WO2000071555A2 - 509rr - Google Patents

Abstract

The invention provides 509RR polypeptides and polynucleotides encoding 509RR polypeptides and methods for producing such polypeptides by recombinant techniques. Also provided are methods for utilizing 509RR polypeptides to screen for antibacterial compounds.

Description

509RR

RELATED APPLICATIONS

This application claims benefit to US Provisional Patent Application Number 60/132.935, filed May 6, 1999

FIELD OF THE INVENTION

This invention relates to newly identified polynucleotides and polypeptides. and their production and uses, as well as their vanants, agomsts and antagonists, and their uses In particular, the invention relates to polynucleotides and polypeptides of the Two-Component Signal Transduetion Response

Regulator family, as well as their variants, herein referred to as "509RR," "509RR polynucleotιde(s)," and "509RR polypeptιde(s)" as the case may be

BACKGROUND OF THE INVENTION

It is particularly preferred to employ Staphylococcal genes and gene products as targets tor the development of antibiotics The Staph lococcι make up a medically important genera of microbes Thc\ are known to produce two types of disease, invasive and toxigenic Invasive infections are charactenzed gcncrall} by abscess formation cffcctmg both skin surfaces and deep tissues S aurcus is the second leading cause of bacteremia in cancer patients Osteomyelitis, septic arthritis, septic thrombophlebitis and acute bacterial endocarditis arc also relatively common There arc at least three clinical conditions resulting from the toxigenic properties of Staphylococci The manifestation of these diseases result from the actions of exotoxms as opposed to tissue invasion and bacteremia These conditions include Staphv lococcal food poisoning, scalded skin syndrome and toxic shock syndrome

The frequency of Staphylococciis aurcus infections has nsen 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 Staphylococciis 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 earlier approaches based on "positional cloning" and other methods Functional genomics relies heavily on the various tools of bioinformatics 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 509RR embodiments of the mvention, 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 509RR, m particular 509RR polypeptides and 509RR 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 50 R 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 invenUon relates to 509RR polypeptides and polynucleotides as described in greater detail below In particular, the mvention relates to polypeptides and polynucleotides of a 509RR of Staphylococciis aureus, that is related by ammo acid sequence homology to YhcZ polypeptide The invention relates especially to 509RR having a nucleotide and ammo acid sequences set out m Table 1 as SEQ ID NO 1 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 πbopolynucleotides

TABLE 1 509RR Polynucleotide and Polypeptide Sequences

(A) Staphylococciis aureus 509RR polynucleotide sequence [SEQ ID NO 1] b -

ATGAACAAAGTAATATTAGTAGATGACCATTATATTGTGCGACAAGGATTGCGATTTTTATTATCCACGATTG.∑V

AACAT

AGAAGTTTTACAAGACTTTGCAGATGGAGAAACATTTTTAGAATATTTAAAAGAGCATGAGCACCCTGATATTGT GCTAT

TAGATTTAGTGATGCCTGGCATGAATGGTATTGAAATTACGGAATATATTAAGGCACATTATCCGGAAATTAAAG

TTTTG

GTATTAACAAGTTATGTTGATGATGAACATGTAATTTCAGCAATCAATAAAGGTGCTGATGGTTATGAAATG.\AA

GACGT TGAGCCTCAGCAATTAATTGAAACTATTAGACGAGTTATGAACGGTGAAAAAATGATACATCCTAAGGCACAAGA

TG AT

TCGAAACAGTTAGCCAAAAACCACACTACACGAATAAGTTGTCAAAGAGAGAAATTGAAGTGTTACGTGAAATGG

TTAAA

GGTAAAACAAATAAAGAGATTGCAGAAACTTTATTTGTATCTGAAAAAACAATTAAAACACATGTCAGTCATATA TTTAG

TAAATTACAAGTTAGCGATCGTACACAAGCAGCAATTTATGCAATGGAAAATAAGTTGATTTAG-3 '

(B) Staphylococciis aureus 509RR polypeptide sequence deduced from a polynucleotide sequence in this table [SEQ ID NO:2]. NH₂-

MNKVILVDDHYIVRQGLRFLLSTIEMIEVLQDFADGETFLEYLKEHEHPDIVLLDLVMPGMNGIEITEYIKAHYP

EIKVL

VLTSYVDDEHVISAINKGADGYEMKDVEPQQLIETIRRVMNGEKMIHPKAQDVFETVSQKPHYTNKLSKREIEV

REMVK GKTNKEIAETLFVSEKTIKTHVSHIFSKLQVSDRTQAAIYA ENKLI-COOH

Deposited materials

A deposit comprising a Stuphylocotcm uureits WC H 29 strain lias been deposited w ith the

National Collections of Industrial and Mannc Bactcπa Ltd (herein "NCIMB" ). 23 St Maehar Dru e. AB2 1 RY. Scotland on I I September 1995 and assigned NCIM B Deposit No -1077 1 and referred to as Staphylococciis aureus CUH29 on deposit The Sftψhy/ύt oca atira/^y strain depo_.it it. re eπ ed to heiem as "the deposited strain" or as "the DNA of the deposited strain "

The deposited strain comprises a full length 509RR gene The sequence of the poh nuclcotidcs comprised in the deposited strain, as well as the ammo acid sequence of any poly peptide encoded theι

. .ire controllmg m the

enr of am conflict with any desenpiion of sequences heiein

Die deposit of the deposited strain has been made under the terms of the Budapest

on the International Recognition of the Deposit of Micro-organisms for Purposes of Patent Procedure Tlι_ deposited stram will be irrevocably and without restriction or condition released to the public upon die issuance of a patent. The deposited stram is ided merely as co cmcnce to those of skill in t e art and is not an admission that a deposit is requned for enablement. such as that lequired undei ^'^5 I S (^' § 1 12 A license may be required to make, use or sell the deposited strain, and compounds dern ed theiefrom. and no such license is hereby granted In one aspect of the lm cntion there is provided an isolated nucleic acid molecule encoding a mature polypeptide expressible by the Sraphy/ocoLCU.s aureus \V( l_f I I 29 strain, which polypeptide ι. comprised u the deposited strain Further pro\ ided by the invention are 509RR poly nucicoπdc sequences in the deposited strain, such as DNA and RNA, and ammo acid sequences encoded therebv Also provided by the invention are 509RR polypeptide and polynucleotide sequences isolated fiom the deposited strain.

Polypeptides

509RR 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 mvention there are provided polypeptides of Staphylococciis aureus referred to herein as "509RR" and "509RR polypeptides" as well as biologically, diagnostically, prophylacdcally, clinically or therapeutically useful variants thereof, and compositions comprising the same.

Among the particularly preferred embodiments of the invention are variants of 509RR polypeptide encoded by naturally occurring alleles of a 509RR gene.

The present invention further provides for an isolated polypeptide that: (a) comprises or consists of an amino 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 comprising or consisting of 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: l ; (c) a polypeptide encoded by an isolated polynucleotide comprising or consisting 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 ID NO:2.

