BACKGROUND OF THE INVENTION It is particularly preferred to employ Staphylococcal genes and gene products as targets for the development of antibiotics. The Staphylococci make up a medically important genera of microbes. They are known to produce two tv vg ru ive infections are characterized generally bv abscess formation effecting both skin surfaces and deep tissues. S. aureus is the second leading cause of bacteremia in cancer patients. Osteomvelitis. septic arthritis. septic thrombophlebitis and acute bacterial endocarditis are also relatively common. There are at least three clinical conditions resulting from the toxigeruc properties of Staphylococci. The manifestation of these diseases result from the actions of exotoxins as opposed to tissue invasion and bacteremia. These conditions include: Staphylococcal food poisoning. scalded skin syndrome and toxic shock syndrome.

The frequency of Staphylococcus aureus infections has risen dramatically in the past few decades. This has been attributed to the emergence of multiply antibiotic resistant strains and an increasing population of people with weakened immune systems. It is no longer uncommon to isolate Staphylococcus aureus strains which are resistant to some or all of the standard antibiotics.

This phenomenon has created a demand for both new anti-microbial agents. vaccines, and diagnostic tests for this organism.

Clearly. there exists a need for factors. such as the spoOJ2 embodiments of the invention. that have a present benefit of being useful to screen compounds for antibiotic activity. Such factors are also useful to determine their role in pathogenesis of infection, dysfunction and disease. There is also a need for identification and characterization of such factors and their antagonists and agonists to find ways to prevent. ameliorate or correct such infection. dysfunction and disease.

Certam of the polypeptides of the invention possess amino acid sequence homology to a known B. subtilis hypothetical protein yyaA similar to DNA binding protein spoOJ protein.

SUMMARY OF THE INVENTION It is an object of the invention to provide polypeptides that have been identified as novel spoOJ2 polypeptides by homology between the amino acid sequence set out in Table 1 [SEQ ID NO: 2] and a known amino acid sequence or sequences of other proteins such as B. subtilis hypothetical protein yyaA similar to DNA binding protein spoOJ protein.

It is a further object of the invention to provide polynucleotides that encode spoOJ2 polypeptides, particularly polynucleotides that encode the polypeptide herein designated spoOJ2.

In a particularly preferred embodiment of the invention the polynucleotide comprises a region encoding spoOJ2 polypeptides comprising a sequence set out in Table 1 [SEQ ID NO: 1] which includes a full length gene, or a variant thereof.

In another particularly preferred embodiment of the invention there is a novel spoOJ2 protein from Staphylococcus aureus comprising the amino acid sequence of Table 1 [SEQ ID NO: 2], or a variant thereof.

A further aspect of the invention there are provided isolated nucleic acid molecules encoding spoOJ2. particularly Staphylococcus aureus spoOJ2. including mRNAs. cDNAs. genomic DNAs. Further embodiments of the invention include biologically, diagnostically, prophylacticallv. clinically or therapeutically useful variants thereof, and compositions comprising the same.

In accordance with another aspect of the invention. there is provided the use of a polynucleotide of the invention for therapeutic or prophylactic purposes. in particular genetic immunization. Among the particularly preferred embodiments of the invention are naturally occurring allelic variants of spoOJ2 and polypeptides encoded thereby.

Another aspect of the invention there are provided novel polypeptides of Staphylococcus aureus referred to herein as spoOJ2 as well as biologically, diagnostically, prophylactically, clinically or therapeutically useful variants thereof, and compositions comprising the same.

Among the particularly preferred embodiments of the invention are variants of spoOJ2 polypeptide encoded by naturallv occurring alleles of the spoOJ2 gene.

In a preferred embodiment of the invention there are provided methods for producing the aforementioned spoOJ2 polypeptides.

In accordance with yet another aspect of the invention, there are provided inhibitors to such polypeptides, useful as antibacterial agents, including, for example, antibodies.

In accordance with certain preferred embodiments of the invention, there are provided products. compositions and methods for assessing spoOJ2 expression, treating disease. assaying

genetic variation. and administering a spoOJ2 polypeptide or polynucleotide to an organism to raise an immunological response against a bacteria, especially a Staphylococcus aureus bacteria.

In accordance with certain preferred embodiments of this and other aspects of the invention there are provided polynucleotides that hybridize to spoOJ2 polynucleotide sequences. particularly under stringent conditions.

In certain preferred embodiments of the invention there are provided antibodies against spoOJ2 polypeptides.

In other embodiments of the invention there are provided methods for identifying compounds which bind to or otherwise interact with and inhibit or activate an activity of a polypeptide or polynucleotide of the invention comprising: contacting a polypeptide or polynucleotide of the invention with a compound to be screened under conditions to permit binding to or other interaction between the compound and the polypeptide or polvnucleotide to assess the binding to or other interaction with the compound. such binding or interaction being associated with a second component capable of providing a detectable signal in response to the binding or interaction of the polypeptide or polynucleotide with the compound ; and determming whether the compound binds to or otherwise interacts with and activates or inhibits an activity of the polypeptide or polynucleotide by detecting the presence or absence of a signal generated from the binding or interaction of the compound with the polypeptide or polynucleotide.

In accordance with yet another aspect of the invention. there are provided spoOJ2 agonists and antagonists, preferably bacteriostatic or bactenocidal agonists and antagonists.

In a further aspect of the invention there are provided compositions comprising a spoOJ2 polynucleotide or a spoOJ2 polypeptide for administration to a cell or to a multicellular organism.

Various changes and modifications within the spirit and scope of the disclosed invention will become readily apparent to those skilled in the art from reading the following descriptions and from reading the other parts of the present disclosure.

DESCRIPTION OF THE INVENTION The invention relates to novel spoOJ2 polypeptides and polynucleotides as described in greater detail below. In particular, the invention relates to polypeptides and polynucleotides of a novel spoOJ2 of Staphylococcus aureus. which is related by amino acid sequence homology to B. subtilis hypothetical protein yyaA similar to DNA binding protein spoOJ polypeptide. The invention relates especially to spoOJ2 having the nucleotide and amino acid sequences set out in Table 1 as SEQ ID NO: 1 and SEQ ID NO: 2 respectively.

TABLE 1 spoOJ2 Polynucleotide and Polypeptide Sequences (A) Sequences from Staphylococcus aureus spoOJ2 polynucleotide sequence [SEQ ID NO: 1].

