WO2000068140A1 - Signal transduction response regulator family of staphylococcus - Google Patents

Abstract

The invention provides 623RR polypeptides and polynucleotides encoding 623RR polypeptides and methods for producing such polypeptides by recombinant techniques. Also provided are methods for utilizing 623RR polypeptides to screen for antibacterial compounds.

Description

SIGNAL TRANSDUCTION RESPONSE REGULATOR FAMILY OF STAPHYLOCOCCUS

RELATED APPLICATIONS

This application claims benefit to US Provisional Patent Application Number 60/132,759 filed May 6, 1999

HELD OF THE INVENTION

This invention relates to newly identified polynucleotides and polypeptides. and their production and uses, as well as their variants, agonists and antagonists, and their uses In particular, the invention relates to polynucleotides and polypeptides of the Two Component Signal Transducuon Response Regulator family, as well as their variants, herein referred to as "623RR." "623RR polynucleotide(s)," and "623RR polypeptide(s)" as the case may be

BACKGROUND OF THE INVENTION

It is particularly preferred to emplo\ 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 toxigemc 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 toxigemc properties of Staphylococci The manifestation of these diseases result from the actions of exotoxms 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 that are resistant to some or all of the standard antibiotics This phenomenon has created an unmet medical need and demand for new anti-microbial agents, vaccines, drug screening methods, and diagnostic tests for this organism

Moreover, the drug discovery process is currently undergoing a fundamental revolution as it embraces "functional genomics," that is, high throughput genome- or gene-based biology This approach is rapidly superseding earher approaches based on "positional cloning" and other methods Functional genomics relies heavily on the various tools of bioinforrnatics to identify gene sequences of potential interest from the many molecular biology databases now available as well as from other sources There is a continuing and significant need to identify and characterize further genes and other polynucleotides sequences and their related polypeptides, as targets for drug discovery.

Clearly, there exists a need for polynucleotides and polypeptides, such as the 623 RR embodiments of the invention, that have a present benefit of, among other things, being useful to screen compounds for antimicrobial 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.

SUMMARY OF THE INVENTION The present invention relates to 623RR, in particular 623RR polypeptides and 623RR polynucleotides. recombinant materials and methods for their production. In another aspect, the invention relates to methods for using such polypeptides and polynucleotides, including treatment of microbial diseases, amongst others. In a further aspect, the invention relates to methods for identifying agonists and antagonists using the materials provided by the invention, and for treating microbial infections and conditions associated with such infections with the identified agonist or antagonist compounds. In a still further aspect, the invention relates to diagnostic assays for detecting diseases associated with microbial infections and conditions associated with such infections, such as assays for detecting 623RR expression or activity.

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 623 RR polypeptides and polynucleotides as described in greater detail below. In particular, the invention relates to polypeptides and polynucleotides of a 623R-R oϊ Staphylococcus aureus, that is related by amino acid sequence homology to YesN polypeptide. The invention relates especially to 623RR having a nucleotide and amino acid sequences set out in Table 1 as SEQ ID NO:l and SEQ ID NO:2 respectively. Note that sequences recited in the Sequence Listing below as "DNA" represent an exemplification of the invention, since those of ordinary skill will recognize that such sequences can be usefully employed in polynucleotides in general, including ribopolynucleotides.

TABLE 1 623RR Polynucleotide and Polypeptide Sequences (A) Staphylococcus aureus 623RR polynucleotide sequence [SEQ ID NO 1]

5 ' -

ATGTTTAA.GGTAGTTATTTGTGATGATGAACGAA.TTATTCGAGAA.GGATTAAAGCAAATCATTCCTTGGGGAGAC

TATCA TTTCAATACAATATACACTGCTAAAGATGGTGTTGAAGCATTATCACTAATTCAACAACATCAGCCAGAATTAGT

AATTA

CTGATATTCGTATGCCACGTAAAAATGGCGTCGATTTACTCAATGATATTGCGCATCTAGATTGTAATGTCATCA

TATTA

TCGAGTTACGATGACTTTGAATATATGAAAGCCGGCATTCAACATCATGTTCTTGATTATTTATTAAAACCAGTA GACCA

TGCACAATTAGAAGTTATTTTAGGAAGACTTGTCCGTACCTTATTAGAGCAACAATCACAGAACGGCCGTAGCTT

AGCAT

CTTGTCATGATGCCTTCCAACCACTATTAAAAGTAGAATACGATGATTATTATGTAAACCAGATTGTGGATCAAA

TTAAG CAATCTTATCAAACGAAAGTGACTGTTTCAGATTTAATTCAACATATCGATGTTAGTGAATCGTATGCGATGCGA

ACATT

TAAAGATCATGTTGGTATTACAATTGTCGATTATTTAAATCGCTATCGTATTTTACAATCTCTGCAATTGTTAGA

TCGAC

ATTACAAACACTATGAAATTGCAGACAAAGTAGGCTTTTCAGAGTATAAAATGTTTAGCTATCATTTTAAAAAAT ATTTA

CAAATGTCGCCAAGTGATTATTGTAAGCAAGCAAAATAG-3 '

(B) Staphylococcus aureus 623RR polypeptide sequence deduced from a polynucleotide sequence in this table [SEQ ID NO 2] NH₂-

MFKWICDDERIIREGLKQIIP GDYHFNTIYTAKDGVEALSLIQQHQPELVITDIRMPRKNGVDLLNDIAHLDC

NVIIL

SSYDDFEYMKAGIQHHVLDYLLKPVDHAQLEVILGRLVRTLLEQQSQNGRSLASCHDAFQPL KVEYDDYYVNQI

VDQIK QSYQT VTVSDLIQHIDVSESYAMRTFKDHVGITIVDYLNRYRILQSLQLLDRHYKHYEIADKVGFSEYKMFSYH

FKKYL

QMSPSDYCKQAK-COOH

Deposited materials A deposit compnsing a Staphylococcus aureus WCUH 29 strain has been deposited with the

National Collections of Industπal and Maπne Bacteria Ltd (herein "NCIMB"). 23 St Machar Dnve Aberdeen AB2 1RY, Scotland on 11 September 1995 and assigned NCIMB Deposit No 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 "

- j - The deposited strain comprises a full length 623 RR gene The sequence of the polynucleotides compπsed in the deposited strain, as well as the amino acid sequence of any polypeptide encoded thereby, are controlling in the event of any conflict with anv descπpticn 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 deposited strain will be irrevocably and without restriction or condition released to the public upon the issuance of a patent The deposited strain is provided merelv as convenience to those of skill in the art and is not an admission that a deposit is required for enablemem. 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 deπved therefrom, and no such license is hereby granted

In one aspect of the invention there is provided an isolated nucleic acid molecule encoding a mature polypeptide expressible by the Staphylococcus aureus WCUH 29 strain, which polypeptide is compπsed in the deposited strain Further provided by the invention are 623 RR polynucleotide sequences in the deposited strain, such as DNA and RNA, and amino acid sequences encoded thereby Also provided by the invention are 623RR polypeptide and polynucleotide sequences isolated from the deposited strain

Polypeptides

623RR polypeptide of the invention is substantially phylogenetically related to other proteins of the Two Component Signal Transduction Response Regulator family

In one aspect of the invention there are provided polypeptides oϊ Staphylococcus aureus referred to herein as "623 RR" and "623RR polypeptides" as well as biologically, diagnostically, prophylactically, clinically or therapeutically useful vaπants thereof, and compositions compnsing the same

Among the particularly preferred embodiments of the invention are vaπants of 623 RR polypeptide encoded by naturally occurring alleles of a 623 RR gene

The present invention further provides for an isolated polypeptide that (a) comprises or consists of an ammo acid sequence that has at least 95% identity, most preferably at least 97-99% or exact identity, to that of SEQ ID NO 2 over the entire length of SEQ ID NO 2, (b) a polypeptide encoded by an isolated polynucleotide compnsing or consistmg of a polynucleotide sequence that has at least 95% identity, even more preferably at least 97-99% or exact identit to SEQ ID NO 1 over the entire length of SEQ ID NO 1, (c) a polypeptide encoded by an isolated pohτιucleotιde compnsing or consistmg of a polynucleotide sequence encoding a polypeptide that has at least 95% identity, even more preferably at least 97-99% or exact identity, to the amino acid sequence of SEQ ID NO 2. over the entire length of SEQ ED N02

The polypeptides of the invention include a polypeptide of Table 1 [SEQ ID NO 2] (in particular a mature polypeptide) as well as polypeptides and fragments, particularly those that has a biological activity of 623RR, and also those that have 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 comprising at least 30 amino acids and more preferably at least 50 amino acids

The mvention also mcludes a polypeptide consisting of or compnsing a polypeptide of the formula X-(R₁)_m-(R₂)-(R₃)_n-Y wherein, at the ammo teπ nus, X is hydrogen, a metal or any other moiety descnbed herein for modified polypeptides. and at the carboxvl terminus, Y is hydrogen, a metal or any other moiety descnbed herein for modified polypeptides, Ri and R3 are any amino acid residue or modified ammo 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 mvention, particularly an amino acid sequence selected from Table 1 or modified forms thereof In the formula above, R₂ is onented so that its ammo terminal ammo acid residue is at the left, covalently bound to R_j and its carboxy terminal ammo acid residue is at the nght covalently bound to R3 Any stretch of ammo acid residues denoted bv either Ri or R3, where m and/or n is greater than 1. may be either a heteropolymer or a homopolymer, preferably a heteropolymer Other preferred embodiments of the mvention are provided where m is an integer between 1 and 50, 100 or 500, and n is an integer between 1 and 50, 100, or 500