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 509RR, 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 invention also includes a polypeptide consisting of or comprising a polypeptide of the formula: X-(Rl)_m-(R₂)-(R₃)n-Y wherem, at the ammo terminus, X is hydrogen, a metal or any other moiety descnbed herein for modified polypeptides. and at the carboxyl terminus, Y is hydrogen, a metal or any other moiety descnbed herein for modified polypeptides, Ri and R3 are any ammo acid residue or modified amino acid residue, m is an integer between 1 and 1000 or zero, n is an integer between 1 and 1000 or zero, and R₂ is an ammo acid sequence of the invention, particularly an ammo acid sequence selected from Table 1 or modified forms thereof In the formula above, R₂ is oπented so that its ammo terminal amino acid residue is at the left. covalently bound to Rj and its carboxy terminal ammo acid residue is at the right, covalently bound to

R3 Any stretch of ammo acid residues denoted by either R_j 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 invention 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 invention is denved from Staphylococciis 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 variant polypeptide having an amino acid sequence that is entirely the same as part but not all of any ammo acid sequence of any polypeptide of the invention As with 509RR polypeptides, fragments may be "free-standing," or comprised within a larger polypeptide of which they form a part or region, most preferably as a smgle continuous region m a single larger polypeptide Preferred fragments include, for example, truncation polypeptides having a portion of an amino acid sequence of Table 1 [SEQ ID NO 2], or of variants thereof, such as a continuous series of residues that mcludes an ammo- and/or carboxyl-terminal ammo acid sequence Degradation forms of the polypeptides of the mvention produced by or in a host cell, particularly a Staphylococciis aureus, are also preferred Further preferred are fragments charactenzed by structural or functional attributes such as fragments that comprise alpha-helix and alpha-helix forming regions, beta-sheet and beta-sheet-forming regions, turn and turn-forming regions, coil and coil-forming regions, hydrophihc regions, hydrophobic regions, alpha amphipathic regions, beta amphipathic regions, flexible regions, surface-forming regions, substrate bmdmg region, and high antigemc index regions

Further preferred fragments mclude an isolated polypeptide comprising an amino acid sequence having at least 15, 20, 30, 40, 50 or 100 contiguous amino 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 ammo acid sequence of SEQ ID NO:2. Fragments of the polypeptides of the mvention may be employed for producing the corresponding full-length polypeptide by peptide synthesis, therefore, these variants may be employed as intermediates for producmg the full-length polypeptides of the invention Polynucleotides It is an object of the invention to provide polynucleotides that encode 509RR polypeptides particularly polynucleotides that encode a polypeptide herein designated 09RR

In a particularly preferred embodiment of the invention the polynucleotide comprises a region encoding 509RR polypeptides compnsing a sequence set out in Table 1 [SEQ ID NO 1] 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 Slaphylococcus aureus

As a further aspect of the invention there are provided isolated nucleic acid molecules encoding and/or expressmg 509RR polypeptides and polynucleotides, particularly Slaphylococcus aureus 509RR polypeptides and polynucleotides, including, for example, unprocessed RNAs. nbozyme 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 mvention relates to isolated polynucleotides, including at least one full length gene, that encodes a 509RR polypeptide having a deduced ammo acid sequence of Table 1 [SEQ ID NO 2] and polynucleotides closely related thereto and vanants thereof In another particularly preferred embodiment of the invention there is a 509RR polypeptide from Staphylococciis aureus comprising or consisting of an amino acid sequence of Table 1 [SEQ ID NO 2], or a variant thereof

Usmg the information provided herein, such as a polynucleotide sequence set out in Table 1

[SEQ ID NO 1], a polynucleotide of the invention encodmg 509RR polypeptide may be obtained using standard clonmg and screenmg methods, such as those for clonmg and sequencing chromosomal DNA fragments from bacteπa usmg Slaphylococcus aureus WCUH 29 cells as starting mateπal. 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 1], typically a library of clones of chromosomal DNA of Slaphylococcus aureus WCUH 29 in E co 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 pπmers designed from the original polypeptide or polynucleotide sequence it is then possible to extend the polynucleotide sequence in both directions to determine a full length gene sequence Conveniently, such sequencing is performed, for example, usmg denatured double stranded DNA prepared from a plasmid clone Suitable techniques are described by Maniatis. T , Fπtsch, E F and Sambrook et al , MOLECULAR CLONING, A LABORATORY MANUAL. 2nd Ed Cold Spring Harbor Laboratory Press, Cold Spπng Harbor, New York (1989) (see in particular Screening By Hybridization 1 90 and Sequencing Denatured Double-Stranded DNA Templates 13 70) Direct genomic DNA sequencing may also be performed to obtain a full length gene sequence Illustrative of the invention, each polynucleotide set out in Table 1 [SEQ ID NO 1 ] was discovered m a DNA library deπved from Staphylococciis aureus WCUH 29

Moreover, each DNA sequence set out in Table 1 [SEQ ID NO 1] contains an open reading frame encodmg a protein having about the number of ammo 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 1 and the stop codon that begins at nucleotide number 622 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 comprising or consisting 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 Slaphylococcus aureus, may be obtained by a process that comprises the steps of screemng an appropπate library under stringent hybridization conditions with a labeled or detectable probe consisting of or comprising the sequence of SEQ ID NO 1 or a fragment thereof, and isolating a full-length gene and/or genomic clones compnsing said polynucleotide sequence

The invention provides a polynucleotide sequence identical over its entire length to a coding sequence (open readmg frame) m Table 1 [SEQ ID NO 1] Also provided by the invention 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 readmg frame with another codmg sequence, such as a sequence encodmg a leader or secretory sequence, a pre-, or pro- or prepro-protem sequence The polynucleotide of the invention may also comprise at least one non-codmg sequence, including for example, but not limited to at least one non-codmg 5' and 3' sequence, such as the transcribed but non-translated sequences, termination signals (such as rho-dependent and rho-mdependent termination signals), πbosome binding sites, Kozak sequences, sequences that stabilize mRNA, introns, and polyadenylation signals The polynucleotide sequence may also compπse additional coding sequence encodmg additional amino acids

For example, a marker sequence that facilitates purification of a fused polypeptide can be encoded In certam embodiments of the mvention, the marker sequence is a hexa-histidine peptide, as provided m the pQE vector (Qiagen, Inc ) and descnbed m 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 mclude, but are not limited to, polynucleotides compπsmg a structural gene and its naturally associated sequences that control gene expression A preferred embodiment of the invention is a polynucleotide of consisting of or comprising nucleotide 1 to the nucleotide immediately upstream of or including nucleotide 622 set forth in SEQ ID NO 1 of Table 1, both of that encode a 509RR polypeptide

The invention also mcludes a polynucleotide consisting of or compnsmg a polynucleotide of the formula X-(R₁)_m-(R₂)-(R₃)_n-Y wherein, 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 Rj and

