WO1999027073A1

WO1999027073A1 - deoD

Info

Publication number: WO1999027073A1
Application number: PCT/US1998/024797
Authority: WO
Inventors: Magdalena Zalacain; Christopher M. Traini; Richard L. Warren; James R. Brown; Karen A. Ingraham; Alison F. Chalker; David J. Holmes; Alexander P. Bryant; Andrea Marra; Sanjoy Biswas; Lisa K. Shilling
Original assignee: Smithkline Beecham Corporation
Priority date: 1997-11-20
Filing date: 1998-11-20
Publication date: 1999-06-03
Also published as: EP1032649A1; JP2003526316A

Abstract

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

Description

deoD

RELATED APPLICATIONS

This application claims benefit of US Provisional Patent Application Number 60/084,195 filed November 20. 1997

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 deoD (puπne nucleoside phosphorylase) family, as well as their variants, hereinafter referred to as "deoD," "deoD polynucleotιde(s)," and "deoD 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 cerebrospmal 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 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 pneumoniae, 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 pneumoniae infections has πsen 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 charactenze further genes and other polynucleotides sequences and their related polypeptides, as targets for drug discovery Puπne nucleotides may be derived from exogenous purmes by the so-called salvage pathways, or they may be synthesised de novo from simpler precursors The salvage pathways fulfill several functions One is to scavenge exogenous, preformed bases and nucleosides for nucleotide s}Tithesis. and another is to reutihse bases and nucleosides produced endogenoush as a result of nucleotide turnover A third is catabohc, whereby the pentose moieties of exogenous nucleosides and the amino groups of adenine compounds are made available as sources of carbon and nitrogen, respectively Adenine is converted to AMP by adenine phosphonbosyltransferase (encoded by apt) and to adenosine by puπne nucleoside phosphorylase (deoD gene) These enzymes play a key role in bacterial metabolism and therefore inhibitors of these proteins could prevent the bacterium from establishing and maintaining infection of the host and thereby have utility in anti-bacterial therapy

Clearly, there exists a need for polynucleotides and polypeptides, such as the deoD 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 antagomsts and agomsts to find ways to prevent, ameliorate or correct such infection, dysfunction and disease

SUMMARY OF THE INVENTION

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

Various changes and modifications within the spirit and scope of the disclosed mvention will become readily apparent to those skilled m the art from readmg the following descriptions and from readmg the other parts of the present disclosure

DESCRIPTION OF THE INVENTION

The mvention relates to deoD polypeptides and polynucleotides as described m greater detail below In particular, the mvention relates to polypeptides and polynucleotides of a deoD of Streptococcus pneumomae. which is related by ammo acid sequence homology to Streptococcus thermophilus deoD polypeptide The mvention relates especially to deoD havmg the nucleotide and ammo acid sequences set out m Table 1 as SEQ ID NO 1 or 3 and SEQ ID NO 2 or 4 respectively Note that sequences recited in the Sequence Listing below as "DNA" represent an exemplification of the mvention, since those of ordinary skill will recognize that such sequences can be usefully employed in polynucleotides in general, including πbopolynucleotides

TABLE 1 DeoD Polynucleotide and Polypeptide Sequences

(A) Streptococcus pneumoniae deoD polynucleotide sequence [SEQ ID NO 1] 5 ' -

ATGTCTATCCATATTGCTGCTCAGCAGGGTGAAATTGCTGATAAAATTCTTCTTCCTGGGGATCCTCTTC GTGCTAAGTTTATTGCGGAGAATTTCCTTGATGATGCTGTTTGTTTTAACGAAGTGCGTAACATGTTTGG TTACACTGGTACTTACAAGGGTCACTGTGTATCTGTCATGGGAACTGGGATGGGAATGCCATCTATTTCG ATTTATGCGCGTGAGTTAATCGTAGACTACGGTGTGAAGAAATTGATTCGTGTGGGAACTGCAGGTTCTT TGAATGAAGAGGTTCATGTTCGTGAATTAGTTTTGGCGCAGGCGGCTGCAACCAACTCAAACATCGTTCG TAATGACTGGCCACAGTACGATTTTCCACAAATTGCTAGCTTTGATTTGCTTGATAAAGCTTACCATATC GCCAAAAAACTTGGTATGACTACTCACGTTGGGAACGTTTTGTCATCTGATGTCTTTTACTCAAATTACT TTGAAAAGAATATCGAGCTTGGTAAATGGGGAGTCAAGGCTGTGGAAATGGAAGCAGCAGCTCTTTACTA TCTTGCTGCCCAATACCATGTTGATGCGCTAGCTATCATGACCATCTCTGATAGCTTGGTCAATCCAGAC GAAGACACAACTGCAGAAGAACGTCAAAATACCTTCACTGATATGATGAAGGTTGGTTTGGAAACCTTGA TTGCAGAATAA-3 '

