CA2233681A1 - Novel div1b - Google Patents

Abstract

The invention provides Div1b polypeptides and DNA (RNA) encoding Div1b polypeptides and methods for producing such polypeptides by recombinant techniques. Also provided are methods for utilizing Div1b polypeptides to screen for antibacterial compounds.

Description

- NOVEL Div1b FIELD OF T~ ~NVENTION

This invention relates to newly identified polynucleotides and polypeptides, and their produchon and uses, as well as their variants, agonists and antagonists, and their uses. In particular, in these and in other regards, the invention relates to novel polynucleotides and polypeptidles of the div (cell division) family, ~hereinafter referred to as "Divlb".

BACKGROUND OF TE~: 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 types of disease, invasive and toxigenic. Invasive infections are characterized generally by abscess formation effecting both skin surfaces and deep tissues. S. aureus is the second leading cause of bacteremia in cancer patients. Osteomyelitis, septic arthritis, septic thrombophlebitis and acute bacterial endocarditis are also relatively common. There are at least three clinical conditions resulting from the toxigenic 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 20 years. This has been attributed to the emergence of multiply antibiotic resistant strains and an increasin~ 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 has created a demand for both new anti-microbial agents and diagnostic tests for this organism.
Divlb (~1e~ign~t~?d ftsQ in E.coli and divlb in B.subtilus) is an e~centi~l gene involved in bacterial cell division. E.coli FtsQ has been identified as a DivlB homolog, being 18% identical and 44% similar (Harry et al., (1994) Gene 147 85-89). It has been shown in E.coli that FtsQ is required through out the whole process of septum formation during cell division. FtsQ is a simple - cytoplasmic membrane protein with approx 21 amino acids in the cytoplasmic domain, 25 amino acids in the cytoplasmic membrane and approx. 230 amino acids in the periplasmic domain (Carson et al., 1991, J. Bacteriol. 173: 2187-2195). It is estim~tcd that FtsQ is present at about 20 copies/cell (Carson et al., 1991, J. Bacteriol. 173: 2187-2195). DivlB of B.subtilus 168 is also 5 ec~enti~l for viability at 37~C and above and is required at all temperatures for the normal rate of cell division (Beall and ~ lltkenh~llc, 1989, J.Bacteriol. 171: 6821-6834). An FtsQ-MalG fusion protein, where the cytoplasmic and the most of the membrane sp~nning regions of FtsQ were replaced ~rith similar domains of Mal G, has been shown to complement an FtsQ l~ ,eldture sensitive rnutant. This suggests that the cytoplasmic and membrane spanning regions of FtsQ
may not be required for protein function (Dai et al., 1996, 178, 1328-1334). However, a portion of the mernbrane sp~nning region of FtsQ was required to transport the protein to the periplasm.
In contrast, a report by Guzman et al., 1997 (J. Bacteriol. 179, 5094) suggests that only the membrane-~p~nning segment of ftsQ can be replaced and the cytoplasmic domain is ec.~.onli~l for function. Descoteaux and Drapeau et al., 1987 (J. Bacteriol 169, 1938) have suggested that FtsQ
15 may intera~ct with FtsZ. The biochemical function of FtsQ is not known..
C'learly, there is a need for factors, such as the novel compounds 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 20 agonists ~rhich can play a role in plcvel~ lg~ ameliorating or correcting infections, dysfunctions or diseases.
The polypeptides of the invention have amino acid sequence homology to a known FtsQ
in E.coli and Divlb in B.subtilus protein.

SUMMARY OF THE INVENTION

It is an object of the invention to provide polypeptides that have been iflentified as novel Divlb po'lypeptides by homology between the amino acid sequence set out in Table I [SEQ ID
NO: 2] and a known amino acid sequence or sequences of other proteins such as FtsQ in E.coli 30 and Divlb in B.subtilus protein.

-is a further object of thc invention to provide polynucleotides that encode Divlb polypeptides, particularly polynucleotides that encode the polypeptide herein designated Divlb.
~ n a particularly preferred embodiment of the invention the polynucleotide comprises a region encoding Divlb polypeptides comprising the sequence set out in Table 1 [SEQ ID NO:1]
5 which inc ludes a full length gene, or a variant thereof.
In another particularly preferred embodiment of the invention there is a novel Divlb protein fi-om Staphylococcus aureus comprising the amino acid sequence of Table I [SEQ ID
NO:2], o:r a variant thereof.
In accordance with another aspect of the invention there is provided an isolated nucleic 10 acid molecule encoding a mature polypeptide expressible by the Staphylococcus aureus W C U H
29 strain contained in the deposited strain.
A further aspect of the invention there are provided isolated nucleic acid molecules encoding Divlb, particularly Staphylococcus aureus Divlb, including mRNAs, cDNAs, genomic DNAs. Further embodiments of the invention include biologically, ~ nosti~ ~lly, 15 prophylactically, 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 immllni7~tion. Among the particularly preferred embodiments of the invention are naturally 20 occurrin~s allelic variants of Divlb and polypeptides encoded thereby.
Another aspect of the invention there are provided novel polypeptides of Staphylococcus aureus referred to herein as Divlb 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 Divlb 25 polypeptide encoded by naturally occurring alleles of the Divlb gene.
]n a ~r~ ,d ~mbo-lim~nt of the invention there are provided methods for producing the af.,l.,..,..lioned Divlb polypeptides.
]:n accordance with yet another aspect of the invention, there are provided inhibitors to such polypeptides, useful as antibacterial agents, including, for example, antibodies.
30]n accoldance with certain preferred embodiments of the invention, there are provided products, compositions and m.-th~ for ~c.sec.cing Divlb expression, treating disease, for .

example, 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, retroperitoneal abscess), CNS (e.g., cerebral abscess), eye (e.g., blepharitis, conjunctivitis, 5 keratitis, endophthalmitis, preseptal and orbital cellulitis, darcryocystitis), kidney and urinary tract (e.g., epididymitis, intrarenal and perinephric absces, toxic shock syndrome), skin (e.g., impetigo, folliculitis, cutaneous abscesses, cellulitis, wound infection, bacterial myositis) bone and joint (e.g., sepl:ic arthritis, osteomyelitis)~ assaying genetic variation, and a-lmini~t~ring a Divlb polypeptide or polynucleotide to an organism to raise an immunological response against a 10 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 Divlb polynucleotide sequen~n, particular].y under stringent conditions.
In certain preferred embodiments of the invention there are provided antibodies against 15 Divlb polypeptides.
In other embodiments of the invention there are provided methods for identifyingcompounds which bind to or othenvise 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 20 binding to or other interaction between the compound and the polypeptide or polynucleotide to assess the binding to or other interaction with the compound, such binding or interaction being associated with a second compon~nt capable of providing a detectable signal in response to the binding or interaction of the polypeptide or polynucleotide with the compound; and d~ l..inillg whether t]he compound binds to or otherwise interacts with and activates or inhibits an activity of 25 the po1ypeptide or polynucleotide by detecting the presence or absence of a signal ge~lcl~led from the binding or interaction of the compound with the polypeptide or polynucleotide.
In accc,.~cc with yet another aspect of the invention, there are provided Divlb agonists and antagonists, preferably bacteriostatic or bacteriocidal agonists and antagonists.
In a further aspect of the invention there are provided compositions comprising a Divlb 30 polynucleotide or a Divlb polypeptide for ~imini~tration to a cell or to a multicellular organism.

