULAR CLONING: A LABORATORY MANUAL, 2nd Ed., Cold Spring
Harbor Laboratory Press, Cold Spring Harbor, N.Y. (1989).
[0073] Polynucleotide constructs in host cells can be used in a
conventional manner to produce the gene product encoded by the
recombinant sequence. Alternatively, the polypeptides of the
invention can be synthetically produced by conventional peptide
synthesizers.
[0074] Mature proteins can be expressed in mammalian cells, yeast,
bacteria, or other cells under the control of appropriate
promoters. Cell-free translation systems can also be employed to
produce such proteins using RNAs derived from the DNA constructs of
the present invention. Appropriate cloning and expression vectors
for use with prokaryotic and eukaryotic hosts are described by
Sambrook et al., MOLECULAR CLONING: A LABORATORY MANUAL, 2nd Ed.,
Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.
(1989).
[0075] In accordance with this aspect of the invention the vector
may be, for example, a plasmid vector, a single or double-stranded
phage vector, a single or double-stranded RNA or DNA viral vector.
Plasmids generally are designated herein by a lower case p preceded
and/or followed by capital letters and/or numbers, in accordance
with standard naming conventions that are familiar to those of
skill in the art. Starting plasmids disclosed herein are either
commercially available, publicly available, or can be constructed
from available plasmids by routine application of well known,
published procedures, given the teachings herein. Many plasmids and
other cloning and expression vectors that can be used in accordance
with the present invention are well known and readily available to
those of skill in the art.
[0076] Preferred among vectors, in certain respects, are those for
expression of polynucleotides and polypeptides of the present
invention. Generally, such vectors comprise cis-acting control
regions effective for expression in a host operatively linked to
the polynucleotide to be expressed. Appropriate trans-acting
factors either are supplied by the host, supplied by a
complementing vector or supplied by the vector itself upon
introduction into the host.
[0077] In certain preferred embodiments in this regard, the vectors
provide for specific expression. Such specific expression may be
inducible expression or expression only in certain types of cells
or both inducible and cell-specific. Particularly preferred among
inducible vectors are vectors that can be induced for expression by
environmental factors that are easy to manipulate, such as
temperature and nutrient additives. A variety of vectors suitable
to this aspect of the invention, including constitutive and
inducible expression vectors for use in prokaryotic and eukaryotic
hosts, are well known and employed routinely by those of skill in
the art.
[0078] A great variety of expression vectors can be used to express
a polypeptide of the invention. Such vectors include, among others,
chromosomal, episomal and virus-derived vectors, e.g., vectors
derived from bacterial plasmids, from bacteriophage, from
transposons, from yeast episomes, from insertion elements, from
yeast chromosomal elements, from viruses such as baculoviruses,
papova viruses, such as SV40, vaccinia viruses, adenoviruses, fowl
pox viruses, pseudorabies viruses and retroviruses, and vectors
derived from combinations thereof, such as those derived from
plasmid and bacteriophage genetic elements, such as cosmids and
phagemids, all may be used for expression in accordance with this
aspect of the present invention. Generally, any vector suitable to
maintain, propagate or express polynucleotides to express a
polypeptide in a host may be used for expression in this
regard.
[0079] The appropriate DNA sequence may be inserted into the vector
by any of a variety of well-known and routine techniques, such as,
for example, those set forth in Sambrook et al., MOLECULAR CLONING,
A LABORATORY MANUAL, 2nd Ed.; Cold Spring Harbor Laboratory Press,
Cold Spring Harbor, N.Y. (1989).
[0080] The DNA sequence in the expression vector is operatively
linked to appropriate expression control sequence(s), including,
for instance, a promoter to direct mRNA transcription.
Representatives of such promoters include, but are not limited to,
the phage lambda PL promoter, the E. coli lac, trp and tac
promoters, the SV40 early and late promoters and promoters of
retroviral LTRs.
[0081] In general, expression constructs will contain sites for
transcription initiation and termination, and, in the transcribed
region, a ribosome binding site for translation. The coding portion
of the mature transcripts expressed by the constructs will include
a translation initiating AUG at the beginning and a termination
codon appropriately positioned at the end of the polypeptide to be
translated.
[0082] In addition, the constructs may contain control regions that
regulate as well as engender expression. Generally, in accordance
with many commonly practiced procedures, such regions will operate
by controlling transcription, such as transcription factors,
repressor binding sites and termination, among others.
[0083] Vectors for propagation and expression generally will
include selectable markers and amplification regions, such as, for
example, those set forth in Sambrook et al., MOLECULAR CLONING, A
LABORATORY MANUAL, 2nd Ed.; Cold Spring Harbor Laboratory Press,
Cold Spring Harbor, N.Y. (1989).
[0084] Representative examples of appropriate hosts include
bacterial cells, such as streptococci, staphylococci, E. coli,
streptomyces and Bacillus subtilis cells; fungal cells, such as
yeast cells and Aspergillus cells; insect cells such as Drosophila
S2 and Spodoptera Sf9 cells; animal cells such as CHO, COS, HeLa,
C127, 3T3, BHK, 293 and Bowes melanoma cells; and plant cells.
[0085] The following vectors, which are commercially available, are
provided by way of example. Among vectors preferred for use in
bacteria are pQE70, pQE60 and pQE-9, available from Qiagen; pBS
vectors, Phagescript vectors, Bluescript vectors, pNH8A, pNH16a,
pNH18A, pNH46A, available from Stratagene; and ptrc99a, pKK223-3,
pKK233-3, pDR540, pRIT5 available from Pharmacia, and pBR322 (ATCC
37017). Among preferred eukaryotic vectors are pWLNEO, pSV2CAT,
pOG44, pXT1 and pSG available from Stratagene; and pSVK3, pBPV,
pMSG and pSVL available from Pharmacia. These vectors are listed
solely by way of illustration of the many commercially available
and well known vectors that are available to those of skill in the
art for use in accordance with this aspect of the present
invention. It will be appreciated that any other plasmid or vector
suitable for, for example, introduction, maintenance, propagation
or expression of a polynucleotide or polypeptide of the invention
in a host may be used in this aspect of the invention.