It is most preferred that a polypeptide of the mvention is deπved from Staphylococcus aureus, however, it may preferably be obtained from other organisms of the same taxonomic genus A polypeptide of the invention may also be obtained, for example, from organisms of the same taxonomic family or order

A fragment is a vaπant polypeptide having an ammo acid sequence that is entirely the same as part but not all of any ammo acid sequence of any polypeptide of the mvention As with 623RR polypeptides, fragments may be "free-standing," or compπsed within a larger polypeptide of which they form a part or region, most preferably as a single continuous region in a smgle larger polypeptide

Preferred fragments include, for example, truncation polypeptides having a portion of an ammo acid sequence of Table 1 [SEQ ID NO 2], or of vanants thereof, such as a continuous senes of residues that mcludes an ammo- and/or carboxyl-terminai ammo acid sequence Degradation forms of the polypeptides of the mvention produced by or m a host cell, particularly a Staphylococcus aureus, are also prefened Further preferred are fragments characterized by structural or functional attnbutes such as fragments that compπse alpha-helix and alpha-helix forming regions, beta-sheet and beta-sheet-forming regions, turn and turn-formmg regions, coil and coύ-forming regions, hydrophihc regions, hydrophobic regions, alpha amphipathic regions, beta amphipathic regions, flexible regions, surface-forming regions, substrate binding region, and high antigenic mdex regions

Further preferred fragments mclude an isolated polypeptide comprising an amino acid sequence having at least 15, 20, 30, 40, 50 or 100 contiguous ammo acids from the amino acid sequence of SEQ ID NO 2, or an isolated polypeptide comprising an amino acid sequence having at least 15, 20, 30, 40, 50 or 100 contiguous amino acids truncated or deleted from the amino acid sequence of SEQ ID NO:2.

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. Polynucleotides It is an object of the invention to provide polynucleotides that encode 623RR polypeptides, particularly polynucleotides that encode a polypeptide herein designated 623 RR.

In a particularly prefened embodiment of the invention the polynucleotide comprises a region encoding 623RR polypeptides comprising a sequence set out in Table 1 [SEQ ID NO:l] that includes a full length gene, or a variant thereof. The Applicants believe that this full length gene is essential to the growth and/or survival of an organism that possesses it, such as Staphylococcus aureus.

As a further aspect of the invention there are provided isolated nucleic acid molecules encoding and/or expressing 623 RR polypeptides and polynucleotides, particularly Staphylococcus aureus 623RR polypeptides and polynucleotides, including, for example, unprocessed RNAs, ribozyme RNAs, mRNAs, cDNAs, genomic DNAs, B- and Z-DNAs. Further embodiments of the invention include biologically, diagnostically, prophylactically, clinically or therapeutically useful polynucleotides and polypeptides, and variants thereof, and compositions comprising the same.

Another aspect of the invention relates to isolated polynucleotides, including at least one full length gene, that encodes a 623RR polypeptide having a deduced amino acid sequence of Table 1 [SEQ ID NO:2] and polynucleotides closely related thereto and variants thereof.

In another particularly preferred embodiment of the invention there is a 623RR polypeptide from Staphylococcus aureus comprising or consisting of an amino acid sequence of Table 1 [SEQ ID NO: 2], or a variant thereof. Using the information provided herein, such as a polynucleotide sequence set out in Table 1 [SEQ ID

NO:l], a polynucleotide of the invention encoding 623RR 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 polynucleotide sequence given in Table 1 [SEQ ID NO: l], 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 hybridization conditions. By sequencing the individual clones thus identified by hybridization with sequencing primers designed from the onginal polypeptide or polynucleotide sequence it is then possible to extend the polynucleotide sequence m both directions to determine a full length gene sequence Convemently, such sequencmg is performed, for example, usmg denatured double stranded DNA prepared from a plasmid clone Suitable techniques are descnbed by Maniatis. T . Fπtsch, E F and Sambrook et al , MOLECULAR CLONING, A LABORATORY MANUAL, 2nd Ed . Cold Spπng Harbor Laboratory Press, Cold Spπng Harbor, New York (1989) (see m particular Screening By Hybπdization 1 90 and Sequencmg Denatured Double-Stranded DNA Templates 13 70) Direct genoπuc DNA sequencmg may also be performed to obtain a full length gene sequence Illustrative of the mvention, each polynucleotide set out in Table 1 [SEQ ID NO 1] was discovered in a DNA hbrarv deπved from Staphylococcus aureus WCUH 29 Moreover, each DNA sequence set out in Table 1 [SEQ ID NO 1] contains an open reading frame encoding a protein having about the number of a mo acid residues set forth in Table 1 [SEQ ID NO 2] with a deduced molecular weight that can be calculated using ammo acid residue molecular weight values well known to those skilled in the art The polynucleotide of SEQ ID NO 1, between nucleotide number 757 and the stop codon that begins at nucleotide number 1 of SEQ ID NO 1, encodes the polypeptide of SEQ ID NO 2

In a further aspect, the present mvention provides for an isolated polynucleotide compnsmg or consistmg of (a) a polynucleotide sequence that has at least 95% identity, even more preferably at least 97-99% or exact identity to SEQ ID NO 1 over the entire length of SEQ ID NO 1, (b) a polynucleotide sequence encoding a polypeptide that has at least 95% identity, even more preferably at least 97-99% or 100% exact, to the ammo acid sequence of SEQ ID NO 2, over the entire length of SEQ ID NO 2

A polynucleotide encoding a polypeptide of the present mvention, including homologs and orthologs from species other than Staphylococcus aureus, may be obtamed by a process that compnses the steps of screening an appropnate library under stringent hybπdization conditions with a labeled or detectable probe consistmg of or compnsmg the sequence of SEQ ED NO 1 or a fragment thereof, and isolating a full-length gene and/or genomic clones compnsmg said polynucleotide sequence

The mvention provides a polynucleotide sequence identical over its entire length to a coding sequence (open reading frame) m Table 1 [SEQ ID NO 1] Also provided by the mvention is a coding sequence for a mature polypeptide or a fragment thereof, by itself as well as a coding sequence for a mature polypeptide or a fragment m reading frame with another coding sequence, such as a sequence encoding a leader or secretory sequence, a pre-, or pro- or prepro-protem sequence The polynucleotide of the mvention may also compnse at least one non-coding sequence, including for example, but not limited to at least one non-coding 5' and 3' sequence, such as the transcnbed but non-translated sequences, terrnination signals (such as rho-dependent and rho-rndependent termination signals), nbosome binding sites, Kozak sequences, sequences that stabilize mRNA inarms, and polyadenylation signals The polynucleotide sequence may also compnse additional coding sequence encoding additional ammo acids For example, a marker sequence that facilitates purification of a fused polypeptide can be encoded In certain embodiments of the mvention, the marker sequence is a hexa-histidine peptide, as provided m the pQE vector (Qiagen, Inc ) and descnbed in Gentz et al , Proc Natl Acad Set , USA 86 821-824 (1989), or an HA peptide tag (Wilson et al , Cell 37 767 (1984), both of that may be useful in purifying polypeptide sequence fused to them Polynucleotides of the mvention also include, but are not limited to, polynucleotides compnsmg a structural gene and its naturally associated sequences that control gene expression

A preferred embodiment of the mvention is a polvnucleoude of consisting of or compnsmg nucleotide 757 to the nucleotide immediately upstream of or mcluding nucleotide 1 set forth m SEQ ED NO 1 of Table 1. both of that encode a 623RR polypeptide

The mvention also mcludes a polynucleotide consisting of or compnsmg a polynucleotide of the formula

X-(R₁)_m-(R₂)-(R₃)_n-Y wherem, at the 5' end of the molecule, X is hydrogen, a metal or a modified nucleotide residue, or together with Y defines a covalent bond, and at the 3' end of the molecule, Y is hydrogen, a metal, or a modified nucleotide residue, or together with X defines the covalent bond, each occurrence of R and R3 is mdependently any nucleic acid residue or modified nucleic acid residue, m is an mteger between 1 and 3000 or zero , n is an mteger between 1 and 3000 or zero, and R₂ is a nucleic acid sequence or modified nucleic acid sequence of the mvention, particularly a nucleic acid sequence selected from Table 1 or a modified nucleic acid sequence thereof In the polynucleotide formula above, R is onented so that its 5' end nucleic acid residue is at the left, bound to R\ and its 3' end nucleic acid residue is at the πght, bound to R3 Any stretch of nucleic acid residues denoted by either R^ and/or R , where m and/or n is greater than 1, may be either a heteropolymer or a homopolymer, preferably a heteropolymer Where, m a preferred embodiment, X and Y together define a covalent bond, the polynucleotide of the above formula is a closed, circular polynucleotide, that can be a double-stranded polynucleotide wherem the formula shows a first strand to which the second strand is complementary In another preferred embodiment m and or n is an mteger between 1 and 1000. Other prefened embodiments of the mvention are provided where m is an mteger between 1 and 50, 100 or 500, and n is an mteger between 1 and 50, 100, or 500 It is most preferred that a polynucleotide of the mvention is denved from Staphylococcus aureus, however, it may preferably be obtamed from other organisms of the same taxonomic genus A polynucleotide of the mvention may also be obtamed, for example, from organisms of the same taxonomic family or order

