WO2000044764A1 - Mvd - Google Patents

Abstract

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

Description

mvd

FIELD 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 tlieir uses. In particular, the invention relates to polynucleotides and polypeptides of the mvd (mevalonate diphosphate decarboxylase) family, as well as their variants, herein referred to as "mvd," "mvd polynucleotide(s)," and "mvd polypeptide(s)" as the case may be.

BACKGROUND OF THE INVENTION

The Streptococci make up a medically important genera of microbes known to cause several types of disease in humans, including, for example, otitis media, conjunctivitis, pneumonia, bacteremia, meningitis, sinusitis, pleural empyema and endocarditis, and most particularly meningitis, such as for example infection of cerebrospinal fluid. Since its isolation more than 100 years ago, Streptococcus pneumoniae has been one of the more intensively studied microbes. For example, much of our early understanding that DNA is, in fact, the genetic material was predicated on the work of Griffith and of Avery, Macleod and McCarty using this microbe. Despite the vast amount of research with S. pneumoniae, many questions concerning the vinilence of this microbe remain. It is particularly preferred to employ Streptococcal genes and gene products as targets for the development of antibiotics. The frequency of Streptococcus pneumoniae 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 Streptococcus pneumoniae 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 tlieir related polypeptides, as targets for drag discovery.

Clearly, there exists a need for polynucleotides and polypeptides, such as the mvd 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 mvd, in particular mvd polypeptides and mvd polynucleotides, recombinant matenals 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 mvd expression or activity Nanous changes and modifications within the spint and scope of the disclosed invention will become readily apparent to those skilled in the art from reading the following descnptions and from reading the other parts ofthe present disclosure

DESCRIPTION OF THE INVENTION The invention relates to mvd polypeptides and polynucleotides as descnbed in greater detail below

In particular, the invention relates to polypeptides and polynucleotides of a mvd of Streptococcus pneumoniae, which is related by amino acid sequence homology to Borreha burgdorfen mvd polypeptide The invention relates especially to mvd having a nucleotide and ammo acid sequences set out in 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 mvd Polynucleotide and Polypeptide Sequences

(A) Streptococcus pneumoniae mvd polynucleotide sequence [SEQ ID NO 1]

5 ' -ATGGATAGAGAGCCTGTAACAGTACGTTCCTACGCAAATATTGCTATTAT CAAATATTGGGGAAAGAAAAAAGAAAAAGAGATGGTGCCTGCTACTAGCA GTATTTCTCTAACTTTGGAAAATATGTATACAGAGACGACCTTGTCGCCT TTACCAGCCAATGTAACAGCTGACGAATTTTACATCAATGGTCAGCTACA

AAATGAGGTCGAGCATGCCAAGATGAGTAAGATTATTGACCGTTATCGTC

CAGCTGGTGAGGGCTTTGTCCGTATCGATACTCAAAACAATATGCCTACG

GCAGCGGGCCTGTCCTCAAGTTCTAGTGGTTTGTCCGCCCTGGTCAAGGC TTGTAATGCTTATTTCAAGCTTGGATTGGATAGAAGTCAGTTGGCACAGG

AAGCCAAATTTGCCTCAGGCTCTTCTTCTCGGAGTTTTTATGGACCACTA

GGAGCCTGGGATAAGGATAGTGGAGAAATTTACCCTGTAGAGACAGACTT

GAAACTAGCTATGATTATGTTGGTGCTAGAGGACAAGAAAAAACCAATCT

CTAGCCGTGACGGGATGAAACTTTGTGTGGAAACCTCGACGACTTTTGAC GACTGGGTTCGTCAGTCTGAGAAGGACTATCAGGATATGCTGATTTATCT

CAAGGAAAATGATTTTGCCAAGATTGGAGAATTAACGGAGAAAAATGCTC

TGGCTATGCATGCTACGACAAAGACTGCTAGTCCAGCCTTTTCTTATCTG

ACGGATGCCTCTTATGAGGCTATGGCCTTTGTTCGCCAGCTTCGTGAGAA

AGGAGAGGCCTGCTACTTTACCATGGATGCTGGTCCCAATGTTAAGGTCT TCTGTCAGGAGAAAGACTTGGAGCATTTGTCAGAAATTTTCGGTCAGCGT

TATCGCTTGATTGTGTCAAAAACAAAGGATTTGAGTCAAGATGATTGCTGTTAA

-3'

(B) Streptococcus pneumoniae mvd polypeptide sequence deduced from a polynucleotide sequence in this table [SEQ ID NO:2] .

NH₂-MDREPVTVRSYANIAI I KYWGKKKEKEMVPATSS I SLTLEN YTETTLSP

LPi VTADEFYINGQLQNEVEHAKMSKIIDRYRPAGEGFVRIDTQNNMPT AAGLSSSSSGLSALVKACNAYFKLGLDRSQLAQEAKFASGSSSRSFYGPL GA DKDSGEIYPVETDLKLAMIMLVLEDKKKPISSRDGMKLCVETSTTFD D VRQSEKDYQDMLIYLKENDFAKIGE TEKNA AMHATTKTASPAFSYL TDASYEAMAFVRQLREKGEACYFTMDAGPNVKVFCQEKDLEHLSEIFGQR YRLIVSKTKDLSQDDCC* -COOH

Deposited materials

A deposit comprising a Streptococcus pneumoniae 0100993 strain has been deposited with the National Collections of Industrial and Marine Bacteria Ltd. (herein "NCIMB"), 23 St. Machar Drive, Aberdeen AB2 IRY, Scotland on 11 April 1996 and assigned deposit number 40794. The deposit was described as Streptococcus pneumoniae 0100993 on deposit. On 17 April 1996 a Streptococcus pneumoniae 0100993 DNA library in E. coli was similarly deposited with the NCIMB and assigned deposit number 40800. The Streptococcus pneumoniae strain deposit is referred to herein as "the deposited strain" or as "the DNA of the deposited strain." The deposited strain comprises a full length mvd gene. The sequence of the polynucleotides comprised 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 any description of sequences herein.

The deposit ofthe 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 merely as convenience to those of skill in the art and is not an admission that a deposit is required for enablement, such as that required under 35 U.S.C. §112. A license may be required to make, use or sell the deposited strain, and compounds derived therefrom, and no such license is hereby granted.