[0086] Promoter regions can be selected from any desired gene using
vectors that contain a reporter transcription unit lacking a
promoter region, such as a chloramphenicol acetyl transferase
("CAT") transcription unit, downstream of restriction site or sites
for introducing a candidate promoter fragment; i.e., a fragment
that may contain a promoter. As is well known, introduction into
the vector of a promoter-containing fragment at the restriction
site upstream of the cat gene engenders production of CAT activity,
which can be detected by standard CAT assays. Vectors suitable to
this end are well known and readily available, such as pKK232-8 and
pCM7. Promoters for expression of polynucleotides of the present
invention include not only well known and readily available
promoters, but also promoters that readily may be obtained by the
foregoing technique, using a reporter gene.
[0087] Among known prokaryotic promoters suitable for expression of
polynucleotides and polypeptides in accordance with the present
invention are the E. coli lacI and lacZ and promoters, the T3 and
T7 promoters, the gpt promoter, the lambda PR, PL promoters and the
trp promoter.
[0088] Among known eukaryotic promoters suitable in this regard are
the CMV immediate early promoter, the HSV thymidine kinase
promoter, the early and late SV40 promoters, the promoters of
retroviral LTRs, such as those of the Rous sarcoma virus ("RSV"),
and metallothionein promoters, such as the mouse metallothionein-I
promoter.
[0089] Recombinant expression vectors will include, for example,
origins of replication, a promoter preferably derived from a
highly-expressed gene to direct transcription of a downstream
structural sequence, and a selectable marker to permit isolation of
vector containing cells after exposure to the vector.
[0090] Polynucleotides of the invention, encoding the heterologous
structural sequence of a polypeptide of the invention generally
will be inserted into the vector using standard techniques so that
it is operably linked to the promoter for expression. The
polynucleotide will be positioned so that the transcription start
site is located appropriately 5' to a ribosome binding site. The
ribosome binding site will be 5' to the AUG that initiates
translation of the polypeptide to be expressed. Generally, there
will be no other open reading frames that begin with an initiation
codon, usually AUG, and lie between the ribosome binding site and
the initiation codon. Also, generally, there will be a translation
stop codon at the end of the polypeptide and there will be a
polyadenylation signal in constructs for use in eukaryotic hosts.
Transcription termination signal appropriately disposed at the 3'
end of the transcribed region may also be included in the
polynucleotide construct.
[0091] For secretion of the translated protein into the lumen of
the endoplasmic reticulum, into the periplasmic space or into the
extracellular environment, appropriate secretion signals may be
incorporated into the expressed polypeptide. These signals may be
endogenous to the polypeptide or they may be heterologous
signals.
[0092] The polypeptide may be expressed in a modified form, such as
a fusion protein, and may include not only secretion signals but
also additional heterologous functional regions. Thus, for
instance, a region of additional amino acids, particularly charged
amino acids, may be added to the N- or C-terminus of the
polypeptide to improve stability and persistence in the host cell,
during purification or during subsequent handling and storage.
Also, regions also may be added to the polypeptide to facilitate
purification. Such regions may be removed prior to final
preparation of the polypeptide. The addition of peptide moieties to
polypeptides to engender secretion or excretion, to improve
stability or to facilitate purification, among others, are familiar
and routine techniques in the art. A preferred fusion protein
comprises a heterologous region from immunoglobulin that is useful
to solubilize or purify polypeptides. For example, EP-A-0 464 533
(Canadian counterpart 2045869) discloses fusion proteins comprising
various portions of constant region of immunoglobin molecules
together with another protein or part thereof. In drug discovery,
for example, proteins have been fused with antibody Fc portions for
the purpose of high-throughput screening assays to identify
antagonists. See, D. Bennett et al., Journal of Molecular
Recognition, Vol. 8 52-58 (1995) and K. Johanson et al., The
Journal of Biological Chemistry, Vol. 270, No. 16, pp 9459-9471
(1995).
[0093] Cells typically then are harvested by centrifugation,
disrupted by physical or chemical means, and the resulting crude
extract retained for further purification.
[0094] Microbial cells employed in expression of proteins can be
disrupted by any convenient method, including freeze-thaw cycling,
sonication, mechanical disruption, or use of cell lysing agents;
such methods are well known to those skilled in the art.
[0095] Mammalian expression vectors may comprise expression
sequences, such as an origin of replication, a suitable promoter
and enhancer, and also any necessary ribosome binding sites,
polyadenylation regions, splice donor and acceptor sites,
transcriptional termination sequences, and 5' flanking
non-transcribed sequences that are useful or necessary for
expression.
[0096] The polypeptide can be recovered and purified from
recombinant cell cultures by well-known methods including ammonium
sulfate or ethanol precipitation, acid extraction, anion or cation
exchange chromatography, phosphocellulose chromatography,
hydrophobic interaction chromatography, affinity chromatography,
hydroxylapatite chromatography and lectin chromatography. Most
preferably, high performance liquid chromatography is employed for
purification. Well known techniques for refolding protein may be
employed to regenerate active conformation when the polypeptide is
denatured during isolation and or purification.
[0097] Polynucleotide Assays
[0098] This invention is also related to the use of the
polynucleotides of the present invention to detect complementary
polynucleotides such as, for example, as a diagnostic reagent.