The term "polynucleotide encoding a polypeptide" as used herein encompasses polynucleotides that mclude a sequence encoding a polypeptide of the mvention, particularly a bacteπal polypeptide and more particularly a polypeptide of the Staphylococcus aureus 623 RR having an ammo acid sequence set out in Table 1 [SEQ ID NO 2] The term also encompasses polynucleotides that mclude a single continuous region or discontinuous regions encoding the polypeptide (for example, polynucleotides interrupted by integrated phage, an integrated insertion sequence, an integrated vector sequence, an integrated transposon sequence, or due to RNA editing or genomic DNA reorganization) together with additional regions, that also may compnse coding and or non-coding sequences

The mvention further relates to vanants of the polynucleotides descnbed herein that encode vaπants of a polypeptide having a deduced ammo acid sequence of Table 1 [SEQ ED NO 2] Fragments of polynucleotides of the mvention may be used, for example, to synthesize full-length polynucleotides of the mvention

Further particularly prefened embodiments are polynucleotides encoding 623RR vanants, that have the ammo acid sequence of 623RR polypeptide of Table 1 [SEQ ED NO 2] m which several, a few, 5 to 10, 1 to 5, 1 to 3, 2, 1 or no ammo acid residues are substituted, modified, deleted and or added, m any combination Especially prefened among these are silent substitutions, additions and deletions, that do not alter the properties and activities of 623RR polypeptide

Prefened isolated polynucleotide embodiments also mclude polynucleotide fragments, such as a polynucleotide comprising a nuclic acid sequence having at least 15, 20, 30, 40, 50 or 100 contiguous nucleic acids from the polynucleotide sequence of SEQ ID NO: l, or an polynucleotide comprising a nucleic acid sequence having at least 15, 20, 30, 40, 50 or 100 contiguous nucleic acids truncated or deleted from the 5' and/or 3' end of the polynucleotide sequence of SEQ ID NO:l.

Further prefened embodiments of the mvention are polynucleotides that are at least 95% or 97% identical over their entire length to a polynucleotide encoding 623 RR polypeptide having an a mo acid sequence set out m Table 1 [SEQ ID NO 2], and polynucleotides that are complementary to such polynucleotides. Most highly prefened are polynucleotides that compnse a region that is at least 95% are especially prefened. Furthermore, those with at least 97% are highly prefened among those with at least 95%, and among these those with at least 98% and at least 99% are particularly highly prefened, with at least 99% being the more prefened

Prefened embodiments are polynucleotides encoding polypeptides that retain substantially the same biological function or activity as a mature polypeptide encoded by a DNA of Table 1 [SEQ ID NO 1]

In accordance with certain prefened embodiments of this mvention there are provided polynucleotides that hybπdize. particularly under stringent conditions, to 623RR polynucleotide sequences, such as those polynucleotides m Table 1 The mvention further relates to polynucleotides that hybndize to the polynucleotide sequences provided herein In this regard, the mvention especially relates to polynucleotides that hybπdize under strmgent conditions to the polynucleotides descnbed herein As herem used, the terms "stringent conditions" and "stringent hybndization conditions" mean hybπdization occurring only if there is at least 95% and preferably at least 97% identity between the sequences A specific example of strmgent hybndization conditions is overnight incubation at 42°C m a solution compnsmg 50% formamide, 5x SSC (150mM NaCl, 15mM tnsodium citrate), 50 mM sodium phosphate (pH7 6), 5x Denhardt's solution, 10% dextran sulfate, and 20 micrograms/ml of denatured, sheared salmon sperm DNA, followed by washing the hybndization support m 0 lx SSC at about 65°C Hybndization and wash conditions are well known and exemplified in Sambrook, et al Molecular Cloning A Laboratory Manual, Second Edition, Cold Spπng Harbor, N Y , (1989), particularly Chapter 11 therem Solution hybndization may also be used with the polynucleotide sequences provided bv the mvention

The mvention also provides a polynucleotide consistmg of or compnsmg a polynucleotide sequence obtamed by screening an appropπate library compnsmg a complete gene for a polynucleotide sequence set forth m SEQ ID NO 1 under strmgent hybndization conditions with a probe havmg the sequence of said polynucleotide sequence set forth m SEQ ID NO 1 or a fragment thereof, and isolating said polynucleotide sequence Fragments useful for obtaining such a polynucleotide mclude. for example, probes and primers fully descnbed elsewhere herem

As discussed elsewhere herem regarding polynucleotide assays of the mvention, for instance, the polynucleotides of the mvention, may be used as a hybπdization probe for RNA, cDNA and genomic DNA to isolate full-length cDNAs and genomic clones encoding 623 RR and to isolate cDNA and genomic clones of other genes that have a high identity, particularly high sequence identity, to a 623RR gene Such probes generally will compnse at least 15 nucleotide residues or base parrs Preferably, such probes will have at least 30 nucleotide residues or base pairs and may have at least 50 nucleotide residues or base pairs Particularly prefened probes will have at least 20 nucleotide residues or base pairs and will have lee than 30 nucleotide residues or base pairs

A coding region of a 623RR gene may be isolated by screening using a DNA sequence provided m Table 1 [SEQ ID NO 1] to synthesize an oligonucleotide probe A labeled oligonucleoude having a sequence complementary to that of a gene of the mvention is then used to screen a library of cDNN genomic DNA or mRNA to determine which members of the library the probe hybndizes to

There are several methods available and well known to those skilled m the art to obtam full- length DNAs, or extend short DNAs, for example those based on the method of Rapid Amplification of cDNA ends (RACE) (see, for example, Frohman, et al , PNAS USA 85 8998-9002, 1988) Recent modifications of the technique, exemplified by the Marathon™ technology (Clontech Laboratones Inc ) for example, have significantly simplified the search for longer cDNAs In the Marathon™ technology, cDNAs have been prepared from mRNA extracted from a chosen tissue and an 'adaptor' sequence hgated onto each end Nucleic acid amplification (PCR) is then earned out to amplify the "missing" 5' end of the DNA usmg a combmation of gene specific and adaptor specific ohgonucleotide primers The PCR reaction is then repeated usmg "nested" primers, that is, primers designed to anneal within the amplified product (typically an adaptor specific pπmer that anneals further 3' m the adaptor sequence and a gene specific primer that anneals further 5' m the selected gene sequence) The products of this reaction can then be analyzed by DNA sequencmg and a full-length DNA constructed either by joining the product directly to the existing DNA to give a complete sequence, or carrying out a separate full- length PCR usmg the new sequence information for the design of the 5' pπmer

The polynucleotides and polypeptides of the mvention may be employed, for example, as research reagents and mateπals for discovery of treatments of and diagnostics for diseases, particularly human diseases, as further discussed herem relating to polynucleotide assays

The polynucleotides of the mvention that are o gonucleotides denved from a sequence of Table 1 [SEQ ID NOS 1 or 2] may be used m the processes herem as descnbed, but preferably for PCR, to determine whether or not the polynucleotides identified herem m whole or m part are transcnbed m bacteπa in infected tissue It is recognized that such sequences will also have utility m diagnosis of the stage of infection and type of infection the pathogen has attained

The mvention also provides polynucleotides that encode a polypeptide that is a mature protem plus additional ammo or carboxyl-terminai ammo acids, or ammo acids mtenor to a mature polypeptide (when a mature form has more than one polypeptide chain, for instance) Such sequences may play a role in processmg of a protem from precursor to a mature form, may allow protem transport, may lengthen or shorten protem half-life or may facilitate rnanipulation of a protem for assay or production, among other things As generally is the case in vivo, the additional ammo acids may be processed away from a mature protem by cellular enzymes

For each and every polynucleotide of the mvention there is provided a polynucleotide complementary to it It is prefened that these complementary polynucleotides are fully complementary to each polynucleotide with which they are complementary

A precursor protem havmg a mature form of the polypeptide fused to one or more prosequences ma}' be an inactive form of the polypeptide When prosequences are removed such inactive precursors generalh are activated Some or all of the prosequences may be removed before activation Generally, such precursors are called proprote s

As w ll be recognized, the entire polypeptide encoded by an open reading frame is often not required for activity Accordingly, it has become routine m molecular biology to map the boundanes of the pnmarv structure required for activity with N-terminal and C-terrninal deletion experiments These experiments utihze exonuclease digestion or convenient restriction sites to cleave coding nucleic acid sequence For example, Promega (Madison, WE) sell an Erase-a-base™ system that uses Exonuclease HI designed to facilitate analysis of the deletion products (protocol available at www promega com) The digested endpoints can be repaired (e g , by hgation to synthetic hnkers) to the extent necessary to preserve an open readmg frame In this way, the nucleic acid of SEQ ED NO 1 readily provides contiguous fragments of SEQ ID NO 2 sufficient to provide an activity, such as an enzymatic, binding or antibody-inducing activity Nucleic acid sequences encoding such fragments of SEQ ED NO 2 and vanants thereof as descnbed herem are withmthe mvention, as are polypeptides so encoded In sum, a polynucleotide of the mvention may encode a mature protein, a mature protem plus a leader sequence (which may be refened to as a preprotem), a precursor of a mature protem havmg one or more prosequences that are not the leader sequences of a preprotem, or a preproprotein, that is a precursor to a proprotein, havmg a leader sequence and one or more prosequences. that generally are removed duπng processing steps that produce active and mature forms of the polypeptide Vectors, Host Cells, Expression Systems