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

NH₂-

MS IHIAAQQGEIADKI LLPGDPLRAKFIAENFLDDAVCFNEVRNMFGYTGTYKGHCVSλ GTGMGMPS I S IYARELIVDYGVKKLI RVGTAGSLNEEVHVRELVLAQAAATNSNIVRNDWPQYDFPQIAS FDLLDKAYHI AKKLGMTTHVGNVLS SDVFYSNYFEKNIELGI WGVKAVEMEAAALYYLAAQYHVDALAIMTI SDSLVNPD EDTTAEERQNTFTDMMKVGLETLIAE-COOH

(C) Streptococcus pneumoniae deoD ORF sequence [SEQ ID NO.3] 5 ' -

ATGTCTATCCATATTGCTGCTCAGCAGGGTGAAATTGCTGATAAAATTCTTCTTCCTGGGGATCCCCTTC GTGCTAAGTTTATTGCGGAGAATTTCCTTGATGATGCTGTTTGTTTTAACGAAGTGCGTAACATGTTTGG TTACACTGGTACTTACAAGGGTCACTGTGTATCTGTCATGGGAACTGGGATGGGAATGCCATCTATTTCG ATTTATGCGCGTGAGTTAATCGTAGACTACGGTGTGAAGAAATTGATTCGTGTGGGAACTGCAGGTTCTT TGAATGAAGAGGTTCATGTTCGTGAATTAGTTTTGGCGCAGGCGGCTGCAACCAACTCAAACATCGTTCG TAATGACTGGCCACAGTACGATTTTCCACAAATTGCTAGCTTTGATTTGCTTGATAAAGCTTACCATATC GCCAAAAAACTTGGTATGACTACTCACGTTGGGAACGTTTTGTCATCTGATGTCTTTTACTCAAATTACT TTGAAAAG7^ATATCGAGCTTGGTAAATGGGGAGTCAAGGCTGTGGAAATGGAAGCAGCAGCTCTTTACTA TCTTGCTGCCCAATACCATGTTGATGCGCTAGCTATCATGACCATCTCTGATAGCTTGGTCAATCCAGAC GAAGACACAACTGCAGAAGAACGTCAAAATACCTTCACTGATATGATGAAGGTTGGTTTGGAAACCTTGA TTGCAGAATAA -3'

(D) Streptococcus pneumoniae deoD polypeptide sequence deduced from a polynucleotide ORF sequence m this table [SEQ ID N0 4]

NH₂-

MSIHIAAQQGEIADKILLPGDPLRAKFIAENFLDDAVCFNEVRNMFGYTGTYKGHCVSVMGTGMGMPSIS IYARELIVDYGVKKLIRVGTAGSLNEEVHVRELVLAQAAATNSNIVRNDWPQYDFPQIASFDLLDKAYHI AKKLGMTTHVGNVLSSDVFYSNYFEKNIELGKWGVKAVEMEAAALYYLAAQYHVDALAIMTISDSLVNPD EDTTAEERQNTFTDMMKVGLETLIAE-COOH

Deposited materials A deposit containing a Streptococcus pneumoniae 0100993 stram has been deposited with the National Collections of Industrial and Marine Bactena Ltd (herem "NCIMB"), 23 St Machar Dnve, Aberdeen AB2 1RY, 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 m E coh was similarly depositedwith the NCIMB and assigned deposit number 40800 The Streptococcus pneumoniae stram deposit is refened to herem as "the deposited stram" or as "the DNA of the deposited strain "

The deposited stram contains a full length deoD gene The sequence of the polynucleotides contamed in the deposited stram. as well as the ammo acid sequence of any polypeptide encoded thereby, are controlling m the event of any conflict with any descnption of sequences herem

The deposit of the deposited stram 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 stram will be irrevocably and without restnction or condition released to the public upon the issuance of a patent The deposited stram is provided merely as convemence to those of skill m 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 denved therefrom, and no such license is hereby granted