Detection of complementary nucleotides in a eukaryote, particularly
a mammal, and especially a human, will provide a diagnostic method
for diagnosis of a disease. Eukaryotes (herein also
"individual(s)"), particularly mammals, and especially humans,
infected with S. epidermidis may be detected at the DNA level by a
variety of techniques. By selecting regions of nucleic acids that
vary among strains of S. epidermidis, preferred candidates for
distinguishing a specific strain of S. epidermidis can be obtained.
Furthermore, by selecting regions of nucleic acids that vary
between S. epidermidis and other organisms, preferred candidates
for distinguishing a S. epidermidis from other organisms can be
obtained. Nucleic acids for diagnosis may be obtained from an
infected individual's cells and tissues, such as bone, blood,
muscle, cartilage, and skin. Genomic DNA may be used directly for
detection or may be amplified enzymatically by using PCR (Saiki et
al., Nature, 324: 163-166 (1986) prior to analysis. RNA or cDNA may
also be used in the same ways. As an example, PCR primers
complementary to the nucleic acid forming part of the
polynucleotide of the present invention can be used to identify and
analyze for its presence and/or expression. Using PCR,
characterization of the strain of S. epidermidis present in a
mammal, and especially a human, may be made by an analysis of the
genotype of the prokaryote gene. For example, deletions and
insertions can be detected by a change in size of the amplified
product in comparison to the genotype of a reference sequence.
Point mutations can be identified by hybridizing amplified DNA to
radiolabeled RNA or alternatively, radiolabeled antisense DNA
sequences. Perfectly matched sequences can be distinguished from
mismatched duplexes by RNase A digestion or by differences in
melting temperatures.
[0099] Sequence differences between a reference gene and genes
having mutations also may be revealed by direct DNA sequencing. In
addition, cloned DNA segments may be employed as probes to detect
specific DNA segments. The sensitivity of such methods can be
greatly enhanced by appropriate use of PCR or another amplification
method. For example, a sequencing primer can be used with
double-stranded PCR product or a single-stranded template molecule
generated by a modified PCR. The sequence determination is
performed by conventional procedures with radiolabeled nucleotide
or by automatic sequencing procedures with fluorescent-tags.
[0100] Genetic characterization based on DNA sequence differences
may be achieved by detection of alteration in electrophoretic
mobility of DNA fragments in gels, with or without denaturing
agents. Small sequence deletions and insertions can be visualized
by high resolution gel electrophoresis. DNA fragments of different
sequences may be distinguished on denaturing formamide gradient
gels in which the mobilities of different DNA fragments are
retarded in the gel at different positions according to their
specific melting or partial melting temperatures (see, e.g., Myers
et al., Science, 230: 1242 (1985)).
[0101] Sequence changes at specific locations also may be revealed
by nuclease protection assays, such as RNase and S1 protection or
the chemical cleavage method (e.g., Cotton et al., Proc. Natl.
Acad. Sci., USA, 85: 4397-4401 (1985)).
[0102] Thus, the detection of a specific DNA sequence may be
achieved by methods such as hybridization, RNase protection,
chemical cleavage, direct DNA sequencing or the use of restriction
enzymes, e.g., restriction fragment length polymorphisms (RFLP) and
Southern blotting of genomic DNA.
[0103] In addition to more conventional gel-electrophoresis and DNA
sequencing, mutations also can be detected by in situ analysis.
[0104] Cells carrying mutations or polymorphisms in the gene of the
present invention may also be detected at the DNA level by a
variety of techniques, to allow for serotyping, for example. For
example, RT-PCR can be used to detect mutations. It is particularly
preferred to use RT-PCR in conjunction with automated detection
systems, such as, for example, GeneScan. RNA or cDNA may also be
used for the same purpose, PCR or RT-PCR. As an example, PCR
primers complementary to the nucleic acid encoding the polypeptide
of the present invention can be used to identify and analyze
mutations. The primers may be used to amplify the gene isolated
from the individual such that the gene may then be subject to
various techniques for elucidation of the DNA sequence. In this
way, mutations in the DNA sequence may be diagnosed.
[0105] The invention provides a process for diagnosing disease,
arising from infection with S. epidermidis, comprising determining
from a sample isolated or derived from an individual an increased
level of expression of a polynucleotide having the sequence of a
polynucleotide set forth in the Sequence Listing. Increased
expression of polynucleotide can be measured using any on of the
methods well known in the art for the quantitation of
polynucleotides, such as, for example, PCR, RT-PCR, RNase
protection, Northern blotting and other hybridization methods.
[0106] Polypeptide Assays
[0107] The present invention also relates to diagnostic assays such
as quantitative and diagnostic assays for detecting levels of the
polypeptide of the present invention in cells and tissues,
including determination of normal and abnormal levels. Thus, for
instance, a diagnostic assay in accordance with the invention for
detecting over-expression of the polypeptide compared to normal
control tissue samples may be used to detect the presence of an
infection, for example, and to identify the infecting organism.
Assay techniques that can be used to determine levels of a
polypeptide, in a sample derived from a host are well-known to
those of skill in the art. Such assay methods include
radioimmunoassays, competitive-binding assays, Western Blot
analysis and ELISA assays. Among these, ELISAs frequently are
preferred. An ELISA assay initially comprises preparing an antibody
specific to the polypeptide, preferably a monoclonal antibody. In
addition, a reporter antibody generally is prepared which binds to
the monoclonal antibody. The reporter antibody is attached to a
detectable reagent such as radioactive, fluorescent or enzymatic
reagent, such as horseradish peroxidase enzyme.
[0108] Antibodies
[0109] The polypeptides, their fragments or other derivatives, or
analogs thereof, or cells expressing them can be used as an
immunogen to produce antibodies thereto. The present invention
includes, for example, monoclonal and polyclonal antibodies,
chimeric, single chain, and humanized antibodies, as well as Fab
fragments, or the product of an Fab expression library.