The mvention also relates to vectors that compnse a polynucleotide or polynucleotides of the mvention, host cells that are genetically engmeered with vectors of the mvention and the production of polypeptides of the mvention by recombinant techniques Cell-free translation systems can also be employed to produce such proteins using RNAs denved from the DNA constructs of the mvention Recombinant polypeptides of the present mvention may be prepared by processes well known m those skilled m the art from genetically engmeered host cells compnsmg expression systems Accordingly, m a further aspect, the present mvention relates to expression systems that compnse a polynucleotide or polynucleotides of the present mvention, to host cells that are genetically engmeered with such expression systems, and to the production of polypeptides of the mvention by recombinant techniques For recombinant production of the polypeptides of the mvention, host cells can be genetically engmeered to incorporate expression systems or portions thereof or polynucleotides of the mvention Introduction of a polynucleotide into the host cell can be effected by methods descnbed in many standard laboratory manuals, such as Davis, et al , BASIC METHODS IN MOLECULAR BIOLOGY, (1986) and Sambrook, et al , MOLECULAR CLONING A LABORATORY MANUAL, 2nd Ed , Cold Spπng Harbor Laboratory Press. Cold Spnng Harbor, N Y (1989), such as, calcium phosphate transfection, DEAE-dextran mediated transfection, transvection. micromjection. cationic hpid-mediated transfection. electroporation, transduction, scrape loading, ballistic introduction and infection

Representative examples of appropπate hosts mclude bacteπal cells, such as cells of streptococci, staphylococci, enterococci E coll, streptor ces, cyanobactena, Bacillus subtilis, and Staphylococcus aureus, fungal cells, such as cells of a yeast, Kluveromyces, Saccharomyces, a basidiomycete, Candida albicans and Aspergillus, insect cells such as cells of Drosophila S2 and Spodoptera Sf9, animal cells such as CHO, COS, HeLa, C127, 3T3, BHK, 293, CV-1 and Bowes melanoma cells, and plant cells, such as cells of a gymnosperm or angjosperm A great vanety of expression systems can be used to produce the polypeptides of the mvention Such vectors mclude, among others, chromosomal-, episomal- and vrrus-denved vectors, for example, vectors denved from bacteπal plasmids, from bactenophage. from transposons, from yeast episomes, from insertion elements, from yeast chromosomal elements, from viruses such as baculoviruses. papova viruses, such as SV40, vaccinia viruses, adenovmises, fowl pox viruses, pseudorabies viruses, picornaviruses and retrovrruses, and vectors deπved from combinations thereof, such as those deπved from plasmid and bactenophage genetic elements, such as cosmids and phagemids The expression system constructs may compnse control regions that regulate as well as engender expression Generally, any system or vector suitable to maintain, propagate or express polynucleotides and/or to express a polypeptide m a host may be used for expression m this regard The appropnate DNA sequence may be inserted mto the expression system by any of a vanety of well-known and routine techmques, such as, for example, those set forth m Sambrook et al , MOLECULAR CLONING, A LABORATORY MANUAL, (supra)

In recombinant expression systems m eukarvotes, for secretion of a translated protem mto the lumen of the endoplasmic reticulum, mto the penplasmic space or mto the extracellular environment, appropnate secretion signals may be incorporated mto the expressed polypeptide These signals may be endogenous to the polypeptide or they may be heterologous signals

Polypeptides of the mvention can be recovered and purified from recombinant cell cultures by well- known methods including ammonium sulfate or ethanol precipitation, acid extraction, anion or cation exchange chromatography, phosphocellulose chromatography, hydrophobic interaction chromatography, affinity chromatography, hydroxylapatite chromatography, and lectm chromatography Most preferably, high performance hqmd chromatography is employed for purification Well known techmques for refoldmg protem may be employed to regenerate active conformation when the polypeptide is denatured duπng isolation and or purification

Diagnostic, Prognostic, Serotyping and Mutation Assays

This mvention is also related to the use of 623 RR polynucleotides and polypeptides of the mvention for use as diagnostic reagents Detection of 623 RR polynucleotides and/or polypeptides m a eukaryote, particularly a mammal, and especially a human, will provide a diagnostic method for diagnosis of disease, staging of disease or response of an infectious organism to drugs Eukarvotes. particularly mammals, and especially humans, particularly those infected or suspected to be infected with an organism compnsmg the 623RR gene or protein, may be detected at the nucleic acid or amino acid level by a variety of well known techniques as well as by methods provided herein.

Polypeptides and polynucleotides for prognosis, diagnosis or other analysis may be obtained from a putatively infected and/or infected individual's bodily materials. Polynucleotides from any of these sources, particularly DNA or RNA, may be used directly for detection or may be amplified enzymatically by using PCR or any other amplification technique prior to analysis. RNA, particularly mRNA, cDNA and genomic DNA may also be used in the same ways. Using amplification, characterization of the species and strain of infectious or resident organism present in an individual, may be made by an analysis of the genotype of a selected polynucleotide of the organism. Deletions and insertions can be detected by a change in size of the amplified product in comparison to a genotype of a reference sequence selected from a related organism, preferably a different species of the same genus or a different strain of the same species. Point mutations can be identified by hybridizing amplified DNA to labeled 623 RR polynucleotide sequences. Perfectly or significantly matched sequences can be distinguished from imperfectly or more significantly mismatched duplexes by DNase or RNase digestion, for DNA or RNA respectively, or by detecting differences in melting temperatures or renaturation kinetics. Polynucleotide sequence differences may also be detected by alterations in the electrophoretic mobility of polynucleotide fragments in gels as compared to a reference sequence. This may be carried out with or without denaturing agents. Polynucleotide differences may also be detected by direct DNA or RNA sequencing. See, for example, Myers et al, Science, 230: 1242 (1985). Sequence changes at specific locations also may be revealed by nuclease protection assays, such as RNase, VI and S 1 protection assay or a chemical cleavage method. See, for example, Cotton et al, Proc. Natl. Acad. Sci., USA, 85: 4397-4401 (1985).

In another embodiment, an array of oligonucleotides probes comprising 623RR nucleotide sequence or fragments thereof can be constructed to conduct efficient screening of, for example, genetic mutations, serotype, taxonomic classification or identification. Array technology methods are well known and have general applicability and can be used to address a variety of questions in molecular genetics including gene expression, genetic linkage, and genetic variability (see, for example, Chee et al, Science, 274: 610 (1996)).

Thus in another aspect, the present invention relates to a diagnostic kit that comprises: (a) a polynucleotide of the present invention, preferably the nucleotide sequence of SEQ ID NO:l, or a fragment thereof ; (b) a nucleotide sequence complementary to that of (a); (c) a polypeptide of the present invention, preferably the polypeptide of SEQ ID NO:2 or a fragment thereof; or (d) an antibody to a polypeptide of the present invention, preferably to the polypeptide of SEQ ID NO:2. It will be appreciated that in any such kit, (a), (b), (c) or (d) may comprise a substantial component. Such a kit will be of use in diagnosing a disease or susceptibility to a Disease, among others. This mvention also relates to the use of polynucleotides of the present mvention as diagnostic reagents Detection of a mutated form of a polynucleotide of the mvention, preferable, SEQ ED NO 1, that is associated w th a disease or pathogenicity will provide a diagnostic tool that can add to, or define, a diagnosis of a disease, a prognosis of a course of disease, a determination of a stage of disease, or a susceptibility to a disease, that results from under-expression, over-expression or altered expression of the polynucleotide Organisms, particularly infectious organisms, carrying mutations m such polynucleotide may be detected at the polynucleotide level by a vanety of techmques. such as those descnbed elsewhere herem

The differences m a polynucleotide and or polypeptide sequence between organisms possessmg a first phenotype and organisms possessmg a different, second different phenotype can also be determined If a mutation is observed m some or all organisms possessmg the first phenotype but not m any organisms possessmg the second phenotype. then the mutation is likely to be the causative agent of the first phenotype

Cells from an organism carrying mutations or polymorphisms (allehc vaπations) m a polynucleotide and/or polypeptide of the mvention may also be detected at the polynucleotide or polypeptide level by a vanety of techmques, to allow for serotyping, for example For example, RT-PCR can be used to detect mutations m the RNA It is particularly prefened to use RT-PCR m conjunction with automated detection systems, such as, for example, GeneScan RNA, cDNA or genomic DNA may also be used for the same purpose, PCR As an example, PCR primers complementary' to a polynucleotide encoding 623 RR polypeptide can be used to identify and analyze mutations The mvention further provides these pnmers with 1, 2, 3 or 4 nucleotides removed from the 5' and or the 3' end These pnmers may be used for, among other thmgs, amphfymg 623RR DNA and/or RNA isolated from a sample denved from an mdividual, such as a bodily matenal The pnmers may be used to amplify a polynucleotide isolated from an infected mdividual, such that the polynucleotide may then be subject to vaπous techmques for elucidation of the polynucleotide sequence In this way, mutations in the polynucleotide sequence may be detected and used to diagnose and/or prognose the infection or its stage or course, or to serotype and/or classify the infectious agent

