WO1999061452A2 - acpS - Google Patents

Abstract

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

Description

acpS

FIELD OF THE INVENTION

This invention relates to newly identified polynucleotides and polypeptides, and their production and uses, as well as their vanants, agonists and antagonists, and their uses In particular, the invention relates to polynucleotides and polypeptides of the acyl earner protern synthase family, as well as their vanants, herein referred to as "acpS," "acpS polynucleotιde(s)," and "acpS polypeptιde(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 pneumomae has been one of the more intensively studied microbes For example, much of our early understanding that DNA is, in fact, the genetic matenal was predicated on the work of Griffith and of Avery, Macleod and McCarty using this microbe Despite the vast amount of research with S pneumonwe, many questions concerning the virulence 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 pneumomae 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 Streptococcus pneumomae 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 vanous 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 Clearh. there exists a need for polynucleotides and polypeptides, such as the acpS 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, amehorate or correct such infection, dysfunction and disease

SUMMARY OF THE INVENTION

The present invention relates to acpS, in particular acpS polypeptides and acpS 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 acpS expression or activity

Vanous changes and modifications within the spmt 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 of the present disclosure

DESCRIPTION OF THE INVENTION

The invention relates to acpS polypeptides and polynucleotides as descnbed in greater detail below In particular, the invention relates to polypeptides and polynucleotides of a acpS of Streptococcus pneumomae, which is related b\ ammo acid sequence homology to H pylon acpS polypeptide The invention relates especially to acpS having a nucleotide and amino 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 m polynucleotides in general, including πbopolynucleotides

TABLE 1 acpS Polynucleotide and Polypeptide Sequences

(A) Streptococcus pneumomae acpS polynucleotide sequence [SEQ ID NO 1]

5 ' -

ATGATAGTTGGACACGGAATTGACATCGAAGAATTGGCTTCGATAGAAAGCGCAGTTACACGACATGAAGGATTTG CTAAGCGTGTACTGACCGCTCAGGAAATGGAGCGCTTCACCAGTCTCAAAGGACGCAGGCAAATAGAATATTTAGC TGGTCGCTGGTCGGCTAAGGAGGCCTTTTCCAAGGCTATGGGAACGGGCATTAGCAAGCTCGGTTTTCAGGATTTG GAAGTCTTGAAC

AATGAACGTGGGGCGCCTTATTTTAGTCAGGCACCATTTTCAGGAAAGATTTGGCTGTCTATCAGCCACACCGATC AGTTTGTGACAGCCAGTGTCATTTTGGAGGAAAATCATGAAAGCTAG-3 '

(B) Streptococcus pneumomae acpS polypeptide sequence deduced from a polynucleotide sequence in this table [SEQ ID NO 2]

NH₂-

MIVGHGI DI EELAS I ESAλ TRHEGFAKRVLTAQEMERFTSLKGRRQIEYLAG WSAKEAFSKAMGTGI SKLGFQDL EVLNNERGAPYFSQAP FSGKIWLS I SHTDQFVTASVI LEENHES * -COOH

Deposited materials

A deposit compnsing a Streptococcus pneumomae 0100993 strain has been deposited with the National Collections of Industnal and Marine Bactena Ltd (herein "NCIMB"), 23 St Machar Dnve. Aberdeen AB2 IRY, Scotland on 11 Apnl 1996 and assigned deposit number 40794 The deposit was descnbed as Streptococcus pneumomae 0100993 on deposit On 17 Apnl 1996 a Streptococcus pneumomae 0100993 DNA library in E coh v. as similarly deposited with the NCIMB and assigned deposit number 40800 The Streptococcus pneumomae strain deposit is referred to herein as "the deposited strain" or as "the DNA of the deposited strain " The deposited strain compnses a full length acpS gene The sequence of the polynucleotides compnsed in the deposited strain, as well as the ammo acid sequence of any polypeptide encoded thereby, are controlling in the event of any conflict with any descnption of sequences herein

The deposit of the deposited strain has been made under the terms of the Budapest Treaty on the International Recognition of the Deposit of Micro-organisms for Purposes of Patent Procedure The deposited strain will be irrevocabK and without restnction 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 ma) be required to make, use or sell the deposited strain, and compounds denved therefrom, and no such license is hereby granted In one aspect of the invention there is provided an isolated nucleic acid molecule encoding a mature polypeptide expressible

the Streptococcus pneumomae 0100993 strain, which polypeptide is compnsed in the deposited strain Further provided by the invention are acpS polynucleotide sequences in the deposited strain, such as DNA and RNA, and amino acid sequences encoded thereby Also provided by the invention are acpS polypeptide and polynucleotide sequences isolated from the deposited strain Polypeptides acpS polypeptide of the invention is substantially phylogeneucaliy related to other proteins of the acyl earner protein synthase familv

In one aspect of the invention there are provided polypeptides of Streptococcus pneumomae referred to herem as "acpS" and "acpS polypeptides" as well as biologically, diagnostically, prophylactically, clinically or therapeutically useful vanants thereof, and compositions compnsing the same

Among the particularly preferred embodiments of the invention are vanants of acpS polypeptide encoded by naturally occurring alleles of a acpS gene

The present invention further provides for an isolated polypeptide which (a) compnses or consists of an ammo acid sequence which has at least 95% identity, most preferably at least 97-99% or exact ldentits , 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% identitv. even more preferably at least 97-99% or exact identity to SEQ ID NO 1 over the entire length of SEQ ID NO 1 , (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 ammo 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] (m particular a mature polypeptide) as well as polypeptides and fragments, particularly those which have a biological activity of acpS, and also those which have at least 95% identity to a polypeptide of Table 1 [SEQ ID NO 2] and also mclude portions of such polypeptides with such portion of the polypeptide generally compnsing at least 30 ammo acids and more preferably at least 50 ammo acids