In one aspect ofthe invention there is provided an isolated nucleic acid molecule encoding a mature polypeptide expressible by the Streptococcus pneumoniae 0100993 strain, which polypeptide is comprised in the deposited strain. Further provided by the invention are mvd polynucleotide sequences in the deposited strain, such as DNA and RNA, and amino acid sequences encoded thereby. Also provided by the invention are mvd polypeptide and polynucleotide sequences isolated from the deposited strain.

Polypeptides mvd polypeptide of the invention is substantially phylogenetically related to other proteins of the mvd (mevalonate diphosphate decarboxylase) family.

In one aspect of the invention there are provided polypeptides of Streptococcus pneumoniae referred to herein as "mvd" and "mvd polypeptides" as well as biologically, diagnostically, prophylactically, clinically or therapeutically useful variants thereof, and compositions comprising the same.

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

The present invention further provides for an isolated polypeptide which: (a) comprises or consists of an amino acid sequence which 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 which has at least 95% identity, even more preferably at least 97-99% or exact identity to SEQ ID NO:l 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 which 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 ofthe invention include a polypeptide of Table 1 [SEQ ID NO:2] (in particular a mature polypeptide) as well as polypeptides and fragments, particularly those which have a biological activity of mvd, and also those which 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 ofthe polypeptide generally compnsing at least 30 ammo acids and more preferably at least 50 ammo acids

The invention also includes a polypeptide consisting of or compnsmg a polypeptide ofthe formula X-(R,)_m-(R₂)-(R₃)_n-Y wherein, at the amino 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 herem for modified polypeptides, Ri and R3 are any ammo 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 R is an amino acid sequence of the invention, particularly an ammo 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 Ri and its carboxy terminal am o acid residue is at the ngl t, covalently bound to R3 Any stretch of ammo acid residues denoted by 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 ofthe mvention is denved from Streptococcus pneumoniae, however, it may preferably be obtained from other organisms ofthe same taxonomic genus A polypeptide ofthe mvention may also be obtained, for example, from organisms ofthe same taxonomic family or order A fragment is a variant 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 mvd polypeptides, fragments may be "free-standing," or compnsed within a larger polypeptide of which they form a part or region, most preferably as a smgle contmuous region m a single larger polypeptide

Preferred fragments mclude, for example, truncation polypeptides havmg a portion of an amino acid sequence of Table 1 [SEQ ID NO 2], or of vanants thereof, such as a contmuous senes of residues that includes an ammo- and/or carboxyl-terminal ammo acid sequence Degradation forms of the polypeptides ofthe mvention produced by or m a host cell, particularly a Streptococcus pneumoniae, are also preferred Further preferred are fragments characterized by structural or functional attributes such as fragments that compnse alpha-helix and alpha-helix forming regions, beta-sheet and beta-sheet-forming regions, turn and turn-forming regions, coil and coil-forming regions, hydrophilic regions, hydrophobic regions, alpha amphipathic regions, beta amphipathic regions, flexible regions, surface-forming regions, substrate binding region, and high antigenic index regions

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 ammo acid sequence of SEQ ID NO 2, or an isolated polypeptide compπsmg 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 ofthe polypeptides ofthe mvention may be employed for producmg the corresponding full-length polypeptide by peptide synthesis, therefore, these vanants may be employed as mtermediates for producmg the full-length polypeptides ofthe mvention Polynucleotides

It is an object ofthe mvention to provide polynucleotides that encode mvd polypeptides, particularly polynucleotides that encode a polypeptide herein designated mvd In a particularly preferred embodiment of the mvention the polynucleotide compnses a region encoding mvd polypeptides compnsmg a sequence set out in Table 1 [SEQ ID NO 1] which mcludes 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 Streptococcus pneumoniae

As a further aspect ofthe mvention there are provided isolated nucleic acid molecules encoding and/or expressmg mvd polypeptides and polynucleotides, particularly Streptococcus pneumoniae mvd polypeptides and polynucleotides, including, for example, unprocessed RNAs, nbozyme RNAs, mRNAs, cDNAs, genomic DNAs, B- and Z-DNAs Further embodiments of the mvention mclude biologically, diagnostically, prophylactically, clinically or therapeutically useful polynucleotides and polypeptides, and vanants thereof, and compositions compnsmg the same Another aspect of the mvention relates to isolated polynucleotides, including at least one fLill length gene, that encodes a mvd 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 ofthe invention there is a mvd polypeptide from Streptococcus pneumoniae comprising or consisting of an ammo 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 m Table 1 [SEQ ID NO 1], a polynucleotide of the mvention encoding mvd polypeptide may be obtained usmg standard cloning and screening methods, such as those for cloning and sequencmg chromosomal DNA fragments from bactena using Streptococcus pneumoniae 0100993 cells as starting matenal, followed by obtaining a full length clone For example, to obtain a polynucleotide sequence of the mvention, such as a polynucleotide sequence given in Table 1 [SEQ ID NO 1], typically a library of clones of chromosomal DNA of Streptococcus pneumoniae 0100993 in E coli or some other suitable host is probed with a radiolabeled o gonucleotide, 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 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, using denatured double stranded DNA prepared from a plasmid clone. Suitable techniques are described by Maniatis, T., Fritsch, E.F. and Sambrook et al., MOLECULAR CLONING, A LABORATORY MANUAL, 2nd Ed.; Cold Spring Harbor Laboratory Press, Cold Spring 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:l] was discovered in a DNA library derived from Streptococcus pneumoniae 0100993.

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 amino acid residues set forth in Table 1 [SEQ ID NO:2] with a deduced molecular weight that can be calculated using amino acid residue molecular weight values well known to those skilled in the art. The polynucleotide of SEQ ID NO:l, between nucleotide number 1 and the stop codon which begins at nucleotide number 952 of SEQ ID NO:l, encodes the polypeptide of SEQ ID NO:2.