R3 is mdependently any nucleic acid residue or modified nucleic acid residue, m is an integer between 1 and 3000 or zero , n is an mteger between 1 and 3000 or zero, and R is a nucleic acid sequence or modified nucleic acid sequence of the invention, particularly a nucleic acid sequence selected from Table 1 or a modified nucleic acid sequence thereof In the polynucleotide formula above, R is oπented so that its 5' end nucleic acid residue is at the left, bound to Rj and its 3' end nucleic acid residue is at the nght, bound to R3 Any stretch of nucleic acid residues denoted by either Rj and/or R₂, where m and/or n is greater than 1, may be either a heteropolymer or a homopolymer, preferably a heteropolymer Where, in a prefened 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 wherein the formula shows a first strand to which the second strand is complementary In another preferred embodiment m and/or n is an integer between 1 and 1000. Other preferred 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 deπved from Slaphylococcus auieus, however, it may preferably be obtained from other organisms of the same taxonomic genus A polynucleotide of the mvention may also be obtained, for example, from organisms of the same taxonomic family or order

The term "polynucleofrde encoding a polypeptide" as used herem encompasses polynucleotides that include a sequence encodmg a polypeptide of the invention, particularly a bacterial polypeptide and more particularly a polypeptide of the Slaphylococcus aureus 509RR having an ammo acid sequence set out in Table 1 [SEQ ID NO 2] The term also encompasses polynucleotides that include a smgle contmuous region or discontmuous regions encodmg the polypeptide (for example, polynucleotides interrupted by integrated phage, an integrated insertion sequence, an mtegrated vector sequence, an integrated transposon sequence, or due to RNA editmg or genomic DNA reorganization) together with additional regions, that also may compnse coding and/or non-codmg sequences

The invention further relates to variants of the polynucleotides described herein that encode variants of a polypeptide having a deduced ammo acid sequence of Table 1 [SEQ ID NO 2] Fragments of polynucleotides of the invention may be used, for example, to synthesize full-length polynucleotides of the invention

Further particularly preferred embodiments are polynucleotides encoding 509RR variants, that have the ammo acid sequence of 509RR polypeptide of Table 1 [SEQ ID NO 2] in which several, a few, 5 to 10, 1 to 5, 1 to 3, 2, 1 or no amino acid residues are substituted, modified, deleted and/or added, in any combination Especially prefened among these are silent substitutions, additions and deletions, that do not alter the properties and activities of 509RR polypeptide

Preferred isolated polynucleotide embodiments also mclude polynucleotide fragments, such as a polynucleotide comprising a nuchc 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 preferred embodiments of the mvention are polynucleotides that are at least 95% or 97% identical over their entire length to a polynucleotide encodmg 509RR polypeptide having an ammo acid sequence set out m Table 1 [SEQ ID NO 2], and polynucleotides that are complementary to such polynucleotides Most highly preferred are polynucleotides that compnse a region that is at least 95% are especially preferred Furthermore, those with at least 97% are highly preferred among those with at least

95%. and among these those with at least 98% and at least 99% are particularly highly preferred, with at least 99% being the more preferred

Preferred embodiments are polynucleotides 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 l]

In accordance with certam preferred embodiments of this mvention there are provided polynucleotides that hybπdize, particularly under strmgent conditions, to 509RR polynucleotide sequences, such as those polynucleotides in Table 1

The mvention further relates to polynucleotides that hybndize to the polynucleotide sequences provided herem In this regard, the mvention especially relates to polynucleotides that hybndize under strmgent conditions to the polynucleotides described herem As herem used, the terms "stringent conditions" and "strmgent hybndization conditions" mean hybndization occurrmg only if there is at least

95% and preferably at least 97% identity between the sequences A specific example of stnngent hybndization conditions is overnight incubation at 42°C m a solution compnsing 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 washmg the hybndization support m 0 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 Harbor, N Y , (1989), particularly Chapter 11 therein Solution hybridization may also be used with the polynucleotide sequences provided by the invention

The invention also provides a polynucleotide consisting of or comprising a polynucleotide sequence obtained by screening an appropriate library comprising a complete gene for a polynucleotide sequence set forth in SEQ ID NO 1 under strmgent hybridization conditions with a probe having the sequence of said polynucleotide sequence set forth in SEQ ID NO 1 or a fragment thereof, and isolating said polynucleotide sequence Fragments useful for obtaining such a polynucleotide mclude, for example, probes and pnmers fully described elsewhere herein

As discussed elsewhere herem regarding polynucleotide assays of the mvention, for instance, the polynucleotides of the mvention, may be used as a hybndization probe for RNA, cDNA and genomic DNA to isolate full-length cDNAs and genomic clones encoding 509RR and to isolate cDNA and genomic clones of other genes that have a high identity, particularly high sequence identity, to a 509RR gene Such probes generally will compnse at least 15 nucleotide residues or base pairs 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 pan's

A codmg region of a 509RR gene may be isolated by screenmg usmg a DNA sequence provided in Table 1 [SEQ ID NO 1] to synthesize an oligonucleotide probe A labeled oligonucleotide having a sequence complementary to that of a gene of the mvention is then used to screen a library of cDNA genomic DNA or mRNA to determine which members of the library the probe hybridizes to

There are several methods available and well known to those skilled in the art to obtain 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 ligated onto each end Nucleic acid amplification (PCR) is then carried out to amplify the "missing" 5' end of the DNA using a combmation of gene specific and adaptor specific oligonucleotide pnmers The PCR reaction is then repeated using "nested" pnmers, that is, primers designed to anneal within the amplified product (typically an adaptor specific pnmer that anneals further 3' m the adaptor sequence and a gene specific pnmer that anneals further 5' in the selected gene sequence) The products of this reaction can then be analyzed by DNA sequencing 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' primer

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

The polynucleotides of the invention that are ohgonucleotides derived from a sequence of Table 1 [SEQ ID NOS 1 or 2] may be used the processes herein as described, but preferably for PCR, to determine whether or not the polynucleotides identified herein in whole or in part are transcπbed 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 mfection the pathogen has attained

The mvention also provides polynucleotides that encode a polypeptide that is a mature protein plus additional ammo or carboxyl-terminal ammo acids, or am o acids mtenor to a mature polypeptide (when a mature form has more than one polypeptide chain, for instance) Such sequences may play a role m processmg of a protein from precursor to a mature form, may allow protein transport, may lengthen or shorten protem half-life or may facilitate manipulation of a protein for assay or production, among other things As generally is the case m vivo, the additional ammo acids may be processed away from a mature protein 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. having a mature form of the polypeptide fused to one or more prosequences may be an inactive form of the polypeptide When prosequences are removed such inactive precursors generally are activated Some or all of the prosequences may be removed before activation Generally, such precursors are called proprotems

As will be recognized, the entire polypeptide encoded by an open readmg frame is often not required for activity Accordmgly, it has become routine m molecular biology to map the boundanes of the primary structure required for activity with N-terminal and C-terminal deletion experiments These experiments utilize exonuclease digestion or convement restriction sites to cleave codmg nucleic acid sequence For example, Promega (Madison, WI) sell an Erase-a-base™ system that uses Exonuclease III 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 linkers) to the extent necessary to preserve an open readmg frame In this way, the nucleic acid of SEQ ID NO 1 readily provides contiguous fragments of SEQ ID NO 2 sufficient to provide an activity, such as an enzvmatic, binding or antibody-inducing activity Nucleic acid sequences encodmg such fragments of SEQ ID NO 2 and vanants thereof as descnbed herem are within the mvention, as are polypeptides so encoded