The mvention further provides a process for diagnosing, disease, preferably bactenal infections, more preferably infections caused by Staphylococcus aureus, compnsing determining from a sample denved from an mdividual, such as a bodily matenal. an mcreased level of expression of polynucleotide havmg a sequence of Table 1 [SEQ ID NO 1] Increased or decreased expression of a 623 RR polynucleotide can be measured usmg any on of the methods well known m the art for the quantitation of polynucleotides, such as, for example, amplification, PCR, RT-PCR, RNase protection, Northern blotting, spectrometry and other hybndization methods

In addition, a diagnostic assay m accordance with the mvention for detectmg over-expression of 623 RR polypeptide compared to normal control tissue samples may be used to detect the presence of an infection, for example Assay techmques that can be used to determine levels of a 623RR polypeptide. m a sample denved from a host, such as a bodily matenal. are w ell-known to those of skill m the art Such assay methods mclude radioimmunoassays, competitive-binding assays, Western Blot analysis, antibody sandwich assays, antibody detection and ELISA assays

Antagonists and Agonists - Assays and Molecules

Polypeptides and polynucleotides of the mvention may also be used to assess the binding of small molecule substrates and hgands in, for example, cells, cell-free preparations, chemical hbranes, and natural product mixtures These substrates and hgands may be natural substrates and hgands or may be structural or functional mimetics See, e g , Cohgan etal , Current Protocols in Immunology 1(2) Chapter 5 (1991) Polypeptides and polynucleotides of the present mvention are responsible for many biological functions, mcluding many disease states, m particular the Diseases herem mentioned It is therefore desirable to devise screening methods to identify compounds that agonize (e g , stimulate) or that antagonize (e g .inhibit) the function of the polypeptide or polynucleotide Accordingly, m a further aspect, the present mvention provides for a method of screening compounds to identify those that agonize or that antagonize the function of a polypeptide or polynucleotide of the mvention. as well as related polypeptides and polynucleotides In general, agomsts or antagonists (e g , inhibitors) may be employed for therapeutic and prophylactic purposes for such Diseases as herem mentioned Compounds may be identified from a vanety of sources, for example, cells, cell-free preparations, chemical hbranes, and natural product mixtures Such agomsts and antagomsts so-identified may be natural or modified substrates, hgands, receptors, enzymes, etc , as the case may be, of 623RR polypeptides and polynucleotides, or may be structural or functional mimetics thereof (see Cohgan et al , Current Protocols in Immunology 1 (2) Chapter 5 (1991))

The screening methods may simply measure the binding of a candidate compound to the polypeptide or polynucleotide, or to cells or membranes bearing the polypeptide or polynucleotide, or a fusion protem of the polypeptide by means of a label directly or mdirectly associated with the candidate compound Alternatively, the screening method may mvolve competition with a labeled competitor Further, these screening methods may test whether the candidate compound results m a signal generated by activation or inhibition of the polypeptide or polynucleotide, usmg detection systems appropnate to the cells compnsmg the polypeptide or polynucleotide Inhibitors of activation are generally assayed m the presence of a known agomst and the effect on activation by the agomst by the presence of the candidate compound is observed Constitutively active polypeptide and/or constitutively expressed polypeptides and polynucleotides may be employed m screemng methods for mverse agonists, m the absence of an agomst or antagonist, by testmg whether the candidate compound results m inhibition of activation of the polypeptide or polynucleotide, as the case may be Further, the screemng methods may simply compnse the steps of mixing a candidate compound with a solution compnsmg a polypeptide or polynucleotide of the present mvention, to form a mixture, measuring 623RR polypeptide and/or polynucleotide activity m the mixture, and comparing the 623 RR polypeptide and/or polynucleotide activity of the mixture to a standard Fusion proteins, such as those made from Fc portion and 623RR polypeptide, as herem descnbed, can also be used for high-throughput screemng assays to identify antagomsts of the polypeptide of the present mvention, as well as of phylogenetically and and/or functionally related polypeptides (see D Bennett et al . J Mol Recognition, 8 52-58 (1995), and K Johanson et al , J Biol Chem, 270(16) 9459-9471 (1995))

The polynucleotides, polypeptides and antibodies that bmd to and/or mteract with a polypeptide of the present mvention may also be used to configure screemng methods for detectmg the effect of added compounds on the production of mRNA and/or polypeptide m cells For example, an ELISA assay may be constructed for measuring secreted or cell associated levels of polypeptide usmg monoclonal and polyclonal antibodies by standard methods known m the art This can be used to discover agents that may inhibit or enhance the production of polypeptide (also called antagonist or agomst, respectively) from suitably manipulated cells or tissues

The mvention also provides a method of screemng compounds to identify those that enhance (agomst) or block (antagonist) the action of 623RR polypeptides or polynucleotides, particularly those compounds that are bacteπstatic and/or bactencidal The method of screemng may mvolve high-throughput techmques For example, to screen for agonists or antagonists, a synthetic reaction mix, a cellular compartment, such as a membrane, cell envelope or cell wall, or a preparation of any thereof, compnsing 623RR polypeptide and a labeled substrate or hgand of such polypeptide is incubated m the absence or the presence of a candidate molecule that may be a 623RR agomst or antagonist The ability of the candidate molecule to agonize or antagonize the 623RR polypeptide is reflected in decreased binding of the labeled hgand or decreased production of product from such substrate Molecules that bmd gratuitously, < e , without inducing the effects of 623 RR polypeptide are most likely to be good antagomsts Molecules that bmd well and, as the case may be, mcrease the rate of product production from substrate, mcrease signal transduction, or mcrease chemical channel activity are agomsts Detection of the rate or level of, as the case may be, production of product from substrate, signal transduction, or chemical channel activity may be enhanced by usmg a reporter system Reporter systems that may be useful m this regard mclude but are not limited to coloπmetπc, labeled substrate converted mto product, a reporter gene that is responsive to changes m 623RR polynucleotide or polypeptide activity, and binding assays known m the art

Polypeptides of the mvention may be used to identify membrane bound or soluble receptors, if any, for such polypeptide, through standard receptor bmdmg techmques known m the art These techmques mclude, but are not limited to, hgand bmdmg and crosslinking assavs m which the polypeptide is labeled with a radioactive isotope (for instance. ^1), chemically modified (for instance, biotinylated), or fused to a peptide sequence suitable for detection or purification, and incubated with a source of the putative receptor (e.g., cells, cell membranes, cell supernatants, tissue extracts, bodily materials). Other methods include biophysical techniques such as surface plasmon resonance and spectroscopy. These screening methods may also be used to identify agomsts and antagonists of the polypeptide that compete with the binding of the polypeptide to its receptor(s), if any. Standard methods for conducting such assays are well understood in the art.

The fluorescence polarization value for a fluorescently-tagged molecule depends on the rotational conelation time or tumbling rate. Protein complexes, such as formed by 623RR polypeptide associating with another 623 RR polypeptide or other polypeptide. labeled to comprise a fluorescently-labeled molecule will have higher polarization values than a fluorescently labeled monomeric protein. It is prefened that this method be used to characterize small molecules that disrupt polypeptide complexes.

Fluorescence energy transfer may also be used characterize small molecules that interfere with the formation of 623RR polypeptide dimers, trimers, tetramers or higher order structures, or structures formed by 623RR polypeptide bound to another polypeptide. 623RR polypeptide can be labeled with both a donor and acceptor fluorophore. Upon mixing of the two labeled species and excitation of the donor fluorophore, fluorescence energy transfer can be detected by observing fluorescence of the acceptor. Compounds that block dimerization will inhibit fluorescence energy transfer.

Surface plasmon resonance can be used to momtor the effect of small molecules on 623RR polypeptide self-association as well as an association of 623RR polypeptide and another polypeptide or small molecule. 623RR polypeptide can be coupled to a sensor chip at low site density such that covalently bound molecules will be monomeric. Solution protein can then passed over the 623RR polypeptide -coated surface and specific binding can be detected in real-time by monitoring the change in resonance angle caused by a change in local refractive index. This technique can be used to characterize the effect of small molecules on kinetic rates and equilibrium binding constants for 623RR polypeptide self-association as well as an association of 623RR polypeptide and another polypeptide or small molecule. A scintillation proximity assay may be used to characterize the interaction between an association of 623RR polypeptide with another 623RR polypeptide or a different polypeptide . 623RR polypeptide can be coupled to a scintillation-filled bead. Addition of radio-labeled 623RR polypeptide results in binding where the radioactive source molecule is in close proximity to the scintillation fluid. Thus, signal is emitted upon 623 RR polypeptide binding and compounds that prevent 623RR polypeptide self-association or an association of 623RR polypeptide and another polypeptide or small molecule will diminish signal