[0110] Antibodies generated against the polypeptides corresponding
to a sequence of the present invention can be obtained by direct
injection of the polypeptides into an animal or by administering
the polypeptides to an animal, preferably a nonhuman. The antibody
so obtained will then bind the polypeptide itself. In this manner,
even a sequence encoding only a fragment of the polypeptides can be
used to generate antibodies binding the whole native polypeptides.
Such antibodies can then be used to isolate the polypeptide from
tissue expressing that polypeptide.
[0111] For preparation of monoclonal antibodies, any technique
known in the art which provides antibodies produced by continuous
cell line cultures can be used. Examples include various
techniques, such as those in Kohler, G. and Milstein, C., Nature
256: 495-497 (1975); Kozbor et al., Immunology Today 4: 72 (1983);
Cole et al., pg. 77-96 in MONOCLONAL ANTIBODIES AND CANCER THERAPY,
Alan R. Liss, Inc. (1985); U.S. Pat. No. 5,545,403; U.S. Pat. No.
5,545,405; U.S. Pat. No. 5,654,403; U.S. Pat. No. 5,792,838; U.S.
Pat. No. 5,316,938; U.S. Pat. No. 5,633,162; U.S. Pat. No.
5,644,036; U.S. Pat. No. 5,858,725.
[0112] Techniques described for the production of single chain
antibodies (U.S. Pat. No. 4,946,778) can be adapted to produce
single chain antibodies to immunogenic polypeptide products of this
invention. Also, transgenic mice, or other organisms such as other
mammals, may be used to express humanized antibodies to immunogenic
polypeptide products of this invention.
[0113] Alternatively, phage display technology could be utilized to
select antibody genes with binding activities towards the
polypeptide either from repertoires of PCR amplified v-genes of
lymphocytes from humans screened for possessing anti-Fbp or from
naive libraries (McCafferty, J. et al., (1990), Nature 348,
552-554; Marks, J. et al., (1992) Biotechnology 10, 779-783). The
affinity of these antibodies can also be improved by chain
shuffling (Clackson, T. et al., (1991) Nature 352, 624-628).
[0114] If two antigen binding domains are present, each domain may
be directed against a different epitope--termed `bispecific`
antibodies.
[0115] The above-described antibodies may be employed to isolate or
to identify clones expressing the polypeptide or purify the
polypeptide of the present invention by attachment of the antibody
to a solid support for isolation and/or purification by affinity
chromatography.
[0116] Thus, among others, antibodies against the polypeptide of
the present invention may be employed to inhibit and/or treat
infections, particularly bacterial infections and especially
infections arising from S. epidermidis.
[0117] Polypeptide derivatives include antigenically, epitopically
or immunologically equivalent derivatives which form a particular
aspect of this invention. The term "antigenically equivalent
derivative" as used herein encompasses a polypeptide or its
equivalent which will be specifically recognized by certain
antibodies which, when raised to the protein or polypeptide
according to the present invention, interfere with the immediate
physical interaction between pathogen and mammalian host. The term
"immunologically equivalent derivative" as used herein encompasses
a peptide or its equivalent which when used in a suitable
formulation to raise antibodies in a vertebrate, the antibodies act
to interfere with the immediate physical interaction between
pathogen and mammalian host.
[0118] The polypeptide, such as an antigenically or immunologically
equivalent derivative or a fusion protein thereof can be used as an
antigen to immunize a mouse or other animal such as a rat or
chicken. The fusion protein may provide stability to the
polypeptide. The antigen may be associated, for example by
conjugation, with an immunogenic carrier protein, for example
bovine serum albumin (BSA) or keyhole limpet haemocyanin (KLH).
Alternatively, a multiple antigenic peptide comprising multiple
copies of the protein or polypeptide, or an antigenically or
immunologically equivalent polypeptide thereof, may be sufficiently
antigenic to improve immunogenicity so as to obviate the use of a
carrier.
[0119] Preferably the antibody or derivative thereof is modified to
make it less immunogenic in the individual. For example, if the
individual is human the antibody may most preferably be
"humanized," wherein the complementarity determining region(s) of
the hybridoma-derived antibody has been transplanted into a human
monoclonal antibody, for example as described in Jones, P. et al.
(1986), Nature 321, 522-525 or Tempest et al., (1991) Biotechnology
9, 266-273.
[0120] The use of a polynucleotide of the invention in genetic
immunization will preferably employ a suitable delivery method such
as direct injection of plasmid DNA into muscle (Wolff et al., Hum
Mol Genet 1992, 1:363, Manthorpe et al., Hum. Gene Ther. 1963: 4,
419), delivery of DNA complexed with specific protein carriers (Wu
et al., J Biol Chem 1989: 264, 16985), coprecipitation of DNA with
calcium phosphate (Benvenisty & Reshef, PNAS, 1986: 83, 9551),
encapsulation of DNA in various forms of liposomes (Kaneda et al.,
Science 1989: 243, 375), particle bombardment (Tang et al., Nature
1992, 356:152, Eisenbraun et al., DNA Cell Biol 1993, 12:791) and
in vivo infection using cloned retroviral vectors (Seeger et al.,
PNAS 1984: 81, 5849).
[0121] Binding Molecules and Assays
[0122] This invention also provides a method for identification of
molecules, such as binding molecules, that bind to the polypeptide
of the present invention. Genes encoding proteins that bind to the
polypeptide can be identified by numerous methods known to those of
skill in the art, for example, ligand panning and FACS sorting.
Such methods are described in many laboratory manuals such as, for
instance, Coligan et al., Current Protocols in Immunology 1(2):
Chapter 5 (1991). Also, a labeled ligand can be photoaffinity
linked to a cell extract. Polypeptides of the invention also can be
used to assess the binding capacity of a binding molecule, in cells
or in cell-free preparations.