The mvention also includes a polypeptide consisting of or compnsing a polypeptide of the formula

X-(R₁)_m-(R₂)-(R₃)_n-Y wherein, at the ammo terminus, X is hydrogen, a metal or any other moiety descnbed herem 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 ammo acid residue, m is an mteger between 1 and 1000 or zero, n is an mteger between 1 and 1000 or zero, and R₂ is an ammo acid sequence of the mvention 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 ammo acid residue is at the nght. 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 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 polypeptide of the mvention is denved from Streptococcus pneumomae, however, it may preferably be obtained from other organisms of the same taxonomic genus A polypeptide of the mvention may also be obtained, for example, from organisms of the same taxonomic family or order

A fragment is a vanant 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 acpS 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 continuous region in a s gle larger polypeptide

Preferred fragments include, for example, truncation polypeptides having a portion of an ammo acid sequence of Table 1 [SEQ ID NO 2], or of vanants thereof, such as a continuous senes of residues that mcludes an ammo- and/or carboxyl-termmal ammo acid sequence Degradation forms of the polypeptides of the mvention produced by or in a host cell, particularly a Streptococcus pneumomae, are also preferred Further preferred are fragments charactenzed 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-foiming regions, hydrophi c regions, hydrophobic regions, alpha amphipathic regions, beta amphipathic regions, flexible regions, surface-forming regions, substrate b dmg region, and high antigenic mdex regions

Further preferred fragments include an isolated polypeptide comprismg an ammo 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 compnsmg an ammo acid sequence having at least 15, 20, 30, 40, 50 or 100 contiguous ammo 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 vanants may be employed as intermediates for producmg the full-length polypeptides of the mvention

Polynucleotides It is an object of the mvention to provide polynucleotides that encode acpS polypeptides, particularly polynucleotides that encode a polypeptide herem designated acpS

In a particularly preferred embodiment of the mvention the polynucleotide compnses a region encoding acpS polypeptides compnsmg a sequence set out m 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 pneumomae

As a further aspect of the mvention there are provided isolated nucleic acid molecules encoding and/or expressmg acpS polypeptides and polynucleotides, particularly Streptococcus pneumomae acpS polypeptides and polynucleotides, including, for example, unprocessed RNAs, nbozyme RNAs. mRNAs, cDNAs. genomic DNAs, B- and Z-DNAs Further embodiments of the mvention include biologically, diagnostically, prophylactically, clmically 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 full length gene, that encodes a acpS polypeptide havmg 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 acpS polypeptide from Streptococcus pneumomae compnsmg or consistmg of an ammo acid sequence of Table 1 [SEQ ID NO 2], or a variant thereof

Usmg the information provided herem, such as a polynucleotide sequence set out in Table 1 [SEQ ID NO 1]. a polynucleotide of the mvention encoding acpS polypeptide may be obtained usmg standard cloning and screening methods, such as those for cloning and sequencmg chromosomal DNA fragments from bactena usmg Streptococcus pneumomae 0100993 cells as starting matenal, 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 Streptococcus pneumomae 0100993 m E coh or some other suitable host is probed with a radiolabeled ohgonucleotide, 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 b\ hybridization with sequencing primers designed from the oπgmal 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 sequencmg is performed, for example, usmg denatured double stranded DNA prepared from a plasmid clone Suitable techniques are described by Ma atis, T , Fπtsch, 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 Bv 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 mvention, each polynucleotide set out in Table 1 [SEQ ID NO 1] was discovered m a DNA library denved from Streptococcus pneumomae 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 ammo acid residues set forth in Table 1 [SEQ ID NO 2] with a deduced molecular weight that can be calculated usmg ammo acid residue molecular weight values well known to those skilled m the art The polvnucleotide of SEQ ID NO 1, between nucleotide number 1 and the stop codon which begms at nucleotide number 361 of SEQ ID NO 1, encodes the polypeptide of SEQ ID NO 2

In a further aspect, the present mvention provides for an isolated polynucleotide compnsmg or 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 1 over the entire length of SEQ ID NO 1. or the entire length of that portion of SEQ ID NO 1 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 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 Streptococcus pneumomae, may be obtained by a process which compnses the steps of screening an appropnate library under stringent hybndrzation conditions with a labeled or detectable probe consisting of or compnsmg the sequence of SEQ ID NO 1 or a fragment thereof, and isolating a full-length gene and/or genomic clones compnsmg said polynucleotide sequence

The mvention provides a polynucleotide sequence identical over its entire length to a codmg sequence (open readmg frame) m Table 1 [SEQ ID NO 1] Also provided by the mvention is a codmg sequence for a mature polypeptide or a fragment thereof, by itself as well as a codmg sequence for a mature polypeptide or a fragment in readmg frame with another codmg sequence, such as a sequence encoding a leader or secretory sequence, a pre- or pro- or prepro-protein sequence The polynucleotide of the mvention may also compnse at least one non-coding sequence, including for example, but not limited to at least one non-coding 5' and 3' sequence, such as the transcnbed but non-translated sequences, termination signals (such as rho-dependent and rho-independent termination signals), nbosome bmdmg sites, Kozak sequences, sequences that stabilize mRNA, mtrons and polyadenylation signals The polynucleotide sequence may also compnse additional codmg sequence encoding additional ammo acids For example, a marker sequence that facilitates punficauon of a fused polypeptide can be encoded In certain embodiments of the mvention, the marker sequence is a hexa- histidme 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 which may be useful m punfymg polypeptide sequence fused to them Polynucleotides of the mvention also mclude. but are not limited to, polynucleotides compnsmg a structural gene and its naturally associated sequences that control gene expression A preferred embodiment of the mvention is a polynucleotide of consisting of or compnsing nucleotide 1 to the nucleotide immediately upstream of or including nucleotide 361 set forth m SEQ ID NO 1 of Table 1, both of which encode a acpS polypeptide