In other embodiments of the mvention there are provided methods for identifying compounds that bmd to or otherwise mteract with and inhibit or activate an activity or expression of a polypeptide and/or polynucleotide of the mvention compnsmg contacting a polypeptide and/or polynucleotide of the mvention with a compound to be screened under conditions to permit bmdmg to or other mteraction between the compound and the polypeptide and/or polynucleotide to assess the bmdmg to or other mteraction with the compound, such bmdmg or mteraction preferably being associated with a second component capable of providing a detectable signal m response to the bmdmg or mteraction of the polypeptide and/or polynucleotide with the compound, and determining whether the compound bmds to or otherwise interacts with and activates or inhibits an activity or expression of the polypeptide and/or polynucleotide by detecting the presence or absence of a signal generated from the bmdmg or mteraction of me compound with the polypeptide and/or polynucleotide Another example of an assay for 623RR agonists is a competitive assay that combines 623RR and a potential agonist with 623RR-bιndιng molecules, recombinant 623RR binding molecules, natural substrates or hgands, or substrate or hgand mimetics, under appropnate conditions for a competitive inhibition assay 623RR can be labeled, such as by radioactivity or a coloπmetnc compound, such that the number of 623RR molecules bound to a bmdmg molecule or converted to product can be determined accurately to assess the effectiveness of the potential antagonist

It will be readily appreciated by the skilled artisan that a polypeptide and/or polynucleotide of the present mvention may also be used m a method for the structure-based design of an agomst or antagonist of the polypeptide and/or polynucleotide. by (a) determining m the first instance the three- dunensional structure of the polypeptide and/or polynucleotide, or complexes thereof, (b) deducmg the three-dimensional structure for the likely reactive sιte(s), bmdmg sιte(s) or motιf(s) of an agomst or antagonist, (c) synthesizing candidate compounds that are predicted to bmd to or react with the deduced bmdmg sιte(s), reactive sιte(s), and/or mouf(s), and (d) testmg whether the candidate compounds are mdeed agomsts or antagonists It will be further appreciated that this will normally be an iterative process, and this iterative process may be performed usmg automated and computer-controlled steps

In a further aspect, the present inversion provides methods of treatmg abnormal conditions such as, for instance, a Disease, related to either an excess of. an under-expression of, an elevated activity of, or a decreased activity of 623RR polypeptide and'or polynucleotide If the expression and/or activity of the polypeptide and/or polynucleotide is m excess, several approaches are available One approach compnses admrnisteπng to an mdividual m need thereof an inhibitor compound (antagonist) as herem descnbed, optionally m combination with a pharmaceutically acceptable earner, m an amount effective to inhibit the function and/or expression of the polypeptide and/or polynucleotide, such as, for example, by blockmg the bmdmg of hgands, substrates, receptors, enzymes, etc , or by inhibiting a second signal, and thereby alleviating the abnormal condition In another approach, soluble forms of the polypeptides still capable of bmdmg the hgand, substrate, enzymes, receptors, etc m competition with endogenous polypeptide and or polynucleotide may be administered Typical examples of such competitors mclude fragments of the 623RR polypeptide and/or polypeptide

In still another approach, expression of the gene encodmg endogenous 623RR polypeptide can be inhibited usmg expression blockmg techmques This blockmg may be targeted against any step m gene expression, but is preferably targeted agamst transcnption and/or translation An examples of a known technique of this sort mvolve the use of antisense sequences, either internally generated or separately administered (see, for example, O'Connor, J Neurochem (1991) 56 560 m

Oligodeoxynucleotides as Antisense Inhibitors of Gene Expression, CRC Press, Boca Raton, FL (1988)) Alternatively, oligonucleotides that form tnple helices with the gene can be supplied (see, for example, Lee et al , Nucleic Acids Res (1979) 6 3073, Cooney et al , Science (1988) 241 456, Dervan et al , Science (1991) 251 1360) These ohgomers can be administered per se or the relevant o gomers can be expressed in vivo

Each of the polynucleotide sequences provided herem may be used m the discovery and development of antibacteπal compounds The encoded protein, upon expression, can be used as a target for the screemng of antibacteπal drugs Additionally, the polynucleotide sequences encodmg the ammo terminal regions of the encoded protem 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 mterest

The mvention also provides the use of the polypeptide, polynucleotide, agonist or antagonist of the mvention to interfere with the initial physical mteraction between a pathogen or pathogens and a eukaryotic, preferably mammalian, host responsible for sequelae of infection In particular, the molecules of the mvention may be used m the prevention of adhesion of bacteria, m particular gram positive and/or gram negative bactena. to eukaryotic, preferably mammalian, extracellular matπx protems on in-dwelling devices or to extracellular matrix proteins m wounds, to block bactenal adhesion between eukaryotic, preferably mammalian, extracellular matrix protems and bacterial 623RR protems that mediate tissue damage and/or. to block the normal progression of pathogenesis m infections initiated other than bv the implantation of in-dwelling devices or by other surgical techmques

In accordance with \et another aspect of the mvention. there are provided 623 RR agomsts and antagomsts, preferably bacteπstatic or bactencidal agonists and antagomsts The antagomsts and agomsts of the mvention may be employed, for instance, to prevent, inhibit and/or treat diseases

Hehcobacter pylori (herem "H pylori") bactena infect the stomachs of over one-third of the world's population causmg stomach cancer, ulcers, and gastntis (International Agency for Research on Cancer (1994) Schistosomes, Liver Flukes and Hehcobacter 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 gastnc adenocarcinoma, classifying the bacteπum as a Group I (definite) carcmogen Prefened antimicrobial compounds of the mvention (agomsts and antagomsts of 623RR polypeptides and/or polynucleotides) found usmg screens provided by the mvention, or known m the art, particularly narrow-spectrum antibiotics, should be useful m the treatment of H pylori mfection Such treatment should decrease the advent of H pylori-mduc-d cancers, such as gastrointestinal carcinoma Such treatment should also prevent, inhibit and/or cure gastnc ulcers and gastntis

All publications and references, including but not limited to patents and patent applications, cited m this specification are herem incorporated by reference m their entirety as if each mdividual publication or reference were specifically and individually indicated to be incorporated by reference herem as bemg fully set forth Any patent application to which this application claims pnonty is also incorporated by reference herem m its entirety m the manner descnbed above for publications and references

GLOSSARY

The following definitions are provided to facilitate understanding of certain terms used frequently herem

"Bodily matenal(s) means any matenal denved from an mdividual or from an organism infecting, infesting or inhabiting an mdividual, mcludmg but not limited to, cells, tissues and waste, such as, bone, blood, serum, cerebrospinal fluid, semen, saliva, muscle, cartilage, organ tissue, skin, urine, stool or autopsy mateπals

"Dιsease(s)" means any disease caused by or related to infection by a bactena, mcludmg , for example, disease, such as. infections of the upper respiratory tract (e g , otitis media, bactenal tracheitis, acute epiglottitis, thyroiditis), lower respiratory (e g , empyema. lung abscess), cardiac (e g , infective endocarditis), gastrointestinal (e g , secretory dianhoea, splenic absces. retropeπtoneal abscess), CNS (e g , cerebral abscess), eye (e g , blephaπtis, conjunctivitis, keratitis, endophthalmitis, preseptal and orbital celluhtis, darcryocystitis), kidney and urinary tract (e g , epiώdvrnitis. lntrarenal and peπnephπc absces, toxrc shock syndrome), slαn (e g , impetigo, folhcuhtis, cutaneous abscesses, celluhtis. wound infection, bactenal myositis) bone andjomt (e g , septic arthπtis, osteomvehtis)

"Host cell(s)" is a cell that has been introduced (e g , transformed or transfected) or is capable of mtroduction (e g , transformation or transfection) b\ an exogenous polynucleotide sequence

"Identity," as known m the art, is a relationship between two or more polypeptide sequences or two or more polynucleotide sequences, as the case may be as detenruned by companng 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 by known methods, mcludmg but not limited to those descnbed m (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 Gnffin, H G , eds , Humana Press, New Jersey, 1994, Sequence Analysis in Molecular Biology, von Hemje, G , Academic Press, 1987, and Sequence Analysis Primer, Gnbskov, M and Devereux, J , eds , M Stockton Press, New York, 1991, and Canllo, H , and Lipman, D , S AM J Applied Math , 48 1073 (1988) Methods to determme identity are designed to give the largest match between the sequences tested Moreover, methods to determme identity are codified m publicly available computer programs Computer program methods to determme identity between two sequences mclude, but are not limited to, the GCG program package (Devereux, J , et al , Nucleic Acids Research 12(1) 387 (1984)), BLASTP, BLASTN, and FASTA (Altschul, S F et al , J Molec Bwl 215 403-410 (1990) The BLAST X program is publicly available from NCBI and other sources (BLAST Manual, Altschul, S , et al , NCBI NLM NEH Bethesda, MD 20894, Altschul, S , et al , J Mol Bwl 215 403-410 (1990) The well known Smith Waterman algonthm may also be used to determme identity

Parameters for polypeptide sequence companson mclude the following Algonthm Needleman and Wunsch, J Mol Biol 48 443-453 (1970) Companson matπx 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 pubhch available as the "gap" program from Genetics Computer Group, Madison WI The aforementioned parameters are the default parameters for peptide compansons (along with no penalty for end gaps)

Parameters for polynucleotide companson mclude the following Algonthm Needleman and Wunsch, J Mol Biol 48 443-453 (1970)