In a further aspect, the present invention provides for an isolated polynucleotide comprising or consisting of: (a) a polynucleotide sequence which has at least 95% identity, even more preferably at least 97-99% or exact identity to SEQ ID NO:l over the entire length of SEQ ID NO: l, or the entire length of that portion of SEQ ID NO: l which encodes SEQ ID NO:2; (b) a polynucleotide sequence encoding a polypeptide which has at least 95% identity, even more preferably at least 97-99% or 100% exact, to the amino acid sequence of SEQ ID NO:2, over the entire length of SEQ ID NO:2.

A polynucleotide encoding a polypeptide ofthe present invention, including homologs and orthologs from species other than Streptococcus pneumoniae, may be obtained by a process which comprises the steps of screening an appropriate library under stringent hybridization conditions with a labeled or detectable probe consisting of or comprising the sequence of SEQ ID NO:l or a fragment thereof; and isolating a full- length gene and/or genomic clones comprising said polynucleotide sequence.

The invention provides a polynucleotide sequence identical over its entire length to a coding sequence (open reading frame) in Table 1 [SEQ ID NO:l]. 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 in reading frame with another coding sequence, such as a sequence encoding a leader or secretory sequence, a pre-, or pro- or prepro-protein sequence. The polynucleotide ofthe invention may also comprise 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 transcribed but non-translated sequences, termination signals (such as rho-dependent and rho-independent termination signals), ribosome binding sites, Kozak sequences, sequences that stabilize mRNA, introns, and polyadenylation signals. The polynucleotide sequence may also comprise additional coding sequence encoding additional amino acids. For example, a marker sequence that facilitates purification of a fused polypeptide can be encoded. In certain embodiments ofthe invention, the marker sequence is a hexa-histidine peptide, as provided in the pQE vector (Qiagen, Inc.) and described in Gentz et al, Proc. Natl. Acad. Sci., USA 86: 821-824 (1989), or an HA peptide tag (Wilson et al, Cell 37: 767 (1984), both of which may be useful in purifying polypeptide sequence fused to them. Polynucleotides ofthe invention also include, but are not limited to, polynucleotides comprising a structural gene and its naturally associated sequences that control gene expression.

A prefened embodiment of the invention is a polynucleotide of consisting of or comprising nucleotide 1 to the nucleotide immediately upstream of or including nucleotide 952 set forth in SEQ ID NO: 1 of Table 1, both of which encode a mvd polypeptide.

The invention also includes a polynucleotide consisting of or comprising a polynucleotide of the formula:

X-(Rι )_m-(R₂)-(R₃)_n-Y wherein, at the 5' end ofthe molecule, X is hydrogen, a metal or a modified nucleotide residue, or together with Y defines a covalent bond, and at the 3' end ofthe 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 independently any nucleic acid residue or modified nucleic acid residue, m is an integer between 1 and 3000 or zero , n is an integer between 1 and 3000 or zero, and R₂ is a nucleic acid sequence or modified nucleic acid sequence ofthe invention, particularly a nucleic acid sequence selected from Table 1 or a modified nucleic acid sequence thereof. In the polynucleotide formula above, R₂ is oriented so that its 5' end nucleic acid residue is at the left, bound to Ri and its 3' end nucleic acid residue is at the right, bound to R3. Any stretch of nucleic acid residues denoted by either Ri and/or R , where m and/or n is greater than 1, may be either a heteropolymer or a homopolymer, preferably a heteropolymer. Where, in a preferred embodiment, X and Y together define a covalent bond, the polynucleotide ofthe above formula is a closed, circular polynucleotide, which 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 ofthe 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 polynucleotide of the invention is derived from Streptococcus pneumoniae, however, it may preferably be obtained from other organisms ofthe same taxonomic genus. A polynucleotide ofthe invention may also be obtained, for example, from organisms ofthe same taxonomic family or order.

The term "polynucleotide encoding a polypeptide" as used herein encompasses polynucleotides that include a sequence encoding a polypeptide of the invention, particularly a bacterial polypeptide and more particularly a polypeptide ofthe Streptococcus pneumoniae mvd having an amino acid sequence set out in Table 1 [SEQ ID NO:2]. The term also encompasses polynucleotides that include a single continuous region or discontinuous regions encoding the polypeptide (for example, 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 comprise coding and/or non-coding sequences.

The invention further relates to variants ofthe polynucleotides described herein that encode variants of a polypeptide having a deduced amino acid sequence of Table 1 [SEQ ID NO:2]. Fragments of polynucleotides ofthe invention may be used, for example, to synthesize full-length polynucleotides of the invention. Further particularly prefened embodiments are polynucleotides encoding mvd variants, that have the amino acid sequence of mvd 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 preferred among these are silent substitutions, additions and deletions, that do not alter the properties and activities of mvd polypeptide. Further preferred embodiments of the invention are polynucleotides that are at least 95% or 97% identical over tlieir entire length to a polynucleotide encoding mvd polypeptide having an amino acid sequence set out in Table 1 [SEQ ID NO:2], and polynucleotides that are complementary to such polynucleotides. Most highly preferred are polynucleotides that comprise a region that is at least 95% are especially preferred. 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 preferred.

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 invention there are provided polynucleotides that hybridize, particularly under stringent conditions, to mvd polynucleotide sequences, such as those polynucleotides in Table 1.

The invention further relates to polynucleotides that hybridize to the polynucleotide sequences provided herein. In this regard, the invention especially relates to polynucleotides that hybridize under stringent conditions to the polynucleotides described herein. As herein used, the terms "stringent conditions" and "stringent hybndization conditions" mean hybndization occurring only if there is at least 95% and preferably at least 97% identity between the sequences A specific example of stringent hybridization conditions is overnight incubation at 42°C in a solution comprising 50% formamide, 5x SSC (150mM NaCl, 15mM tnsodium citrate), 50 inM 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 hybridization support m 0 lx SSC at about 65°C Hybndization and wash conditions are well known and exemplified m Sambrook, et al , Molecular Cloning A Laboratory Manual, Second Edition, Cold Spnng 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 stringent hybridization conditions with a probe having the sequence of said polynucleotide sequence set forth in SEQ ID NO 1 or a fragment thereof, and isolating said polynucleotide sequence Fragments useful for obtaining such a polynucleotide include, for example, probes and primers fully described elsewhere herein