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

GLOSSARY

The following definitions are provided to facilitate understanding of certain terms used frequentl y herein.
"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 determined by comparing the seq~lenres.
In the art, "identity" also means the degree of sequence relatedness bet~veen polypeptide or polynucleotide sequences, as the case may be, as determined by the match between strings of such sequences. "Identity" and "similarity" can be readily calculated by 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 Griffin, H.G., eds., Humana Press, New Jersey, 1994; Sequence ~nalysis 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 I,ipman, D., SIAM J. Applied Math., 48: 1073 (1988). Preferred methods to del~ e identity are designed to give the largest match between the sequences tested. Methods to determine identity and similarity are codified in publicly available computer programs.
Preferrecl computer program methods to determine identity and similarity between two sequences include, but are not lirnited to, the GCG program package (Devereux, J., et al., Nucleic,4cids 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 (BLASTManual, Altschul, S., et al., NCBI NLM NIH Bethesda, MD
20894; Altschul, S., et al., J. Mol. Biol. 215: 403-410 (1990). As an illustration, by a polynucleotide having a nucleotide sequence having at least, for example, 95% "identity" to a - reference nucleotide sequence of SEQ ID NO: I it is intended that the nucleotide sequence of ~ the polyrlucleotide is identical to the reference sequence except that the polynucleotide sequence may include up to five point mutations per each 100 nucleotides of the reference nucleotide sequence of SEQ ID NO: 1. In other words, to obtain a polynucleotide having a 5 nucleotide sequence at least 95% identical to a reference nucleotide sequence, up to 5% of the nucleotides in the reference sequence may be deleted or substituted with another nucleotide, or a number of nucleotides up to 5% of the total nucleotides in the reference sequence may be inserted into the reference sequence. These mutations of the reference sequence may occur at the 5 or :3 terminal positions of the reference nucleotide sequence or anywhere between those 10 terminal positions, interspersed either individually among nucleotides in the reference sequence or in one or more contiguous groups within the reference sequence. Analogously, by a polypeptide having an amino acid sequence having at least, for example, 95% identity to a reference amino acid sequence of SEQ ID NO:2 is intended that the amino acid sequence of the polypeptide is identical to the reference sequence except that the polypeptide sequence may 15 include up to five amino acid alterations per each 100 amino acids of the reference amino acid of SEQ I~D NO: 2. In other words, to obtain a polypeptide having an amino acid sequence at least 95~/(, identical to a reference amino acid sequence, up to 5% of the amino acid residues in the reference sequence may be deleted or substituted with another amino acid, or a number of amino acids up to 5% of the total amino acid residues in the reference sequence may be 20 inserted into the reference sequence. These alterations of the reference sequence may occur at the amino or carboxy terminal positions of the reference amino acid sequence or anywhere between those terminal positions, interspersed either individually among residues in the reference sequence or in one or more contiguous groups within the reference sequence.
":[solated" means altered "by the hand of man" from its natural state, i.e., if it occurs in 25 nature, it has been changed or removed from its original ~,,vi,ulllllcllt, 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 te"" is employed herein.
"Polynucleotide(s)" generally refers to any polyribonucleotide or polydeoxribonucleotide, 30 which may be ~ mo-lified 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-sb-anded regions or single-, double- and b-iple-stranded regions, single- and double-sbranded RNA, and RNA that is mixblre of single- and double-sbranded regions, hybrid molecules comprising DNA and RNA that may be single-sbranded or, more typically, double-sbranded, or briple-sbranded regions, or a mixblre of single- and double-stranded regions. In addition, 5 "polynucleotide" as used herein refers to briple-sbranded regions comprising RNA or DNA or both RNA and DNA. The sbrands 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 briple-helical region often is an oligonucleotide. As used herein, the term "polynucleotide(s)" also includes DNAs or 10 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 britylated 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 15 DNA ancl RNA that serve many useful purposes known to those of skill in the art. The term "polynucleotide(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 chai.ns, 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 those modified either by natural processes, such as processin~ 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 they 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 several sites in a given polypeptide. Also, a given polypeptide may contain rnany types of modifications. Modifications can occur anywhere in a polypeptide, including the peptide backbone, the amino acid side-chains, and the amino or carboxyl termini.

- Modificat-ions include, for example, acetylation, acylation, ADP-ribosylation, amidation, covalent ~ att lr.hm~nt 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, 5 formation of covalent cross-links, forrnation of cysteine, formation of pyroglutamate, formylation, gamma-carboxylation, glycosylation, GPI anchor formation, hydroxylation, iodination, methylation, myristoylation, oxidation, proteolytic processing, phosphorylation, prenylation, racemi7.~tion, glycosylation, lipid attachment, sulfation, gamma-carboxylation of glutamic acid residues, :hydroxylation and ADP-ribosylation, selenoylation, sulfation, transfer-RNA m~ ted 10 addition of amino acids to proteins, such as arginylation, and ubiquitination. See, for instance, PROTEmrS - STRUCTURE AND MOLECULAR PROPERTIES, 2nd Ed., T. E. Creighton, W. H.
Freeman .md Company, New York (1993) and Wold, F., Posttranslational Protein Modifications:
Perspectives and Prospects, pgs. 1-12 in POSITRANSLATIONAL COVALENTMODIFICATION
OF PRO-rElNS, 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 Modificat,ions and Aging, Ann. N.Y. Acad. Sci. 663: 48-62 (1992). Polypeptides may be branched or cyclic, 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.
"'~ariant(s)" as the term is used herein, is a polynucleotide or polypeptide that differs from a rei:erence polynucleotide or polypeptide respectively, but retains ess~nti~l properties. A
typical variant of a polynucleotide differs in nucleotide sequence firom 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 polynucleotide. Nucleotide changes rnay 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. Generally, differences are limited so that the sequences of the reference polypeptide 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 in any combinatiion. A substituted or inserted amino acid residue may or may not be one encoded by g CA 0223368l l998-04-Ol --the genetic code. A variant of a polynucleotide or polypeptide may be a naturally occurring such as a:n allelic variant, or it may be a variant that is not known to occur naturally. Non-naturally occurring variants of polyrlucleotides and polypeptides may be made by mutagenesis techniques, by direct synthesis, and by other recombinant methods known to skilled artisans.

DESCRIPTION OF T~ INVENTION
The invention relates to novel Divlb polypeptides and polynucleotides as described in greater detail below. In particular, the invention relates to polypeptides and polynucleotides of a novel Divlb of Staphylococcus aureus, which is related by amino acid sequence homology to 10 FtsQ in E;.coli and Divlb in B.subtilus polypeptide. The invention relates especially to Divlb having the nucleotide and amino acid sequences set out in Table 1 [SEQ ID NO: 1] and Table 1 [SEQ ID NO: 2] respectively, and to the Divlb nucleotide sequences of the DNA in the deposited strain and amino acid sequences encoded thereby.

Divlb Polynucleotide and Polypeptide Sequences (A) Sequences from Staphylococcus aureus Divlb polynucleotide sequence [SEQ ID
NO:I].
20 5'-1 ATJGATGATA AAACGAAGAA (-GATCAACAA GAATCAAATG AAGATAAAGA

51 TGAATTAGAA TTATTTACGA Gi,AATACATC TAAGAAAAGA CGGCAAAGAA

201 AAAAGAAGAA AGCAATGATG .~AAATAATGG TTCTGCTTCT AGTCATGCGA

251 ATvATAATAA TATCGATGAT TCTACAGACT CTAATATTGA AAATGAGGAT

.