The mvention 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 Ri 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 integer between 1 and 3000 or zero, and R₂ 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, R₂ is oriented 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 right, bound to R3 Any stretch of nucleic acid residues denoted by either Ri and/or R₂, where m and/or n is greater than 1, may be either a heteropolymer or a homopolymer, preferably a heteropolymer Where, m a preferred embodiment, X and Y together define a covalent bond, the polynucleotide of the above formula is a closed, circular polynucleotide, which can be a double-stranded polynucleotide wherem the formula shows a first strand to which the second strand is complementary In another preferred embodiment m and/or n is an mteger between 1 and 1000. Other 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 denved from Streptococcus pneumomae, 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 "polynucleotide encoding a polypeptide" as used herem encompasses polynucleotides that mclude a sequence encoding a polypeptide of the mvention, particularly a bactenal polypeptide and more particularly a polypeptide of the Streptococcus pneumomae acpS having an ammo acid sequence set out in Table 1 [SEQ ID NO 2] The term also encompasses polynucleotides that mclude a single continuous region or discontinuous regions encoding the polypeptide (for example, polynucleotides interrupted by mtegrated phage, an mtegrated insertion sequence, an mtegrated vector sequence, an mtegrated transposon sequence, or due to RNA editing or genomic DNA reorganization) together with additional regions, that also may compnse codmg and/or non-coding sequences

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

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

Further preferred embodiments of the mvention are polynucleotides that are at least 95 % or 97% identical over their entire length to a polynucleotide encoding acpS 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 polvnucleotides 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 preferred embodiments of this mvention there are provided polynucleotides that hybndize, particularly under strmgent conditions, to acpS 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 descnbed herem As herem used, the terms "strmgent conditions" and "strmgent 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 strmgent hybridization conditions is overnight incubation at 42°C in a solution comprising 50% formamide, 5x SSC (150mM NaCl, 15mM tnsodium citrate), 50 mM sodium phosphate (pH7 6), 5x Denhardt's solution, 10% dextran sulfate, and 20 micrograms/ml of denatured, sheared salmon sperm DNA, followed by washing the hybridization support in 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 therem Solution hybndization may also be used with the polynucleotide sequences provided by the mvention The mvention also provides a polynucleotide consisting of or compnsmg a polynucleotide sequence obtained by screemng an appropπate library compnsmg 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 herem

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 acpS and to isolate cDNA and genomic clones of other genes that have a high identity, particularly high sequence identity, to a acpS 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 preferred probes will have at least 20 nucleotide residues or base pairs and will have lee than 30 nucleotide residues or base parrs

A codmg region of a acpS gene may be isolated by screemng usmg a DNA sequence provided in Table 1 [SEQ ID NO 1] to synthesize an ohgonucleotide probe A labeled ohgonucleotide havmg a sequence complementary to that of a gene of the mvention is then used to screen a libraiy of cDNA, genomic DNA or mRNA to determine which members of the library the probe hybndizes to

There are several methods available and well known to those skilled m the art to 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 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 hgated onto each end Nucleic acid amplification (PCR) is then earned out to amplify the "missing" 5' end of the DNA using a combination of gene specific and adaptor specific ohgonucleotide pnmers The PCR reaction is then repeated using "nested" primers, that is, primers designed to anneal withm 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 sequencmg and a full-length DNA constructed either by joining the product directly to the existing DNA to give a complete sequence, or carrymg out a separate full-length PCR using the new sequence information for the design of the 5' primer

The polynucleotides and polypeptides of the mvention 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 herem relatmg to polynucleotide assays The polynucleotides of the invention that are o gonucleotides denved from a sequence of Table 1

[SEQ ID NOS 1 or 2] may be used m the processes herem as descnbed. but preferably for PCR. to determine whether or not the polynucleotides identified herein in whole or m part are transcnbed in bacteria m infected tissue It is recognized that such sequences will also have utility in diagnosis of the stage of infection and type of infection the pathogen has attained The mvention also provides polynucleotides that encode a polypeptide that is a mature protem plus additional ammo or carboxyl-termmal ammo acids, or ammo acids intenor to a mature polypeptide (when a mature form has more than one polypeptide chain, for instance) Such sequences may play a role m processing of a protem from precursor to a mature form, may allow protem transport, may lengthen or shorten protem half- life or may facilitate manipulation of a protem for assay or production, among other things As generally is the case in vivo, the additional ammo acids may be processed away from a mature protem by cellular enzymes

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

A precursor protem, havmg a mature form of the polypeptide fused to one or more prosequences 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

In sum, a polynucleotide of the mvention may encode a mature protem, a mature protem plus a leader sequence (which may be referred to as a preprotem), a precursor of a mature protem havmg one or more prosequences that are not the leader sequences of a preprotem, or a preproprotein, which is a precursor to a proprotein, havmg a leader sequence and one or more prosequences, which generally are removed during processmg steps that produce active and mature forms of the polypeptide