1 ATGAAAAAAC CTTTTTCAAA ATTATTTGGT TTGAAAAACA AAGATGACAT 51 CATTGGACAT ATTGAAGAAG ATCGCAATAG TAATGTTGAA TCCATTCAAA 101 TTGAACGTAT CGTTCCCAAC CGTTATCAAC CAAGACAGGT GTTTGAACCA 151 AATAAAATTA AAGAACTTGC TGAATCAATA CATGAACATG GTTTACTACA 201 ACCTATTGTT GTAAGACCGA TTGAAGAAGA TATGTTTGAA ATTATTGCTG 251 GAGAGCGCCG ATTTAGAGCA ATACAATCAC TAAATTTACC TCAAGCAGAC 301 GTTATTATTC GTGATATGGA TGAAGAAGAG ACGGCTGTTG TTGCATTAAT 351 TGAGAATATT CAAAGAGAAA ATTGGTCTGT TGTTGAAGAA GCGGAAGCCT 401 ATAAGAAATT ATTGGAAATT GGTGATACAA CGCAAAGTGA ATTGGCAAAA 451 AGTTTAGGTA AAAGTCAAAG CTTTATTGCA AATAAGTTGC GTTTATTGAA 501 GTTGGCGCCG AAAGTACTAC TTCGCTTAAG AGAAGGTAAA ATTACTGAAC 551 GCCATGCGAG AGCGGTATTA TCATTGTCTG ATAGTGAACA AGAAGCGTTG 601 ATTGAGCAAG TCATTGCACA AAAACTAAAT GTGAAACAGA CTGAAGATAG 651 AGTACGCCAA AAAACGGGGC CCGAAAAAGT CAAAGCACAA AACCTTCGCT 701 TTGCACAAGA TGTCACTCAA GCACGAGATG AGGTAGGCAA AAGTATCCAA 751 GCGATTCAAC AAACAGGACT ACATGTTGAG CATAAAGACA AAGATCATGA

801 AGATTATTAT GAAATAAAAA TTCGAATATA TAAACGTTAG 3 (B) Staphylococcus aureus spoOJ2 polypeptide sequence deduced from the polvnucleotide sequence in this table [SEQ ID NO: 2].

NH2- 1 MKKPFSKLFG LKNKDDIIGH IEEDRNSNVE SIQIERIVPN RYQPRQVFEP 51 NKIKELAESI HEHGLLQPIV VRPIEEDMFE IIAGERRFRA IQSLNLPQAD 101 VIIRDMDEEE TAWALIENI QRENWSWEE AEAYKKLLEI GDTTQSELAK 151 SLGKSQSFIA NKLRLLKLAP KVLLRLREGK ITERHARAVL SLSDSEQEAL 201 IEQVIAQKLN VKQTEDRVRQ KTGPEKVKAQ NLRFAQDVTQ ARDEVGKSIQ 251 AIQQTGLHVE HKDKDHEDYY EIKIRIYKR -COOH (C) Polynucleotide sequences comprising Staphylococcus aiireits spoOJ2 ORF sequence [SEQ ID NO: 3].

1 GTTATTATTC GTGATATGGA TGATGAAGAG ACGGCTGTTG TTGCATTAAT 51 TGAGAATATT CAAAGAGAAA ATTTGTCTGT TGTTGAAGAA GCGGAAGCCT 101 ATAAGAAATT ATTGGAAATT GGTGATACAA CGCAAAGTGA ATTGGCAAAA 151 AGTTTAGGTA AAAGTCAAAG CTTTATTGCA AATAAGTTGC GTTTATTGAA 201 GTTGGCGCCG AAAGTACTAC TTCGCTTAAG AGAAGGTAAA ATTACTGAAC 251 GCCATGCGAG AGCGGTATTA TCATTGTCTG ATAGTGAACA AGAAGCGTTG 301 ATTGAGCAAG TCATTGCACA AAAACTAAAT GTGAAACAGA CTGAAGATAG 351 AGTACGCCAA AAAACGGGGC CCGAAAAAGT CAAAGCACAA AACCTTCGCT

401 TTGCACAAGA TGTCACTCAA GCACGAGATG AGGTAGGCAA AAGTATCCAA 451 GCGATTCAAC AAACAGGACT ACATGTTGAG CATAAAGACA AAGATCATGA 501 AGATTATTAT GAAATAAAAA TTCGAATATA TAAACGTTAG <BR> <BR> <BR> <BR> <BR> -3'<BR> <BR> <BR> <BR> <BR> <BR> <BR> <BR> <BR> <BR> <BR> <BR> <BR> <BR> (D) Staphylococcus aure11s spoOJ2 polvpeptide sequence deduced from the polynucleotide ORF sequence in this table [SEQ ID NO: 4].

NH2- 1 VIIRDMDDEE TAWALIENI QRENLSWEE AEAYKKLLEI GDTTQSELAK 51 SLGKSQSFIA NKLRLLKLAP KVLLRLREGK ITERHARAVL SLSDSEQEAL 101 IEQVIAQKLN VKQTEDRVRQ KTGPEKVKAQ NLRFAQDVTQ ARDEVGKSIQ 151 AIQQTGLHVE HKDKDHEDYY EIKIRIYKR -COOH Deposited materials A deposit containing a Staphylococcus aiireiis WCUH 29 strain has been deposited with <BR> <BR> <BR> <BR> the National Collections of Industrial and Marine Bacteria Ltd. (herein"NCIMB"). 23 St. Machar Drive. Aberdeen AB2 1RY. Scotland on 11 September 1995 and assigned NCIMB Deposit No. <BR> <BR> <BR> <BR> <P>40771, and referred to as Staphylococcus aureus WCUH29 on deposit. The Staphylococcus aureus strain deposit is referred to herein as"the deposited strain"or as"the DNA of the deposited strain." The deposited strain contains the full length spoOJ2 gene. The sequence of the polynucleotides contained in the deposited strain, as well as the ammo acid sequence of the polypeptide encoded thereby, are controlling in the event of any conflit with any description of sequences herein.

The deposit of the deposited strain has been made under the terms of the Budapest Treaty on the International Recognition of the Deposit of Micro-organisms for Purposes of Patent Procedure. The strain will be irrevocably and without restriction or condition released to the public upon the issuance of a patent. The deposited strain is provided merely as convenience to those of skill in the art and is not an admission that a deposit is required for enablement, such as that required under 35 U. S. C. §112.

A license may be required to make. use or sell the deposited strain. and compounds derived therefrom. and no such license is hereby granted.

One aspect of the invention there is provided an isolated nucleic acid molecule encoding a mature polypeptide expressible by the Staphylococcus aurens WCUH 29 strain contained in the deposited strain. Further provided by the invention are spoOJ2 nucleotide sequences of the DNA in the deposited strain and ammo acid sequences encoded thereby. Also provided by the invention are spoOJ2 polypeptide sequences isolated from the deposited strain and amino acid sequences derived therefrom.

Polypeptides The polypeptides of the invention include a polypeptide of Table 1 [SEQ ID NO: 2] (in <BR> <BR> <BR> <BR> <BR> particular the mature polypeptide) as well as polypeptides and fragnents. particularly those which have the biological activity of spoOJ2, and also those which have at least 70% identity to a polypeptide of Table 1 [SEQ ID NO: I] or the relevant portion. preferably at least 80% identity to a polypeptide of Table 1 [SEQ ID NO: 2and more preferably at least 90% similarity (more preferably at least 90% identity) to a polypeptide of Table 1 [SEQ ID NO: 2] and still more preferably at least 95% similarity (still more preferablv at least 95% identity) to a polypeptide of Table 1 [SEQ ID NO: 2] 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.

The invention also includes polypeptides of the formula : X- (RI) m- (R2)- (R3) n-Y wherein, at the amino terminus. X is hydrogen. and at the carboxyl terminus. Y is hydrogen or a metal. RI and R3 are any amino acid residue, m is an integer between 1 and 1000 or zero. n is an integer between 1 and 1000 or zero. and R2 is an amino acid sequence of the invention, particularly an amino acid sequence selected from Table 1. In the formula above R2 is oriented so that its amino terminal residue is at the left. bound to RI and its carboxy terminal residue is at the right. bound to R3. Any stretch of amino acid residues denoted by either R group, where m and/or n is greater than 1, may be either a heteropolymer or a homopolymer, preferably a heteropolymer.