CA 0223368l l998-04-Ol 451 CAATTAAAGC CATTAACACT ~GAAGAAAAG CGGAAGTTAA GACGTAAGCG
s 551 CTGTTATATT AATTTACATG ~TTTCACCAC TTAGTAAAAT TGCGCATGTA

601 AATATAAATG GAAATAATCA ~GTTAGTACT TCAAAGATAA ACAAAGTTTT

651 AGGTGTTAAA AATGATTCGA ~GATGTATAC GTTTAGTAAA AAAAATGCTA

1301 AATCAAGTAA GAATAATTAA-3' CA 0223368l l998-04-0l (B) Divlb polypeptide sequence deduced from the polynucleotide sequence in this table [SEQ ID N0:2].
NH2-lMDDKTKNDQQ ESNEDKDELE LFTRNTSKKR RQRKRSKATH FSNQNKDDTS

101 YRYNQEIDDQ NESNGIAVAN l.QPQSAPKEQ NSDSNDEETV TKKERKSKVT

151 QLKPLTLEEK RKLRRKRQKR :[QYSVITILV LLIAVILIYM FSPLSKIAHV

251 QF:?NTLNVDI TENEIIALVK YKGKYLPLLE NGKLLKGSND VKINDAPVMD

351 IGDISTISKK MKYYPQMSQS ].SRDSSGKLK TRGYIDLSVG ASFIPYRGNT

~01 SSQSESDKNV TKSSQEENQA KEELQSVLNK INKQSSKNN-COOH

(C) Polynucleotide sequence embodiments [SEQ ID N0: 1].
X- (Rl) n~l ATGGATGATA AAACGAAGAA CGATCAACAA GAATCAAATG AAGATAAAGA

51 TGAATTAGAA TTATTTACGA G(,AATACATC TAAGAAAAGA CGGCAAAGAA

201 AA,~AGAAGAA AGCAATGATG AAAATAATGG TTCTGCTTCT AGTCATGCGA

CA 0223368l l998-04-Ol ..

1001 A~AACAAACA TAGCAGAATT GAATTGTTTA CGACAGATGG ACTTCAAGTA

1151 Al'ATTGATTT ATCAGTCGGT GCTTCATTTA TCCCATACCG TGGAAACACG

1201 TC'TAGTCAAT CAGAAAGCGA TAAAAATGTG ACTAAATCAT CTCAAGAGGA

1301 AATCAAGTAA GAATAATTAA-(R2)n-Y

CA 0223368l l998-04-Ol - (D) Polypeptide sequence embodiments [SEQ ID NO:2].
X- (Rl ) n-l MDDKTKNDQQ ESNEDKDELE LFTRNTSKKR RQRKRSKATH FSNQNKDDTS

101 YRYNQEIDDQ NESNGIAVAN :EQPQSAPKEQ NSDSNDEETV TKKERKSKVT

351 IGDISTISKK MKYYPQMSQS :LSRDSSGKLK TRGYIDLSVG ASFIPYRGNT

401 SSQSESDKNV TKSSQEENQA :KEELQSVLNK INKQSSKNN-(R2~n-Y
(E) Sequences from Staphylococcus aureus Divlb polynucleotide ORF sequence [SEQ ID
NO:3].
5'-1 ATGAATCGGA ATGGAATTGC AAGTCGGCAA CCGGACCAAC CTCAATCAGC

251 CACTTAGTAA AATTGCGCAT ~TAAATATAA ATGGAAATAA TCACGTTAGT

CA 0223368l l998-04-Ol .

401 TC'AAAAGTGT TGAGATACAC AAGCAATTAC CAAACACATT AAACGTAGAT

451 Al'CACAGAAA ATGAAATTAT TGCTTTAGTG AAATATAAAG GTAAATATTT

5 501 AC'CTTTATTA GAAAATGGTA AATTGCTTAA AGGTTCAAAT GATGTCAAAA

551 Tl'AATGATGC ACCTGTCATG GATGGTTTCA AAGGTACAAA AGAAGATGAT

601 Al'GATTAAGG CGTTATCTGA AATGACACCT GAAGTTAGAC GATATATTGC

701 Tl'ACGACAGA TGGACTTCAA GTAATCGGTG ATATTTCGAC GATATCTAAG

801 GC;GTAAACTA AAAACAAGAG GCTATATTGA TTTATCAGTC GGTGCTTCAT

851 Tl'ATCCCATA CCGTGGAAAC ACGTCTAGTC AATCAGAAAG CGATAAAAAT
901 Gl'GACTAAAT CATCTCAAGA GGAAAATCAA GCAAAAGAAG AATTACAAAG

951 CC;TTTTAAAC AAAATTAACA AACAATCAAG TAAGAATAAT -3' CA 0223368l l998-04-0l - (F) Divlb polypeptide sequence deduced from the polynucleotide ORF sequence in this - table [SEQ ID NO:4].
NH2-lMNRNGIASRQ PDQPQSAPKE QNSDSNDEET VTKKERKSKV TQLKPLTLEE

51 KRK:LRRKRQK RIQYSVITIL V:;LIAVILIY MFSPLSKIAH VNINGNNHVS

101 TS:~INKVLGV KNDSRMYTFS ]~KNAINDLEE DPLIKSVEIH KQLPNTLNVD

151 IT:_NEIIALV KYKGKYLPLL ENGKLLKGSN DVKINDAPVM DGFKGTKEDD
201 MI:~ALSEMTP EVRRYIAEVT YAPSKNKHSR IELFTTDGLQ VIGDISTISK

251 KM:~YYPQMSQ SLSRDSSGKL ]~TRGYIDLSV GASFIPYRGN TSSQSESDKN

301 VT:~SSQEENQ AKEELQSVLN I~INKQSSKNN-COOH

D~p c ~ t~ 1' materials A deposit containing a Staphylococcus aureus WCUH 29 strain has been deposited with the National Collections of Indusbrial and Marine Bacteria Ltd. (herein "NCIMB"), 23 St. Machar Drive, Aberdeen AB2 lRY, Scotland on 11 September 1995 and ~csign~d NCIMB Deposit No.
40771, and is referred to as Staphylococcus aureus WCUH29 on deposit. The Staphylococcus aureus sb-ain deposit is referred to herein as "the deposited strain" or as "the DNA of the deposited sb-ain."
The deposited strain contains the full length Divlb gene. The sequence of the polynucleotides cont~inçd in the deposited strain, as well as the amino acid sequence of the polypeptide encoded thereby, are conbrolling in the event of any conflict with any descripbion of sequences herem.
The deposit of the deposited sbrain has been made under the terrns of the Budapest Treaty on the Tnt~nqti-~nql Recognition of the Deposit of Micro-organisms for Purposes of Patent Procedure. The strain will be irrevocably and without resbriction or condition released to the public upon the issuance of a patent. The deposited sb-ain 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 thatrequiredunder35 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.
P~ F S
The polypeptides of the invention include the polypeptide of Table I [SEQ ID NO:2] (in 5 particular lhe mature polypeptide) as well as polypeptides and fr~gm~nt~, particularly those which have the biological activity of Divlb, and also those which have at least 70% identity to a polypeptide of Table 1 [SEQ ID NOS:2 and 4] or the relevant portion, preferably at least 80%
identity to a polypeptide of Table I [SEQ ID NOS:2 and 4], and more preferably at least 90%
similarity (more preferably at least 90~/O identity) to a polypeptide of Table 1 [SEQ ID NOS:2 and 4] and still more preferably at least 95% similarity (still more preferably at least 95% identity) to a polypepl:ide of Table I [SEQ ID NOS:2 and 4] 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.
T]he invention also includes polypeptides of the formula set forth in Table I (D) [SEQ ID
15 NO:2] wherein, at the amino tenni-nl~c, X is hydrogen, and at the carboxyl terminus, Y is hydrogen or a metal, Rl and R2 is any amino acid residue, and n is an integer between 1 and l000. A~y stretch of amino acid residues denoted by either R group, where R is greater than l, 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 20 same as part but not all of the amino acid sequence of the aforementioned polypeptides. As with Divlb polypeptides fragments may be "free-st~n-ling," or comprised within a larger polypeptide of which they form a part or region, most preferably as a single continllous region, a single larger polypeptide.
Preferred fragments include, for example, truncation polypeptides having a portion of an 25 amino acid sequ~nce of Table I [SEQ ID NOS:2 and 4], or of variants thereof, such as a co~ o~.A series of residues that includes the amino terminus, or a continuous series of residues that includes the carboxyl tenninlls 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 attributes such as fragments that comprise alpha-helix 30 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.
Also preferred are biologically active fragments which are those fragments that mediate activities of Divlb, including those with a similar activity or an improved activity, or with a 5 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 ability to initiate, or m~int~in cause disease in an individual, particularl y 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 intemlediates for producing the full-length polypeptides of the mventlon.
Polynucleoffdes Another aspect of the invention relates to isolated polynucleotides, including the full length gen.e, that encode the Divlb polypeptide having a deduced amino acid sequence of Table 1 [SEQ ID NOS:2 and 4] and polynucleotides closely related thereto and variants thereof.
Using the information provided herein, such as a polynucleotide sequence set out in Table I [SEQ ID NOS:I and 3], a polynucleotide of the invention encoding Divlb polypeptide may be 20 obtained using standard cloning and screening methods, such as those for cloning and sequencing chromosomal DNA fragments from bacteria using S~aphylococcus aureus WCUH 29 cells as starting rnaterial, 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 NOS: I
and 3], typically a library of clones of chromosomal DNA of Staphylococcus aureus WCUH 29 25 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 identic~l 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.
30 Conveniently, such sequencing is performed using denatured double stranded DNA prepared from a plasmid clone. Suitable techniques are described by Maniatis, T., Fritsch, E.F. and Sambrook et al., MOI,ECULAR CLOiVING, A LABORATORY MANUAL, 2nd Ed.; Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York (1989). (see in particular Screening By Hybriclization 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 5 in a DNA library derived from Staphylococcus aureus WCUH, 29.
Tlle DNA sequence set out in Table 1 [SEQ ID NOS:I] contains an open reading frame encoding a, protein having about the number of amino acid residues set forth in Table I [SEQ ID
NO:2] wil:h 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 I through number 1317 encodes the polypeptide of SEQ ID NO:2. The stop codon begins at nucleotide number 1318 of SEQ ID NO: I .
Divlb of the invention is structurally related to other proteins of the div (cell division) family, as shown by the results of sequencing the DNA encoding Divlb of the deposited strain.
The protein exhibits greatest homology to FtsQ in E. coli and Divlb in B. subtilis protein among known proteins. Divlb of Table 1 [SEQ ID NO:2] has about 27.9% over its entire length and about 52.5% similarity over its entire length with the amino acid sequence of Divlb in B. subtilis polypepticle.
T]le invention provides a polynucleotide sequence identical over its entire length to the coding sequence in Table 1 [SEQ ID NO:l]. Also provided by 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 secretory sequence, a pre-, or pro- or prepro- protein sequence.
The polynucleotide may also contain non-coding sequ~n~es, including for example, but not limited to non-coding 5' and 3' sequences, such as the transcribed, non-translated sequences, l~ nillalion signals, ribosome binding sites, sequences that stabilize mRNA, introns, polyaden~/lation signals, and additional coding sequence which encode additional amino acids.
For ~ . le, a marker sequence that facilitates purification of the fused polypeptide can be enco~letl In certain embodiments of the invention, the marker seq~ence is a hexa-histidine peptide, a.s provided in the pQE vector (Qiagen, Inc.) and described in Gentz et al., Proc. Natl.
Acad. Sci., USA 86: 821-824 (1989), or an HA tag (Wilson et al., Cell 37: 767 (1984).