Companson matnx 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 compansons

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

(1) Polynucleotide embodiments further mclude an isolated polynucleotide compnsmg a polynucleotide sequence havmg at least a 95, 97 or 100% identity to the reference sequence of SEQ ID NO 1, wherem said polynucleotide sequence may be identical to the reference sequence of SEQ ID NO 1 or may mclude up to a certain mteger number of nucleotide alterations as compared to the reference sequence, wherem said alterations are selected from the group consisting of at least one nucleotide deletion, substitution, mcludmg transition and transversion, or insertion, and wherem said alterations may occur at the 5 ' or 3' terminal positions of the reference nucleotide sequence or anywhere between those termmal positions, mterspersed either mdividually among the nucleotides m the reference sequence or in one or more contiguous groups within the reference sequence, and wherem said number of nucleotide alterations is determined by multiplying the total number of nucleotides m SEQ ID NO 1 by the mteger defining the percent identity divided by 100 and then subtractmg that product from said total number of nucleotides m SEQ ID NO 1, or

n_n < x_n (^χ _n • y)>

wherem n_n is the number of nucleotide alterations, x_n is the total number of nucleotides in SEQ ID NO 1, y is 0 95 for 95%, 0 97 for 97% or 1 00 for 100%, and • is the symbol for the multiplication operator, and wherem any non-mteger product of x_n and y is rounded down to the nearest mteger pnor to subtractmg it from x_n Alterations of a polynucleotide sequence encodmg the polypeptide of SEQ ID NO 2 may create nonsense, missense or frameshift mutations m this codmg sequence and thereby alter the polypeptide encoded by the polynucleotide following such alterations (2) Polypeptide embodiments further mclude an isolated polypeptide compnsmg a polypeptide havmg at least a 95, 97 or 100% identity to a polypeptide reference sequence of SEQ ID NO 2, wherem said polypeptide sequence may be identical to the reference sequence of SEQ ID NO 2 or may mclude up to a certain mteger number of ammo acid alterations as compared to the reference sequence, wherem said alterations are selected from the group consistmg of at least one ammo acid deletion, substitution, mcludmg conservative and non-conservative substitution, or insertion, and wherem said alterations may occur at the ammo- or carboxy-terminal positions of the reference polypeptide sequence or anywhere between those terminal positions, mterspersed either mdividually among the ammo acids m the reference sequence or m one or more contiguous groups within the reference sequence, and wherem said number of ammo acid alterations is determined by multiplymg the total number of ammo acids m SEQ ID NO 2 by the mteger defining the percent identity divided by 100 and then subtracting that product from said total number of ammo acids m SEQ ID NO 2, or

ⁿa ≤ ^xa (^χ _a • y)>

wherem n_a is the number of ammo acid alterations. x_a is the total number of ammo acids m SEQ ID NO 2, y is 0 95 for 95%, 0 97 for 97% or 1 00 for 100%, and • is the symbol for the multiplication operator, and wherem any non-mteger product of x_a and y is rounded down to the nearest mteger pnor to subtracting it from x_a "Indιvιdual(s)" means a multicellular eukaryote. mcludmg, but not limited to a metazoan, a mammal, an ovid, a bovid, a simian, a pπmate, and a human

"Isolated" means altered "by the hand of man" from its natural state, i e , if it occurs m nature, it has been changed or removed from its onginal 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 mateπals of its natural state is "isolated", as the term is employed herem Moreover, a polynucleotide or polypeptide that is introduced mto an organism by transformation, genetic mampulation or by any other recombinant method is "isolated" even if it is still present m said organism, which organism may be Irving or non-living

"Orgamsm(s)" means a (I) prokaryote, mcludmg but not limited to, a member of the genus Streptococcus, Staphylococcus, Bordetella, Corynebactenum, Mycobactenum, Neissena, Haemophilus, Actinomycetes, Streptomycetes, Nocardia Enterobacter, Yersinia Fancisella, Pasturella, Moraxella, Acinetobacter, Erysψelothnx, Branhamella Actmobacillus, Streptobacillus, Listena, Calymmatobactenum, Brucella, Bacillus, Clostndium, Treponema, Eschenchia, Salmonella, Kleibsiella, Vibno, Proteus, Erwima, Borrelia, Leptospira Spmllum, Campylobacter, Shigella, Legionella Pseudomonas, Aeromonas, Ricketts a, Chlamydia, Borrelia and Mycoplasma, and further including, but not limited to, a member of the species or group, Group A Streptococcus, Group B Streptococcus, Group C Streptococcus, Group D Streptococcus, Group G Streptococcus, Streptococcus pneumoniae, Streptococcus pyogenes, Streptococcus agalactiae, Streptococcus faeca s. Streptococcus faecium. Streptococcus durans, Neissena gonorrheae, Neissena memngitidis, Staphylococcus aureus, Staphylococcus epidermidis, Corynebactenum dψthenae, Gardnerella vaginahs. Mycobactenum tuberculosis, Mycobactenum bovis, Mycobactenum ulcerans, Mycobactenum leprae, Actinomyctes israelii, Listena monocytogenes, Bordetella pertusis, Boraatella parapertusis, Bordetella bronchiseptica, Eschenchia coh, Shigella dysentenae, Haemophilus influenzae, Haemophilus aegyptius, Haemophilus parainfluenzae, Haemophilus ducreyi, Bordetella, Salmonella typhi, Citrobacter freundu, Proteus mirabilis, Proteus vulgans, Yersima pestis, Kleibsiella pneumoniae, Serratia marcessens, Serratia liquefaciens, Vibno cholera, Shigella dysenteni, Shigella flexnen, Pseudomonas aemginosa, Franscisella tularensis, Brucella abortis, Bacillus anthracis, Bacillus cereus, Clostndium perjhngens, Clostndium tetani, Clostndium botuhnum, Treponema pallidum, Rickettsia nckettsn and Chlamydia trachomitis, (ii) an archaeon, mcluding but not limited to Archaebacter, and (hi) a unicellular or filamentous eukaryote, including but not limited to, a protozoan, a fungus, a member of the genus Saccharomyces, Kluveromyces, or Candida, and a member of the species Saccharomyces cenviseae, Kluveromyces lactis, or Candida albicans.

"Polynucleotide(s)" generally refers to any polyribonucleotide or polydeoxyπbonucleotide, that may be unmodified RNA or DNA or modified RNA or DNA. "Polynucleoude(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, hybnd molecules compnsmg 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 tnple-stranded regions comprising RNA or DNA or both RNA and DNA. The strands in such regions may be from the same molecule or from different molecules. The regions may include all of one or more of the molecules, but more typically involve only a region of some of the molecules. One of the molecules of a triple-hehcal region often is an oligonucleotide. As used herein, the term "polynucleotide(s)" also includes DNAs or RNAs as described above that comprise 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 tπtylated bases, to name just two examples, are polynucleotides as the term is used herein. It will be appreciated that a great variety of modifications have been made to DNA and RNA that serve many useful purposes known to those of skill in the art. The term "polynucleotide(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 charactenstic of viruses and cells, mcludmg, for example, simple and complex cells "Polynucleotide(s)" also embraces short polynucleotides often refened to as ohgonucleotide(s)

"Polypeptide(s)" refers to any peptide or protem compnsmg two or more ammo acids joined to each other by peptide bonds or modified peptide bonds "Polypeptide(s)" refers to both short chains, commonly refened to as peptides, ohgopeptides and ohgomers and to longer chains generally refened to as proteins Polypeptides may compnse ammo acids other than the 20 gene encoded ammo acids "Polypeptide(s)" mclude those modified either by natural processes, such as processmg and other post-translational modifications, but also by chemical modification techmques Such modifications are well descnbed m basic texts and m 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 m the same or varying degree at several sites m a given polypeptide Also, a given polypeptide may compnse many types of modifications Modifications can occur anywhere m a polypeptide. mcludmg the peptide backbone, the ammo acid side-chains, and the ammo or carboxyl termini Modifications mclude, for example, acetylation, acylation, ADP-nbosylation, amidation. covalent attachment of flavin, covalent attachment of a heme moiety, covalent attachment of a nucleotide or nucleotide denvative, covalent attachment of a pid or pid denvative, covalent attachment of phosphotidyhnositol, cross-linking, cychzation, disulfide bond formation, demethylation, formation of covalent cross-hnks, formation of cysteine, formation of pyroglutamate, formylation, garnrna-carboxylation, GPI anchor formation, hydroxylation, lodination, methylation, myπstoylation, oxidation, proteolytic processmg, phosphorylation, prenylation, racemization, glycosylation, hpid attachment, sulfabon, garnma-carboxyiation of glutamic acid residues, hydroxylation and ADP-nbosylation, selenoylation, sulfation. transfer-RNA mediated addition of ammo acids to proteins, such as arginylation, and ubiquitination See, for instance, PROTEINS - STRUCTURE AND MOLECULAR PROPERTIES, 2nd Ed , T E Creighton, W H Freeman and Company, New York (1993) and Wold, F , Posttranslational Protem Modifications Perspectives and Prospects, pgs 1-12 m POSTTRANSLATIONAL COVALENT MODIFICATION OF PROTEINS, B C Johnson, Ed , Academic Press, New York (1983), Seifter et al , Meth Enzymol 182 626-646 (1990) and Rattan et al , Protein Synthesis Posttranslational Modifications and Aging, Ann N Y Acad Sci 663 48-62 (1992) Polypeptides may be branched or cyclic, with or without branching Cychc, branched and branched circular polypeptides may result from post- translational natural processes and may be made by entirely synthetic methods, as well