In sum, a polynucleotide of the mvention may encode a mature protem. a mature protein plus a leader sequence (which may be refened to as a preprotem), a precursor of a mature protein having one or more prosequences that are not the leader sequences of a preprotem, or a preproprotein, diat is a precursor to a proprotein, having 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 invention, host cells that are genetically engmeered 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 usmg RNAs denved from the DNA constructs of the invention

Recombinant polypeptides of the present mvention may be prepared by processes well known m those skilled in the art from genetically engmeered host cells compπsmg 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 engineered with such expression systems, and to the production of polypeptides of the invention by recombinant techniques

For recombinant production of the polypeptides of the invention, 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 manuals, such as Davis, et al , BASIC METHODS IN MOLECULAR BIOLOGY, (1986) and Sambrook, et al , MOLECULAR CLONING A LABORATORY MANUAL, 2nd Ed , Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N Y (1989), 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 bactenal cells, such as cells of streptococci, staphylococci, enterococci E coll, streptomyces, cyanobacteπa, Bacillus subtihs, and Staphylococciis aureus, fungal cells, such as cells of a yeast, Kluveromyces, Saccharomyces, a basidiomycete, Candida albicans and Aspergil/us, msect 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 angiosperm

A great vaπety of expression systems can be used to produce the polypeptides of the invention Such vectors mclude, among others, chromosomal-, episomal- and virus-denved vectors, for example vectors denved from bactenal 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, adenoviruses, fowl pox viruses, pseudorabies viruses, picornaviruses and retrovrruses, and vectors denved from combinations thereof, such as those derived 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 in a host may be used for expression in this regard The appropriate DNA sequence may be inserted mto the expression system by any of a vaπety of well-known and routme techniques, such as. for example, those set forth m Sambrook et al , MOLECULAR CLONING, A LABORATORY MANUAL, (supra)

In recombinant expression systems in eukaryotes, for secretion of a translated protein into the lumen of the endoplasmic reticulum, into the penplasmic space or into the extracellular environment, appropπate secretion signals may be incorporated into the expressed polypeptide These signals may be endogenous to the polypeptide or they may be heteroiogous signals Polypeptides of the invention can be recovered and purified from recombinant cell cultures by well-known methods including ammonium sulfate or ethanol precipitation, acid extraction, anion or cation exchange chromatography, phosphocellulose chromatography, hydrophobic interaction chromatography, affinity chromatography, hydroxylapatite chromatography, and lectin chromatography Most preferably, high performance liquid chromatography is employed for puπfication Well known techniques for refoldmg protein may be employed to regenerate active conformation when the polypeptide is denatured dunng isolation and or puπfication

Diagnostic, Prognostic, Serotyping and Mutation Assays

This invention is also related to the use of 509RR polynucleotides and polypeptides of the invention for use as diagnostic reagents Detection of 509RR polynucleotides and/or polypeptides in 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 orgamsm to drugs Eukaryotes, particularly mammals, and especially humans, particularly those infected or suspected to be infected with an organism comprising the 509RR gene or protem, may be detected at the nucleic acid or ammo acid level by a vaπety of well known techniques as well as by methods provided herem Polypeptides and polynucleotides for prognosis, diagnosis or other analysis may be obtained from a putatively infected and/or infected individual's bodily matenals Polynucleotides from any of these sources, particularly DNA or RNA, may be used directly for detection or may be amplified enzymatically by usmg PCR or any other amplification technique prior to analysis RNA, particularly mRNA, cDNA and genomic DNA may also be used m the same ways Usmg amplification, characterization of the species and stram of infectious or resident orgamsm present in an mdividual, may be made by an analysis of the genotype of a selected polynucleotide of the orgamsm Deletions and insertions can be detected by a change m size of the amplified product m companson to a genotype of a reference sequence selected from a related orgamsm, preferably a different species of the same genus or a different stram of the same species Pomt mutations can be identified by hybridizing amplified DNA to labeled 09RR 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 meltmg temperatures or renaturation kinetics Polynucleotide sequence differences may also be detected by alterations m the electrophoretic mobility of polynucleotide fragments in gels as compared to a reference sequence This may be earned 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 SI 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 anay of o gonucleotides probes compπsmg 5 9RR nucleotide sequence or fragments thereof can be constructed to conduct efficient screemng 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 vaπabihty (see, for example, Chee et al , Science, 274 610 (1996))

Thus m 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 1, 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 mvention, preferably to the polypeptide of SEQ ID NO 2 It will be appreciated that m any such kit, (a), (b), (c) or (d) may compnse a substantial component Such a kit will be of use in diagnosing a disease or susceptibility to a Disease, among others

This invention 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 ID NO 1, that is associated with 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 vaπety of techniques, such as those described elsewhere herem

The differences in a polynucleotide and/or polypeptide sequence between orgamsms possessing a first phenotype and organisms possessing 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 in any organisms possessing the second phenotype, then the mutation is likely to be the causative agent of the first phenotype

Cells from an orgamsm carrying mutations or polymorphisms (allehc vaπations) in a polynucleotide and/or polypeptide of the invention 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 in conjunction with automated detection systems, such as, for example, GeneScan RNA, cDNA or genomic DNA may also be used for the same purpose, PCR As an example, PCR pnmers complementary to a polynucleotide encoding 509RR polypeptide can be used to identify and analyze mutations The invention further provides these primers with 1, 2, 3 or 4 nucleotides removed from the 5' and/or the 3' end These pnmers may be used for, among other things, amplifying 509RR 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 vanous techniques 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 invention further provides a process for diagnosing, disease, preferably bactenal mfections, more preferably infections caused by Staphylococciis aureus, compπsmg determining from a sample derived from an individual, such as a bodily matenal, an increased level of expression of polynucleotide having a sequence of Table 1 [SEQ ID NO 1] Increased or decreased expression of a 509RR polynucleotide can be measured using any on of the methods well known in 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 invention for detecting over-expression of 509RR 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 509RR polypeptide, m a sample denved from a host, such as a bodily matenal, are well-known to those of skill in 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 invention may also be used to assess the bmdmg of small molecule substrates and ligands in, for example, cells, cell-free preparations, chemical hbraπes, and natural product mixtures These substrates and ligands may be natural substrates and ligands or may be structural or functional mimetics See, e g , Cohgan et al , Current Protocols m Immunology 1(2) Chapter 5 (1991)