[0123] Polypeptides of the invention may also be used to assess the
binding or small molecule substrates and ligands in, for example,
cells, cell-free preparations, chemical libraries, and natural
product mixtures. These substrates and ligands may be natural
substrates and ligands or may be structural or functional
mimetics.
[0124] The invention further provides a complex of a polypeptide
and a binding molecule which comprises a polypeptide as described
herein and a binding molecule capable of modulating the activity of
the polypeptide. A complex of this kind can arise in vivo upon
administration to a patient of a binding molecule as described
herein.
[0125] Antagonists and Agonists--Assays and Molecules
[0126] The invention also provides a method of screening compounds
to identify those which enhance (agonist) or block (antagonist) the
function of polypeptides or polynucleotides of the present
invention, such as its interaction with a binding molecule. The
method of screening may involve high-throughput.
[0127] For example, to screen for agonists or antagonists, a
synthetic reaction mix, a cellular compartment, such as a membrane,
cell envelope or cell wall, or a preparation of any thereof, may be
prepared from a cell that expresses a molecule that binds to the
polypeptide of the present invention. The preparation is incubated
with labeled polypeptide in the absence or the presence of a
candidate molecule which may be an agonist or antagonist. The
ability of the candidate molecule to bind the binding molecule is
reflected in decreased binding of the labeled ligand. Molecules
which bind gratuitously, i.e., without inducing the functional
effects of the polypeptide, are most likely to be good antagonists.
Molecules that bind well and elicit functional effects that are the
same as or closely related to the polypeptide are good
agonists.
[0128] The functional effects of potential agonists and antagonists
may by measured, for instance, by determining activity of a
reporter system following interaction of the candidate molecule
with a cell or appropriate cell preparation, and comparing the
effect with that of the polypeptide of the present invention or
molecules that elicit the same effects as the polypeptide. Reporter
systems that may be useful in this regard include but are not
limited to colorimetric labeled substrate converted into product, a
reporter gene that is responsive to changes in the functional
activity of the polypeptide, and binding assays known in the
art.
[0129] Another example of an assay for antagonists is a competitive
assay that combines the polypeptide of the present invention and a
potential antagonist with membrane-bound binding molecules,
recombinant binding molecules, natural substrates or ligands, or
substrate or ligand mimetics, under appropriate conditions for a
competitive inhibition assay. The polypeptide can be labeled, such
as by radioactivity or a colorimetric compound, such that the
number of polypeptide molecules bound to a binding molecule or
converted to product can be determined accurately to assess the
effectiveness of the potential antagonist.
[0130] Potential antagonists include small organic molecules,
peptides, polypeptides and antibodies that bind to a polypeptide of
the invention and thereby inhibit or extinguish its activity.
Potential antagonists also may be small organic molecules, a
peptide, a polypeptide such as a closely related protein or
antibody that binds to the same sites on a binding molecule without
inducing functional activity of the polypeptide of the
invention.
[0131] Potential antagonists include a small molecule which binds
to and occupies the binding site of the polypeptide thereby
preventing binding to cellular binding molecules, such that normal
biological activity is prevented. Examples of small molecules
include but are not limited to small organic molecules, peptides or
peptide-like molecules.
[0132] Other potential antagonists include antisense molecules (see
Okano, J. Neurochem. 56: 560 (1991); OLIGODEOXYNUCLEOTIDES AS
ANTISENSE INHIBITORS OF GENE EXPRESSION, CRC Press, Boca Raton,
Fla. (1988), for a description of these molecules).
[0133] Preferred potential antagonists include derivatives of the
polypeptide of the invention.
[0134] In a particular aspect, the invention provides the use of
the polypeptide, polynucleotide or inhibitor of the invention to
interfere with the initial physical interaction between a pathogen
and mammalian host responsible for sequelae of infection. In
particular the molecules of the invention may be used: i) in the
prevention of adhesion of S. epidermidis to mammalian extracellular
matrix proteins on in-dwelling devices or to extracellular matrix
proteins in wounds; ii) to block protein mediated mammalian cell
invasion by, for example, initiating phosphorylation of mammalian
tyrosine kinases (Rosenshine et al., Infect. Immun. 60:2211
(1992)); iii) to block bacterial adhesion between mammalian
extracellular matrix proteins and bacterial proteins which mediate
tissue damage; iv) to block the normal progression of pathogenesis
in infections initiated other than by the implantation of
in-dwelling devices or by other surgical techniques.
[0135] Each of the DNA coding sequences provided herein may be used
in the discovery and development of antibacterial compounds. The
encoded protein upon expression can be used as a target for the
screening of antibacterial drugs. Additionally, the DNA sequences
encoding the amino terminal regions of the encoded protein or
Shine-Delgarno or other translation facilitating sequences of the
respective mRNA can be used to construct antisense sequences to
control the expression of the coding sequence of interest.
[0136] The antagonists and agonists may be employed, for instance,
to inhibit diseases arising from infection with Staphylococcus,
especially S. epidermidis, such as sepsis and endocarditis.
[0137] Vaccines
[0138] Another aspect of the invention relates to a method for
inducing an immunological response in an individual, particularly a
mammal, which comprises inoculating the individual with the
polypeptide of the invention, or a fragment or variant thereof,
adequate to produce antibody to protect said individual from
infection, particularly bacterial infection and most particularly
Staphylococcus infections. Yet another aspect of the invention
relates to a method of inducing immunological response in an
individual which comprises, through gene therapy or otherwise,
delivering a nucleic acid functionally encoding the polypeptide, or
a fragment or a variant thereof, for expressing the polypeptide, or
a fragment or a variant thereof in vivo in order to induce an
immunological response to produce antibodies or a cell mediated T
cell response, either cytokine-producing T cells or cytotoxic T
cells, to protect said individual from disease, whether that
disease is already established within the individual or not. One
way of administering the gene is by accelerating it into the
desired cells as a coating on particles or otherwise.