As discussed elsewhere herem regarding polynucleotide assays of the mvention, for instance, the polynucleotides ofthe mvention, may be used as a hybndization probe for RNA, cDNA and genomic DNA to isolate full-length cDNAs and genomic clones encoding mvd and to isolate cDNA and genomic clones of other genes that have a high identity, particularly high sequence identity, to a mvd gene Such probes generally will compnse at least 15 nucleotide residues or base pairs Preferably, such probes wall 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 mvd gene may be isolated by screening usmg a DNA sequence provided in Table 1 [SEQ ID NO 1] to synthesize an oligonucleotide probe A labeled oligonucleotide havmg a sequence complementary to that of a gene ofthe invention is then used to screen a library of cDNA, genomic DNA or mRNA to determine which members ofthe library the probe hybndizes to

There are several methods available and well known to those skilled in 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 ofthe technique, exemplified by the Marathon™ technology (Clontech Laboratories 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 gated onto each end Nucleic acid amplification (PCR) is then carried out to amplify the "missing" 5' end ofthe DNA using a combination of gene specific and adaptor specific oligonucleotide primers. The PCR reaction is then repeated using "nested" primers, that is, primers designed to anneal within the amplified product (typically an adaptor specific primer that anneals further 3' in the adaptor sequence and a gene specific primer 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 using the new sequence information for the design ofthe 5' primer.

The polynucleotides and polypeptides ofthe invention may be employed, for example, as research reagents and materials 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 oligonucleotides derived from a sequence of Table 1 [SEQ ID NOS: l or 2] may be used in the processes herein as described, but preferably for PCR, to determine whether or not the polynucleotides identified herein in whole or in part are transcribed in bacteria in infected tissue. It is recognized that such sequences will also have utility in diagnosis ofthe stage of infection and type of infection the pathogen has attained.

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

For each and every polynucleotide of the invention 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 protein, having a mature form ofthe 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 ofthe prosequences may be removed before activation. Generally, such precursors are called proproteins. In sum, a polynucleotide of the invention may encode a mature protein, a mature protein plus a leader sequence (which may be referred to as a preprotein), a precursor of a mature protein having one or more prosequences that are not the leader sequences of a preprotein, or a preproprotein, which is a precursor to a proprotein, having a leader sequence and one or more prosequences, which generally are removed during processing steps that produce active and mature fomis ofthe 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 ofthe mvention by recombinant techniques Cell-free translation systems can also be employed to produce such proteins usmg RNAs denved from the DNA constructs ofthe mvention

Recombinant polypeptides ofthe present mvention may be prepared by processes well known m those skilled m the art from genetically engmeered host cells compnsmg expression systems Accordingly, in a further aspect, the present mvention relates to expression systems which compnse a polynucleotide or polynucleotides ofthe present mvention, to host cells which are genetically engmeered with such expression systems, and to the production of polypeptides ofthe mvention by recombinant techniques

For recombinant production of the polypeptides of the mvention, host cells can be genetically engineered 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 m 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, catiomc lipid-mediated transfection, electroporation, transduction, scrape loading, ballistic mtroduction and infection

Representative examples of appropnate hosts mclude bactenal cells, such as cells of streptococci, staphylococci, enterococci E coli, streptomyces, cyanobactena, Bacillus subtilis, and Streptococcus pneumoniae, fungal cells, such as cells of a yeast, Kluveromyces, Saccharomyces, a basidiomycete, Candida albicans and Aspergύlus, 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 gymnospeim or angiosperm A great vanety of expression systems can be used to produce the polypeptides of the mvention

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 retroviruses, and vectors denved from combinations thereof, such as those denved 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 in this regard The appropnate DNA sequence may be inserted mto the expression system by any of a variety of well-known and routine techniques, such as, for example, those set forth in Sambrook et al, MOLECULAR CLONING, A LABORATORY MANUAL, (supra).

In recombinant expression systems in eukaryotes, for secretion of a translated protein into the lumen of the endoplasmic reticulum, into the periplasmic space or into the extracellular environment, appropriate secretion signals may be incorporated into the expressed polypeptide. These signals may be endogenous to the polypeptide or they may be heterologous signals.

Polypeptides ofthe 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 purification. Well known techniques for refolding protein may be employed to regenerate active conformation when the polypeptide is denatured during isolation and or purification.

Diagnostic, Prognostic, Serotyping and Mutation Assays This invention is also related to the use of mvd polynucleotides and polypeptides of the invention for use as diagnostic reagents. Detection of mvd 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 organism to drugs. Eukaryotes, particularly mammals, and especially humans, particularly those infected or suspected to be infected with an organism comprising the mvd 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 ofthe 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 ofthe amplified product in comparison to a genotype of a reference sequence selected from a related organism, preferably a different species ofthe same genus or a different strain of the same species. Point mutations can be identified by hybridizing amplified DNA to labeled mvd 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 SI protection assay or a chemical cleavage method. See, for example, Cotton et al, Proc. Natl. Acad. Set, USA, 85: 4397-4401 (1985).

In another embodiment, an anay of oligonucleotides probes comprising mvd 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 which comprises: (a) a polynucleotide ofthe 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 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 invention also relates to the use of polynucleotides ofthe present invention as diagnostic reagents. Detection of a mutated form of a polynucleotide ofthe invention, preferable, SEQ ID NO: 1, which 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, which results from under-expression, over-expression or altered expression ofthe polynucleotide. Organisms, particularly infectious organisms, carrying mutations in such polynucleotide may be detected at the polynucleotide level by a variety of techniques, such as those described elsewhere herein.

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

Cells from an organism carrying mutations or polymorphisms (allelic variations) in a polynucleotide and/or polypeptide ofthe invention may also be detected at the polynucleotide or polypeptide level by a variety of techniques, to allow for serotyping, for example. For example, RT-PCR can be used to detect mutations in 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 primers complementary to a polynucleotide encoding mvd 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 primers may be used for, among other things, amplifying mvd DNA and/or RNA isolated from a sample derived from an individual, such as a bodily material. The primers may be used to amplify a polynucleotide isolated from an infected individual, such that the polynucleotide may then be subject to various 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 bacterial infections, more preferably infections caused by Streptococcus pneumoniae, comprising determining from a sample derived from an individual, such as a bodily material, an increased level of expression of polynucleotide having a sequence of Table 1 [SEQ ID NO:l]. Increased or decreased expression of a mvd 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 hybridization methods.