Polypeptides and polynucleotides of the present mvention are responsible for many biological functions, including many disease states, in particular the Diseases herein mentioned It is therefore desirable to devise screenmg methods to identify compounds that agomze (e g , stimulate) or that antagonize (e g ,ιnhιbιt) the function of the polypeptide or polynucleotide Accordingly, in a further aspect, the present invention provides for a method of screening compounds to identify those that agonize or that antagonize the function of a polypeptide or polynucleotide of the invention, as well as related polypeptides and polynucleotides In general, agonists or antagonists (e g , inhibitors) mav be employed for therapeutic and prophylactic purposes for such Diseases as herein mentioned Compounds may be identified from a vanety of sources, for example, cells, cell-free preparations, chemical hbraπes, and natural product mixtures Such agonists and antagonists so-identified may be natural or modified substrates, ligands, receptors, enzymes, etc , as the case may be, of 509RR 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 bmdmg of a candidate compound to the polypeptide or polynucleotide, or to cells or membranes bearing the polypeptide or polynucleotide, or a fusion protein of the polypeptide by means of a label directly or indirectly associated with the candidate compound Alternatively, the screening method may involve competition with a labeled competitor Further, these screening methods may test whether the candidate compound results in a signal generated by activation or inhibition of the polypeptide or polynucleotide, usmg detection systems appropπate to the cells compns g the polypeptide or polynucleotide Inhibitors of activation are generally assayed in the presence of a known agonist and the effect on activation by the agonist bv the presence of the candidate compound is observed Constitutively active polypeptide and/or constitutively expressed polypeptides and polynucleotides may be employed in screening methods for inverse agonists, in the absence of an agonist or antagonist, by testing whether the candidate compound results m inhibition of activation of the polypeptide or polynucleotide, as the case may be Further, the screening methods may simply compnse the steps of mixing a candidate compound with a solution compnsmg a polypeptide or polynucleotide of the present invention, to form a mixture, measuring 509RR polypeptide and/or polynucleotide activity m the mixture, and comparing the

509RR polypeptide and/or polynucleotide activity of the mixture to a standard Fusion proteins, such as those made from Fc portion and 509RR polypeptide, as herein descnbed, can also be used for high- throughput screemng assays to identify antagonists of the polypeptide of the present invention, 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 interact with a polypeptide of the present invention may also be used to configure screening methods for detecting the effect of added compounds on the production of mRNA and/or polypeptide in cells For example, an ELISA assay may be constructed for measunng secreted or cell associated levels of polypeptide using monoclonal and polyclonal antibodies by standard methods known in the art This can be used to discover agents that may inhibit or enhance the production of polypeptide (also called antagonist or agonist, respectively) from suitably manipulated cells or tissues

The invention also provides a method of screenmg compounds to identify those that enhance (agonist) or block (antagonist) the action of 509RR polypeptides or polynucleotides, particularly those compounds that are bactenstatic and/or bactericidal The method of screening may involve 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, comprising 509RR polypeptide and a labeled substrate or hgand of such polypeptide is incubated in the absence or the presence of a candidate molecule that may be a 509RR agonist or antagonist The ability of the candidate molecule to agonize or antagomze the 509RR polypeptide is reflected in decreased binding of the labeled gand or decreased production of product from such substrate Molecules that bind gratuitously, e , without inducing the effects of 09RR polypeptide are most likely to be good antagonists Molecules that bmd well and, as the case may be, increase the rate of product production from substrate, increase signal transduction, or increase chemical channel activity are agonists 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 in this regard include but are not limited to colonmetnc, labeled substrate converted into product, a reporter gene that is responsive to changes m 509RR polynucleotide or polypeptide activity, and binding assays known m the art Polypeptides of the invention may be used to identify membrane bound or soluble receptors, if any, for such polypeptide, through standard receptor binding techmques known m the art These techniques include, but are not limited to, hgand binding and crosshnkmg assays 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 mateπals) Other methods include biophysical techniques such as surface plasmon resonance and spectroscopy These screenmg methods may also be used to identify agomsts and antagonists of the polypeptide that compete with the bmding of the polypeptide to its receptor(s), if any Standard methods for conducting such assays are well understood in the art The fluorescence polaπzation value for a fluorescently-tagged molecule depends on the rotational correlation time or tumbling rate Protein complexes, such as formed by 509RR polypeptide associating with another 509RR polypeptide or other polypeptide, labeled to comprise a fluorescently-labeled molecule will have higher polarization values than a fluorescently labeled monomeπc protein It is prefened that this method be used to charactenze small molecules that disrupt polypeptide complexes

Fluorescence energy transfer may also be used charactenze small molecules that interfere with the formation of 509RR polypeptide dimers, tr mers, tetramers or higher order structures, or structures formed by 509RR polypeptide bound to another polypeptide 509RR 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 dimeπzation will inhibit fluorescence energy transfer

Surface plasmon resonance can be used to momtor the effect of small molecules on 509RR polypeptide self-association as well as an association of 509RR polypeptide and another polypeptide or small molecule 09RR polypeptide can be coupled to a sensor chip at low site density such that covalently bound molecules will be monomenc Solution protein can then passed over the 5 9RR 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 charactenze the effect of small molecules on kinetic rates and equilibnum binding constants for 509RR polypeptide self-association as well as an association of 509RR polypeptide and another polypeptide or small molecule

A scintillation proximity assay may be used to characterize the interaction between an association of 509RR polypeptide with another 509RR polypeptide or a different polypeptide 509RR polypeptide can be coupled to a scintillation-filled bead Addition of radio-labeled 509RR polypeptide results in binding where the radioactive source molecule is m close proximity to the scintillation fluid Thus, signal is emitted upon 509RR polypeptide binding and compounds that prevent 509RR polypeptide self-association or an association of 509RR polypeptide and another polypeptide or small molecule will dimmish signal

In other embodiments of the invention there are provided methods for identifying compounds that bind to or otherwise interact 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 interaction between the compound and the polypeptide and/or polynucleotide to assess the bmdmg to or other interaction with the compound, such bmdmg or mteraction preferably being associated with a second component capable of providmg a detectable signal m response to the binding or interaction of the polypeptide and/or polynucleotide with the compound, and determining whether the compound binds 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 bindmg or interaction of the compound with the polypeptide and/or polynucleotide

Another example of an assay for 509RR agonists is a competitive assay that combines 509RR and a potential agonist with 509RR-bιndιng molecules, recombinant 509RR binding molecules, natural substrates or ligands, or substrate or hgand mimetics, under appropriate conditions for a competitive inhibition assay 509RR can be labeled, such as by radioactivity or a coloπmetπc compound, such that the number of 509RR 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 invention may also be used in a method for the structure-based design of an agonist or antagonist of the polypeptide and/or polynucleotide, by (a) determining in the first instance the three- dimensional structure of the polypeptide and/or polynucleotide, or complexes thereof, (b) deducing the three-dimensional structure for the likely reactive sιte(s), binding sιte(s) or motif(s) of an agonist or antagonist, (c) synthesizing candidate compounds that are predicted to bmd to or react with the deduced binding sιte(s), reactive sιte(s), and/or motιf(s), and

(d) testing whether the candidate compounds are indeed agonists or antagonists

It will be further appreciated that this will normally be an iterative process, and this iterative process may be performed using automated and computer-controlled steps

In a further aspect, the present invention provides methods of treating 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 509RR polypeptide and/or polynucleotide