Polynucleotides 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 polynucleotide of comprising nucleotide 1 to 1317 or 1320 set forth in SEQ ID NO: I of Table 1 which encode the Divlb polypeptide.
S The invention also includes polynucleotides of the formula set forth in Table I (C)[SEQ
ID NO: I] 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, Rl and R2 is any nucleic acid residue, and n is an integer between 1 and 1000. Any stretch of nucleic acid residues denoted by either R group, where R is greater than 1, may be eilher a heteropolymer or a homopolymer, preferably a heteropolymer.
Tlhe 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 Staphylococcus aureus Divlb having the amino acid sequence set out in Table I [SEQ ID NO:2]. The term also encompasses polynucleotides that include a single continuous region or discontinuous regions encoding the 15 polypeptide (for example, interrupted by integrated phage or an insertion sequence or editing) together with additional regions, that also may contain coding andlor non-coding sequences.
The invention further relates to variants of the polynucleotides described herein that encode for variants of the polypeptide having the deduced amino acid sequence of Table I [SEQ
ID NO:2]1. Variants that are fragments of the polynucleotides of the invention may be used to 20 synthesize full-length polynucleotides of the invention.
Further particularly plcr~ cd embodiments are polynucleotides encoding Divlb variants, that have the amino acid sequence of Divlb polypeptide of Table I [SEQ ID NO:2] in which several, a few, 5 to 10, I to 5, I 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 25 and deletions, that do not alter the properties and activities of Divlb.
Further pl~r~ ,d embodiments of the invention are polynucleotides that are at least 70%
identical over their entire length to a polynucleotide encodinE Divlb polypeptide having an amino acid seql~en~e set out in Table I [SEQ ID NOS:2 and 4], and polynucleotides that are compl~ll,c~ll~y to such polynucleotides. Alternatively, most highly pl~fc.l~d are polynucleotides 30 that comprise a region that is at least 80% identical over its entire length to a polynucleotide encoding Divlb polypeptide of the deposited strain and polynucleotides complelllcnl~ thereto.

In this re~;ard, polynucleotides at least 90% idf ntic~l over their entire length to the same are particularly preferred, and among these particularly preferred polynucleotides, those with at least 95% are especially preferred. Furthelmore, 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 retain substantia]ly the same biological function or activity as the mature polypeptide encoded by the DNAofTable 1 [SEQIDNO:1].
The invention further relates to polynucleotides that hybridize to the herein above-10 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 hybridization conditions" mean hybridization will occur only if there is at least 95% and preferably at least 97% identity between the sequences.
An example of stringent hybridization conditions is overnight incubation at 42~C in a solution 15 comprising: 50% formamide, 5x SS(' (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.lx 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 20 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:I or SEQ ID NO:3 under stringent hybridization conditions with a probe having the sequence of said polynucleotide sequence set 25 forth in SEQ ID NO:I or a fragment thereof; and isolating said DNA sequence. Fragrnentc useful for obtaining such a polynucleotide include, for example, probes and primers described elsewhere herein.
As fii.ccuC~e~ additionally herein regarding polynucleotide assays of the invention, for in~t~nce, polynucleotides of the invention as discussed above, may be used as a hybridization 30 probe for RNA, cDNA and genomic DNA to isolate full-length cDNA~s and genomic clones encoding Divlb and to isolate cDNA and genomic clones of other genes that have a high sequence similarity to the Divlb 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 Lllcrcll~,d probes will have at least 30 bases and will have 50 bases or less.
For example, the coding region of the Divlb gene may be isolated by screening using the DNA sequence provided in SEQ ID NO: 1 to synthesize an oligonucleotide probe. A labeled oligonucleotide having a sequence cornplementary 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 treatm~nt~ of and diagnostics for disease, particularl~y human disease, as further discussed herein relating to polynucleotide assays.
Polynucleotides of the invention that are oligonucleotides derived from the sequences of SEQ IEI NOS: I and/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 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 sequ~nces may play a role in processing of a protein from precursor to a mature form, ma~/ allow protein ll~lsl,ull, rnay 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 prosequ~nces may be an inactive form of the polypeptide. When prosequences are removed such inactive plc~ u~ generally are activated. Some or all of the proseql~nces may be removed before activation. Generally, such precursors are called ylu~loleills ~ sum, a polynucleotide of the invention may encode a mature protein, a mature protein plus a leader sequence (which may be referred to as a pl~,~lulci1l), a precursor of a mature protein having one or more prosequences that are not the leader sequences of a p~cl~olcill, or a p,e~ otein, 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 genetically engineered to incorporate expression systems or portions thereof or polynucleotides of the invention. Introduction of a polynucleotide into the host cell can be effected by methods described in many standard laboratory m~n~ , such as Davis et al., BASIC METHODS lN MOrECULAR B OLOGY, (1986) and Sambrook et al., MOLECULAR CLON~JG: A LABOR~TORY MANUAL, 2nd Ed., Cold 15 Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (1989), such as, calcium phosphate sre-;lion, DEAE-dextran mediated transfection, transvection, microinjection, cationic lipid-m.~ ted transfection, ele~ o~)ul~lion, transduction, scrape loading, ballistic introduction and infection.
Representative examples of apl..opliate hosts include bacterial cells, such as streptococci, 20 staphylococci, enterococci E. coli, ~ )tolllyces 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 25 invention. Such vectors include, among others, chromosomal, episomal and virus-derived vectors, ~.g, vectors derived from bacterial plasmids, from bacteriophage, from transposons, from yeast episomes, from insertion cle~ t~, 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 30 those derived from plasmid and bacteriophage genetic clclll.,ll~, such as cosmids and phagemil1~
The expression system constructs may contain control regions that regulate as well as ~ngenller expression. Generally, any system or vector suitable to m~int~in, 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 system by any of a variety of well-known and routine techniques, such as, for example, those set forth in Sambrook 5 et al., MO~LECULAR CLONING, A L~BORATORYMANUAL, (supra).
For secretion of the translated protein into the lumen of the endoplasmic reticulum, into the periplasmic space or into the extracellular envi,vlllllcnt, appropriate secretion signals may be incorporated into the expressed polypeptide. These signals may be endogenous to the polypeptide or they m~y be heterologous signals.
Polypeptides of the invention can be recovered and purified from recombinant cell cultures by well-known methods including ammonium sulfate or ethanol ~lecipil~tion, acid extraction, anion or cation exchange chromatography, phosphocellulose chromatography, hydrophobic interaction chromatography, affinity chromatography, hydroxylapatitechromatography, and lectin chrornatography. 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.
Diqgr~sti~ Assays lhis invention is also related to the use of the Divlb polynucleotides of the invention for use as diagnostic reagents. Detection of Divlb in a eukaryote, particularly a m~mm~l, and especially a human, will provide a diagnostic method for ~ gn~si~ of a disease. Eukaryotes (herein also "individual(s)"), particularly m~mm~l~, and especially humans, infected with an organism colll~ lg the Divlb 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 directly for detection or may be amplified enzyrnatically by using PCR or other amplification technique prior to analysis. RNA or cDNA 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 l~;r~ .lce seq~lenre.