[0139] A further aspect of the invention relates to an
immunological composition which, when introduced into a host
capable of having induced within it an immunological response,
induces an immunological response in such host, wherein the
composition comprises recombinant DNA which codes for and expresses
an antigen of the polypeptide of the present invention. The
immunological response may be used therapeutically or
prophylactically and may take the form of antibody immunity or
cellular immunity such as that arising from CTL or CD4+ T
cells.
[0140] The polypeptide of the invention or a fragment thereof may
be fused with co-protein which may not by itself produce
antibodies, but is capable of stabilizing the first protein and
producing a fused protein which will have immunogenic and
protective properties. This fused recombinant protein preferably
further comprises an antigenic co-protein, such as
Glutathione-S-transferase (GST) or beta-galactosidase, relatively
large co-proteins which solubilise the protein and facilitate
production and purification thereof. Moreover, the co-protein may
act as an adjuvant in the sense of providing a generalized
stimulation of the immune system. The co-protein may be attached to
either the amino or carboxy terminus of the first protein.
[0141] Provided by this invention are compositions, particularly
vaccine compositions, and methods comprising the polypeptides or
polynucleotides of the invention and immunostimulatory DNA
sequences, such as those described in Sato, Y. et al. Science 273:
352 (1996).
[0142] Also, provided by this invention are methods using the
described polynucleotide or particular fragments thereof which have
been shown to encode non-variable regions of bacterial cell surface
proteins in DNA constructs used in such genetic immunization
experiments in animal models of infection with S. epidermidis. Such
fragments will be particularly useful for identifying protein
epitopes able to provoke a prophylactic or therapeutic immune
response. This approach can allow for the subsequent preparation of
monoclonal antibodies of particular value from the requisite organ
of the animal successfully resisting or clearing infection for the
development of prophylactic agents or therapeutic treatments of S.
epidermidis infection in mammals, particularly humans.
[0143] The polypeptide may be used as an antigen for vaccination of
a host to produce specific antibodies which protect against
invasion of bacteria, for example by blocking adherence of bacteria
to damaged tissue. Examples of tissue damage include wounds in skin
or connective tissue caused e.g. by mechanical, chemical or thermal
damage or by implantation of indwelling devices, or wounds in the
mucous membranes, such as the mouth, mammary glands, urethra or
vagina.
[0144] The present invention also includes a vaccine formulation
which comprises the immunogenic recombinant protein together with a
suitable carrier. Since the protein may be broken down in the
stomach, it is preferably administered parenterally, including, for
example, administration that is subcutaneous, intramuscular,
intravenous, or intradermal. Formulations suitable for parenteral
administration include aqueous and non-aqueous sterile injection
solutions which may contain anti-oxidants, buffers, bacteriostats
and solutes which render the formulation isotonic with the bodily
fluid, preferably the blood, of the individual; and aqueous and
non-aqueous sterile suspensions which may include suspending agents
or thickening agents. The formulations may be presented in
unit-dose or multi-dose containers, for example, sealed ampoules
and vials, and may be stored in a freeze-dried condition requiring
only the addition of the sterile liquid carrier immediately prior
to use. The vaccine formulation may also include adjuvant systems
for enhancing the immunogenicity of the formulation, such as
oil-in-water systems and other systems known in the art. The dosage
will depend on the specific activity of the vaccine and can be
readily determined by routine experimentation.
[0145] While the invention has been described with reference to
certain polypeptides, it is to be understood that this covers
fragments of the naturally occurring protein and similar proteins
with additions, deletions or substitutions which do not
substantially affect the immunogenic properties of the recombinant
protein.
[0146] Compositions
[0147] The invention also relates to compositions comprising the
polynucleotide or the polypeptides discussed above or the agonists
or antagonists. Thus, the polypeptides of the present invention may
be employed in combination with a non-sterile or sterile carrier or
carriers for use with cells, tissues or organisms, such as a
pharmaceutical carrier suitable for administration to a subject.
Such compositions comprise, for instance, a media additive or a
therapeutically effective amount of a polypeptide of the invention
and a pharmaceutically acceptable carrier or excipient. Such
carriers may include, but are not limited to, saline, buffered
saline, dextrose, water, glycerol, ethanol and combinations
thereof. The formulation should suit the mode of
administration.
[0148] Kits
[0149] The invention further relates to diagnostic and
pharmaceutical packs and kits comprising one or more containers
filled with one or more of the ingredients of the aforementioned
compositions of the invention. The ingredient(s) can be present in
a useful amount, dosage, formulation or combination. Associated
with such container(s) can be a notice in the form prescribed by a
governmental agency regulating the manufacture, use or sale of
pharmaceuticals or biological products, reflecting approval by the
agency of the manufacture, use or sale of the product for human
administration.
Administration
[0150] Polypeptides and other compounds of the present invention
may be employed alone or in conjunction with other compounds, such
as therapeutic compounds.
[0151] The pharmaceutical compositions may be administered in any
effective, convenient manner including, for instance,
administration by topical, oral, anal, vaginal, intravenous,
intraperitoneal, intramuscular, subcutaneous, intranasal or
intradermal routes among others.
[0152] The pharmaceutical compositions generally are administered
in an amount effective for treatment or prophylaxis of a specific
indication or indications. In general, the compositions are
administered in an amount of active agent of at least about 10
.mu.g/kg body weight. In most cases they will be administered in
one or more doses in an amount not in excess of about 8 mg/kg body
weight per day. Preferably, in most cases, dose is from about 10
.mu.g/kg to about 1 mg/kg body weight, daily. For administration
particularly to mammals, and particularly humans, it is expected
that the daily dosage level of the active agent will be from 0.01
mg/kg to 10 mg/kg and typically around 1 mg/kg. For example, a dose
may be 1 mg/kg daily. It will be appreciated that optimum dosage
will be determined by standard methods for each treatment modality
and indication, taking into account the indication, its severity,
route of administration, complicating conditions and the like. The
physician in any event will determine the actual dosage which will
be most suitable for an individual and will vary with the age,
weight and response of the particular individual. The above dosages
are exemplary of the average case. There can, of course, be
individual instances where higher or lower dosage ranges are
merited, and such are within the scope of this invention.