If the expression and/or activity of the polypeptide and/or polynucleotide is in excess, several approaches are available One approach compnses administering 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 ligands, 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 binding the hgand, substrate, enzymes, receptors, etc m competition with endogenous polypeptide and/or polynucleotide may be admimstered Typical examples of such competitors include fragments of the 509RR polypeptide and/or polypeptide

In still another approach, expression of the gene encoding endogenous 509RR polypeptide can be inhibited using expression blocking techniques This blocking may be targeted agamst any step in gene expression, but is preferably targeted against transcnption and/or translation An examples of a known technique of this sort involve the use of antisense sequences, either internally generated or separately administered (see, for example, O'Connor, J Neurochem (1991) 56 560 in Ohgodeoxynucleotides as Antisense Inhibitors of Gene Expression, CRC Press, Boca Raton, FL (1988)) Alternatively, ohgonucleotides 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 ohgomers can be expressed in vivo

Each of the polynucleotide sequences provided herein may be used in the discovery and development of antibactenal compounds The encoded protein, upon expression, can be used as a target for the screening of antibactenal drags Additionally, the polynucleotide sequences encoding the amino terminal regions of the encoded protein or Shine-Delgarno or other translation facilitating sequences of the respective mRNA can be used to construct antisense sequences to control the expression of the coding sequence of interest The invention also provides the use of the polypeptide, polynucleotide, agonist or antagonist of the invention to interfere with the initial physical interaction between a pathogen or pathogens and a eukaryotic, preferably mammalian, host responsible for sequelae of infection In particular, the molecules of the invention may be used in the prevention of adhesion of bactena, in particular gram positive and/or gram negative bactena, to eukaryotic, preferably mammalian, extracellular matrix proteins on in-dwelling devices or to extracellular matπx proteins in wounds, to block bactenal adhesion between eukaryotic, preferably mammalian, extracellular matnx proteins and bacterial 509RR proteins that mediate tissue damage and/or, to block the normal progression of pathogenesis in infections initiated other than by the implantation of in-dwelling devices or by other surgical techniques In accordance with yet another aspect of the mvention, there are provided 509RR agonists and antagonists, preferably bactenstatic or bactencidal agonists and antagonists

The antagonists and agonists of the mvention may be employed, for instance, to prevent, inhibit and/or treat diseases

Helicobacter pylori (herein "H pylori") bactena infect the stomachs of over one-third of the world's population causing stomach cancer, ulcers, and gastntis (International Agency for Research on Cancer (1994) Schistosomes, Liver Flukes and Helicobacter Pylori (International Agency for Research on Cancer, Lyon, France, http //www uicc ch/ecp/ecp2904 htm) Moreover, the International Agency for Research on Cancer recently recognized a cause-and-effect relationship between H pylori and gastric adenocarcinoma, classifying the bacterium as a Group I (definite) carcmogen Preferred antimicrobial compounds of the invention (agonists and antagonists of 509RR polypeptides and/or polynucleotides) found using screens provided by the invention, or known in the art, particularly narrow-spectrum antibiotics, should be useful in the treatment of H pylori infection Such treatment should decrease the advent of H pylon-mduced cancers, such as gastrointestinal carcinoma Such treatment should also prevent, inhibit and/or cure gastric ulcers and gastntis

All publications and references, including but not limited to patents and patent applications, cited in this specification are herein incorporated by reference m their entirety as if each individual 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 pnoπty is also incorporated by reference herein in its entirety in the manner described above for publications and references

GLOSSARY

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

"Bodily mateπal(s) means any material derived from an individual or from an organism infecting, infesting or inhabiting an individual, including 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 bacteria, including , for example, disease, such as, infections of the upper respiratory tract (c g , otitis media, bactenal trachcitis. acute cpiglottitis. thyroiditis). lo er respiratory (c g , cmpycma, lung abscess), cardiac (c g , infective endocarditis), gastrointestinal (e g , secretory diarrhoea, splenic absces. retropeπtoneal abscess). CNS (e g . cerebral abscess), eye (c g , blcphaπtis. conjunctivitis, kcratitis. cndophthalmitis. prcseptal and orbital cellulitis, darcryocystitis), kidney and urinary⁷ tract (e g , epididvmitis, intrarenal and perinephric absces, toxic shock syndrome), skin (c g , nnpetigo, folhculitis. cutaneous abscesses, cellulitis ound infection, bacterial myositis) bone and joint (e g , septic arthntis, osteom elitis)

"Host cell(s)" is a cell that has been introduced (e g , transformed or transfected) or is capable of introduction (e g , transformation or transfection) by 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 determined 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, including but not limited to those described in (Computational Molecular Biology, Lesk, A M , ed , Oxford University Press, New York, 1988 Bwcomputing Informatics and Genome Projects, Smith, D W , ed , Academic Press, New York 1993. Computer Analysis of Sequence Data, Part I, Griffin. A M , and Griffin, H G . eds , Humana Press, New Jersey, 1994, Sequence Analysis in Molecular Biology, von Heinje, G , Academic Press, 1987. and Sequence Analysis Primer, Gnbskov, M and Devereux, J , eds , M Stockton Press, New York, 1991, and Caπllo, H , and Lipman, D , SIAM J Applied Math 48 1073 (1988) Methods to determine identity are designed to give the largest match between the sequences tested Moreover methods to determine identity are codified in publicly available computer programs Computer program methods to determme identity between two sequences include, 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 Biol 215 403-410 (1990) The BLAST X program is publicly available from NCBI and other sources (BLAST Manual, Altschul, S , et a/ , NCBI NLM NIH Bethesda, MD 20894, Altschul, S , et al , J Mol Biol 215 403-410 (1990) The well known Smith Waterman algoπthm may also be used to determine identity Parameters for polypeptide sequence comparison include the following Algorithm

Needleman and Wunsch, J Mol Biol 48 443-453 (1970)

Comparison 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 publicly available as the "gap" program from Genetics Computer Group, Madison WI The aforementioned parameters are the default parameters for peptide comparisons (along with no penalty for end gaps)

Parameters for polynucleotide comparison include the following 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 preferred meaning for "identity" for polynucleotides and polypeptides. as the case may be. are provided in (1) and (2) below

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

ⁿn ≤ ^xn " (^xn ^# y),

wherein 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 wherein any non-integer product of x_n and y is rounded down to the nearest integer prior to subtractmg it from x_n Alterations of a polynucleotide sequence encoding the polypeptide of SEQ

ID NO 2 may create nonsense, missense or frameshift mutations in this coding sequence and thereby alter the polypeptide encoded by the polynucleotide following such alterations

(2) Polypeptide embodiments further include an isolated polypeptide comprising a polypeptide having at least a 95, 97 or 100% identity to a polypeptide reference sequence of SEQ ID NO 2, wherein said polypeptide sequence may be identical to the reference sequence of SEQ ID NO 2 or may include up to a certam mteger number of amino acid alterations as compared to the reference sequence, wherem said alterations are selected from the group consisting of at least one ammo acid deletion, substitution, including conservative and non-conservative substitution, or insertion, and wherein said alterations may occur at the amino- or carboxy-terminal positions of the reference polypeptide sequence or anywhere between those terminal positions, interspersed either individually among the amino acids in the reference sequence or in one or more contiguous groups within the reference sequence, and wherein said number of ammo acid alterations is determined by multiplying the total number of amino acids in SEQ ID NO 2 by the integer defining the percent identity divided by 100 and then subtractmg that product from said total number of amino acids m SEQ ID NO 2, or ⁿa ≤ ^xa " (^xa • ϊ

wherein n_a is the number of amino acid alterations, x_a is the total number of amino acids in 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 wherein any non-integer product of x_a and y is rounded down to the nearest integer prior to subtracting it from x_a.