A fragment is a variant polypeptide having an amino acid sequence that entirely is the same as part but not all of the amino acid sequence of the aforementioned polypeptides. As with spoOJ2 polypeptides fragments may be"free-standing,"or comprised withm a larger polypeptide of which they form a part or region, most preferably as a single continuous region, a single larger polypeptide.

Preferred fragments include, for example. truncation polypeptides having a portion of an amino acid sequence of Table 1 [SEQ ID NO: 2], or of variants thereof such as a continuous serines of residues that includes the amino terminus, or a continuous serines of residues that includes the

carboxyl terminus. Degradation forms of the polypeptides of the invention in a host cell. particularly a Staphylococcus aureus. are also preferred. Further preferred are fragments characterized by structural or functional attributs such as fragments that comprise alpha-helix and alpha-helix forming regions, beta-sheet and beta-sheet-fornung 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.

Also preferred are biologicallv active fragments which are those fragments that mediate activities of spoOJ2, including those with a similar activity or an improved activity, or with a decreased undesirable activity. Also included are those fragments that are antigenic or immunogenic in an animal. especially in a human. Particularly preferred are fragments comprising receptors or domains of enzymes that confer a function essential for viability of Staphylococcus aureus or the abilitv to initiate, or maintain cause disease in an individual, particularly a human.

Variants that are fragments of the polypeptides of the invention may be employed for producing the corresponding full-length polypeptide by peptide synthesis; therefore, these variants may be employed as intermediates for producing the full-length polypeptides of the invention.

In addition to the standard single and triple letter representations for amino acids. the term"X"or"Xaa"may also be used in describing certain polypeptides of the invention."X" and"Xaa"mean that anv of the twentv naturally occuring amino acids may appear at such a designated position in the polypeptide sequence.

Polynucleotides Another aspect of the invention relates to isolated polynucleotides, including the full length gene. that encode the spoOJ2 polypeptide having a deduced amino acid sequence of Table 1 [SEQ ID NO: 2] and polynucleotides closelv related thereto and variants thereof.

Using the information provided herein. such as a polynucleotide sequence set out in Table I [SEQ ID NO: 1], a polynucleotide of the invention encoding spoOJ2 polypeptide may be obtained using standard cloning and screening methods, such as those for cloning and sequencing chromosomal DNA fragments from bacteria using Staphylococcus aureus WCUH 29 cells as starting material, followed by obtaining a full length clone. For example, to obtain a polynucleotide sequence of the invention, such as a sequence given in Table 1 [SEQ ID NO: 1], typically a library of clones of chromosomal DNA of Staphylococcus aureus WCUH 29 in E. coli or some other suitable host is probed with a radiolabeled oligonucleotide. preferably a 17-mer or longer. derived from a partial sequence. Clones carrying DNA identical to that of the probe can then be distinguished using stringent conditions. By sequencing the individual clones thus identified

with sequencing primers designed from the original sequence it is then possible to extend the sequence in both directions to determine the full gene sequence. Conveniently. such sequencing is performed using denatured double stranded DNA prepared from a plasmid clone. Suitable techniques are described by Maniais, T.. Fritsch, E. F. and Sambrook et al.. MOLECULAR CLONING, A LABORATORY MANUAL, 2nd Ed.. Cold Spring Harbor Laboratorv Press. Cold Spring Harbor, New York (1989). (see in particular Screening By Hybridization 1.90 and Sequencing Denatured Double-Stranded DNA Templates 13.70). Illustrative of the invention. the polynucleotide set out in Table 1 [SEQ ID NO: 1] was discovered in a DNA library derived from Staphylococcus aureus WCUH 29.

The DNA sequence set out in Table 1 [SEQ ID NO: 1] contains an open reading frame encoding a protein having about the number of amino acid residues set forth in Table 1 [SEQ ID NO: 2] with a deduced molecular weight that can be calculated using amino acid residue molecular weight values well known in the art. The polynucleotide of SEQ ID NO: 1, between nucleotide number 1 and the stop codon which begins at nucleotide number 838 of SEQ ID NO: 1. encodes the polypeptide of SEQ ID NO: 2.

SpoOJ2 of the invention is structurallv related to other proteins of the spoOJ family.

The invention provides a polvnucleotide sequence identical over its entire length to a coding sequence in Table 1 [SEQ ID NO: 1]. Also provided bv the invention is the coding sequence for the mature polypeptide or a fragment thereof by itself as well as the coding sequence for the mature polypeptide or a fragment in reading frame with other coding sequence, such as those encoding a leader or secretorv sequence, a pre-, or pro-or prepro-protein sequence. The polynucleotide may also contain non-coding sequences. including for example. but not limited to non-coding 5'and 3' sequences. such as the transcribed. non-translated sequences. terrnination signals. nbosome binding sites, sequences that stabilize mRNA. introns. polyadenylation signals. and additional coding sequence which encode additional amino acids. For example. a marker sequence that facilitates purification of the fused polypeptide can be encoded. In certain embodiments of the invention, the marker sequence is a hexa-histidine peptide, as provided in the pQE vector (Qiagen, Inc.) and described in Gentz et al., Proc. Natl. Acacl. Sci., USA 86: 821-824 (1989), or an HA tag (Wilson et al., Cell 37 767 (1984). Polvnucleotides of the invention also include, but are not limited to, polynucleotides comprising a structural gene and its naturally associated sequences that control gene expression.

A preferred embodiment of the invention is a polvnucleotide of comprising nucleotide 1 to the nucleotide immediately upstream of or including nucleotide 838 set forth in SEQ ID N0 : 1 of Table 1. both of which encode the spoOJ2 polypeptide.

The invention also includes polynucleotides of the formula: X-(Rl) m-(R2)-(R3) n~Y wherein. at the 5'end of the molecule. X is hydrogen or together with Y defines a covalent bond, and at the 3'end of the molecule. Y is hydrogen or a metal or together with X defines the covalent bond, each occurance of R I and R3 is independentlv any nucleic acid residue. m is an integer between I and 3000 or zero. n is an integer between 1 and 3000 or zero. and R is a nucleic acid sequence of the invention particularly a nucleic acid sequence selected from Table 1. In the polynucleotide formula above R2 is oriente so that its 5'end residue is at the left, bound to Ri and its 3'end residue is at the right, bound to R3. Any stretch of nucleic acid residues denoted by either R group, where m and/or n is greater than 1, may be either a heteropolymer or a homopolymer, preferably a heteropolymer. Where. in a preferred embodiment, X and Y together define a covalent bond, the polynucleotide of the above formula is a closed, circular polynucleotide. which can be a double-stranded polvnucleotide wherein the formula shows a first strand to which the second strand is complementary. In another preferred embodiment m and/or n is an integer between 1 and 1000.

It is most preferred that the polynucleotides of the inventions are derived from <BR> <BR> <BR> <BR> Staphylococcus aureus, however, they may preferably be obtained from organisms of the same taxonomic genus. They may also be obtained, for example, from organisims of the same taxonomic family or order.