Point mutations can be identified by hybridizing amplified DNA to labeled Divlb polynucleotide sequences. Perfectly matched sequences can be distinguished from mismatched duplexes by 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 5 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 a~ the DNA level by a variety of techniques, to allow for serotyping, for example. For example, ~T-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 or cDNA
may also be used for the same purpose, PCR or RT-PCR. As an example, PCR primers15 compl~ll,c.l~y to a nucleic acid encoding Divlb can be used to identify and analyze mutations.
Examples of representative primers are shown below in Table 2.

Table 2 Primers for amplification of Divlb polynu~ lPoti~lPs 5'-atggatgataaaacgaagaa-3' 6 5'-ttattct:tacttgattgttt-3' The invention fur~er 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 Divlb DNA isolated from a sample derived from an individual. The primers may be used to amplify the gene isolated from an infected 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 seqllence 30 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 bactenal infections, more preferably infections by Staphylococcus aureus, and most preferably 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 5 endocardilis), gastrointestinal (e.g., secretory diarrhoea, splenic absces, ~cllol)cliloneal abscess), CNS (e.g., cerebral abscess), eye (e.g., blepharitis, conjunctivitis, keratitis, endophth~lmiti~
preseptal and orbital cellulitis, darcryocystitis), kidney and urinary tract (e.g., epididymitis, inll~cl~al and perinephric absces, toxic shock syndrome), skin (e.g., impetigo, folliculitis, cutaneous abscesses, cellulitis, wound infection, bacterial myositis) bone and joint (e.g., septic 10 arthritis, osteomyelitis), comprising determining from a sample derived from an individual a increased level of expression of polynucleotide having the sequence of Table I [SEQ ID NO:
1]. Increased or decreased expression of Divlb 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, Northem blotting and other 15 hybridization methods.
Ln addition, a diagnostic assay in accordance with the invention for detecting over-expression of Divlb 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 Divlb protein, in a sample derived from a host are well-known to those of skill in the art. Such 20 assay methods include radioimmn~o~c.s~ys, competitive-binding assays, Western Blot analysis and ELISA assays.
Antibodies lhe polypeptides of the invention or variants thereof, or cells c~ es~ g them can be used as an immllnogen to produce antibodies immlmospecific for such polypeptides.
25 "Antibodies" as used herein includes monoclonal and polyclonal antibodies, chimeric, single chain, ~ d antibodies and hllm~ni~d antibodies, as well as Fab fr~rnenti, including the products of an Fab immunolglobulin expression library.
Antibodies generated against the polypeptides of the invention can be obtained by admini~tering the polypeptides or epitope-bearing fr~gm~nt.~, analogues or cells to an animal, 30 preferably a nonhl-m~n, using routine protocols. For plc~ lion of monoclonal antibodies, any technique known in the art that provides antibodies produced by continuous cell line cultures can be used. Examples include various l;echniques, such as those in Kohler, G. and Milstein, C., Nature 25~S: 495-497 (1975); Kozbor et al., Immunology Today 4. 72 (1983); Cole et al., pg. 77-96 inMONOCLONAL ANTIBODIES~IND CANCER THERAPY, Alan R. Liss, Inc. (1985).
Techniques for the production of single chain antibodies (U.S. Patent No. 4,946,778) can 5 be adapted. to produce single chain antibodies to polypeptides of this invention. Also, transgenic mice, or other organisms such as other m~mm~lc, may be used to express hllm~ni7Pd antibodies.
Alternatively phage display t:echnology may be utilized to select antibody genes with binding activities towards the polypeptide either from repertoires of PCR amplifled v-genes of Iymphocytes from humans screened for possessing anti-Divlb or from naive libraries 10(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 ~hnffling (Clackson, T. et al., ( l991) Nature 352, 624-628).
If two antigen binding domains are present each domain may be directed against adifferent epitope - termed 'bispecific' antibodies.
15The 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 Divlb- polypeptide may be employed to treat infections, particularly bacterial infections and especially disease, such as, infections of the upper respiratory tract (e.g., otitis media, bacterial tracheitis, acute epiglottitis, thyroiditis), lower 20 respiratory (e.g., empyema, lung abscess), cardiac (e.g., infective endocarditis), ga~ ,t~
(e.g., secretory diarrhoea, splenic absces, r~llop~,liloneal abscess), CNS (e.g., cerebral abscess), eye (e.g., blepharitis, Co~ljullClivi~is, keratitis, endophth~lmitis, preseptal and orbital cellulitis, darcryocystitis), kidney and urinary tract (e.g., epididymitis, int~ al and perinephric absces, toxic shock syndrome), skin (e.g., impetigo, folliculitis, cutaneous abscesses, cellulitis, wound 25 infection, bacterial myositis) bone and joint (e.g., septic arthritis, osteomyelitis).
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 30 according to the invention, hlle.~l~; with the imme~ te physical interaction between pathogen and m~mm~ n host. The term l'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 m:~mm~ n host.
The polypeptide, such as an cmtigenically 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 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 10 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 "h~ ni7.ed"; where the complimentarity determining region(s) of the hybridoma-derived 15 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 immlmi7~tion will preferably employ a suitable delivery method such as direct injection of plasmid DNA into muscles 20 (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
USA, 1986:83,9551), encapsulation of DNA in various forms of liposomes (Kaneda et al., Science 1989:243,375), particle bomba.dlllcllt (Tang et al., Nature 1992, 356:152, Eisenbraun 25 et al., DNA Cell Biol 1993, 12:791) and in vivo infection using cloned retroviral vectors (Seeger et al., PNAS USA 1984:81,5849).
AntL~ 'o'~ and agonists - assays and _Dlee~
Polypeptides of the invention may also be used to assess the binding of small molecule substrates and ligands in, for example, cells, cell-free pl~pdl~lions, chemical libraries, and natural 30 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., Curren~ Pro~ocols 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 Divlb polypeptides or polynucleotides, 5 particularlv 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 co~ ~tlllent, such as a membrane, cell envelope or cell wall, or a ~,-epa,alion of any thereof, comprising Divlb polypeptide and a labeled substrate or ligand of such polypeptide is incubated in the absence or the presence of a c~n~iid~te molecule 10 that may be a Divlb agonist or antagonist. The ability of the candidate molecule to agonize or antagonize the Divlb polypeptide is reflected in decreased binding of the labeled ligand or decreased production of product from such substrate. Molecules that bind gratuitously, ie., without inducing the effects of Divlb polypeptide are most likely to be good antagonists.
Molecules that bind well and increase the rate of product production from substrate are agonists.
15 Detection of the rate or level of production of product from substrate may be enh~n~ed 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 Divlb polynucleotide or polypeptide activity, and binding assays known in the art.
Another example of an assay for Divlb antagonists is a conl~liliv~ assay that combines 20 Divlb ancl a potential antagonist with Divlb-binding molecules, recombinant Divlb binding molecules, natural substrates or ligands, or substrate or ligand mimetics, under appropriate conditions for a competitive inhibition assay. Divlb can be labeled, such as by radioactivity or a colorimetric compound, such that the number of Divlb molecules bound to a binding molecule or converted to product can be d~t~,.l.-i..cd accurately to assess the effectiveness of the potential 25 antagonist.