Vectors, Host Cells, Expression Systems

The mvention also relates to vectors that compnse a polynucleotide or polynucleotides of the mvention, host cells that are genetically engmeered with vectors of the mvention and the production of polypeptides of the mvention by recombinant techniques Cell-free translation systems can also be employed to produce such proteins usmg RNAs denved from the DNA constructs of the mvention

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

For recombinant production of the polypeptides of the mvention, host cells can be genetically engmeered to incorporate expression systems or portions thereof or polynucleotides of the mvention Introduction of a polynucleotide mto 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, cationic hpid-mediated transfection, electroporation, transduction, scrape loading, ballistic introduction and infection

Representative examples of appropnate hosts mclude bactenal cells, such as cells of streptococci, staphylococci. enterococci E coh, streptomyces, cyanobactena, Bacillus subtihs, and Streptococcus pneumomae, fungal cells, such as cells of a yeast, Kluveromyces, Saccharomyces, a basidiomycete, Candida albicans and Aspergύlus. insect cells such as cells of Drosophύa 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 gvmnosperm or angiosperm

A great variety 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, vaccmia 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 phagermds 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 m this regard The appropnate DNA sequence may be inserted mto the expression system by any of a vanety 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 m eukaryotes, for secretion of a translated protem mto the lumen of the endoplasmic reticulum, mto the penplasmic space or mto the extracellular environment, appropnate secretion signals may be incorporated mto the expressed polypeptide These signals may be endogenous to the polypeptide or they may be heterologous signals

Polypeptides of the mvention can be recovered and punfied 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 protem may be employed to regenerate active conformation when the polypeptide is denatured during isolation and or punfication Diagnostic, Prognostic, Serotyping and Mutation Assays

This invention is also related to the use of acpS polynucleotides and polypeptides of the mvention for use as diagnostic reagents Detection of acpS polynucleotides and/or polypeptides m a eukaryote, particularly a mammal, and especially a human, will provide a diagnostic method for diagnosis of disease, staging of disease or response of an infectious organism to drugs Eukaryotes, particularly mammals, and especially humans, particularly those infected or suspected to be infected with an organism compnsmg the acpS gene or protem, may be detected at the nucleic acid or ammo acid level by a vanety 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 an\ other amplification technique pnor to analysis RNA, particularly mRNA, cDNA and genomic DNA may also be used m the same ways Usmg amplification, characterization of the species and strain of infectious or resident organism present in an individual, may be made by an analysis of the genotype of a selected polynucleotide of the organism Deletions and insertions can be detected by a change 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 strain of the same species Pomt mutations can be identified by hybndizing amplified DNA to labeled acpS polynucleotide sequences Perfect 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 m 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 earned out with or without denaturing agents Polynucleotide differences may also be detected by direct DNA or RNA sequencmg 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 array of oligonucleotides probes compnsmg acpS 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 vanety of questions m molecular genetics including gene expression, genetic linkage, and genetic vanability (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 polvnucleotide 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 in 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 mvention also relates to the use of polynucleotides of the present mvention as diagnostic reagents Detection of a mutated form of a polynucleotide of the mvention, preferable, SEQ 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 of the polynucleotide Organisms, particularly infectious organisms, carrymg mutations in such polynucleotide may be detected at the polynucleotide level by a vanety of techniques, such as those descnbed elsewhere herem

The differences in a polvnucleotide and/or polypeptide sequence between organisms possessmg 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 m any organisms possessing the second phenotype, then the mutation is likely to be the causative agent of the first phenotype Cells from an organism carrying mutations or polymorphisms (allelic variations) in a polynucleotide and/or polypeptide of the 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 preferred 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 acpS 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 acpS 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 acpS 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 acpS 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 acpS 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 of the invention may also be used to assess the binding of small molecule substrates and ligands in, for example, cells, cell-free preparations, chemical libraries, and natural product mixtures. These substrates and ligands may be natural substrates and ligands or may be structural or functional mimetics. See, e.g., Coligan et al. Current Protocols in Immunology 1(2): Chapter 5 (1991).

Polypeptides and polynucleotides of the 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 of the mvention, as well as related polypeptides and polynucleotides In general, agomsts or antagomsts (e g , inhibitors) may be employed for therapeutic and prophylactic purposes for such Diseases as herem mentioned Compounds may be identified from a vanety of sources, for example, cells, cell-free preparations, chemical branes, and natural product mixtures Such agomsts and antagomsts so-identified may be natural or modified substrates, ligands. receptors, enzymes, etc , as the case may be, of acpS polypeptides and polynucleotides; or may be structural or functional mimetics thereof (see Coligan et al , Current Protocols in Immunology 1(2) Chapter 5 (1991))