The term"polynucleotide encoding a polypeptide"as used herein encompasses polynucleotides that include a sequence encoding a polypeptide of the invention. particularly a bacterial polypeptide and more particularly a polypeptide of the Staphvlococcus s auretf. s spo0J2 having an amino acid sequence set out in Table 1 [SEQ ID NO: 2]. The term also encompasses polynucleotides that include a single continuous region or discontinuous regions encoding the polypeptide (for example, interrupted by integrated phage or an insertion sequence or editing) together with additional regions, that also may contain coding and/or non-coding sequences.

The invention further relates to variants of the polynucleotides described herein that encode for variants of the polypeptide having a deduced amino acid sequence of Table 1 [SEQ ID NO : 2].

Variants that are fragments of the polynucleotides of the invention may be used to synthesize full- length polynucleotides of the invention.

Further particularly preferred embodiments are polynucleotides encoding spoOJ2 variants. that have the amino acid sequence of spoOJ2 polypeptide of Table 1 [SEQ ID NO: 2] 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. Especiallv preferred among these are silent substitutions, additions and deletions, that do not alter the properties and activities of spoOJ2.

Further preferred embodiments of the invention are polynucleotides that are at least 70% identical over their entire length to a polynucleotide encoding spoOJ2 polypeptide having an amino acid sequence set out in Table 1 [SEQ ID NO: 2]. and polynucleotides that are complementary to such polynucleotides. Alternatively. most highly preferred are polynucleotides that comprise a region that is at least 80% identical over its entire length to a polynucleotide encoding spoOJ2 polypeptide and polynucleotides complementary thereto. In this regard, polynucleotides at least 90% identical over their entire length to the same are particularly preferred, and among these particularly preferred polynucleotides. 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% being the more preferred.

Preferred embodiments are polynucleotides that encode polypeptides that retam substantiallv the same biological function or activity as the mature polypeptide encoded by a DNA of Table 1 [SEQ ID NO: 1].

The invention further relates to polynucleotides that hybridize to the herein above-described sequences. In this regard, the invention especially relates to polynucleotides that hybridize under stringent conditions to the herein above-described polynucleotides. As herein used. the terms "stringent conditions"and"stringent hvbridization conditions"mecul hybndization will occur only if there is at least 95% and preferably at least 97% identitv between the sequences. An example of stringent hybridization conditions is overnight incubation at 42°C in a solution comprising: 50% formamide, 5x SSC (150mM NaCI. 15mM trisodium citrate) 50 mM sodium phosphate (pH7.6), 5x Denhardt's solution. 10% dextran sulfate. and 20 micrograms/ml denatured. sheared salmon sperm DNA, followed by washing the hybridization support in O. 1 x SSC at about 65°C. Hybridization and wash conditions are well known and exemplified in Sambrook, et al.. Molecular Cloning: A Laboratory Manual, Second Edition, Cold Spring Harbor, N. Y., (1989), particularly Chapter 11 therein.

The invention also provides a polynucleotide consisting essentially of a polynucleotide sequence obtainable by screening an appropriate library containing the complete gene for a polynucleotide sequence set forth in SEQ ID NO: 1 under stringent hybridization conditions with a probe having the sequence of said polynucleotide sequence set forth in SEQ ID NO: 1 or a fragment thereof ; and isolating said DNA sequence. Fragments useful for obtaining such a polynucleotide include, for example, probes and primers described elsewhere herein.

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 spoOJ2 and to isolate cDNA and genomic clones of other genes that have a high sequence similarity to the spoOJ2 gene. Such probes generally will comprise at least 15 bases. Preferably. such probes will have 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.

For example, the coding region of the spoOJ2 gene may be isolated by screening using a DNA sequence provided in Table 1 [SEQ ID NO: 1] to synthesize an oligonucleotide probe. A labeled oligonucleotide having a sequence complementary to that of a gene of the invention is then used to screen a library of cDNA, genomic DNA or mRNA to determine which members of the library the probe hybridizes to.

The polynucleotides and polypeptides of the invention may be employed, for example, as research reagents and materials for discovery of treatments of and diagnostics for disease, particularly human disease, as further discussed herein relating to polynucleotide assays.

Polynucleotides of the invention that are oligonucleotides derived from the sequences of Table 1 [SEQ ID NOS: 1 or 2] may be used in the processes herein as described. but preferably for PCR, to determine whether or not the polynucleotides identified herein in whole or in part are transcribed in bacteria in infected tissue. 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.

The invention also provides polynucleotides that may encode a polypeptide that is the mature protein plus additional amino or carboyl-ternlinal 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.

A precursor protein, having the mature form of the polypeptide fused to one or more prosequences ive peptide. 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.

In addition to the standard A, G, C. T/U representations for nucleic acid bases. the term"N"may also be used in describing certain polynucleotides of the invention."N"means that any of the four DNA or RNA bases may appear at such a designated position in the DNA

or RNA sequence. except it is preferred that N is not a base that when taken in combination with adjacent nucleotide positions when read in the correct reading frame, would have the effect of generating a premature termination codon in such reading frame.

In sum, a polvnucleotide of the 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 that 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.

Vectors, host cells, expression The invention also relates to vectors that comprise a polynucleotide or polynucleotides of the invention, host cells that are genetically engineered with vectors of the invention and the production of polypeptides of the invention by recombinant techniques. Cell-free translation systems can also be employed to produce such proteins using RNAs derived from the DNA constructs of the invention.

For recombinant production, host cells can be geneticallv engineered to incorporate expression systems or portions thereof or polynucleotides of the invention. Introduction of a polynucleotide into the host cell can be effected bv methods described in manv standard laboratory manuals, such as Davis et al., BASIC METHODS IN MOLECULAR BIOLOGY. (1986) and Sambrook et al., MOLECULAR CLONING: A LABORATORYMANUAL, 2nd Ed.. Cold Spring Harbor Laboratorv Press. Cold Spring Harbor. N. Y. (1989). such as. calcium phosphate transfection. DEAE-dextran mediated transfection. transvection. microinjection. cationic lipid- mediated transfection, electroporation. transduction. scrape loading, ballistic introduction and infection.

Representative examples of appropriate hosts include bacterial cells, such as streptococci, staphylococci, enterococci 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.

A great variety of expression systems can be used to produce the polypeptides 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 visses adenovimses. 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. The expression system constructs may contain control regions that regulate as well as engender expression. Generally. anv svstem or vector suitable to maintain. propagate or express polynucleotides and/or to express a polypeptide in a host may be used for expression in this regard.

The appropriate DNA sequence may be inserted into the expression svstem bv any of a variety of well-known and routine techniques, such as. for example, those set forth in Sambrook et al., MOLECULAR CLONING. A LABORATORYMANUAL (sllpra).

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.

Polypeptides of the invention can be recovered and purifie 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 emploved to regenerate active conformation when the polypeptide is denatured during isolation and or purification.

Diagnostic Assays This invention is also related to the use of the spoOJ2 polynucleotides of the invention for use as diagnostic reagents. Detection of spoOJ2 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 especiallv humans, particularly those infected or suspected to be infected with an organism comprising the spoOJ2 gene may be detected at the nucleic acid level by a variety of techniques.

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 directlv for detection or may be amplified enzvmaticallv by using PCR or other amplification technique prior to analysis.