In one aspect of the mvention there is provided an isolated nucleic acid molecule encodmg a mature polypeptide expressible by the Streptococcus pneumoniae 0100993 stram, which polypeptide is contamed m the deposited stram Further provided by the mvention are deoD polynucleotide sequences m the deposited strain, such as DNA and RNA. and ammo acid sequences encoded thereby Also provided by the mvention are deoD polypeptide and polynucleotide sequences isolated from the deposited stram

Polypeptides DeoD polypeptide of the mvention is substantially phylogenetically related to other proteins of the deoD (punne nucleoside phosphorylase) family

In one aspect of the mvention there are provided polypeptides of Streptococcus pneumoniae refened to herem as "deoD" and "deoD polypeptides" as well as biologically, diagnostically, prophylactically, clinically or therapeutically useful vanants thereof, and compositions compnsmg the same

Among the particularly prefened embodiments of the mvention are vanants of deoD polypeptide encoded by naturally occurnng alleles of the deoD gene The present invention further provides for an isolated polypeptide which (a) comprises or consists of an amino acid sequence which has at least 70% identity, preferably at least 80% identity, more preferably at least 90% identity, yet more preferably at least 95% identity, most preferably at least 97. 98 or 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 70% identity, preferably at least 80% identity, more preferably at least 90% identity, yet more preferably 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, (c) a polypeptide encoded by an isolated polynucleotide comprising or consisting of a polynucleotide sequence encodmg a polypeptide which has at least 70% identity, preferably at least 80% identity, more preferably at least 90% identity, yet more preferably 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, or (d) a polypeptide encoded by an isolated polynucleotide compnsmg or consisting of a polynucleotide sequence which has at least 70% identity, preferably at least 80% identity, more preferably at least 90% identity, yet more preferably 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 3, (e) a polypeptide encoded by an isolated polynucleotide compnsmg or consisting of a polynucleotide sequence which has at least 70% identity, preferably at least 80% identity, more preferably at least 90% identity, yet more preferably at least 95% identity, even more preferably at least 97-99%. or exact identity to SEQ ID NO 3 over the entire length of SEQ ID NO 3, or (f) a polypeptide encoded by an isolated polynucleotide compnsmg or consistmg of a polynucleotide sequence encoding a polypeptide which has at least 70% identity, preferably at least 80% identity, more preferably at least 90% identity, yet more preferably at least 95% identity, even more preferably at least 97-99% or exact identity, to the ammo acid sequence of SEQ ID NO 4, over the entire length of SEQ ID NO 4, (g) comprises or consists of an ammo acid sequence which has at least 70%> identity, preferably at least 80% identity, more preferably at least 90% identity, yet more preferably at least 95% identity, most 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 4 The polypeptides of the mvention mclude a polypeptide of Table 1 [SEQ ID NO 2 or 4] (m particular the mature polypeptide) as well as polypeptides and fragments, particularly those which have the biological activity of deoD, and also those which have at least 70% identity to a polypeptide of Table 1 [SEQ ID NO 1 or 3]or the relevant portion, preferably at least 80% identity to a polypeptide of Table 1 [SEQ ID NO 2 or 4and more preferably at least 90% identity to a polypeptide of Table 1 [SEQ ID NO 2 or 4] and still more preferably at least 95% identity to a polypeptide of Table 1 [SEQ ID NO 2 or 4] and also mclude portions of such polypeptides with such portion of the polypeptide generally containing at least 30 ammo acids and more preferably at least 50 ammo acids

The n ention also mcludes a polypeptide consistmg of or compnsmg a polypeptide of the formula

X-(R!)_m-(R₂)-(R3)_n-Y wherem. at the ammo termmus. X is hydrogen, a metal or any other moiety descnbed herem for modified polypeptides, and at the carboxyl termmus, 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 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 prefened embodiments of the mvention are provided where m is an mteger between 1 and 50, 100 or 500. and n is an mteger between 1 and 50, 100, or 500 It is most prefened that a polypeptide of the mvention is denved from Streptococcus pneumoniae. 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 havmg 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 deoD polypeptides, fragments may be "free-standing," or compπsed within a larger polypeptide of which they form a part or region, most preferably as a smgle contmuous region m a smgle larger polypeptide

Preferred fragments mclude, for example, truncation polypeptides havmg a portion of an ammo acid sequence of Table 1 [SEQ ID NO 2 or 4], or of vanants thereof, such as a contmuous senes of residues that mcludes an ammo- and or carboxyl-terminal ammo acid sequence Degradation forms of the polypeptides of the mvention produced by or m a host cell, particularly a Streptococcus pneumoniae, are also prefened Further prefened are fragments characterized by structural or functional attnbutes such as fragments that compnse alpha-hehx and alpha-he x forming regions beta-sheet and beta-sheet-formmg regions, turn and turn-forming regions, coil and coil-forming regions, hydrophiiic regions, hydrophobic regions, alpha amphipathic regions, beta amphipathic regions, flexible regions, surface-forming regions, substrate bmdmg region, and high antigenic mdex regions