[0153] In therapy or as a prophylactic, the active agent may be
administered to an individual as an injectable composition, for
example as a sterile aqueous dispersion, preferably isotonic.
[0154] Alternatively the composition may be formulated for topical
application, for example in the form of ointments, creams, lotions,
eye ointments, eye drops, ear drops, mouthwash, impregnated
dressings and sutures and aerosols, and may contain appropriate
conventional additives, including, for example, preservatives,
solvents to assist drug penetration, and emollients in ointments
and creams. Such topical formulations may also contain compatible
conventional carriers, for example cream or ointment bases, and
ethanol or oleyl alcohol for lotions. Such carriers may constitute
from about 1% to about 98% by weight of the formulation; more
usually they will constitute up to about 80% by weight of the
formulation.
[0155] The pharmaceutical composition may be administered in
conjunction with an in-dwelling device. In-dwelling devices include
surgical implants, prosthetic devices and catheters, i.e., devices
that are introduced to the body of an individual and remain in
position for an extended time. Such devices include, for example,
artificial joints, heart valves, pacemakers, vascular grafts,
vascular catheters, cerebrospinal fluid shunts, urinary catheters,
continuous ambulatory peritoneal dialysis (CAPD) catheters,
etc.
[0156] The composition of the invention may be administered by
injection to achieve a systemic effect against relevant bacteria
shortly before insertion of an in-dwelling device. Treatment may be
continued after surgery during the in-body time of the device. In
addition, the composition could also be used to broaden
perioperative cover for any surgical technique to prevent
Staphylococcus wound infections.
[0157] Many orthopaedic surgeons consider that humans with
prosthetic joints should be considered for antibiotic prophylaxis
before dental treatment that could produce a bacteremia. Late deep
infection is a serious complication sometimes leading to loss of
the prosthetic joint and is accompanied by significant morbidity
and mortality. It may therefore be possible to extend the use of
the active agent as a replacement for prophylactic antibiotics in
this situation.
[0158] In addition to the therapy described above, the compositions
of this invention may be used generally as a wound treatment agent
to prevent adhesion of bacteria to matrix proteins exposed in wound
tissue and for prophylactic use in dental treatment as an
alternative to, or in conjunction with, antibiotic prophylaxis.
[0159] Alternatively, the composition of the invention may be used
to bathe an indwelling device immediately before insertion. The
active agent will preferably be present at a concentration of 1
.mu.g/ml to 10 mg/ml for bathing of wounds or indwelling
devices.
[0160] A vaccine composition is conveniently in injectable form.
Conventional adjuvants may be employed to enhance the immune
response. A suitable unit dose for vaccination is 0.5-5 .mu.g/kg of
antigen, and such dose is preferably administered 1-3 times and
with an interval of 1-3 weeks.
[0161] With the indicated dose range, no adverse toxicological
effects should be observed with the compounds of the invention
which would preclude their administration to suitable
individuals.
[0162] The antibodies described above may also be used as
diagnostic reagents to detect the presence of bacteria containing
the protein.
[0163] In order to facilitate understanding of the following
example certain frequently occurring methods and/or terms are
explained in the foregoing glossary.
[0164] The present invention is further described by the following
examples. While illustrating certain specific aspects of the
invention, the examples do not portray the limitations or
circumscribe the scope of the disclosed invention.
[0165] All examples were carried out using routine molecular
biology techniques as generally described in standard laboratory
manuals, such as Sambrook et al., MOLECULAR CLONING: A LABORATORY
MANUAL, 2nd Ed.; Cold Spring Harbor Laboratory Press, Cold Spring
Harbor, N.Y. (1989).
EXAMPLES
[0166] A small insert plasmid library was generated in the minimal
sequencing vector pOT2a (O. Hubbard, C. Martin, and M. Palazzolo,
unpublished). pOT2a vector was prepared by BstXI digestion of the
parent plasmid pOT2a-sacB followed by preparative agarose gel
electrophoresis to separate the 1.6 kb vector fragment from a B.
subtilis sacB gene fragment. To prepare inserts for library
construction S. epidermidis SR1 strain genomic DNA was sonicated
and the resulting random fragments were end-repaired with klenow
and T4 polymerase and phosphorylated with T4 polynucleotide kinase.
Oligos (5'-CTCTAAAG-3',5'-CTTTAGAGCACA-3') (SEQ ID NO.:4465) to
create BstX1 adaptors were annealed and ligated to the blunt-ended
fragments. The configuration of the BstXI sites in pOT2a and the
sequence of the adaptors allowed a ligation strategy that minimized
the recovery of clones without insert (Seed, 1987). DNA samples
were electrophoresed on a low-melting-temperature agarose gel and
fragments of 3000-4000 bp were isolated and purified. The
linearized vector and random DNA fragments were ligated overnight
using T4 DNA ligase at 16 C and transformed into DH10B competent
cells (Life Technologies Inc., Gaithersburg, Md.) by
electroporation. Transformed bacteria were selected on LB agar
plates containing 5% sucrose and 12.5 ug/ml chloramphenicol.