The screenmg 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 m a signal generated by activation or inhibition of the polypeptide or polynucleotide, usmg 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 of the candidate compound is observed Constitutively active polypeptide and/or constitutively expressed polypeptides and polynucleotides may be employed in screening methods for inverse agomsts, 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 screemng 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, measurmg acpS polypeptide and/or polynucleotide activity m the mixture, and comparing the acpS polypeptide and/or polynucleotide activity of the mixture to a standard Fusion proteins, such as those made from Fc portion and acpS polypeptide, as herem descnbed, can also be used for high-throughput screenmg assays to identify antagomsts 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 bind to and/or interact with a polypeptide of the present mvention may also be used to configure screenmg 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 measurmg secreted or cell associated levels of polypeptide usmg monoclonal and polyclonal antibodies by standard methods known m the art This can be used to discover agents which may inhibit or enhance the production of polypeptide (also called antagonist or agonist, respectively) from suitably manipulated cells or tissues The mvention also provides a method of screenmg compounds to identify those which enhance (agonist) or block (antagonist) the action of acpS polypeptides or polynucleotides, particularly those compounds that are bactenstatic and/or bactencidal The method of screenmg may mvolve high-throughput techniques For example, to screen for agomsts or antagomsts. a synthetic reaction mix, a cellular compartment, such as a membrane, cell envelope or cell wall or a preparation of any thereof, compnsmg acpS polypeptide and a labeled substrate or ligand of such polypeptide is incubated m the absence or the presence of a candidate molecule that may be a acpS agomst or antagonist The ability of the candidate molecule to agonize or antagonize the acpS polypeptide is reflected m decreased bmdmg of the labeled ligand or decreased production of product from such substrate Molecules that bmd gratuitously, i e , without mducmg the effects of acpS polypeptide are most likely to be good antagomsts Molecules that bmd well and, as the case may be, mcrease the rate of product production from substrate, mcrease signal transduction, or mcrease chemical channel activity are agomsts Detection of the rate or level of, as the case may be, production of product from substrate, signal transduction or chemical channel activity may be enhanced by usmg a reporter system Reporter systems that may be useful m this regard mclude but are not limited to colonmetnc, labeled substrate converted mto product, a reporter gene that is responsive to changes m acpS polynucleotide or polypeptide activity, and bmdmg 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 bindmg techniques known m the art These techniques include, but are not limited to, ligand bmdmg and crosshnkmg assays in which the polypeptide is labeled with a radioactive isotope (for instance, ^^1), chemically modified (for instance, biotmylated), or fused to a peptide sequence suitable for detection or punfication, and mcubated with a source of the putative receptor (e g , cells, cell membranes, cell supernatants, tissue extracts, bodily matenals) Other methods mclude biophysical techniques such as surface plasmon resonance and spectroscopy These screemng methods may also be used to identify agonists and antagomsts of the polypeptide which compete with the bmdmg of the polypeptide to its receptor(s), if any Standard methods for conducting such assays are well understood m the art

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

Fluorescence energv transfer may also be used characterize small molecules that interfere with the formation of acpS polypeptide dimers, tπmers. tetramers or higher order structures, or structures formed by acpS polypeptide bound to another polypeptide acpS 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 monitor the effect of small molecules on acpS polypeptide self-association as well as an association of acpS polypeptide and another polypeptide or small molecule acpS 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 acpS polypeptide -coated surface and specific bmdmg can be detected m real-time by monitoring the change in resonance angle caused by a change in local refractive index This technique can be used to characterize the effect of small molecules on kinetic rates and equihbnum bmdmg constants for acpS polypeptide self-association as well as an association of acpS polypeptide and another polypeptide or small molecule

A scintillation proximity assay may be used to characterize the interaction between an association of acpS polypeptide with another acpS polypeptide or a different polypeptide acpS polypeptide can be coupled to a scintillation-filled bead Addition of radio-labeled acpS polypeptide results in bind g where the radioactive source molecule is m close proximity to the scintillation fluid Thus, signal is emitted upon acpS polypeptide bmdmg and compounds that prevent acpS polypeptide self-association or an association of acpS polypeptide and another polypeptide or small molecule will diminish signal In other embodiments of the mvention there are provided methods for identifying compounds which bmd 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 bemg associated with a second component capable of providing a detectable signal m response to the bmdmg or mteraction of the polypeptide and/or polynucleotide with the compound, and determining whether the compound 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 bmdmg or mteraction of the compound with the polypeptide and/or polynucleotide

Another example of an assay for acpS agomsts is a competitive assay that combmes acpS and a potential agomst with acpS-bmdmg molecules recombinant acpS bmdmg molecules, natural substrates or ligands. or substrate or ligand mimetics, under appropnate conditions for a competitive inhibition assay acpS can be labeled, such as by radioactivity or a colonmetnc compound, such that the number of acpS molecules bound to a bmdmg molecule or converted to product can be determmed 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 agomst or antagonist of the polypeptide and/or polynucleotide, by (a) determimng m the first instance the three-dimensional structure of the polypeptide and/or polynucleotide, or complexes thereof, (b) deducmg the three- dimensional structure for the likely reactive sιte(s), bmdmg sιte(s) or motιf(s) of an agonist or antagonist, (c) synthesizing candidate compounds that are predicted to bmd to or react with the deduced bmdmg sιte(s). reactive sιte(s), and/or motιf(s), and (d) testing whether the candidate compounds are indeed agonists or antagomsts

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 mvention provides methods of treatmg abnormal conditions such as. for instance, a Disease, related to either an excess of, an under-expression of, an elevated activity of, or a decreased activity of acpS polypeptide and/or polynucleotide

If the expression and/or activity of the polypeptide and/or polynucleotide is m excess, several approaches are available One approach compnses administering to an individual in 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 ligand, substrate, enzymes, receptors, etc m competition with endogenous polypeptide and/or polynucleotide may be administered Typical examples of such competitors include fragments of the acpS polypeptide and/or polypeptide In still another approach, expression of the gene encodmg endogenous acpS polypeptide can be inhibited using expression blocking techniques This blockmg 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 JNeurochem (1991) 56 560 in Ohgodeoxynucleotides as Antisense Inhibitors of Gene Expression, CRC Press, Boca Raton, FL (1988)) Alternatively, o gonucleotides 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 per se or the relevant ohgomers can be expressed in vivo