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

Also prefened are biologically active fragments that are those fragments that mediate activities of deoD, mcludmg those with a similar activity or an improved activity, or with a decreased undesirable activity Also mcluded are those fragments that are antigenic or lmmunogenic m an animal, especially m a human Particularly prefened are fragments compnsmg receptors or domains of enzymes that confer a function essential for viability of Streptococcus pneumoniae or the ability to initiate, or maintain cause Disease in an mdividual, particularly a human

Fragments of the polypeptides of the mvention may be employed for producmg the conespondmg full-length polypeptide by peptide synthesis therefore, these vanants may be employed as intermediates for producmg the full-length polypeptides of the mvention In addition to the standard single and triple letter representations for ammo acids, the term "X" or "Xaa" may also be used in describing certain polypeptides of the invention "X" and "Xaa" mean that any of the twenty naturally occurring ammo acids may appear at such a designated position in the polypeptide sequence Polynucleotides It is an object of the mvention to provide polynucleotides that encode deoD polypeptides, particularly polynucleotides that encode the polypeptide herem designated deoD

In a particularly prefened embodiment of the mvention the polynucleotide compnses a region encodmg deoD polypeptides compnsmg a sequence set out m Table 1 [SEQ ID NO 1 or 3] 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 of the mvention there are provided isolated nucleic acid molecules encodmg and/or expressmg deoD polypeptides and polynucleotides, particularly Streptococcus pneumoniae deoD polypeptides and polynucleotides, mcludmg, 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, mcludmg at least one full length gene, that encodes a deoD polypeptide havmg a deduced ammo acid sequence of Table 1 [SEQ ID NO 2 or 4] and polynucleotides closely related thereto and vanants thereof

In another particularly preferred embodiment of the invention there is a deoD polypeptide from Streptococcus pneumomae comprising or consisting of an ammo acid sequence of Table 1 [SEQ ID NO 2 or 4]. or a variant thereof

Usmg the information provided herem, such as a polynucleotide sequence set out m Table 1 [SEQ ID NO 1 or 3], a polynucleotide of the mvention encodmg deoD polypeptide may be obtamed usmg standard cloning and screening methods, such as those for cloning and sequencmg chromosomal DNA fragments from bactena usmg 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 or 3], typically a library of clones of chromosomal DNA of Streptococcus pneumoniae 0100993 in E coh or some other suitable host is probed with a radiolabeled oligonucleotide, preferably a 17-mer or longer, derived from a partial sequence Clones carrying DNA identical to that of the probe can then be distinguished usmg stringent hybridization conditions By sequencing the individual clones thus identified by hybridization with sequencing primers designed from the origmal polypeptide or polynucleotide sequence it is then possible to extend the polynucleotide sequence m both directions to determine a full length gene sequence Conveniently, such sequencmg 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 Sprmg Harbor Laboratory Press, Cold Sprmg 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 mvention, each polynucleotide set out m Table 1 [SEQ ID NO 1 or 3] was discovered in a DNA library denved from Streptococcus pneumoniae 0100993

Moreover, each DNA sequence set out m Table 1 [SEQ ID NO 1 or 3] contains an open readmg frame encodmg a protem having about the number of ammo acid residues set forth m Table 1 [SEQ ID NO 2 or 4] 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 polynucleotide of SEQ ID NO 1 between nucleotide number 1 and the stop codon which begms at nucleotide number 709 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 70% identity, preferably at least 80% identity, more preferably at least 90% identity, yet more preferably at least 95% identity, even more preferably at least 97-99%, 99 5% or exact identity to SEQ ID NO 1 over the entire length of SEQ ID NO 1, (b) a polynucleotide sequence encodmg a polypeptide which has at least 70% identity, preferably at least 80% identity, more preferably at least 90% identity, yet more preferably 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, or (c) a nucleotide sequence which has at least 70% identity, preferably at least 80% identity, more preferably at least 90% identity, yet more preferably at least 95% identity, even more preferably at least 97-99%, 99 5% or 100% identity, to SEQ ID NO 1 over the entire length of SEQ ID NO 3, (d) a nucleotide sequence which has at least 70% identity, preferably at least 80% identity more preferably at least 90% identity, yet more preferably at least 95% identity, even more preferably at least 97-99%, 99 5% or exact identity to SEQ ID NO 3 over the entire length of SEQ ID NO 3, or (e) a polynucleotide sequence encodmg a polypeptide which has at least 70% identity, preferably at least 80% identity, more preferably at least 90% identity, yet more preferably at least 95%. identity, even more preferably at least 97-99%, 99 5% or exact identity, to the ammo acid sequence of SEQ ID NO 4, over the entire length of SEQ ID NO 4