"Indιvidual(s)" means a multicellular eukaryote, including, 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, .e., if it occurs in nature, it lias been changed or removed from its original environment, or both. For example, a polynucleotide or a polypeptide naturally present in a livmg orgamsm is not "isolated," but the same polynucleotide or polypeptide separated from the coexisting materials of its natural state is "isolated", as the term is employed herem. Moreover, a polynucleotide or polypeptide that is introduced into an organism by transformation, genetic manipulation or by any other recombinant method is "isolated" even if it is still present m said organism, which organism may be living or non-living.

"Organism(s)" means a (i) prokaryote, including but not limited to, a member of the genus Streptococcus, Slaphylococcus, Bordetella, Corynebacterium, Mycobactenum, Neissena, Haemophύus, Actinomycetes, Streptomycetes, Nocardia, Enterobacter, Yersinia, Fancisella, Pasturella, Moraxella, Acinetobacter, Erysipelothnx, Branhamella, Actinobacillus, Streptobacillus, Listena, Calymmatobactenum, Brucella, Bacillus, Clostndium, Treponema, Eschenchia, Salmonella, Kleibsiella, Vibrio, Proteus, Erwinia, Borreha, Leptospira, Spirillum, Campylobacter, Shigella, Legionella, Pseudomonas, Aeromonas, Rickettsia, Chlamydia, Borreha 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 pneumomae, Streptococcus pyogenes, Streptococcus agalactiae, Streptococcus faecalis, Streptococcus faecium, Streptococcus durans, Neissena gonorrheae, Neissena meningitidis, Staphylococcus aureus, Staphylococcus epidermidis, Corynebactenum dipthenae, Gardnerella vaginalis, Mycobactenum tuberculosis, Mycobactenum bovis, Mycobactenum ulcerans, Mycobactenum leprae, Actmomyctes israelii, Listena monocytogenes, Bordetella pertusis, Bordatella parapertusis, Bordetella bronchiseptica, Eschenchia coll, Shigella dysentenae, Haemophilus influenzae, Haemophilus aegyptius, Haemophilus parainfluenzae, Haemophilus ducreyi, Bordetella, Salmonella typhi, Citrobacter freundu, Proteus mirabilis, Proteus vulgans, Yersinia pestis, Kleibsiella pneumomae, Serratia marcessens, Serratia iiquejaciens, Vibno cholera, Shigella dysentern, Shigella flexnen, Pseudomonas aeruginosa, Franscisella tularensis, Bruce/la aborhs, Bacillus anthracis, Bacillus cereus, Clostndium perfrmgens Clostndium tetani, Clostndium botuhnum, Treponema pallidum, Rickettsia nckettsn and Chlamydia trachomitis, (n) an archaeon. including but not limited to Archaebacter, and (in) 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

"Polynucleotιde(s)" generally refers to any polyπbonucleotide or polydeoxyπbonucleotide. that may be unmodified RNA or DNA or modified RNA or DNA "Polynucleotιde(s)" include, without limitation, smgle- and double-stranded DNA, DNA that is a mixture of single- and double-stranded regions or single-, double- and triple-stranded regions, smgle- and double-stranded RNA, and RNA that is mixture of single- and double-stranded regions, hybrid molecules comprising DNA and RNA that may be single-stranded or, more typically, double-stranded, or tπple-stranded regions, or a mixture of single- and double-stranded regions In addition, "polynucleotide" as used herein refers to triple-stranded regions compnsmg 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 mclude 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 tπple-hehcal region often is an oligonucleotide As used herem, the term "polynucleotιde(s)" also includes DNAs or RNAs as descnbed above that comprise one or more modified bases Thus, DNAs or RNAs with backbones modified for stability or for other reasons are "polynucleotιde(s)" as that term is intended herem Moreover, DNAs or RNAs comprising unusual bases, such as mosme, 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 vanety of modifications have been made to DNA and RNA that serve many useful purposes known to those of skill in the art The term "polynucleotιde(s)" as it is employed herein embraces such chemically, enzymatically or metabohcally modified forms of polynucleotides. as well as the chemical forms of DNA and RNA charactenstic of viruses and cells, including, for example, simple and complex cells "Polynucleotιde(s)" also embraces short polynucleotides often referred to as ohgonucleotιde(s)

"Polypeptιde(s)" refers to any peptide or protem compnsmg two or more ammo acids jomed to each other by peptide bonds or modified peptide bonds "Polypeptιde(s)" refers to both short chains, commonly refened to as peptides, oligopeptides and ohgomers and to longer chains generally referred to as protems Polypeptides may comprise ammo acids other than the 20 gene encoded amino acids "Polypeptιde(s)" include those modified either by natural processes, such as processmg and other post- translational modifications, but also by chemical modification techniques Such modifications are well descnbed m basic texts and m more detailed monographs, as well as m a voluminous research literature, and they are well known to those of skill m 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 in a polypeptide, mcludmg the peptide backbone, the amino acid side-chains, and the ammo or carboxvl termini Modifications include, for example, acetylation, acylation. ADP-πbosylation. amidation. covalent attachment of flavin, covalent attachment of a heme moiety, covalent attachment of a nucleotide or nucleotide denvative, covalent attachment of a hpid or pid derivative, covalent attachment of phosphotidylinositol, cross-linking, cyclization, disulfide bond formation, demethylation, formation of covalent cross-links, formation of cysteine, formation of pyroglutamate, formylation, gamma- carboxylation, GPI anchor formation, hydroxylation, lodination, methylation, myπstoylation. oxidation proteolytic processing, phosphorylation, prenylation, racemization, glycosylation, hpid attachment, sulfation, gamma-carboxylation of glutamic acid residues, hydroxylation and ADP-πbosylation, 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 in POSTTRANSLATIONAL COVALENT MODIFICATION OF PROTEINS, B C Johnson, Ed , Academic Press, New York (1983), Seifter et al , Meth Enzymol 182 Glβ-G^β (1990) and Rattan et al , Protein Synthesis Posttranslational Modifications and Aging, Aim 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 "Recombinant expression system(s)" refers to expression systems or portions thereof or polynucleotides of the invention introduced or transformed mto a host cell or host cell lysate for the production of the polynucleotides and polypeptides of the invention