Potential ~nt~goni~tS include small organic molecules, peptides, polypeptides and antibodies that bind to a polynucleotide or polypeptide of the invention and thereby inhibit or ~xtingui~h its activity. Potential antagonists also may be small organic molecules, a peptide, a polypepticle such as a closely related protein or antibody that binds the same sites on a binding 30 molecule, such as a binding molecule, without inducing Divlb-induced activities, thereby ,thlg the action of Divlb by excluding Divlb 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. Exarnples of small molecules include but are not limited to small organic molecules, peptides or peptide-like molecules. Other potential antagonists include 5 ~nti~.-n.~e molecules (see Okano, J. Neurochem. 56: 560 (1991); OLIGODEOXYNUCLEOTIDES
AS ANTISENSE INHIBITORS O~ GENE EXPRESSION, CRC Press, Boca Raton, FL (1988), for a description of these molecules). Preferred potential antagonists include compounds related to and variants of Divlb.
Each of the DNA sequences provided herein may be used in the discovery and 10 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 ~nti~en~e 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 m~mm~ n 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 m~mm~ n extracellular matrix proteins on in-dwelling devices or to extracellular matrix proteins in wounds; to block Divlb protein-mediated m~mn~ n cell invasion by, for example, initiating phosphorylation of m~mm~ n tyrosine kinases (Ros~n~hine et al., Infect.
Immun. 60:2211 (1992); to block bacterial adhesion between m~mm~ n extracellular matrix proteins ~md bacterial Divlb proteins that mediate tissue damage and; to block the normal progression of pathogenesis in infections initiated other than by the implantation of in-dwelling devices or by other surgical techniques.
The antagonists and agonists of the invention may be employed, for in~t~n~e, to inhibit and treat disease, such as, infections of the upper lc~yilal~ tract (e.g., otitis media, bacterial tracheitis, acute epiglottitis, thyroiditis), lower respiratory (e.g., empyema, lung abscess), cardiac (e.g., infective endocarditis), ga~llu;~testin~l (e.g., secretory diarrhoea, splenic absces, ~ uyc~ik)lleal abscess), CNS (e.g., cerebral abscess), eye (e.g., blepharitis, conju,~;livilis, keratitis, endophth~lmiti~, preseptal and orbital cellulitis, darcryocystitis), kidney and urinary tract (e.g., epididymitis, intrarenal and pennephric absces, toxic shock syndrome), skin (e.g., impetigo, folliculitis, cutaneous abscesses, cellwlitis, wound infection, bacterial myositis) bone and joint (e.g., septic arthritis, osteomyelitis).
Vaccines S Another aspect of the invention relates to a method for inducing an immunological response in an individual, particularly a m~mm~l which comprises inoculating the individual with Divlb, 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 10 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 Divlb, or a fragment or a variant thereof, for expressing Divlb, 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, 15 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 ~-lmini.ctering 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 Divlb or protein coded therefrom, wherein the composition comprises a recombinant Divlb or protein coded therefromcomprising DNA which codes for and expresses an antigen of said Divlb or protein coded 25 th~,leli.~,l. The immunological response may be used therapeutically or prophylactically and may take the form of antibody hllll,~ ily or cellular immunity such as that arising from CTL or CD4+ T cells.
A Divlb polypeptide or a fragment thereof may be fused with co-protein which maynot by itself produce antibodies, but is capable of stabilizing the first protein and producing a 30 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 S 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 fragm~nt.~ thereof which have been shown to encode non-variable regions of bacterial cell surface proteins in DNA constructs used in such genetic imm~ tionexperiments 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 plcl)al~lion 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 treatm~nt~ of bacterial infection, particularly Staphylococcus aureus infection, in m~mm~
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, m~mm~ry glands, urethra or vagina.
The invention also includes a vaccine formulation which comprises an immunogenicrecombinant 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 s~hcut~neous, intramuscular, intravenous, or intradermal. Formulations suitable for parclltel~l administration include aqueous and non-aqueous sterile injection solutions which may contain anti-oxidants, buffers, bacteriostats and solutes which render the formulati~n insotonic with the bodily fluidt 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 5 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 Divlb protein, it is to 10 be understood that this covers fragml nt.c 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 admi...~ .lion The invention also relates to compositions COln~ illg the polynucleotide or the polypeptides 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 org~nicmc, such as a pharmaceutical carrier suitable for a~minictration 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 a~minictration. The invention further relates to diagnostic and pharm~ce~ltic~l 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 pll= ".~c~ ltic~l compositions may be a~lminictered in any effective, convenient manner including, for inct~n~e, a~lmini.ctration by topical, oral, anal, vaginal, intravenous, iloneal~ intramuscular, subcutaneous, il~ nasal or intradermal routes among others.
In therapy or as a prophylactic, the active agent may be a~lminicteted to an individual as an injectable composition, for example as a sterile aqueous dispersion, preferably isotonic.

~- All:ernatively 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 5 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 ~(lministration to m~mm~l~, and particularly hum~n~, it is expected that the daily dosage level of the active agent will be from 0.01 mg/kg to 10 mg/kg, typically around I
mg/kg. I'he physician in any event will determinç 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 15 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, pacem~k~rs, vascular 20 grafts, vascular catheters, cerebrospinal fluid shunts, urinary catheters, continuous ambulatory peritoneal dialysis (CAPD) catheters.
The composition of the invention may be a-1mini~tered 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, 25 the composition could also be used to broaden perioperative cover for any surgical technique to prevent bacterial wound infections, especially Staphylococcus aureus wound infections.
Many orthopaedic surgeons consider that humans with prosthetic joints should be considered for antibiotic prophylaxis before dental tl~at~ t that could produce a bacteremia.
Late deep infection is a serious complication sometimes leading to loss of the prosthetic joint 30 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 rnay be used to bathe an indwelling device irnmediately before insertion. The active agent will preferably be present at a concentration of 1 llg/ml to lOmg/ml for bathing of wounds or indwelling devices.
A vaccine composition is conveniently in injectable forrn. Conventional adjuvants may be employed to enhance the immune response. A suitable unit dose for vaccination is 0.5-5 10 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 15 patent application to which this application claims priority is also mcorporated by reference herein in its entirety.