RNA, cDNA and genomic DNA may also be used in the same ways. Using amplification. characterization of the species and strain of prokaryote present in an individual, may be made by an analysis of the genotype of the prokaryote gene. 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 bv hybridizing amplified DNA to labeled spoOJ2 polynucleotide sequences. Perfectly matched sequences can be distinguished from mismatched duplexes bv RNase

digestion or by differences in melting temperatures. DNA sequence differences may also be detected by alterations in the electrophoretic mobility of the DNA fragments in gels, with or without denaturing agents, or by direct DNA sequencing. See. e. g., Myers et al.. Science. 230: 1242 (1985).

Sequence changes at specific locations also may be revealed by nuclease protection assays. such as RNase and S1 protection or a chemical cleavage method. See, e. g.. Cotton et al., Proc. Natl. Acad.

Sci.. USA. 85: 4397-4401 (1985).

Cells carrying mutations or polymorphisms in the gene of the invention may also be detected at the DNA level by a varietv of techniques, to allow for serotyping, for example. For example, RT- PCR can be used to detect mutations. It is particularly preferred to used RT-PCR in conjunction with automated detection systems. such as. for example, GeneScan. RNA. cDNA or genomic DNA may also be used for the same purpose, PCR or RT-PCR. As an example, PCR primers complementary to a nucleic acid encoding spoOJ2 can be used to identify and analyze mutations.

Examples of representative primers are shown below in Table 2.

Table 2 Primers for amplification of spoOJ2 polynucleotides SEQ ID NO PRIMER SEQUENCE 5 5'-GTGATATGGATGATGAAGAGACGG-3' 6 5'-TGCAAAGCGAAGGTTTTGTGC-3' The invention also includes primers of the formula: X- (Ri) n,- (R2)- (R3) n-Y wherein, at the 5'end of the molecule, X is hydrogen, and at the 3'end of the molecule. Y is hydrogen or a metal, RI and R3 is any nucleic acid residue, m is an integer between 1 and 20 or zero. n is an integer between 1 and 20 or zero. and R2 is a primer sequence of the invention, particularly a primer sequence selected from Table 2. In the polynucleotide formula above R2 is oriented so that its 5'end residue is at the left, bound to RI and its 3'end residue is at the right, bound to R3. Any stretch of nucleic acid residues denoted by either R group, where m and/or n is greater than 1, may be either a heteropolymer or a homopolymer, preferably a heteropolymer being complementary to a region of a polynucleotide of Table 1. In a preferred embodiment m and/or n is an integer between 1 and 10.

The invention further provides these primers with 1.2.3 or 4 nucleotides removed from the 5'and/or the 3'end. These primers may be used for. among other things, amplifying spoOJ2 DNA isolated from a sample derived from an individual. The primers may be used to anplify the gene

isolated from an infected individual such that the gene mav then be subject to various techniques for elucidation of the DNA sequence. In this wav, mutations in the DNA sequence may be detected and used to diagnose infection and to serotype and/or classify the infectious agent.

The invention further provides a process for diagnosing, disease, preferably bacterial infections. more preferably infections by Staphylococcus aureus comprising determining from a sample derived from an individual a increased level of expression of polynucleotide having a sequence of Table 1 [SEQ ID NO: 1]. Increased or decreased expression of spoOJ2 polynucleotide can be measured using any on of the methods well known in the art for the quantation of polynucleotides. such as. for example. amplification, PCR, RT-PCR, RNase protection, Northern blotting and other hybridization methods.

In addition, a diagnostic assay in accordance with the invention for detecting over- expression of spoOJ2 protein compared to normal control tissue samples may be used to detect the presence of an infection for example. Assay techniques that can be used to determine levels of a spoOJ2 protein. 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.

Antibodies The polypeptides of the invention or variants thereof. or cells expressing them can be used as an en to produce antibodies immunospecific for such polypeptides."Antibodies"as used herein includes monoclonal and polyclonal antibodies chimeric. single chain, simianized antibodies and humanized antibodies, as well as Fab fragments, including the products of an Fab immunolglobulin expression library.

Antibodies peptides of the invention can be obtained by administering the polypeptides or epitope-bearing fragments, analogues or cells to an animal preferably a nonhuman. using routine protocols. For preparation of monoclonal antibodies. any technique known in the art that 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 ANTIBODIESAND CANCER THERAPY, Alan R. Liss, Inc. (1985).

Techniques for the production of single chain antibodies (U. S. Patent No. 4,946,778) can be adapted to produce single chain antibodies to polypeptides of this invention. Also. transgenic notice. or other organisms such as other mammals, may be used to express humanized antibodies.

Alternatively phage display technology mav 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-spoOJ2 or from naive libraries (McCafferty, J. et al.. (1990). Nature 348. 552-554 : Marks. J. et al.. (1992) Biotechnology 10.

779-783). The affinitv of these antibodies can also be improved by chain shuffling (Clackson, T. et al., (1991) Nature 352.624-628).

If two antigen binding domains are present each domain may be directed against a different epitope-termed'bispecific'antibodies.

The above-described antibodies may be employed to isolate or to identify clones expressing the polypeptides to purify the polypeptides by affinity chromatography.

Thus, among others, antibodies against spoOJ2-polypeptide may be employed to treat infections, particularly bacterial infections.

Polypeptide variants include antigenically. epitopically or immunologically equivalent variants that 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 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.

The polypeptide, such as an antigenically or immunologically equivalent derivative or a fusion protein thereof is used as an antigen to immunize a mouse or other animal such as a rat or chicken. The fusion protein may provide stabilitv to the polypeptide. The antigen mav be associated, for example by conjugation, with an immunogenic carrier protein for example bovine serum albumin (BSA) or kevhole limpet haemocvanin (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.

Preferably, the antibody or variant 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" ; where the complimentaritv 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.

The use of a polynucleotide of the invention in genetic immunization will preferably employ a suitable deliverv method such as direct injection of plasmid DNA into muscles (Wolff et al.. Hum Mol Genet 1992.1: 363. Manthorpe et al.. Hum. Gene Ther. 1963: 4.419). delivery of DNA complexe with specific protein carriers (Wu et al.. J Biol Chem. 1989: 264.16985), coprecipitation of DNA with calcium phosphate (Benvenisty & Reshef, PNAS USA.

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 USA 1984: 81.5849).

Antagonists and agonists-assays and molecules Polypeptides of the invention may also be used to assess the binding of 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. See, e. g.. Coligan et al.. Current Protocols in immunology 1 (2).

Chapter 5 (1991).

The invention also provides a method of screening compounds to identify those which enhance (agonist) or block (antagonist) the action of spoOJ2 polypeptides or polynucleotides. particularly those compounds that are bacteriostatic and/or bacteriocidal. The method of screening may involve high-throughput techniques. For example, to screen for agonists or antagoists, a synthetic reaction mix. a cellular compartment. such as a membrane, cell envelope or cell wall, or a preparation of any thereof, comprising spoOJ2 polypeptide and a labeled substrate or ligand of such polypeptide is incubated in the absence or the presence of a candidate molecule that may be a spoOJ2 agonist or antagonist. The ability of me candidate molecule to agonize or antagonize the spoOJ2 polypeptide is reflected in decreased binding of the labeled ligand or decreased production of product from such substrate. Molecules that bind gratuitously, i. e., without inducing the effects of spoOJ2 polypeptide are most likely to be good antagonists. Molecules that bind well and increase the rate of product production from substrate are agonists. Detection of the rate or level of production of product from substrate may be enhanced by using a reporter system. 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 spoOJ2 polynucleotide or polypeptide activity, and binding assays known in the art.