"Recombinant expression system(s)" refers to expression systems or portions thereof or polynucleotides of the mvention introduced or transformed mto a host cell or host cell lysate for the production of the polynucleotides and polypeptides of the mvention 'Nanant(s)" as the term is used herem, 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 m the nucleotide sequence of the vaπant may or may not alter the ammo acid sequence of a polypeptide encoded by the reference polynucleotide Nucleotide changes may result m ammo acid substitutions, additions, deletions, fusion protems and truncations m the polypeptide encoded by the reference sequence, as discussed below A typical vaπant of a polypeptide differs in ammo acid sequence from another, reference polypeptide Generalh . differences are limited so that the sequences of the reference polypeptide and the vanant are closely similar overall and, m many regions, identical A vanant and reference polypeptide may differ in ammo acid sequence by one or more substitutions, additions, deletions in any combination A substituted or inserted ammo acid residue may or may not be one encoded by the genetic code The present mvention also mcludes mclude vanants of each of the polypeptides of the mvention, that is polypeptides that vary from the referents by conservative ammo acid substitutions, whereby a residue is substituted by another with like characteπstics Typrcal such substitutions are among Ala, Val, Leu and lie, among Ser and Thr, among the acidic residues Asp and Glu, among Asn and Gin, and among the basic residues Lys and Arg, or aromatic residues Phe and Tyr Particularly prefened are vaπants m which several, 5-10, 1-5, 1-3, 1-2 or 1 ammo acids are substituted, deleted, or added m any combination A vanant of a polynucleotide or polypeptide may be a naturally occurring such as an allehc vanant, or it may be a vanant that is not known to occur naturally Non-naturally occurring vaπants of polynucleotides and polypeptides may be made by mutagenesis techniques, by direct synthesis, and by other recombinant methods known to skilled artisans EXAMPLES

The examples below are earned out usmg standard techmques, that are well known and routine to those of skill in the art, except where otherwise descnbed m detail The examples are illustrative, but do not limit the mvention

Example 1 Strain selection, Library Production and Sequencing

The polynucleotide havmg a DNA sequence given m Table 1 [SEQ ID NO 1] was obtamed from a library of clones of chromosomal DNA of Staphylococcus aureus m E coh The sequencmg data from two or more clones compnsing overlapping Staphylococcus aureus DNAs was used to construct the contiguous DNA sequence m SEQ ID NO 1 Libraπes may be prepared by routme 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 m order to size- fractionate accordmg to standard procedures DNA fragments of up to l lkbp m size are rendered blunt by treatment with exonuclease and DNA polymerase, and EcoRI linkers added Fragments are hgated mto the vector Lambda ZapII that has been cut with EcoRI, the library packaged by standard procedures and E coh 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 combmation of restπction enzymes appropnate to generate a senes of fragments for cloning mto library vectors (e g , Rsal, Pall, Alul, Bshl235I), and such fragments are size-fractionated accordmg to standard procedures EcoRI linkers are hgated to the DNA and the fragments then hgated mto the vector Lambda ZapII that have been cut with EcoRI, the library packaged by standard procedures, and E coll infected with the packaged library The library is amplified by standard procedures

Claims

What is claimed is:

1 An isolated polypeptide selected from the group consistmg of

(1) an isolated polypeptide compnsmg an ammo acid havmg at least 95% identity to the ammo acid sequence of SEQ ID NO 2 over the entire length of SEQ ID NO 2, (π) an isolated polypeptide compnsmg the ammo acid sequence of SEQ ID NO 2, (in) an isolated polypeptide that is the ammo acrd sequence of SEQ ID NO 2, and (rv) a polypeptide that rs encoded by a recombinant polynucleotide compnsmg the polyncleotide sequence of SEQ ID NO 1

2 An isolated polynucleotide selected from the group consisting of

(l) an isolated polynucleotide comprising a polynucleotide sequence encoding a polypeptide that has at least 95% identity to the ammo acid sequence of SEQ ED NO 2, over the entire length of SEQ ED NO 2,

(u) an isolated polynucleotide comprising a polynucleotide sequence that has at least 95 % identity over its entire length to a polynucleotide sequence encoding the polypeptide of SEQ ED

NO 2,

(m) an isolated polynucleotide compnsing a nucleotide sequence that has at least 95% identity to that of SEQ ID NO 1 over the entire length of SEQ ID NO 1 ,

(IV) an isolated polynucleotide comprising a nucleotide sequence encoding the polypeptide of SEQ ED

NO 2,

(v) an isolated polynucleotide that is the polynucleotide of SEQ ID NO 1 ,

(vi) an isolated polynucleotide of at least 30 nucleotides m length obtamable by screemng an appropnate lrbrary under str gent hybndization conditions with a probe havmg the sequence of SEQ

ED NO 1 or a fragment thereof of of at least 30 nucleotides m length,

(vu) an isolated polynucleotide encoding a mature polypeptide expressed by the 623RR gene compπsed in the Staphylococcus aureus. and

(v ) a polynucleotide sequence complementary to said isolated polynucleotide of (I), (u), (m), (iv), (v), (v) or (vu)

3 A method for the treatment of an individual

(I) m need of enhanced activity or expression of or lmmunological response to the polypeptide of claim 1 compnsmg the step of administering to the mdividual a therapeutically effective amount of an antagonist to said polypeptide. or (π) havmg need to inhibit activity or expression of the polypeptide of claim 1 compnsmg

(a) administering to the mdividual a therapeutically effective amount of an antagonist to said polypeptide. or

(b) administering to the mdividual a nucleic acid molecule that inhibits the expression of a polynucleotide sequence encodmg said polypeptide,

(c) administering to the mdividual a therapeutically effective amount of a polypeptide that competes with said polypeptide for its hgand, substrate, or receptor, or

(d) administering to the mdividual an amount of a polypeptide that mduces an lmmunological response to said polypeptide m said mdividual

4 A process for diagnosing or prognosing a disease or a susceptibility to a disease m an mdividual related to expression or activity of the polypeptide of claim 1 m an mdividual compnsing the step of

(a) determining the presence or absence of a mutation m the nucleotide sequence encoding said polypeptide in an organism m said mdividual, or

(b) analyzing for the presence or amount of said polypeptide expression m a sample denved from said mdividual

5 A process for producmg a polypeptide selected from the group consistmg of

(l) an isolated polypeptide compnsmg an ammo acid sequence selected from the group having at least 95% identity to the ammo acid sequence of SEQ ID NO 2 over the entire length of SEQ ID NO 2, (n) an isolated polypeptide compnsmg the ammo acid sequence of SEQ ID NO 2, (in) an isolated polypeptide that is the ammo acid sequence of SEQ ID NO 2, and (iv) a polypeptide that is encoded by a recombinant polynucleotide compnsmg the polynucleotide sequence of SEQ ID NO 1, compnsmg the step of cultunng a host cell under conditions sufficient for the production of the polypeptide

6 A process for producmg a host cell compnsmg an expression system or a membrane thereof expressmg a polypeptide selected from the group consistmg of (I) an isolated polypeptide compnsmg an ammo acid sequence selected from the group havmg at least 95% identity to the ammo acid sequence of SEQ ID NO 2 over the entire length of SEQ ID NO 2, (π) an isolated polypeptide compnsmg the ammo acid sequence of SEQ ID NO 2, (in) an isolated polypeptide that is the ammo acid sequence of SEQ ID NO 2, and (iv) a polypeptide that is encoded by a recombinant polynucleotide compnsmg the polynucleotide sequence of SEQ ID NO 1, said process compnsmg the step of transforming or transfecting a cell with an expression system compnsmg a polynucleotide capable of producmg said polypeptide of (l), (n), (m) or (iv) when said expression system is present m a compatible host cell such the host cell, under appropnate culture conditions, produces said polypeptide of (I), (u), (in) or (iv)

7 A host cell or a membrane expressmg a polypeptide selected from the group consistmg of (I) an isolated polypeptide compnsmg an ammo acid sequence selected from the group havmg at least 95% identity to the ammo acid sequence of SEQ ID NO 2 over the entire length of SEQ ID NO 2,

(II) an isolated polypeptide compnsmg the ammo acid sequence of SEQ ID NO 2, (in) an isolated polypeptide that is the ammo acid sequence of SEQ ID NO 2, and (iv) a polypeptide that is encoded by a recombinant polynucleotide compnsmg the polynucleotide sequence of SEQ ID NO 1

8 An antibody lmmunospecific for the polypeptide of claim 1

9 A method for screening to identify compounds that agonize or that inhibit the function of the polypeptide of claim 1 that compnses a method selected from the group consisting of

(a) measuring the bmdmg of a candidate compound to the polypeptide (or to the cells or membranes bearmg the polypeptide) or a fusion protem thereof by means of a label directly or mdirectly associated with the candidate compound,

(b) measuring the bmdmg of a candidate compound to the polypeptide (or to the cells or membranes bearing the polypeptide) or a fusion protem thereof m the presence of a labeled competitor,

(c) testmg whether the candidate compound results m a signal generated by activation or inhibition of the polypeptide, usmg detection systems appropnate to the cells or cell membranes bearmg the polypeptide, (d) mixing a candidate compound with a solution compnsmg a polypeptide of claim 1, to form a mixture, measurmg activity of the polypeptide in the mixture, and companng the activity of the mixture to a standard, or

(e) detectmg the effect of a candidate compound on the production of mRNA encodmg said polypeptide and said polypeptide m cells, usmg for instance, an ELISA assay

10 An agomst or antagonist to the polypeptide of claim 1