EXAMPLES

The examples below are carried out using standard techniques, which are well known and routine to those of shll in the art, except where otherwise described in detail. The examples are illustrative, but do not limit the invention.
Example 1 Strain selection, Library Production and Seqn~--ing The polynucleotide having the DNA sequence given in SEQ ID NO:l was obtained 25 from a library of clones of chromosomal DNA of Staphylococcus aureus in E. coli. The sequencing data from t~,vo or more clones containing overlapping Staphylococcus aureus DNAs was used to construct the contiguous DNA sequence in SEQ ID NO:1. Libraries may be ~epa.ed by routine methods, for example:
Methods 1 and 2 below.
Total cellular DNA is isolated from Staphylococcus aureus W C~nH 29 according tostandard procedures and size-fractionated by either of t vo 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 1 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 ~.coli infected with the packaged library. The library is amplified by standard procedures.
Method 2 Total cellular DNA is partially 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 fragment.~ 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 by standard 1 5 procedures.
Example 2 Divlb Char~ . ~alion E.coli FtsQ, H.influenzae FtsQ, B.subtilis DivIB and S.aureus DivIB have similarhydrophobicity profiles. All four proteins contain a highly hydrophobic region of approximately 20 amino acids in length which is indicative of the membrane ~p~nning region.
These regions are located towards the N-terminus of the mature peptide. Based on the work by Dai et al., 1996 (J. Bacteriology 178, 1328-1334) on the E.coli homolog, the cytoplasmic and membrane sp~nning regions of Staph DivlB are dispensible. Therefore, removal of the cytoplasmlc and membrane spanning regions of the Staph DivlB protein allows for the evaluation of this protein in the assays below.
Interactions of FtsQ with other cell divsion proteins (e.g., Fts A, Z, N, W, Y) are used as the basis of an assay for identifying agonists or antagonists of DivlB. The measurement of the interaction of DivlB protein with additional proteins or peptides, either within a lipid-based membrane system or in solution, provides for a potential assay format. Heterogeneous assays encompassing the use of an imml-no~csqy or surface-coating format in conjunction with either radiolabelled or optically labelled proteins and colllponellt~ can be used. The interaction of unlabelled DivlB with other polypeptides can also be observed directly using surface plasmon - resonance technology in optical biosensor devices. This method is particularly useful for measuring interactions with larger (~5kDa) polypeptides and can be readily adapted to screen for inhibitors of the protein-protein interaction. Solution-based homogeneous assays using fluorescently-labelled components can be configured to report on changes in fluorescence 5 intensity, fluorescence anisotropy, fluorescence energy transfer or correlation fluctuations in intensity as a result of the binding interaction. Binding proteins useful in these types of assay can be identified by 'ligand fishing' using, for example, optical biosensor methods (Bartley et al. 1994, Nature 368: 558) and bacterial extracts followed by affmity capture orchromatography on immobilized DivlB. Following elution of binding proteins from 10 immobilized DivlB using salt, pH changes or chaotropic agents, the eluted protein products can be separated using high-resolution methods such as reverse-phase high performance liquid chromatography and the individual polypeptides characterized by N-terminal amino acid sequencing and/or mass mapping (mass spectrometry combined with molecular ion weight matching against a protein ~1~t~b~e). Yeast two-hybrid and phage display technologies are 15 also be used to identify ligands which bind to DivlB. Interaction of ligands, identified by the methods described above or otherwise, with DivlB will form the basis of an assay to screen for inhibitors and/or agonists of DivlB.
Pure DivlB protein is used to raise antibodies in mice or rabbits or other suitable animal host. Using standard techniques with materials such as gold-antibody conjugates, 20 fluoresently labelled antibodies or GFP the DivlB protein is locali~ed and the effect of compounds is tested.

CA 0223368l l998-04-0l SEQUENCE LISTING

(1) GENERAL INFORMAT:[ON

(i) APPLICANT: Pearce, Kenneth H.
Payne, David J.
Pearson, Stewart C.
Hodgson, John E.

(ii) TITLE OF THE INvENrrIoN NOVEL Divlb (iii) NUMBER OF SEQUENCES: 6 (iv) CORRESPONDENCE ADDRESS:
(A) ADDRESSEE: Dechert Price & Rhoads (B) STREET: 4000 Bell Atlantic Tower, 1717 Arch Stre (C) CITY: Philadelphia (D) STATE: PA

(E) COUNTRY: US
(F) ZIP: 19103 (v) COMPUTER READABLE FORM:
(A) MEDIUM TYPE: Diskette (B) COMPUTER: IBM Compatible (C) OPERATING SYSTEM: DOS
(D) SOFTWARE: FastSEQ for Windows Version 2.0 (vi) CURRENT APPLICATION DATA:
(A) APPLICATION NUMBER:
(B) FILING DATE:
(C) CLASSIFICATION:

CA 0223368l l998-04-0l (vii) PRIOR APPLICATION DATA:
(A) APPLICATION NUMBER:
(B) FILING DATE:

(viii) ATTORNEY/AGENT INFORMATION:
(A) NAME: Dickinson, Todd Q
(B) REGISTRATION NUMBER: 28,354 (C) REFERENCE/DOCKET NUMBER: P50592-1 (ix) TELECOMMUNICATION INFORMATION:
(A) TELEPHONE: 215-994-2252 (B) TELEFAX: 215-994-2222 (C) TELEX:

(2) INFORMATION FOR SEQ ID NO:1:

(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 1316 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: double (D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:

~ ll~lAATC AAAATAAAGA TGATACATCT CAACAAGCTG ATTTTGATGA AGAAATTTAC 180 CA 0223368l l998-04-0l -(2) INFORMATION FOR SEQ ID NO:2:

(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 439 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:

Met Asp Asp Lys Thr Lys Asn Asp Gln Gln Glu Ser Asn Glu Asp Lys Asp Glu Leu Glu Leu Phe Thr Arg Asn Thr Ser Lys Lys Arg Arg Gln Arg Lys Arg Ser Lys Ala Thr His Phe Ser Asn Gln Asn Lys Asp Asp Thr Ser Gln Gln Ala Asp Phe Asp Glu Glu Ile Tyr Leu Ile Asn Lys Asp Phe kys Lys Glu Glu Ser Asn Asp Glu Asn Asn Gly Ser Ala Ser ~er His Ala Asn Asp Asn Asn Ile Asp Asp Ser Thr Asp Ser Asn Ile ~lu Asn Glu Asp Tyr Arg Tyr Asn Gln Glu Ile Asp Asp Gln Asn Glu Ser Asn Gly Ile Ala Val Ala Asn Glu Gln Pro Gln Ser Ala Pro Lys Glu Gln Asn Ser Asp Ser Asn Asp Glu Glu Thr Val Thr Lys Lys Glu Arg Lys Ser Lys Val Thr Gln Leu Lys Pro Leu Thr Leu Glu Glu Lys ~rg Lys Leu Arg Arg Lys Arg Gln Lys Arg Ile Gln Tyr Ser Val Ile ~hr Ile Leu Val Leu Leu Ile Ala Val Ile Leu Ile Tyr Met Phe Ser Pro Leu Ser Lys Ile Ala Hls Val Asn Ile Asn Gly Asn Asn His Val Ser Thr Ser Lys Ile Asn Lys Val Leu Gly Val Lys Asn Asp Ser Arg Met Tyr Thr Phe Ser Lys Lys Asn Ala Ile Asn Asp Leu Glu Glu Asp ~ro Leu Ile Thr Ser Val Glu Ile His Lys Gln Phe Pro Asn Thr Leu ~sn Val Asp Ile Thr Glu Asn Glu Ile Ile Ala Leu Val Lys Tyr Lys Gly Lys Tyr Leu Pro Leu Leu Glu Asn Gly Lys Leu Leu Lys Gly Ser Asn Asp Val Lys Ile Asn Asp Ala Pro Val Met Asp Gly Phe Lys Gly Thr Lys Glu Asp Asp Met Ile Lys Ala Leu Ser Glu Met Thr Pro Glu -Val Arg Arg Tyr Ile Ala Glu Val Thr Tyr Pro Pro Ser Lys Asn Lys His Ser Arg Ile Glu Leu Phe Thr Thr Asp Gly Leu Gln Val Ile Gly Asp Ile Ser Thr Ile Ser Lys Lys Met Lys Tyr Tyr Pro Gln Met Ser Gln Ser Leu Ser Arg Asp Ser Ser Gly Lys Leu Lys Thr Arg Gly Tyr Ile Asp Leu Ser Val Gly Ala Ser Phe Ile Pro Tyr Arg Gly Asn Thr Ser Ser Gln Ser Glu Ser Asp Lys Asn Val Thr Lys Ser Ser Gln Glu Glu Asn Gln Ala Lys Glu Glu Leu Gln Ser Val Leu Asn Lys Ile Asn Lys Gln Ser Ser Lys Asn Asn (2) INFORMATION FOR SEQ ID NO:3:

(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 990 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: double (D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:

Al~LlllCAC CACTTAGTAA AATTGCGCAT GTAAATATAA ATGGAAATAA TCACGTTAGT 300 CA 0223368l l998-04-0l AAAATTAACA AACAATCAAG TAAGAATAAT ggo (2) INFORMATION FOR SEQ ID NO:4:

(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 330 amino acids (~) TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:

Met Asn Arg Asn Gly Ile Ala Ser Arg Gln Pro Asp Gln Pro Gln Ser Ala Pro Lys Glu Gln Asn Ser Asp Ser Asn Asp Glu Glu Thr Val Thr Lys Lys Glu Arg Lys Ser Lys Val Thr Gln Leu Lys Pro Leu Thr Leu Glu Glu Lys Arg Lys Leu Arg Arg Lys Arg Gln Lys Arg Ile Gln Tyr Ser Val Ile Thr Ile Leu Val Leu Leu Ile Ala Val Ile Leu Ile Tyr CA 0223368l l998-04-0l ' Met Phe Ser Pro Leu Ser Lys Ile Ala His Val Asn Ile Asn Gly Asn Asn His Val Ser Thr Ser Lys Ile Asn Lys Val Leu Gly Val Lys Asn Asp Ser Arg Met Tyr Thr Phe Ser Lys Lys Asn Ala Ile Asn Asp Leu Glu Glu Asp Pro Leu Ile Lys Ser Val Glu Ile His Lys Gln Leu Pro Asn Thr Leu Asn Val Asp Ile Thr Glu Asn Glu Ile Ile Ala Leu Val Lys Tyr Lys Gly Lys Tyr Leu Pro Leu Leu Glu Asn Gly Lys Leu Leu Lys Gly Ser Asn Asp Val Lys Ile Asn Asp Ala Pro Val Met Asp Gly Phe Lys Gly Thr Lys Glu Asp Asp Met Ile Lys Ala Leu Ser Glu Met Thr Pro Glu Val Arg Arg Tyr Ile Ala Glu Val Thr Tyr Ala Pro Ser Lys Asn Lys His Ser Arg Ile Glu Leu Phe Thr Thr Asp Gly Leu Gln Val Ile Gly Asp Ile Ser Thr Ile Ser Lys Lys Met Lys Tyr Tyr Pro Gln Met Ser Gln Ser Leu Ser Arg Asp Ser Ser Gly Lys Leu Lys Thr Arg Gly Tyr Ile Asp Leu Ser Val Gly Ala Ser Phe Ile Pro Tyr Arg Gly Asn Thr Ser Ser Gln Ser Glu Ser Asp Lys Asn Val Thr Lys Ser Ser Gln Glu Glu Asn Gln Ala Lys Glu Glu Leu Gln Ser Val Leu Asn Lys Ile Asn Lys Gln Ser Ser Lys Asn Asn (2) INFORMATION FOR SEQ ID NO:5:

(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 20 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO:5:

(2) INFORMATION FOR SEQ ID NO:6:

(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 20 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO:6:

,.. . ... . . .

Claims (24)

1. An isolated polynucleotide comprising a polynucleotide sequence selected fromthe group consisting of:
(a) a polynucleotide having at least a 70% identity to a polynucleotide encoding a polypeptide comprising the amino acid sequence of SEQ ID NO:2;
(b) a polynucleotide having at least a 70% identity to a polynucleotide encoding the same mature polypeptide expressed by the Div1b gene contained in the Staphylococcus aureus of the deposited strain;
(c) a polynucleotide encoding a polypeptide comprising an amino acid sequence which is at least 70% identical to the amino acid sequence of SEQ ID NO:2;
(d) a polynucleotide which is complementary to the polynucleotide of (a), (b) or (c);
and (e) a polynucleotide comprising at least 15 sequential bases of the polynucleotide of (a), (b), (c) or (d).

2. The polynucleotide of Claim 1 wherein the polynucleotide is DNA.

3. The polynucleotide of Claim 1 wherein the polynucleotide is RNA.

4. The polynucleotide of Claim 2 comprising the nucleic acid sequence set forth in SEQ ID NO:1.

5. The polynucleotide of Claim 2 comprising nucleotide 1 to 1317 set forth in SEQ
ID NO:1.

6. The polynucleotide of Claim 2 which encodes a polypeptide comprising the amino acid sequence of SEQ ID NO:2.

7. A vector comprising the polynucleotide of Claim 1.

8. A host cell comprising the vector of Claim 7.

9. A process for producing a polypeptide comprising: expressing from the host cell of Claim 8 a polypeptide encoded by said DNA.

10. A process for producing a Div1b polypeptide or fragment comprising culturinga host of claim 8 under conditions sufficient for the production of said polypeptide or fragment.

11. A polypeptide comprising an amino acid sequence which is at least 70% identical to the amino acid sequence of SEQ ID NO:2.

12. A polypeptide comprising an amino acid sequence as set forth in SEQ ID NO:2.

13. An antibody against the polypeptide of claim 11.

14. An antagonist which inhibits the activity or expression of the polypeptide of claim 11.

15. A method for the treatment of an individual in need of Div1b polypeptide comprising: administering to the individual a therapeutically effective amount of the polypeptide of claim 11.

16.. A method for the treatment of an individual having need to inhibit Div1b polypeptide comprising: administering to the individual a therapeutically effective amount of the antagonist of Claim 14.

17. A process for diagnosing a disease related to expression or activity of the polypeptide of claim 11 in an individual comprising:
(a) determining a nucleic acid sequence encoding said polypeptide, and/or (b) analyzing for the presence or amount of said polypeptide in a sample derived from the individual.

18. A method for identifying compounds which interact with and inhibit or activate an activity of the polypeptide of claim 11 comprising:
contacting a composition comprising the polypeptide with the compound to be screened under conditions to permit interaction between the compound and the polypeptide to assess the interaction of a compound, such interaction being associated with a second component capable of providing a detectable signal in response to the interaction of the polypeptide with the compound;
and determining whether the compound interacts with and activates or inhibits an activity of the polypeptide by detecting the presence or absence of a signal generated from the interaction of the compound with the polypeptide.

19. A method for inducing an immunological response in a mammal which comprises inoculating the mammal with Div1b polypeptide of claim 11, or a fragment or variant thereof, adequate to produce antibody and/or T cell immune response to protect said animal from disease.

20. A method of inducing immunological response in a mammal which comprises delivering a nucleic acid vector to direct expression of Div1b polypeptide of claim 11, or fragment or a variant thereof, for expressing said Div1b polypeptide, or a fragment or a variant thereof in vivo in order to induce an immunological response to produce antibody and/or T cell immune response to protect said animal from disease.

21. The use of a therapeutically effective amount of the polypeptide of claim 11 to treat an individual in need of Div1b polypeptide.

22. The use of a therapeutically effective amount of the antagonist of claim 14 to treat an individual having need to inhibit Div1b polypeptide.

23. The use of Div1b polypeptide of claim 11, or a fragment or variant thereof, adequate to produce antibody and/or T cell immune response to induce an immunological response in a mammal to protect said mammal from disease.

24. The use of a nucleic acid vector to direct expression of Div1b polypeptide of claim 11, or fragment or a variant thereof, for expressing said Div1b polypeptide, or a fragment or a variant thereof in vivo in order to induce an immunological response to produce antibody and/or T cell immune response to protect a mammal from disease.