Sequencing templates were isolated from single colonies and
purified using R.E.A.L. Prep 96 Plasmid Kit (QIAGEN, Chatsworth,
Calif.). Seq01 primer (5'-CACTATAGAACTCGAGCAGCTG-3') (SEQ ID
NO.:4466) and seq02 primer (5'-CGACTCACTATAGGGAGACCG-3') (SEQ ID
NO.:4467) were used to generate end-sequence using ABI Prism BigDye
Terminators (PE Applied Biosystems, Foster City, Calif.).
[0167] Constructs from the pOT2a library were transformed into
POX38 bacteria and selected on LB agar plates containing 12.5 ug/ml
chloramphenicol. A single colony from each construct was used to
inoculate an overnight culture. These POX38 cultures were mated
with a culture of the F-bearing, kanamycin resistant JGM strain by
combining the two strains and shaking for 3 hours at 37 C without
to antibiotics. Each successful mating event resulted in the random
insertion of a single gamma-delta transposon into the pOT2a
construct. This collection of transpositions was captured in the
JGM cells by selection of the mated cultures on LB agar plates
containing 12.5 ug/ml chloramphenicol and 25 ug/ml kanamycin. A
transposon library was created for each of the original pOT2a
library constructs by picking 96 individual colonies. A set of two
PCR reactions was performed on each of the 96 library members to
determine the position of the transposon integration. PM001 primer
(5'-CGTTAGAACGCGGCTACA-3') (SEQ ID NO.:4468) and NGDIR primer
(5'-GTTCCATTGGCCCTCAAAC-3') (SEQ ID NO.:4469) were used to
determine the integration site distance from the left side of the
vector and PM002 primer (5'-GCCGATTCATTAATGCAGGT-3') (SEQ ID
NO.:4470) and NGDIR primer were used to confirm the integration
position by measuring the distance from the right side of the
vector. PCR products were electrophoresed in 1.times.TBE on 1.4%
agarose gels. After gel analysis, a subset of transposon clones was
selected for sequencing based upon obtaining an integration site
about every 300 bp along the full length of the pOT2a insert.
Sequencing templates were purified using R.E.A.L. Prep 96 Plasmid
Kit (QIAGEN, Chatsworth, Calif.). M21 primer
(5'-GTAAAACGACGGCCAGT-3') (SEQ ID NO.:4471) and rev primer
(5'-CAGGAAACAGCTATGAC-3') (SEQ ID NO.:4472) were used to generate
internal sequence using ABI Prism BigDye Terminators (PE Applied
Biosystems, Foster City, Calif.).
[0168] The sequences, including ORFs (nucleic acid sequences within
SEQ ID NOs 1-3334) and non-ORFs (SEQ ID NOs 3335-4464) are set
forth in the Sequence Listing. The non-ORF regions may be
particularly useful as diagnostic sequences. The ribosomal RNA
genes may also be useful to distinguish between species. Also,
intergenic regions generally may be useful diagnostics to establish
genus and species of an unidentified microbe, as there may be less
selective pressure to maintain fidelity of the sequences in these
intergenic regions as compared to intragenic regions.
[0169] About 26 different isolates of S. epidermidis have been
submitted to ATCC listed in their on-line catalog, listed
below:
[0170] 1: ATCC Number: 146 Organism: Staphylococcus epidermidis
[0171] 2: ATCC Number: 33501 Organism: Staphylococcus
epidermidis
[0172] 3: ATCC Number: 49741 Organism: Staphylococcus
epidermidis
[0173] 4: ATCC Number: 51625 Organism: Staphylococcus
epidermidis
[0174] 5: ATCC Number: 29997 Organism: Staphylococcus
epidermidis
[0175] 6: ATCC Number: 19654 Organism: Staphylococcus
epidermidis
[0176] 7: ATCC Number: 14389 Organism: Staphylococcus sp.
deposit
[0177] 8: ATCC Number: 14852 Organism: Staphylococcus
epidermidis
[0178] 9: ATCC Number: 49134 Organism: Staphylococcus
epidermidis
[0179] 10: ATCC Number: 13518 Organism: Staphylococcus
epidermidis
[0180] 11: ATCC Number: 9491 Organism: Staphylococcus
epidermidis
[0181] 12: ATCC Number: 35547 Organism: Staphylococcus
epidermidis
[0182] 13: ATCC Number: 35984 Organism: Staphylococcus
epidermidis
[0183] 14: ATCC Number: 35983 Organism: Staphylococcus
epidermidis
[0184] 15: ATCC Number: 700296 Organism: Staphylococcus
epidermidis
[0185] 16: ATCC Number: 49461 Organism: Staphylococcus
epidermidis
[0186] 17: ATCC Number: 29641 Organism: Staphylococcus
epidermidis
[0187] 18: ATCC Number: 29887 Organism: Staphylococcus
epidermidis
[0188] 19: ATCC Number: 29886 Organism: Staphylococcus
epidermidis
[0189] 20: ATCC Number: 55133 Organism: Staphylococcus
epidermidis
[0190] 21: ATCC Number: 27626 Organism: Staphylococcus sp.
deposit
[0191] 22: ATCC Number: 31874 Organism: Staphylococcus
epidermidis
[0192] 23: ATCC Number: 14990 Organism: Staphylococcus
epidermidis
[0193] 24: ATCC Number: 155 Organism: Staphylococcus sp.
deposit
[0194] 25: ATCC Number: 155-U Organism: Staphylococcus sp.
deposit
[0195] 26: ATCC Number: 12228 Organism: Staphylococcus
epidermidis
[0196] Throughout this application, various publications are
referenced. These publications are hereby incorporated by reference
in their entirety.
[0197] While the invention has been described with respect to
certain specific embodiments, it will be appreciated that many
modifications and changes may be made by those skilled in the art
without departing from the spirit of the invention. It is intended,
therefore, by the appended claims, to cover all such modification
and changes as fall within the true spirit and scope of the
invention.
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=US20100272743A1).
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=US20100272743A1).
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