A polynucleotide encodmg a polypeptide of the present mvention, mcludmg homologs and orthologs from species other than Streptococcus pneumoniae, may be obtained by a process which compnses the steps of screening an appropnate library under stnngent hybndization conditions with a labeled or detectable probe consistmg of or compnsmg the sequence of SEQ ID NO 1 or 3 or a fragment thereof, and isolating a full-length gene and or genomic clones containing 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 or 3] 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 m 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 contain at least one non-coding sequence, mcludmg 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-mdependent termination signals), nbosome bmdmg sites, Kozak sequences, sequences that stabilize mRNA, nitrons, and polyadenylation signals The polynucleotide sequence may also compnse additional codmg sequence encodmg additional ammo acids For example, a marker sequence that facilitates punfication of the fused polypeptide can be encoded In certain embodiments of the mvention. the marker sequence is a hexa-histidine peptide. as provided in the pQE vector (Qiagen, Inc ) and descnbed m Gentz et al . Proc Natl Acad Sc , 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 prefened embodiment of the mvention is a polynucleotide of consistmg of or compnsmg nucleotide 1 to the nucleotide immediately upstream of or mcludmg nucleotide 709 set forth m SEQ ID NO 1 of Table 1, both of which encode the deoD polypeptide

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

X-(R!)_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 R_] 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 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 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 R₂, 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 of the above formula is a closed, circular polynucleotide, which can be a double-stranded polynucleotide wherein the formula show s 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 prefened embodiments of the mvention are provided where m is an mteger between 1 and 50, 100 or 500. and n is an mteger between l and 50, 100. or 500

It is most prefened that a polynucleotide of the mvention is denved from Streptococcus pneumoniae. however, it may preferably be obtamed from other organisms of the same taxonomic genus A polynucleotide of the mvention may also be obtamed, for example, from orgamsms of the same taxonomic family or order

The term "polynucleotide encodmg a polypeptide" as used herem encompasses polynucleotides that mclude a sequence encodmg a polypeptide of the mvention, particularly a bactenal polypeptide and more particularly a polypeptide of the Streptococcus pneumoniae deoD havmg an ammo acid sequence set out m Table 1 [SEQ ID NO 2 or 4] The term also encompasses polynucleotides that mclude a smgle contmuous region or discontmuous regions encoding the polypeptide (for example, polynucleotides interrupted by mtegrated phage, an integrated insertion sequence, an integrated vector sequence, an mtegrated transposon sequence, or due to RNA editing or genomic DNA reorganization) together with additional regions, that also may contain 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 or 4] 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 encodmg deoD vanants, that have the ammo acid sequence of deoD polypeptide of Table 1 [SEQ ID NO 2 or 4] m which several, a few, 5 to 10, 1 to 5, 1 to 3, 2, 1 or no ammo acid residues are substituted, modified, deleted and/or added, m any combination Especially prefened among these are silent substitutions, additions and deletions, that do not alter the properties and activities of deoD polypeptide Further prefened embodiments of the mvention are polynucleotides that are at least 70% identical over their entire length to a polynucleotide encodmg deoD polypeptide havmg an ammo acid sequence set out m Table 1 [SEQ ID NO 2 or 4], and polynucleotides that are complementary to such polynucleotides Alternatively, most highly prefened are polynucleotides that compnse a region that is at least 80% identical over its entire length to a polynucleotide encodmg deoD polypeptide and polynucleotides complementary thereto In this regard, polynucleotides at least 90%> identical over their entire length to the same are particularly prefened, and among these particularly prefened polynucleotides. those with at least 95% are especially prefened Furthermore, those with at least 97% are highly prefened among those with at least 95%. and among these those with at least 98% and at least 99% are particularly highly prefened, with at least 99% bemg the more prefened

Prefened embodiments are polynucleotides encodmg polypeptides that retain substantially the same biological function or activity as the mature polypeptide encoded by a DNA of Table 1 [SEQ ID NO 1 or 3]