Another example of an assay for spoOJ2 antagonists is a competitive assay that combines spoOJ2 and a potential antagonist with spoOJ2-binding molecules, recombinant spoOJ2 binding molecules. natural substrates or ligands, or substrate or ligand mimetics. under appropriate

conditions for a competitive inhibition assay. SpoOJ2 can be labeled. such as by radioactivity or a colorimetric compound, such that the number of spoOJ2 molecules bound to a binding molecule or converted to product can be determined accurately to assess the effectiveness of the potential antagonist.

Potential antagonists include small organic molecules. peptides. polypeptides and antibodies that bind to a polynucleotide or polypeptide of the invention and therebv 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 the same sites on a binding molecule. such as a binding molecule, without inducing spoOJ2-induced activities thereby preventing the action of spoOJ2 by excluding spoOJ2 from binding.

Potential antagonists include a small molecule that 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. Other potential antagonists include antisense <BR> <BR> <BR> <BR> molecules (see Okano, J Neurochem. 56: 560 (1991); OLIGODEOXYNUCLEOTIDES AS<BR> <BR> <BR> <BR> <BR> <BR> <BR> ANTISENSE INHIBITORS OF GENE EXPRESSION, CRC Press. Boca Raton, FL (1988), for a description of these molecules). Preferred potential antagonists include compounds related to and variants of spoOJ2.

Each of the DNA sequences provided herein mav 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.

The invention also 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: in the prevention of adhesion of bacteria, in particular gram positive bacteria, to mammalian extracellular matrix proteins on in-dwelling devices or to extracellular matrix proteins in wounds ; to block spoOJ2 protein-mediated mammalian cell invasion by, for example, initiating phosphorylation of mammalian tyrosine kinases (Rosenshine et al.. Infect. <BR> <BR> <BR> <BR> <P>Immun. 60: 2211 (1992) to block bacterial adhesion between mammalian extracellular matrix proteins and bacterial spoOJ2 proteins that mediate tissue damage and to block the normal

progression of pathogenesis in infections initiated other than bv the implantation of in-dwelling devices or by other surgical techniques.

The antagonists and agonists of the invention mav be employed. for instance. to inhibit and treat diseases.

Helicobacter pylori (herein H. pylori) bacteria infect the stomachs of over one-third of the world's population causing stomach cancer. ulcers. and gastritis (International Agency for Research on Cancer (1994) Schistosomes, Liver Flukes and Helicobacter Pylori (International Agency for Research on Cancer. Lyon, France; http://www. uicc. ch/ecp/ecp2904. htm).

Moreover, the international Agency for Research on Cancer recently recognized a cause-and- effect relationship between H. pylori and gastric adenocarcinoma. classifying the bacterium as a Group I (definite) carcinogen. Preferred antimicrobial compounds of the invention (agonists and antagonists of spoOJ2) found using screens provided by the invention, particularly broad- spectrum antibiotics, should be useful in the treatment of H. pylori infection. Such treatment should decrease the advent of H. pylori-induced cancers, such as gastrointestinal carcinoma.

Such treatment should also cure gastric ulcers and gastritis.

Vaccines 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 spoOJ2, or a fragment or variant thereof, adequate to produce antibody and/or T cell immune response to protect said individual from infection, particularly bacterial infection and most particularly Staphylococcus aureus infection. Also provided are methods whereby such immunological response slows bacterial replication. Yet another aspect of the invention relates to a method of inducing immunological response in an individual which comprises delivering to such individual a nucleic acid vector to direct expression of spoOJ2, or a fragment or a variant thereof, for expressing spoOJ2. or a fragment or a variant thereof in vivo in order to induce an immunological response, such as, to produce antibody and/or T cell immune response, including, for example, 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. Such nucleic acid vector may comprise DNA, RNA, a modified nucleic acid, or a DNA/RNA hybrid.

A further aspect of the invention relates to an immunological composition which, when introduced into an individual capable or having induced within it an immunological response, induces an immunological response in such individual to a spoOJ2 or protein coded therefrom,

wherein the composition comprises a recombinant spoOJ2 or protein coded therefrom comprising DNA which codes for and expresses an antigen of said spoOJ2 or protein coded therefrom. The immunological response may be used therapeutically or prophylactically and may take the form of antibody inununity or cellular immunitv such as that arising from CTL or CD4+ T cells.

A spoOJ2 polypeptide 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. Thus fused recombinant protein, preferably further comprises an antigenic co-protein such as lipoprotein D from Hemophilus influenzae, Glutathione-S-transferase (GST) or beta-galactosidase. relatively large co-proteins which solubilize 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.

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).

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 Staphylococcus aureus will be particularly useful for identifying protein epitopes able to provoke a prophylactic or therapeutic immune response. It is believed that this approach will 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 bacterial infection, particularly Staphylococcus aureus infection, in mammals. particularly humans.

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.

The invention also includes a vaccine formulation which comprises an immunogenic recombinant protein of the invention 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 insotonic with the bodilv 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 ampules 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.

While the invention has been described with reference to certain spoOJ2 protein, 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.

Compositions, kits and administration The invention also relates to compositions comprising the polynucleotide or the pohpeptides discussed above or their agonists or antagonists. The polypeptides of the 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. 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.

Polypeptides and other compounds of the invention may be employed alone or in conjunction with other compounds, such as therapeutic compounds.

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.

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.

Alternatively the composition may be formulated for topical application for example in the form of ointments. creams. lotions, eve ointments. eve 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.

For administration to mammals. and particularly humans. it is expected that the daily dosage level of the active agent will be from U. 01 mg/kg to 10 mg/kg, typically around 1 mg/kg. 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.

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, urinarv catheters, continuous ambulatory peritoneal dialysis (CAPD) catheters.

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 bacterial wound infections, especially Staphylococcus aurens wound infections.

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.

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.

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 lg/ml to l Omg/ml for bathing of wounds or indwelling devices.

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 microgram/kg of antigen, and such dose is preferably administered 1-3 times and with an interval of 1-3 weeks. With the indicated dose range, no adverse toxicological effects will be observed with the compounds of the invention which would preclude their administration to suitable individuals.

Each reference disclosed herein is incorporated by reference herein in its entirety. Any patent application to which this application claims priority is also incorporated by reference herein in its entirety.

GLOSSARY The following definitions are provided to facilitate understanding of certain terms used frequently herein.

"Disease (s)" means and disease caused by or related to infection by a bacteria. including disease, such as, infections of the upper respiratory tract (e. g., otitis media, bacterial tracheitis. acute epiglottitis. thyroiditis). lower respiratory (e. g., empyema, lung abscess), cardiac (e. g.. infective endocarditis), gastrointestinal (e. g., secretory diarrhoea, splenic absces, retropentoneal abscess), CNS (e. g., cerebral abscess), eye (e. g., blepharitis, conjunctivitis, keratitis, endophthalmitis, preseptal and orbital cellulitis. darcryocystitis), kidney and urinary tract (e. g., epidid « nitis. intrarenal and perinephric absces, toxic shock syndrome), skin (e. g., impetigo, folliculitis, cutaneous abscesses, cellulitis, wound infection, bacterial myositis) bone and joint (e. g., septic arthritis, osteomyelitis).