In addition, a diagnostic assay in accordance with the invention for detecting over-expression of mvd polypeptide compared to normal control tissue samples may be used to detect the presence of an infection, for example. Assay techniques that can be used to determine levels of a mvd polypeptide, in a sample derived from a host, such as a bodily material, are well-known to those of skill in the art. Such assay methods include radioimmunoassays, competitive-binding assays, Western Blot analysis, antibody sandwich assays, antibody detection and ELISA assays.

Antagonists and Agonists - Assays and Molecules

Polypeptides and polynucleotides ofthe invention may also be used to assess the binding of small molecule substrates and ligands in, for example, cells, cell-free preparations, chemical libraries, and natural product mixtures. These substrates and ligands may be natural substrates and ligands or may be structural or functional mimetics. See, e.g., Coligan et al, Current Protocols in Immunology 1(2): Chapter 5 (1991).

Polypeptides and polynucleotides ofthe present invention are responsible for many biological functions, including many disease states, in particular the Diseases herein mentioned. It is therefore desirable to devise screening methods to identify compounds which stimulate or which inhibit 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 which stimulate or which inhibit the function of a polypeptide or polynucleotide ofthe invention, as well as related polypeptides and polynucleotides. In general, agonists or antagonists (e.g., inhibitors) may be employed for therapeutic and prophylactic purposes for such Diseases as herein mentioned. Compounds may be identified from a variety of sources, for example, cells, cell-free preparations, chemical libraries, 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 mvd polypeptides and polynucleotides; or may be structural or functional mimetics thereof (see Coligan et al, Current Protocols in Immunology l(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 protein ofthe 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 ofthe polypeptide or polynucleotide, using detection systems appropriate to the cells comprising 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 by the presence ofthe 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 in inhibition of activation ofthe polypeptide or polynucleotide, as the case may be. Further, the screening methods may simply comprise the steps of mixing a candidate compound with a solution comprising a polypeptide or polynucleotide ofthe present invention, to form a mixture, measuring mvd polypeptide and/or polynucleotide activity in the mixture, and comparing the mvd polypeptide and/or polynucleotide activity ofthe mixture to a standard. Fusion proteins, such as those made from Fc portion and mvd polypeptide, as herein described, can also be used for high-throughput screening assays to identify antagonists ofthe polypeptide ofthe 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 bind to and/or interact with a polypeptide ofthe 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 measuring 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 which 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 screening compounds to identify those which enhance (agonist) or block (antagonist) the action of mvd polypeptides or polynucleotides, particularly those compounds that are bacteristatic and/or bactericidal. The method of screening may involve high-throughput techniques. 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 mvd polypeptide and a labeled substrate or ligand of such polypeptide is incubated in the absence or the presence of a candidate molecule that may be a mvd agonist or antagonist. The ability of the candidate molecule to agonize or antagonize the mvd polypeptide is reflected in decreased binding ofthe labeled ligand or decreased production of product from such substrate. Molecules that bind gratuitously, i.e., without inducing the effects of mvd polypeptide are most likely to be good antagonists. Molecules that bind 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 using a reporter system. Reporter systems that may be useful in this regard include but are not limited to colorimetric, labeled substrate converted into product, a reporter gene that is responsive to changes in mvd polynucleotide or polypeptide activity, and binding assays known in the art.

Polypeptides ofthe invention may be used to identify membrane bound or soluble receptors, if any, for such polypeptide, through standard receptor binding techniques known in the art. These techniques include, but are not limited to, ligand binding and crosslinking assays in 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 ofthe putative receptor (e.g., cells, cell membranes, cell supernatants, tissue extracts, bodily materials). Other methods include biophysical teclmiques such as surface plasmon resonance and spectroscopy. These screening methods may also be used to identify agonists and antagonists ofthe polypeptide which compete with the binding ofthe 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 correlation time or tumbling rate. Protein complexes, such as formed by mvd polypeptide associating with another mvd polypeptide or other polypeptide, labeled to comprise a fluorescently- labeled molecule will have higher polarization values than a fluorescently labeled monomeric protein. It is preferred 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 mvd polypeptide dimers, tπmers, tetramers or higher order structures, or structures formed by mvd polypeptide bound to another polypeptide mvd polypeptide can be labeled with both a donor and acceptor fluorophore Upon mixing ofthe two labeled species and excitation ofthe donor fluorophore, fluorescence energy transfer can be detected by observing fluorescence ofthe acceptor Compounds that block dimerization will inhibit fluorescence energy transfer

Surface plasmon resonance can be used to monitor the effect of small molecules on mvd polypeptide self-association as well as an association of mvd polypeptide and another polypeptide or small molecule mvd polypeptide can be coupled to a sensor chip at low site density such that covalently bound molecules will be monomeπc Solution protein can then passed over the mvd polypeptide -coated surface and specific binding can be detected in real-time by monitoring the change in resonance angle caused by a change m local refractive mdex This technique can be used to characterize the effect of small molecules on kinetic rates and equilibrium binding constants for mvd polypeptide self-association as well as an association of mvd polypeptide and another polypeptide or small molecule