In accordance with certam prefened embodiments of this mvention there are provided polynucleotides that hybndize, particularly under stnngent conditions, to deoD polynucleotide sequences, such as those polynucleotides m 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 stnngent conditions to the polynucleotides descnbed herem As herem used, the terms "stnngent conditions" and "stnngent hybndization conditions" mean hybndization occurnng only if there is at least 95% and preferably at least 97% identity between the sequences A specific example of stringent hybndization conditions is overnight incubation at 42°C m a solution compnsmg 50% formamide, 5x SSC (150mM NaCl, 15mM tnsodium citrate), 50 mM sodium phosphate (pH7 6), 5x Denhardt's solution, 10%> dextran sulfate, and 20 micrograms/ml of denatured, sheared salmon sperm DNA, followed by washing the 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 therein Solution hybridization may also be used with the polynucleotide sequences provided by the invention

The mvention also provides a polynucleotide consisting of or comprising a polynucleotide sequence obtamed by screenmg an appropriate library containing the complete gene for a polynucleotide sequence set forth in SEQ ID NO 1 or 3 under stringent hybridization conditions with a probe havmg the sequence of said polynucleotide sequence set forth m SEQ ID NO 1 or 3 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 herein regarding polynucleotide assays of the invention, for instance, the polynucleotides of the invention, may be used as a hybridization probe for RNN cDNA and genomic DNA to isolate full-length cDNAs and genomic clones encoding deoD and to isolate cDNA and genomic clones of other genes that have a high identity, particularly high sequence identity, to the deoD gene. Such probes generally will comprise at least 15 nucleotide residues or base pairs. Preferably, such probes will have at least 30 nucleotide residues or base pairs and may have at least 50 nucleotide residues or base pairs. Particularly prefened probes will have at least 20 nucleotide residues or base pairs and will have lee than 30 nucleotide residues or base pairs.

A coding region of a deoD gene may be isolated by screening using a DNA sequence provided in Table 1 [SEQ ID NO:l or 3] to synthesize an oligonucleotide probe. A labeled oligonucleotide having a sequence complementary to that of a gene of the invention is then used to screen a library of cDNA, genomic DNA or mRNA to determine which members of the library the probe hybridizes to. There are several methods available and well known to those skilled in the art to obtain full-length DNAs, or extend short DNAs, for example those based on the method of Rapid Amplification of cDNA ends (RACE) (see, for example, Frohman, et al, PNAS USA 85: 8998- 9002. 1988). Recent modifications of the technique, exemplified by the Marathon™ technology (Clontech 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 ligated onto each end. Nucleic acid amplification (PCR) is then carried out to amplify the "missing" 5' end of the 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 of the 5' primer. The polynucleotides and polypeptides of the 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 ohgonucleotides derived from a sequence of

Table 1 [SEQ ID NOS 1 or 2 or 3 or 4] may be used in the processes herein as described, but preferably for PCR. to determine whether or not the polynucleotides identified herein m whole or m part are transcribed m 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 the mature protem plus additional ammo or carboxyl-terminal ammo acids, or ammo acids mtenor to the mature polypeptide (when the mature form has more than one polypeptide chain, for instance) Such sequences may play a role m processmg of a protem from precursor to a mature form, may allow protem transport, may lengthen or shorten protem half-life or may facihtate manipulation of a protem for assay or production, among other thmgs As generally is the case in vivo, the additional ammo acids may be processed away from the mature protem by cellular enzymes

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

A precursor protem, havmg a mature form of the polypeptide fused to one or more prosequences may be an inactive form of the polypeptide When prosequences are removed such mactive precursors generally are activated Some or all of the prosequences may be removed before activation Generally, such precursors are called proproteins In addition to the standard A, G, C, T/U representations for nucleotides, the term "N" may also be used in describing certain polynucleotides of the invention "N" means that any of the four DNA or RNA nucleotides may appear at such a designated position m the DNA or RNA sequence, except it is preferred that N is not a nucleic acid that when taken m combination with adjacent nucleotide positions, when read m the correct reading frame, would have the effect of generatmg a premature termination codon in such reading frame