"Host cell"is a cell which has been transformed or transfected. or is capable of transformation or transfection by an exogenous polynucleotide sequence.

"Identity,"as known in the art, is a relationship between two or more polypeptide sequences or two or more polynucleotide sequences, as the case may be, 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 bv known methods.

including but not limited to those described in (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 Griffon. 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 ; and Carillo, H., and Lipman, D.. SIAM J Applied Math.. 48: 1073 (1988).

Methods to determine identitv are designed to give the largest match between the sequences tested. Moreover, methods to determine identity are codified in publicly available computer programs. Computer program methods to determine identity between two sequences include, but are not limited to, the GCG program package (Devereux. J.. et al.. Nlxcleic Acid. Research 12 (1): 387 (1984)), BLASTP, BLASTN, and FASTA (Atschul. S. F. et al.. J. Molec. Biol.

215: 403-410 (1990). The BLAST X program is publicly available from NCBI and other sources (BLAST Manual, Altschul, S.. et al., NCBI NLM NIH Bethesda. MD 20894 : Altschul, S., et al., J. Mol. Biol. 215: 403-410 (1990). The well known Smith Waterman algorithm may also be used to determine identity.

Parameters for polypeptide sequence comparison include the following: 1) Algorithm: Needleman and Wunsch. J. Mol Biol. 48: 443-453 (1970) Comparison matrix: BLOSSUM62 from Hentikoff and Hentikoff, Proc. Natl. Acad. Sci.

USA. 89: 10915-10919 (1992) Gap Penalty: 12 Gap Length Penalty: 4 A program useful with these parameters is publicly available as the"gap"program from Genetics Computer Group, Madison WI. The aforementioned parameters are the default parameters for peptide comparisons (along with no penalty for end gaps).

Parameters for polynucleotide comparison include the following: 1) Algorithm: Needleman and Wunsch, J. Mol Biol. 48: 443-453 (1970) Comparison matrix: matches = + 10. mismatch = 0 Gap Penalty: 50 Gap Length Penalty: 3 Available as: The"gap"program from Genetics Computer Group, Madison WI. These are the default parameters for nucleic acid comparisons.

A preferred meaning for"identity"for polynucleotides and polypeptides. as the case may be, are provided in (1) and (2) below.

(1) Polvnucleotide embodiments further include an isolated polvnucleotide comprising a polynucleotide sequence having at least a 50 6 () 70,80.85,90,95,97 or 100% identity to the reference sequence of SEQ ID NO: 1. wherein said polynucleotide sequence may be identical to the reference sequence of SEQ ID NO: 1 or may include up to a certain integer number of nucleotide alterations as compared to the reference sequence wherein said alterations are selected from the group consisting of at least one nucleotide deletion. substitution, including transition and transversion. or insertion. and wherein said alterations may occur at the 5'or 3'terminal positions of the reference nucleotide sequence or anywhere between those terminal positions, interspersed either individually among the nucleotides in the reference sequence or in one or more contiguous groups within the reference sequence. and wherein said number of nucleotide alterations is determined by multiplying the total number of nucleotides in SEQ ID NO: 1 by the integer defining the percent identity divided by 100 and then subtracting that product from said total number of nucleotides in SEQ ID NO: or nn: Xn- (Xn Y) wherein nn is the number of nucleotide alterations, xn is the total number of nucleotides in SEQ ID NO: 1. y is 0.50 for 50%. 0.60 for 60%, 0.70 for 70%. 0. 80 for 80%. 0.85 for 85%. 0.90 for 90%. 0.95 for 95%, 0.97 for 97% or 1.00 for 100%. and is the symbol for the multiplication operator. and wherein any non-integer product of xn and y is rounded down to the nearest integer prior to subtracting it from xn Alterations of a polynucleotide sequence encoding the polypeptide of SEQ ID NO 2 mat create nonsense, missense or frameshift mutations in this coding sequence and thereby alter the polypeptide encoded by the polynucleotide following such alterations.

By way of example, a polynucleotide sequence of the present invention may be identical to the reference sequence of SEQ ID NO: 2, that is it may be 100% identical. or it may include up to a certain integer number of amino acid alterations as compared to the reference sequence such that the percent identity is less than 100% identity. Such alterations are selected from the group consisting of at least one nucleic acid deletion, substitution, including transition and transversion, or insertion, and wherein said alterations may occur at the 5'or 3'terminal positions of the reference polynucleotide sequence or anywhere between those terminal positions, interspersed either individually among the nucleic acids in the reference sequence or in one or more contiguous groups within the reference sequence. The number of nucleic acid alterations for a given percent identity is determined bv multiplying the total number of amino

acids in SEQ ID NO: 2 bv the integer defining the percent identity divided bv 100 and then <BR> <BR> <BR> subtracting that product from said total number of amino acids in SEQ ID NO: 2. or :<BR> <BR> <BR> <BR> <BR> <BR> <BR> <BR> <BR> <BR> nn ("y)- wherein nn is the number of amino acid alterations. xn is the total number of amino acids in SEQ ID NO: 2 y is. for instance 0.70 for 70%. 0.80 for 80%. 0.85 for 85%> etc is the symbol for the multiplication operator. and wherein any non-integer product of xi, and v is rounded down to the nearest integer prior to subtracting it from xn.

(2) Polypeptide embodiments further include an isolated polypeptide comprising a polypeptide having at least a 50. 60,70.80,85,90,95,97 or 100% identity to a polypeptide reference sequence of SEQ ID NO: 2, wherein said polypeptide sequence may be identical to the reference sequence of SEQ ID NO: 2 or may include up to a certain integer number of amino acid alterations as compared to the reference sequence. wherein said alterations are selected from the group consisting of at least one amino acid deletion, substitution, including conservative and non-conservative substitution, or insertion. and wherein said alterations may occur at the amino-or carboxy-terminal positions of the reference polypeptide sequence or anywhere between those terminal positions, interspersed either individually among the amino acids in the reference sequence or in one or more contiguous groups within the reference sequence, and wherein said number of amino acid alterations is determined by multiplying the total number of amino acids in SEQ ID NO 2 boy the integer defining the percent identity divided by 100 and then subtracting that product from said total number of amino acids in SEQ <BR> <BR> <BR> <BR> ID NO: 2, or :<BR> <BR> <BR> <BR> <BR> <BR> <BR> <BR> <BR> <BR> na! Xa-Xa Y) wherein na is the number of amino acid alterations, xa is the total number of amino acids in SEQ ID NO: 2, y is 0.50 for 50%, 0.60 for 60%, 0.70 for 70%, 0.80 for 80%, 0.85 for 85%, 0.90 for 90%, 0.95 for 95%, 0.97 for 97% or 1.00 for 100%. and is the symbol for the multiplication operator, and wherein any non-integer product of xa and y is rounded down to the nearest integer prior to subtracting it from xa By way of example, a polypeptide sequence of the present invention may be identical to the reference sequence of SEQ ID NO: 2, that is it may be 100% identical, or it may include up to a certain integer number of amino acid alterations as compared to the reference sequence