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

In other embodiments ofthe invention there are provided methods for identifying compounds winch bind to or otherwise mteract with and inhibit or activate an activity or expression of a polypeptide and/or polynucleotide ofthe mvention compnsmg contacting a polypeptide and/or polynucleotide ofthe mvention with a compound to be screened under conditions to permit binding to or other mteraction between the compound and the polypeptide and/or polynucleotide to assess the binding to or other mteraction with the compound, such binding or mteraction preferably being associated with a second component capable of providing a detectable signal m response to the binding or mteraction ofthe 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 detectmg the presence or absence of a signal generated from the binding or mteraction ofthe compound with the polypeptide and/or polynucleotide Another example of an assay for mvd agonists is a competitive assay that combines mvd and a potential agonist with mvd-binding molecules, recombinant mvd binding molecules, natural substrates or ligands, or substrate or ligand mimetics, under appropriate conditions for a competitive inhibition assay, mvd can be labeled, such as by radioactivity or a colorimetric compound, such that the number of mvd molecules bound to a binding molecule or converted to product can be determined accurately to assess the effectiveness ofthe 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 ofthe polypeptide and/or polynucleotide, by: (a) determining in the first instance the three- dimensional structure ofthe polypeptide and/or polynucleotide, or complexes thereof; (b) deducing the three-dimensional structure for the likely reactive site(s), binding site(s) or motif(s) of an agonist or antagonist; (c) synthesizing candidate compounds that are predicted to bind to or react with the deduced binding site(s), reactive site(s), and/or motif(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. hi 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 mvd 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 comprises administering to an individual in need thereof an inhibitor compound (antagonist) as herein described, optionally in combination with a phannaceutically acceptable carrier, in an amount effective to inhibit the function and/or expression of the polypeptide and/or polynucleotide, such as, for example, by blocking the binding of ligands, substrates, receptors, enzymes, etc., or by inliibiting a second signal, and tiiereby alleviating the abnormal condition. In another approach, soluble forms ofthe polypeptides still capable of binding the ligand, substrate, enzymes, receptors, etc. in competition with endogenous polypeptide and/or polynucleotide may be administered. Typical examples of such competitors include fragments ofthe mvd polypeptide and/or polypeptide.

In still another approach, expression ofthe gene encoding endogenous mvd polypeptide can be inhibited using expression blocking techniques. This blocking may be targeted against any step in gene expression, but is preferably targeted against transcription 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 Oligodeoxynucleotides as Antisense Inhibitors of Gene Expression, CRC Press, Boca Raton, FL (1988)) Alternatively, ohgonucleotides which form triple 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 /?er 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 antibacterial compounds The encoded protein, upon expression, can be used as a target for the screenmg of antibacterial drugs Additionally, the polynucleotide sequences encodmg the ammo terminal regions of the encoded protem or Shme-Delgarno or other translation facilitating sequences of the respective mRNA can be used to construct antisense sequences to control the expression ofthe coding sequence of mterest

The invention also provides the use of the polypeptide, polynucleotide, agonist or antagonist ofthe mvention 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 bacteria, in particular gram positive and/or gram negative bacteria, to eukaryotic, preferably mammalian, extracellular matrix proteins on in-dwelling devices or to extracellular matrix proteins in wounds, to block bacterial adhesion between eukaryotic, preferably mammalian, extracellular matrix proteins and bacterial mvd 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 mvd 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 Hehcobacter pylori (herein "H pylon") bacteria infect the stomachs of over one-third of the world's population causing stomach cancer, ulcers, and gastritis (International Agency for Research on Cancer (1994) Schistosomes, Liver Flukes and Hehcobacter Pylon (International Agency for Research on Cancer, Lyon, France, http //www uicc ch/ecp/ecp2904 htm) Moreover, the International Agency for Research on Cancer recently recognized a cause-and-effect relationship between H pylori and gastric adenocarcinoma, classifying the bacterium as a Group I (definite) carcinogen Preferred antimicrobial compounds of the invention (agonists and antagonists of mvd polypeptides and/or polynucleotides) found using screens provided by the invention, or known in the art, particularly narrow-spectrum antibiotics, should be useful m the treatment of H pylori infection Such treatment should decrease the advent of H pylon -induced cancers, such as gastrointestinal carcinoma Such treatment should also prevent, inhibit and/or cure gastric ulcers and gastritis

All publications and references, including but not limited to patents and patent applications, cited m 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 herein as being fully set forth Any patent application to which this application claims priority 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 certain terms used frequently herein

"Bodily mateπal(s) means any matenal denved from an individual or from an organism infecting, mfesting or inhabiting an individual, including but not limited to, cells, tissues and waste, such as, bone, blood, serum, cerebrospmal fluid, semen, saliva, muscle, cartilage, organ tissue, skm, unne. stool or autopsy matenals

"Dιsease(s)" means any disease caused by or related to infection by a bactena, including , for example, otitis media, conjunctivitis, pneumonia, bacteremia, meningitis, sinusitis, pleural empyema and endocarditis, and most particularly meningitis, such as for example infection of cerebrospmal fluid "Host cell(s)" is a cell which has been transformed or transfected, or is capable of 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 comparing the sequences In the art, "identity" also means the degree of sequence relatedness between polypeptide or polynucleotide sequences, as the case may be, as determined by the match between strmgs 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, 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 Carillo, 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 determine 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 al, NCBI NLM NIH Bethesda, MD 20894; Altschul, S., et al, J. Mol. Biol. 215: 403-410 (1990). The well known Smith Waterman algorithm may also be used to determine identity.

Parameters for polypeptide sequence comparison include the following: Algorithm: Needleman and Wunsch, J. Mol Biol. 48: 443-453 (1970) Comparison matrix: BLOSSUM62 from Hentikoff and Hentikoff, Proc. Natl. Acad. Sci. USA. 89: 10915-10919 (1992) Gap Penalty: 12 Gap Length Penalty: 4

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

Parameters for polynucleotide comparison include the following: Algorithm: Needleman and Wunsch, J. Mol Biol. 48: 443-453 (1970) Comparison matrix: matches = +10, mismatch = 0 Gap Penalty: 50 Gap Length Penalty: 3

Available as: The "gap" program from Genetics Computer Group, Madison WI. These are the default parameters for nucleic acid comparisons.

A preferred meaning for "identity" for polynucleotides and polypeptides, as the case may be, are provided in (1) and (2) below. (1) Polynucleotide embodiments further include an isolated polynucleotide comprising a polynucleotide sequence having at least a 50, 60, 70, 80, 85, 90, 95, 97 or 100%, identity to the reference sequence of SEQ ID NO: 1, wherein said polynucleotide sequence may be identical to the reference sequence of SEQ ID NO:l 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 ofthe reference nucleotide sequence or anywhere between those terminal positions, interspersed either individually among the nucleotides in the reference sequence or in one or more contiguous groups within the reference sequence, and wherein said number of nucleotide alterations is determined by multiplying the total number of nucleotides in SEQ ID NO: 1 by the integer defining the percent identity divided by 100 and then subtracting that product from said total number of nucleotides in SEQ ID NO:l, or:

n_n < x_n - (x_n • y),

wherein n_n is the number of nucleotide alterations, x_n is the total number of nucleotides in SEQ ID NO: l, 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 subtracting 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.