In sum, a polynucleotide of the mvention may encode a mature protem, a mature protem plus a leader sequence (which may be refened to as a preprotem), a precursor of a mature protem havmg one or more prosequences that are not the leader sequences of a preprotem, or a preproprotem, which is a precursor to a proprotem, having 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 recombmant techmques Cell-free translation systems can also be employed to produce such proteins usmg RNAs denved from the DNA constructs of the mvention Recombmant polypeptides of the present mvention may be prepared by processes well known m those skilled m the art from genetically engmeered host cells compnsmg expression systems Accordingly, m a further aspect the present mvention relates to expression systems that compnse a polynucleotide or polynucleotides of the present mvention, to host cells which are genetically engmeered with such expression systems, and to the production of polypeptides of the mvention by recombmant 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 Sprmg Harbor, N Y (1989), such as, calcium phosphate transfection, DEAE-dextran mediated transfection, transvection, microinjection, cationic pid-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 Aspergillus, insect cells such as cells of Drosophilα S2 and Spodopterα Sf9, animal cells such as CHO, COS, HeLa, C127, 3T3, BHK, 293, CV-1 and Bowes melanoma cells, and plant cells, such as cells of a gymnosperm or angiosperm A great 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 baculovmises, papova viruses, such as SV40, vaccmia viruses, adenoviruses, fowl pox viruses, pseudorabies viruses, picomaviruses and retroviruses, and vectors denved from combmations thereof, such as those denved from plasmid and bactenophage genetic elements, such as cosmids and phagemids The expression system constructs may contain control regions that regulate as well as engender expression Generally, any system or vector suitable to maintain, propagate or express polynucleotides and or to express a polypeptide m a host may be used for expression m this regard The appropnate DNA sequence may be inserted mto the expression system by any of a vanety of well- known and routme techmques, 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 penplasrmc 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 recombmant cell cultures by well-known methods mcludmg ammonium sulfate or ethanol precipitation, acid extraction, anion or cation exchange chromatography, phosphocellulose chromatography. hydrophobic mteraction chromatograph} . affinity chromatography, hydroxylapatite chromatography, and lectm chromatography Most preferably, high performance liquid chromatography is employed for punfication Well known techmques for refoldmg 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 mvention is also related to the use of deoD polynucleotides and polypeptides of the mvention for use as diagnostic reagents Detection of deoD 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 orgamsm to drugs Eukaryotes, particularly mammals, and especially humans, particularly those infected or suspected to be infected with an organism compnsmg the deoD gene or prote , may be detected at the nucleic acid or ammo acid level by a vanety of well known techmques as well as by methods provided herem Polypeptides and polynucleotides for prognosis, diagnosis or other analysis may be obtamed from a putatively infected and or infected individual's bodily matenals Polynucleotides from any of these sources, particularly DNA or RNA. may be used directly for detection or may be amplified enzymatically by usmg PCR or any other amplification technique 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 m an mdividual, 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 stram of the same species Pomt mutations can be identified by hybndizmg amplified DNA to labeled deoD 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 detectmg differences m meltmg temperatures or renaturation kinetics Polynucleotide sequence differences may also be detected by alterations in the electrophoretic mobility of polynucleotide fragments m 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 arcay of ohgonucleotides probes compnsmg deoD nucleotide sequence or fragments thereof can be constructed to conduct efficient screening of, for example, genetic mutations, serotjpe, 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 mcludmg gene expression, genetic linkage, and genetic vanabihty (see, for example, Chee et al , Science, 274 610 (1996))

Thus m another aspect, the present invention relates to a diagnostic kit which comprises (a) a polynucleotide of the present invention, preferably the nucleotide sequence of SEQ ID NO 1 or 3, 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 4 or a fragment thereof, or (d) an antibody to a polypeptide of the present invention, preferably to the polypeptide of SEQ ID NO 2 or 4 It will be appreciated that in any such kit, (a), (b), (c) or (d) may comprise a substantial component Such a kit will be of use in diagnosing a disease or susceptibility to a Disease, among others

This mvention also relates to the use of polynucleotides of the present mvention as diagnostic reagents Detection of a mutated form of a polynucleotide of the mvention, preferable, SEQ ID NO 1 or 3, which is associated with a disease or pathogemcity 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, carrying mutations m such polynucleotide may be detected at the polynucleotide level by a vanety of techniques, such as those descnbed elsewhere herem