such that the percent identity is less than 100% identity. Such alterations are selected from the group consisting of at least one amino acid deletion, substitution including conservative and non-conservative substitution. or insertion, and wherein said alterations may occur at the amino-or carboxy-terminal positions of the reference polypeptide sequence or anywhere between those terminal positions, interspersed either individually among the amino acids in the reference sequence or in one or more contiguous groups within the reference sequence. The number of amino acid alterations for a given % identity is determined by multiplying the total number of amino acids in SEQ ID NO: 2 by the integer defining the percent identity divided by 100 and then subtracting that product from said total number of amino acids in SEQ ID NO: 2, or : na: Xa'xa'Y), wherein na is the number of amino acid alterations, xa is the total number of amino acids in SEQ ID NO: 2, y is, for instance 0.70 for 70%. 0.80 for 80%, 0.85 for 85% etc.. and is the symbol for the multiplication operator. and wherein any non-integer product of xa and y is rounded down to the nearest integer prior to subtracting it from xa "Isolated"means altered"by the hand of man"from its natural state. i. e.. if it occurs in nature, it has been changed or removed from its original environment, or both. For example, a polynucleotide or a polypeptide naturally present in a living organism 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. Moreover, a polynucleotide or polypeptide that is introduced into an organism by transformation, genetic manipulation or by any other recombinant method is"isolated"even if it is still present in said organism. which organism may be living or non- living.

"Polynucleotide (s)" generally refers to any polyribonucleotide or polydeoxribonucleotide. which may be unmodified RNA or DNA or modified RNA or DNA."Polynucleotide (s)" include. without limitation, 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 regions. 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 tvpicallv mvolve 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 (s)" also 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"polynucleotide (s)" 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 polvnucleotides 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"polvnucleotide (s)" 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, for example, simple and complex cells."Polynucleotide (s)" also embraces short polynucleotides often referred to as oligonucleotide (s).

"Polypeptide (s)" refers to any peptide or protein comprising two or more amino acids joined to each other by peptide bonds or modified peptide bonds."Polypeptide (s)" refers to both short chams, commonly referred to as peptides. oligopeptides and oligomers and to longer chains generally referred to as proteins. Polypeptides may contain amino acids other than the 20 gene encoded amino acids."Polypeptide (s)" include" include those modified either b natural processes, such as processing and other post-translational modifications, but also by chemical modification techniques. Such modifications are well described in basic texts and in more detailed monographs. as well as in a voluminous research literature. and thev are well known to those of skill in the art. It will be appreciated that the same type of modification may be present in the same or varying degree at <BR> <BR> <BR> <BR> several sites in a given polypeptide. Also, a given polypeptide may contain many types of modifications. Modifications can occur anywhere in a polypeptide, including the peptide backbone, the amino acid side-chains, and the amino or carboxyl ternum. Modifications include, for example, 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 phosphotidvlinositol. cross-linking. cyclization. disulfide bond formation demethylation. formation of covalent cross-links, formation of cysteine, formation of pEToglutamate. formylation, gamma-carboxylation glycosvlation. GPI anchor formation, hydroxylation, iodination, methylation, mynstoylation, oxidation, proteolytic processing, phosphorylation, prenylation, racemization, glycosylation, lipid attachment, sulfation, gamma- carboxylation of glutamic acid residues. hvdroxvlation and ADP-ribosylation. selenoylation. sulfation, transfer-RNA mediated addition of amino acids to proteins. such as argmylation. and

ubiquitination. See. for instance. PROTLIN5'-STRUCTUREANDMOLECULAR PROPERTIES, 2nd Ed.. T. E. Creighton, W. H. Freeman and Company. New York (1993) and Wold, F., > Posttranslational Protein Modifications: Perspectives and Prospects, pgs. 1-12 in POSTTRANSLATIONAL COVALENT MODIFICATION OF PROTEINS. B. C. Johnson. Ed., Academic Press, NewYork (1983) : Seifteretal.. Meth. Enzvmol. 182: 626-646 (1990) and Rattan et al.. Protein Synthesis: Posttranslational Modifications and Aging, Ann. N. Y. Acad. Sci. 663: 48- 62 (1992). Polypeptides may be branched or cvclic. with or without branching. Cyclic. branched and branched circular polypeptides may result from post-translational natural processes and may be made by entirely synthetic methods. as well.

"Variant (s)" as the term is used herein. is a polynucleotide or polypeptide that differs from a reference polynucleotide or polypeptide respectively, but retains essential properties. A typical variant of a polynucleotide differs in nucleotide sequence from another. reference polynucleotide. Changes in the nucleotide sequence of the variant may or may not alter the amino acid sequence of a polypeptide encoded by the reference polvnucleotide. Nucleotide changes may result in amino acid substitutions, additions, deletions, fusions and truncations in the polypeptide encoded by the reference sequence. as discussed below. A typical variant of a polypeptide differs in amino acid sequence from another. reference polypeptide. Generallv, differences are limited so that the sequences of the reference polypeptide and the variant are closely similar overall and. in matin, regions, identical. A variant and reference polypeptide may differ in amino acid sequence by one or more substitutions. additions, deletions in any combination. A substituted or inserted amino acid residue mav or may not be one encoded by the genetic code. A variant of a polynucleotide or polypeptide may be a naturally occurring such as an allelic variant. or it mav be a variant that is not known to occur naturally. Non- naturally occurring variants of polvnucleotides and polypeptides may be made by mutagenesis techniques, by direct synthesis. and bv other recombinant methods known to skilled artisans.

EXAMPLES The examples below are carried out using standard techniques, which are well known and routine to those of skill in the art, except where othenvise described in detail. The examples are illustrative, but do not limit the invention.

Example 1 Strain selection, Library Production and Sequencing The polynucleotide having a DNA sequence given in Table 1 [SEQ ID NO: 1] was obtained from a library of clones of chromosomal DNA of Staphylococcus aureus in E. coli.

The sequencing data from two or more clones containing overlapping Staphylococcus aureus

DNAs was used to construct the contiguous DNA sequence in SEQ ID NO: 1. Libraries mav be prepared by routine methods, for example: Methods 1 and 2 below.

Total cellular DNA is isolated from Staphylococcus aureus WCUH 29 according to standard procedures and size-fractionated by either of two methods.

Method 1 Total cellular DNA is mechanically sheared by passage through a needle in order to size-fractionate according to standard procedures. DNA fragments of up to I lkbp in size are rendered blunt by treatment with exonuclease and DNA polymerase. and EcoRI linkers added.

Fragments are ligated into the vector Lambda ZapII that has been cut with EcoRI. the library packaged by standard procedures and E. coli infected with the packaged library. The library is amplified by standard procedures.

Method 2 Total cellular DNA is partiallv hydrolyzed with a one or a combination of restriction enzymes appropriate to generate a series of fragments for cloning into library vectors (e. g..

RsaI, PalI, AluI, Bshl235I), and such fragments are size-fractionated according to standard procedures. EcoRI linkers are ligated to the DNA and the fragments then ligated into the vector Lambda ZapII that have been cut with EcoRI. the library packaged by standard procedures, and E. coli infected with the packaged library. The library is amplified bv standard procedures.