- l i Each of the polynucleotide sequences provided herein may be used m the discovery and development of antibacterial compounds The encoded protem, upon expression, can be used as a target for the screening of antibacterial drugs Additionally, the polynucleotide sequences encoding the ammo terminal regions of the encoded protein or Shme-Delgarno or other translation facilitating sequences of the respective mRNA can be used to construct antisense sequences to control the expression of the codmg sequence of interest

The invention also provides the use of the polypeptide, polynucleotide, agomst or antagonist of the invention to interfere with the initial physical mteraction between a pathogen or pathogens and a eukaryotic, preferably mammalian, host responsible for sequelae of mfection In particular, the molecules of the mvention may be used m the prevention of adhesion of bacteria, in particular gram positive and/or gram negative bacteria, to eukaryotic, preferably mammalian, extracellular matnx protems on m-d ellmg devices or to extracellular matrix proteins m wounds, to block bacterial adhesion between eukaryotic, preferably mammalian, extracellular matrix proteins and bacterial acpS protems that mediate tissue damage and/or, to block the normal progression of pathogenesis in mfections initiated other than by the implantation of m-dwellmg devices or by other surgical techniques

In accordance with yet another aspect of the mvention, there are provided acpS agomsts and antagomsts, preferably bactenstatic or bactencidal agomsts and antagomsts

The antagomsts and agomsts of the mvention may be employed, for instance, to prevent, inhibit and/or treat diseases Helicobacter pylori (herein "H pylori") bacteria mfect 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 gastnc adenocarcmoma, classifymg the bactenum as a Group I (defimte) carcinogen Preferred antimicrobial compounds of the invention (agomsts and antagomsts of acpS polypeptides and/or polynucleotides) found using screens provided by the invention, or known m the art, particularly narrow-spectrum antibiotics, should be useful m the treatment of H pylori infection Such treatment should decrease the advent of H pylori -induced cancers, such as gastrointestinal carcmoma 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 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 being fully set forth Any patent application to which this application claims priority is also incorporated by reference herein m 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 herem "Bodily mateπal(s) means any mateπal denved from an individual or from an orgamsm 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 matenals

"Dιsease(s)" means any disease caused by or related to infection by a bacteπa, including , for example, otitis media, conjunctivitis, pneumoma. bacteremia, meningitis, smusitis, pleural empyema and endocarditis, and most particularly meningitis, such as for example infection of cerebrospinal 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 determmed 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 determmed by the match between strings of such sequences "Identity" can be readily calculated by known methods, including but not limited to those described m (Computational Molecular Biology, Lesk, A M , ed , Oxford University Press, New York, 1988, Bwcomputtng 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 Hemje, G , Academic Press, 1987. and Sequence Analysis Primer, Gnbskov, M and Devereux, J . eds , M Stockton Press, New York, 1991. and Canllo, H , and Lipman, D , 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 m 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 Bwl 215 403-410 (1990) The BLAST X program is publicly available from NCBI and other sources (BLAST Manual, Altschul, S , et al , NCBI NLM 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 Algonthm 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 companson 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 compnsmg 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 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, mcludmg 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 m 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 1. 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 m 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 mteger 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 of the present invention may be identical to the reference sequence of SEQ ID NO 1, 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 consistmg of at least one nucleic acid deletion, substitution, including transition and transversion, or insertion, and wherein said alterations may occur at the 5' or 3' terminal positions of the reference polynucleotide sequence or anywhere between those terminal positions interspersed either individually among the nucleic acids m the reference sequence or in one or more contiguous groups withm 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 1 by the mteger 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

ⁿn ≤ ^xn " (^xn * y)>

wherein n_n is the number of nucleic acid alterations, x_n is the total number of nucleic acids in SEQ ID NO 1, 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 wherem any non-mteger product of x_n and y is rounded down to the nearest integer prior to subtracting it from x_n

(2) Polypeptide embodiments further mclude an isolated polypeptide compnsmg a polypeptide having at least a 95 97 or 100% identity to a polypeptide reference sequence of SEQ ID NO 2, wherem said polypeptide sequence may be identical to the reference sequence of SEQ ID NO 2 or may include up to a certain integer number of ammo acid alterations as compared to the reference sequence, wherein said alterations are selected from the group consistmg of at least one ammo acid deletion, substitution, including conservative and non-conservative substitution, or insertion, and wherein said alterations may occur at the ammo- or carboxy-terminal positions of the reference polypeptide sequence or anywhere between those terminal positions, interspersed either individually among the ammo acids in the reference sequence or m one or more contiguous groups withm the reference sequence, and wherein said number of ammo acid alterations is determined by multiplying the total number of ammo 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 ammo acids in SEQ ID NO 2, or ⁿa ≤ ^xa " (^xa * )>

wherein n_a is the number of ammo acid alterations, x_a is the total number of amino acids m SEQ ID NO 2, y is 0 95 for 95%, 0 97 for 97% or 1 00 for 100%, and • is the symbol for the multiplication operator, and wherem any non-mteger product of x_a and y is rounded down to the nearest integer pnor to subtracting it from x_a

By way of example, a polypeptide sequence of the present mvention may be identical to the reference sequence of SEQ ID NO 2. that is it may be 100% identical, or it may mclude 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-termrnal positions of the reference polypeptide sequence or anywhere between those terminal positions, interspersed either individually among the amino acids in the reference sequence or m one or more contiguous groups withm the reference sequence The number of amino acid alterations for a given % identity is determmed by multiplying the total number of amino acids m SEQ ID NO 2 by the integer defining the percent identity divided by 100 and then subtracting that product from said total number of ammo acids in SEQ ID NO 2, or