By way of example, a polynucleotide sequence ofthe present invention may be identical to the reference sequence of SEQ ID NO:l, that is it may be 100% identical, or it may include up to a certain integer number of nucleic acid alterations as compared to the reference sequence such that the percent identity is less than 100% identity. Such alterations are selected from the group consisting of at least one nucleic acid deletion, substitution, including transition and transversion, or insertion, and wherein said alterations may occur at the 5' or 3' terminal positions ofthe reference polynucleotide sequence or anywhere between those terminal positions, interspersed either individually among the nucleic acids in the reference sequence or in one or more contiguous groups within the reference sequence. The number of nucleic acid alterations for a given percent identity is determined by multiplying the total number of nucleic acids in SEQ ID NO:l by the integer defining the percent identity divided by 100 and then subtracting that product from said total number of nucleic acids in SEQ ID NO: 1, or:

n_n < x_n - (x_n • y),

wherein n_n is the number of nucleic acid alterations, x_n is the total number of nucleic acids in SEQ ID NO: l, y is, for instance 0.95 for 95%, 0.97 for 97% or 1.00 for 100%, etc., • 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 subtracting it from x_n.

(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 certain integer number of amino acid alterations as compared to the reference sequence, wherein said alterations are selected from the group consisting of at least one amino acid deletion, substitution, including conservative and non-conservative substitution, or insertion, and wherein said alterations may occur at the amino- or carboxy-terminal positions ofthe reference polypeptide sequence or anywhere between those terminal positions, interspersed either individually among the amino acids in the reference sequence or in one or more contiguous groups within the reference sequence, and wherein said number of amino acid alterations is determined by multiplying the total number of amino acids in SEQ ID NO:2 by the integer defining the percent identity divided by 100 and then subtracting that product from said total number of amino acids in SEQ ID NO:2, or:

ⁿa ≤ ^xa " (^xa ^# y),

wherein 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.

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

n_a < x_a - (x_a • y),

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, for instance 0.95 for 95%, 0.97 for 97% or 1.00 for 100%, etc., 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 primate, and a human. "Isolated" means altered "by the hand of man" from its natural state, . e , if it occurs in nature, it has been changed or removed from its original environment, or both. For example, a polynucleotide or a polypeptide naturally present in a living organism is not "isolated," but the same polynucleotide or polypeptide separated from the coexisting materials of its natural state is "isolated", as the term is employed herein Moreover, a polynucleotide or polypeptide that is introduced into an organism by transformation, genetic manipulation or by any otlier 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, Staphylococcus, Bordetella, Corynebacterium, Mycobacterium, Neissena, Haemophilus, Actinomycetes, Streptomycetes, Nocardia, Enterobacter, Yersinia, Fancisella, Pasturella, Moraxella. Acinetobacter, Erysipelothnx, Branhamella, Actinobacillus, Streptobacillus, Listeria, Calymmatobactenum, Bntcella, Bacillus, Clostridium, Treponema, Eschenchia, Salmonella, Kleibsiella, Vibrio, Proteus, Erwi a, Borreha, Leptospira, Spirillum, Campylobacter, Shigella, Legwnella, Pseudomonas, Aeromonas, Rickettsia, Chlamydia, Borreha and Mycoplasma, and further including, but not limited to, a member ofthe 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 faecalis, Streptococcus faecium, Streptococcus durans, Neissena gonorrheae, Neissena meningitidis, Staphylococcus aureus, Staphylococcus epidermidis, Corynebacterium dipthenae, Gardnerella vaginahs, Mycobacterium tuberculosis, Mycobacterium bovis, Mycobactenum ulcerans, Mycobactenum leprae, Actinomyctes israeln, Listeria monocytogenes, Bordetella pertusis, Bordatella parapertusis, Bordetella bronchiseptica, Eschenchia coli, Shigella dysentenae, Haemophilus mfluenzae, Haemophilus aegyptius, Haemophilus parainfluenzae, Haemophilus ducreyi, Bordetella, Salmonella typhi, Citrobacter freundu, Proteus mirabihs, Proteus vulgans, Yersinia pestis, Kleibsiella pneumoniae, Serratia marcessens, Serratia bquefaciens, Vibrio cholera, Shigella dysenteru, Shigella flexnen, Pseudomonas aeruginosa, Franscisella tularensis, Brucella abortis, Bacillus anthracis, Bacillus cereus, Clostridium perfnngens, Clostridium tetani, Clostridium botuhnum, Treponema pallidum, Rickettsia nckettsii and Chlamydia trachomitis, (n) an archaeon, mcluding but not limited to Archaebacter, and (iii) a unicellular or filamentous eukaryote. including but not limited to, a protozoan, a fungus, a member ofthe genus Saccharomyces, Kluveromyces, or Candida, and a member of the species Saccharomyces ceriviseae, Kluveromyces lactis, or Candida albicans.

"Polynucleotide(s)" generally refers to any polyribonucleotide or polydeoxyribonucleotide, which may be unmodified RNA or DNA or modified RNA or DNA. "Polynucleotide(s)" include, without limitation, single- and double-stranded DNA, DNA that is a mixture of single- and double-stranded regions or single-, double- and triple-stranded regions, single- and double-stranded RNA, and RNA that is mixture of single- and double-stranded regions, hybrid molecules comprising DNA and RNA that may be single- stranded or, more typically, double-stranded, or triple-stranded regions, or a mixture of single- and double- stranded regions. In addition, "polynucleotide" as used herein refers to triple-stranded regions comprising RNA or DNA or both RNA and DNA. The strands in such regions may be from the same molecule or from different molecules. The regions may include all of one or more of the molecules, but more typically involve only a region of some of the molecules. One of the molecules of a triple-helical region often is an oligonucleotide. As used herein, the tenn "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 tritylated bases, to name just two examples, are polynucleotides as the term is used herein. It will be appreciated that a great variety of modifications have been made to DNA and RNA that serve many useful purposes known to those of skill in the art. The term "polynucleotide(s)" as it is employed herein embraces such chemically, enzymatically or metabolically modified forms of polynucleotides, as well as the chemical forms of DNA and RNA characteristic of viruses and cells, including, for example, simple and complex cells. "Polynucleotide(s)" also embraces short polynucleotides often referred to as oligonucleotide(s).