"Vaπant(s)" as the term is used herein, is a polynucleotide or polypeptide that differs from a reference polynucleotide or polypeptide respectively, but retains essential properties A typical variant of a polynucleotide differs in nucleotide sequence from another, reference polynucleotide

Changes m 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 may result in amino acid substitutions, additions, deletions, fusion proteins 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 vanant are closely similar overall and, m many regions, identical

A variant and reference polypeptide may differ in amino acid sequence by one or more substitutions, additions, deletions m any combination A substituted or inserted amino acid residue may or may not be one encoded by the genetic code The present invention also mcludes include 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 charactenstics Typical 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 variants in 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 naturallv occurring such as an alle c variant, or it may be a vanant that is not known to occur naturally Non- naturally occurrmg vanants 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 carried out using standard techniques, that are well known and routine to tliose of skill in the art, except where otherwise described in detail The examples are illustrative, but do not limit the mvention Example 1 Strain selection, Library Production and Sequencing

The polynucleotide having a DNA sequence given in Table 1 [SEQ ID NO 1] was obtained from a library of clones of chromosomal DNA of Staphylococcus aureus in E coll The sequencing data from two or more clones comprising overlapping Staphylococcus aureus DNAs was used to construct the contiguous DNA sequence in SEQ ID NO 1 Libraries may be prepared by routine methods, for example Methods 1 and 2 below

Total cellular DNA is isolated from Staphylococcus aureus WCUH 29 according to standard procedures and size-fractionated by either of two methods Method 1

Total cellular DNA is mechanically sheared by passage through a needle in order to size- fractionate according to standard procedures DNA fragments of up to 1 lkbp in size are rendered blunt by treatment with exonuclease and DNA polymerase, and EcoRI linkers added Fragments are hgated into the vector Lambda ZapII that has been cut with EcoRI, the library packaged by standard procedures and E coli infected with the packaged library The library is amplified by standard procedures

Method 2

Total cellular DNA is partially hydrolyzed with a one or a combination of restriction enzymes appropriate to generate a senes of fragments for clomng into library vectors (e g , Rsal, Pall, Alul, Bshl235I), and such fragments are size-fractionated according to standard procedures EcoRI linkers are gated to the DNA and the fragments then hgated 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 procedures

Claims

What is claimed is:

1. An isolated polypeptide selected from the group consisting of:

(i) an isolated polypeptide comprising an amino acid having at least 95% identity

to the amino acid sequence of SEQ ID NO:2 over the entire length of SEQ ID NO:2;

(ii) an isolated polypeptide comprising the amino acid sequence of SEQ ID NO:2,

(iii) an isolated polypeptide that is the amino acid sequence of SEQ ID NO:2, and

(iv) a polypeptide that is encoded by a recombinant polynucleotide comprising the polyncleotide sequence of SEQ ID NO: l .

2. An isolated polynucleotide selected from the group consisting of:

(i) an isolated polynucleotide comprising a polynucleotide sequence encoding a polypeptide that has at least 95% identity to the amino acid sequence of SEQ ID NO:2, over the entire length of SEQ ID NO:2;

(ii) 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 ID

NO:2;

(iii) an isolated polynucleotide comprising 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 ID NO:2;

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

(vi) an isolated polynucleotide of at least 30 nucleotides in length obtainable by screening an appropriate library under stringent hybridization conditions with a probe having the sequence of

SEQ ID NO: 1 or a fragment thereof of of at least 30 nucleotides in length;

(vii) an isolated polynucleotide encoding a mature polypeptide expressed by the 509RR gene comprised in the Staphylococcus aureus: and

(viii) a polynucleotide sequence complementary to said isolated polynucleotide of (i), (ii), (iii), (iv), (v), (vi) or (vii). 3 A method for the treatment of an individual

(1) in need of enhanced activity or expression of or lmmunological response to the polypeptide of claim 1 comprising the step of administenng to the individual a therapeutically effective amount of an antagonist to said polypeptide, or

(n) having need to inhibit activity or expression of the polypeptide of claim 1 comprising

(a) administenng to the individual a therapeutically effective amount of an antagonist to said polypeptide, or

(b) administenng to the individual a nucleic acid molecule that inhibits the expression of a polynucleotide sequence encoding said polypeptide.

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

(d) administenng to the individual an amount of a polypeptide that induces an lmmunological response to said polypeptide in said individual

4 A process for diagnosing or prognosing a disease or a susceptibility to a disease in an individual related to expression or activity of the polypeptide of claim 1 in an individual comprising the step of

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

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

5 A process for producing a polypeptide selected from the group consisting of (1) an isolated polypeptide comprising an amino acid sequence selected from the group having at least 95% identity

to the amino acid sequence of SEQ ID NO 2 over the entire length of SEQ ID NO 2,

(n) an isolated polypeptide compnsmg the amino 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 comprising the polynucleotide sequence of SEQ ID NO 1,

comprising the step of cultuπng a host cell under conditions sufficient for the production of the polypeptide

6 A process for producing a host cell compnsmg an expression system or a membrane thereof expressing a polypeptide selected from the group consisting of

(I) an isolated polypeptide comprising an ammo acid sequence selected from the group having at least 95% identity

to the amino acid sequence of SEQ ID NO 2 over the entire length of SEQ ID NO 2,

(π) an isolated polypeptide compnsmg the amino 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 comprising 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 producing said polypeptide of (I), (u), (in) or (iv) when said expression system is present in a compatible host cell such the host cell, under appropriate culture conditions, produces said polypeptide of (l), (n), (in) or (iv)

7 A host cell or a membrane expressing a polypeptide selected from the group consisting of (1) an isolated polypeptide compnsmg an ammo acid sequence selected from the group having at least 95% identity to the amino acid sequence of SEQ ID NO 2 over the entire length of SEQ ID N0 2,

(u) an isolated polypeptide comprising the amino acid sequence of SEQ ID NO 2,

(in) an isolated polypeptide that is the amino acid sequence of SEQ ID NO 2, and

(iv) a polypeptide that is encoded by a recombinant polynucleotide comprising the polynucleotide sequence of SEQ ID NO 1

8 An antibody lmmunospecific for the polypeptide of claim 1

9 A method for screemng to identify compounds that agomze or that inhibit the function of the polypeptide of claim 1 that comprises 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 bearing the polypeptide) or a fusion protein thereof by means of a label directly or indirectly associated with the candidate compound,

(b) measuring the binding of a candidate compound to the polypeptide (or to the cells or membranes beanng the polypeptide) or a fusion protein thereof in the presence of a labeled competitor,

(c) testing whether the candidate compound results in a signal generated by activation or inhibition of the polypeptide, using detection systems appropnate to the cells or cell membranes bearing the polypeptide,

(d) mixing a candidate compound with a solution comprising a polypeptide of claim 1, to form a mixture, measuring activity of the polypeptide m the mixture, and comparmg the activity of the mixture to a standard, or

(e) detecting the effect of a candidate compound on the production of mRNA encoding said polypeptide and said polypeptide in cells, using for instance, an ELISA assay

10 An agomst or antagonist to the polypeptide of claim 1