"a ^{≤ x}a (^χ _a • y)>

wherein n_a is the number of ammo acid alterations, x_a is the total number of ammo 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 "Indrvιdual(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, i e , if it occurs m nature, it has been changed or removed from its onginal environment, or both For example, a polynucleotide or a polypeptide naturally present m a living orgamsm is not "isolated," but the same polynucleotide or polypeptide separated from the coexistmg matenals of its natural state is "isolated", as the term is employed herem Moreover, a polynucleotide or polypeptide that is mtroduced mto an orgamsm by transformation, genetic manipulation or b\ any other recombinant method is "isolated" even if it is still present m said orgamsm, which organism may be Irving or non-Irving "Organιsm(s)" means a (1) prokaryote, mcludmg but not limited to, a member of the genus Streptococcus Staphylococcus, Bordetella, Corynebactenum, Mycobacterium Neisseria, Haemoph us, Actinomycetes Streptomycetes, Nocardia, Enterobacter, Yersmia, Fancisella Pasturella Moraxella Acinetobacter, Erysipelolhnx, Branhamella, Actinobacillus, Streptobacillus, Listena, Calymmatobactenum, Brucella Bacillus Clostridium Treponema, Eschenchia, Salmonella, Kleibsiella, Vibrio, Proteus, Erwinia, Borrelia Leptospira, Spirillum Campylobacter, Shigella, Legionella, Pseudomonas, Aeromonas, Rickettsia, Chlamydia Borrelia and Mycoplasma. and further mcludmg, but not limited to, a member of the species or group. Group A Streptococcus, Group B Streptococcus, Group C Streptococcus, Group D Streptococcus, Group G Streptococcus, Streptococcus pneumoniae, Streptococcus pyogenes, Streptococcus agalactiae Streptococcus faecalis, Streptococcus faecium, Streptococcus durans, Neisseria gonorrheae, Neisseria memngitidis Staphylococcus aureus, Staphylococcus epidermidis, Corynebactenum dψthenae, Gardnerella vaginahs, Mycobactenum tuberculosis, Mycobactenum bovis, Mycobactenum ulcerans, Mycobactenum leprae Actinomyctes israeln Listena monocytogenes, Bordetella pertusis, Bordatella parapertusis, Bordetella bronchiseptica Eschenchia coll Shigella dysentenae, Haemophύus mfluenzae, Haemophύus aegyptius, Haemophilus parainfluenzae, Haemoph us ducreyi, Bordetella, Salmonella typhi, Citrobacter freundn, Proteus mirabihs, Proteus vulgans, Yersmia pestis, Kleibsiella pneumoniae, Serratia marcessens, Serratia liquefaciens, Vibrio cholera, Shigella dysenteru, Shigella flexnen, Pseudomonas aemginosa, Franscisella tularensis, Brucella abortis, Bacillus anthracis, Bacillus cereus, Clostndium perfnngens, Clostridium tetani Clostndium botuhnum, Treponema palhdum, Rickettsia rickettsn and Chlamydia trachomitis (u) an archaeon, mcludmg but not limited to Archaebacter, and (in) a umcellular or filamentous eukaryote. mcludmg 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 polynbonucleotide or polydeoxynbonucleotide, which may be unmodified RNA or DNA or modified RNA or DNA "Polynucleotιde(s)" mclude, without limitation, smgle- and double-stranded DNA, DNA that is a mixture of smgle- and double-stranded regions or single-, double- and tnple-stranded regions, smgle- and double-stranded RNA, and RNA that is mixture of smgle- and double-stranded regions, hybnd molecules compnsmg DNA and RNA that may be single-stranded or. more typically, double-stranded, or tnple-stranded regions, or a mixture of smgle- and double-stranded regions In addition "polynucleotide" as used herem refers to tnple-stranded regions compnsmg RNA or DNA or both RNA and DNA The strands m 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 mvolve only a region of some of the molecules One of the molecules of a tnple-he cal region often is an ohgonucleotide As used herein, the term "polynucleotιde(s)" also mcludes DNAs or RNAs as descnbed above that compnse 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 compnsmg unusual bases, such as mos e, or modified bases, such as tntylated bases, to name just two examples, are polynucleotides as the term is used herem 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 m the art The term "polynucleotιde(s)" as it is employed herem 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, mcludmg, for example, simple and complex cells "Polynucleotιde(s)" also embraces short polynucleotides often refened to as ohgonucleotιde(s)

"Polypeptιde(s)" refers to any peptide or protem compnsmg two or more ammo acids joined to each other by peptide bonds or modified peptide bonds "Polypeptιde(s)" refers to both short chains, commonly refened to as peptides. ohgopeptides and ohgomers and to longer chains generally refened to as proteins Polypeptides may compnse ammo acids other than the 20 gene encoded ammo acids "Polypeptιde(s)" mclude 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 in 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 var ing degree at several sites m a given polypeptide Also, a given polypeptide may compnse many types of modifications Modifications can occur anywhere m a polypeptide, mcludmg the peptide backbone, the amino acid side-chains, and the ammo or carboxyl termini Modifications mclude, for example, acetylauon, acylation, ADP- nbosylation, aπudation, covalent attachment of flavin, covalent attachment of a heme moiety, covalent attachment of a nucleotide or nucleotide denvative. covalent attachment of a lipid or lipid denvative, 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, mynstoylation, oxidation, proteohtic processmg. phosphorylation, prenylation. racemization, glycosylation, lipid attachment, sulfation, gamma-carboxylation of glutamic acid residues, hydroxylation and ADP-nbosylation, selenoylation, sulfation, transfer-RNA mediated addition of ammo 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 , Posttranslauonal 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 182 626-646 (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 of the mvention mtroduced or transformed mto a host cell or host cell lysate for the production of the polynucleotides and polypeptides of the mvention