"Polypeptide(s)" refers to any peptide or protein comprising two or more amino acids joined to each other by peptide bonds or modified peptide bonds. "Polypeptide(s)" refers to both short chains, commonly refened to as peptides, oligopeptides and ohgomers and to longer chains generally refened to as proteins. Polypeptides may comprise amino acids other than the 20 gene encoded amino acids. "Polypeptide(s)" include those modified either by natural processes, such as processing and other post-translational modifications, but also by chemical modification techniques. Such modifications are well described in basic texts and in more detailed monographs, as well as in a voluminous research literature, and they are well known to those of skill in the art. It will be appreciated that the same type of modification may be present in the same or varying degree at several sites in a given polypeptide. Also, a given polypeptide may comprise many types of modifications. Modifications can occur anywhere in a poljpeptide, including the peptide backbone, the amino acid side-chains, and the amino or carboxyl termini. Modifications include, for example, acetylation, acylation, ADP-ribosylation, amidation, covalent attachment of flavin, covalent attachment of a heme moiety, covalent attachment of a nucleotide or nucleotide denvative, covalent attachment of a pid or pid denvative, covalent attachment of phosphotidyhnositol, 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, mynstoylation, oxidation, proteolytic processing, phosphorylation, prenylation, racemization, glycosylation, hpid attachment, sulfation, gamma-carboxylation of glutamic acid residues, hydroxylation and ADP-nbosylation, selenoylation, sulfation, transfer-RNA mediated addition of amino acids to proteins, such as argmylation, and ubiquitmation 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 Protein Modifications Perspectives and Prospects, pgs 1-12 in POSTTRANSLATIONAL COVALENT MODIFICATION OF PROTEINS, B C Johnson, Ed , Academic Press, New York (1983), Seifter et al , Meth Enzymol 182 626-646 (1990) and Rattan et al , Protein Synthesis Posttranslational Modifications and Aging, Ann N Y Acad Sci 663 48- 62 (1992) 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 ofthe mvention introduced or transformed mto a host cell or host cell lysate for the production ofthe polynucleotides and polypeptides ofthe mvention 'Naπant(s)" as the term is used herein, is a polynucleotide or polypeptide that differs from a reference polynucleotide or polypeptide respectively, but retains essential properties A typical variant of a polynucleotide differs in nucleotide sequence from another, reference polynucleotide Changes in the nucleotide sequence of the variant may or may not alter the amino acid sequence of a polypeptide encoded by the reference polynucleotide Nucleotide changes may result m 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 ammo acid sequence from another, reference polypeptide Generally, differences are limited so that the sequences of the reference polypeptide and the variant are closely similar overall and, in many regions, identical A variant and reference polypeptide may differ in ammo 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 mvention also mcludes mclude vanants of each ofthe polypeptides of the invention, 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, Nal, 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 preferred are vanants m which several, 5-10, 1-5, 1-3, 1-2 or 1 ammo acids are substituted, deleted, or added in any combination A variant of a polynucleotide or polypeptide may be a naturally occurnng such as an allehc variant, or it may be a variant that is not known to occur naturally Non- naturally occurrmg variants 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 techniques, which are well known and routme to those of skill m the art, except where otherwise descnbed 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 m Table 1 [SEQ ID NO 1] was obtained from a library of clones of chromosomal DNA of Streptococcus pneumoniae in E coli The sequencing data from two or more clones comprising overlapping Streptococcus pneumoniae 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 Streptococcus pneumoniae 0100993 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 m 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 mfected with the packaged library The library is amplified by standard procedures

Method 2

Total cellular DNA is partially hydrolyzed with a one or a combination of restriction enzymes appropriate to generate a series of fragments for cloning into library vectors (e g , Rsal, Pall, Alul, Bshl235I), and such fragments are size-fractionated according to standard procedures EcoRI linkers are hgated 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 which is the amino acid sequence of SEQ ID NO:2, and (iv) a polypeptide which 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 which 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 which is the polynucleotide of SEQ ID NO: 1 ;

(vi) an isolated polynucleotide 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; (vii) an isolated polynucleotide encoding a mature polypeptide expressed by the mvd gene comprised in the Streptococcus pneumoniae; 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:

(i) in need of enhanced activity or expression of or immunological response to the polypeptide of claim 1 comprising the step of: administering to the individual a therapeutically effective amount of an antagonist to said polypeptide; or (ii) having need to inhibit activity or expression ofthe polypeptide of claim 1 comprising:

(a) administering to the individual a therapeutically effective amount of an antagonist to said polypeptide; or

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

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

(d) administering to the individual an amount of a polypeptide that induces an immunological 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 ofthe 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 in said individual; or

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

5. A process for producing a polypeptide selected from the group consisting of:

(i) 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;

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

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

(iv) a polypeptide which is encoded by a recombinant polynucleotide comprising the polynucleotide sequence of SEQ ID NO:l, comprising the step of culturing a host cell of claim 7 under conditions sufficient for the production of said polypeptide.

6. A process for producing a host cell comprising an expression system expressing a polypeptide selected from the group consisting of: (i) 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;

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

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

(iv) a polypeptide which is encoded by a recombinant polynucleotide comprising the polynucleotide sequence of SEQ ID NO:l, said process comprising the step of transforming or transfecting a cell with an expression system comprising a polynucleotide capable of producing said polypeptide of (i), (ii), (iii) 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 (i), (ii), (iii) or (iv).

7. A host cell expressing a polypeptide selected from the group consisting of:

(i) 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;

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

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

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

8. An antibody immunospecific for the polypeptide of claim 1.

9. A method for screening to identify compounds which stimulate or which inhibit the function of the polypeptide of claim 1 which comprises a method selected from the group consisting of:

(a) measuring the binding 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 bearing 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 ofthe polypeptide, using detection systems appropriate 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 ofthe polypeptide in the mixture, and comparing the activity ofthe 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 agonist or antagonist to the polypeptide of claim 1.