"Vanant(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 m nucleotide sequence from another, reference polynucleotide Changes in the nucleotide sequence of the vanant may or may not alter the ammo acid sequence of a polypeptide encoded by the reference polynucleotide Nucleotide changes may result in ammo acid substitutions, additions, deletions, fusion protems and truncations in the polypeptide encoded by the reference sequence, as discussed below A typical vaπant of a polypeptide differs in ammo acid sequence from another, reference polypeptide Generally differences are limited so that the sequences of the reference polypeptide and the variant are closely similar overall and, m many regions, identical A variant and reference polypeptide may differ m 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 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 vanants m which several, 5-10, 1-5, 1-3, 1-2 or 1 ammo acids are substituted, deleted, or added m any combination A vanant of a polynucleotide or polypeptide may be a naturally occurring such as an allelic vaπant, or it may be a variant that is not known to occur naturally Non-naturally occurring 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 earned out usmg standard techniques, which are well known and routine to those of skill m the art, except where otherwise descnbed m detail The examples are illustrative, but do not limit the mvention Example 1 Strain selection, Library Production and Sequencing The polynucleotide havmg a DNA sequence given m Table 1 [SEQ ID NO 1] was obtamed from a library of clones of chromosomal DNA of Streptococcus pneumoniae in E coh The sequencmg data from two or more clones comprising overlapping Streptococcus pneumoniae DNAs was used to construct the contiguous DNA sequence m 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 accordmg 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 accordmg to standard procedures DNA fragments of up to l lkbp in size are rendered blunt by treatment with exonuclease and DNA polymerase, and EcoRI linkers added Fragments are hgated mto the vector Lambda ZapII that has been cut with EcoRI. the library packaged by standard procedures and

E coh infected with the packaged library The library is amplified by standard procedures

Method 2 Total cellular DNA is partially hydrolyzed with a one or a combination of restriction enzymes appropriate to generate a senes of fragments for cloning mto 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 coh 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 ammo acid having at least 95% identity to the ammo acid sequence of SEQ ID NO 2 over the entire length of SEQ ID NO 2, (n) an isolated polypeptide comprising the ammo acid sequence of SEQ ID NO 2, (in) 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 1

2 An isolated polynucleotide selected from the group consistmg of

(I) an isolated polynucleotide compnsmg a polynucleotide sequence encodmg a polypeptide that has at least 95% identity to the ammo acid sequence of SEQ ID NO 2, over the entire length of SEQ ID NO 2. (n) an isolated polynucleotide compnsmg 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,

(m) an isolated polynucleotide compnsmg 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 compnsmg a nucleotide sequence encodmg 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 screenmg an appropnate library under strmgent hybndization conditions with a probe having the sequence of SEQ ID NO 1 or a fragment thereof

(vn) an isolated polynucleotide encodmg a mature polypeptide expressed by the acpS gene compnsed m the Streptococcus pneumoniae, and (vui) a polynucleotide sequence complementary to said isolated polynucleotide of (I), (u), (m), (iv), (v),

(vi) or (vn)

3 A method for the treatment of an individual

(l) in need of enhanced activity or expression of or mimunological 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

(n) having need to inhibit activit} or expression of the 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 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 m an mdividual compnsmg the step of

(a) determining the presence or absence of a mutation m 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 compnsmg an ammo acid sequence selected from the group havmg 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 comprising the amino acid sequence of SEQ ID NO 2.

(in) 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 1, comprising the step of cultuπng 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 consistmg of

(I) an isolated polypeptide comprising an amino acid sequence selected from the group havmg at least 95% identity to the ammo acid sequence of SEQ ID NO 2 over the entire length of SEQ ID NO 2. (n) an isolated polypeptide comprising the ammo acid sequence of SEQ ID NO 2. (in) an isolated polypeptide which is the ammo acid sequence of SEQ ID NO 2, and (iv) a polypeptide which is encoded by a recombinant polynucleotide compnsmg the polynucleotide sequence of SEQ ID NO 1, said process comprising the step of transforming or transfectmg a cell with an expression system compnsmg a polynucleotide capable of producing said polypeptide of (I), (u), (m) 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), (u), ( ) or (iv)

7 A host cell expressing a polypeptide selected from the group consistmg 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, (n) an isolated polypeptide comprising the ammo acid sequence of SEQ ID NO 2, (in) an isolated polypeptide which is the ammo acid sequence of SEQ ID NO 2, and (iv) a polypeptide which is encoded by a recombinant polynucleotide compnsing the polynucleotide sequence of SEQ ID NO 1

8 An antibody lmmunospecific for the polypeptide of claim 1

9 A method for screening to identify compounds which stimulate or which inhibit the function of the polypeptide of claim 1 which compnses a method selected from the group consistmg of

(a) measuring the bmdmg of a candidate compound to the polypeptide (or to the cells or membranes bearing the polypeptide) or a fusion protem thereof by means of a label directly or indirectly associated with the candidate compound.

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

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

(d) mixmg a candidate compound with a solution compnsmg a polypeptide of claim 1, to form a mixture, measuring activity of the polypeptide in the mixture, and comparing 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 m cells, using for instance, an ELISA assay

10 An agonist or antagonist to the polypeptide of claims 1 to 4