The nucleotide sequences of the present mvention are also valuable for orgamsm chromosome identification The sequence is specifically targeted to, and can hybndize with, a particular location on an organism's chromosome, particularly to a Streptococcus pneumoniae chromosome The mappmg of relevant sequences to chromosomes according to the present mvention may be an important step m conelatmg those sequences with pathogenic potential and/or an ecological mche of an organism and/or drug resistance of an organism, as well as the essentiality of the gene to the orgamsm Once a sequence has been mapped to a precise chromosomal location, the physical position of the sequence on the chromosome can be correlated with genetic map data Such data may be found on-lme m a sequence database The relationship between genes and diseases that have been mapped to the same chromosomal region are then identified through known genetic methods, for example, through linkage analysis (coinhentance of physically adjacent genes) or matmg studies, such as by conjugation The differences in a polynucleotide and/or polypeptide sequence between orgamsms possessing a first phenotype and organisms possessing a different, second different phenotype can also be determined If a mutation is observed 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 of the first phenotype Cells from an organism carrying mutations or polymorphisms (allehc vanations) m a polynucleotide and/or polypeptide of the mvention may also be detected at the polynucleotide or polypeptide level by a vanety of techmques, to allow for serotyping, for example For example, RT- PCR can be used to detect mutations m the RNA It is particularly prefened to use RT-PCR m conjunction with automated detection systems, such as, for example, GeneScan RNA, cDNA or genomic DNA may also be used for the same purpose, PCR As an example, PCR pnmers complementary to a polynucleotide encodmg deoD polypeptide can be used to identify and analyze mutations Examples of representative pnmers are shown below m Table 2

Table 2 Primers for amplification of deoD polynucleotides

SEQ ID NO PRIMER SEQUENCE 5 5 ' -GAGCTTGGTAAATGGGGAGTCA- 3 '

6 5 ' -ACGTTCTTCTGCAGTTGTGTCTTC-3 '

The mvention also mcludes pnmers of the formula X-(R, )_m-(R₂)-(R₃)_n-Y wherem, at the 5' end of the molecule, X is hydrogen, a metal or a modified nucleotide residue, and at the 3' end of the molecule, Y is hydrogen, a metal or a modified nucleotide residue, R_j and R3 are any nucleic acid residue or modified nucleotide residue, m is an mteger between 1 and 20 or zero , n is an mteger between 1 and 20 or zero, and R₂ is a primer sequence of the mvention, particularly a primer sequence selected from Table 2 In the polynucleotide formula above R₂ is onented so that its 5' end nucleotide residue is at the left, bound to Ri and its 3' end nucleotide residue is at the nght, bound to R3 Any stretch of nucleic acid residues denoted by either R group, where m and/or n is greater than 1. may be either a heteropolymer or a homopolymer, preferably a heteropolymer bemg complementary to a region of a polynucleotide of Table 1 In a prefened embodiment m and or n is an mteger between 1 and 10.

The mvention further provides these pnmers with 1, 2, 3 or 4 nucleotides removed from the 5' and/or the 3' end These pnmers may be used for, among other thmgs, amplifying deoD DNA and/or RNA isolated from a sample denved from an mdividual, such as a bodily matenal The pnmers may be used to amplify a polynucleotide isolated from an infected mdividual, such that the polynucleotide may then be subject to vanous techmques for elucidation of the polynucleotide sequence In this way, mutations m the polynucleotide sequence may be detected and used to diagnose and/or prognose the infection or its stage or course, or to serotype and/or classify the infectious agent

The mvention further provides a process for diagnosing, disease, preferably bactenal infections, more preferably infections caused by Streptococcus pneumoniae, comprising determining from a sample denved from an mdividual, such as a bodily material, an mcreased level of expression of polynucleotide having a sequence of Table 1 [SEQ ID NO 1 or 3] Increased or decreased expression of a deoD 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 m accordance with the mvention for detecting over- expression of deoD polypeptide compared to normal control tissue samples may be used to detect the presence of an infection, for example Assay techmques that can be used to determine levels of a deoD polypeptide. m a sample denved from a host, such as a bodily matenal. are well-known to those of skill in the art Such assay methods mclude radioimmunoassays, competitive-binding assays, Western Blot analysis, antibody sandwich assays, antibody detection and ELISA assays Differential Expression

The polynucleotides and polynucleotides of the mvention may be used as reagents for differential screening methods There are many differential screening and differential display methods known m the art m which the polynucleotides and polypeptides of the mvention may be used For example, the differential display technique is described by Chuang et al , J Bacteriol 175 2026-2036 (1993) This method identifies those genes which are expressed in an organism by identifying mRNA present using randomly-primed RT-PCR By comparing pre-infection and post infection profiles, genes up and down regulated during infection can be identified and the RT-PCR product sequenced and matched to ORF "unknowns "

In Vivo Expression Technology (IVET) is described by Camilh et al , Proc Nat'l Acad Sci USA 91 2634-2638 (1994) IVET identifies genes up-regulated during infection when compared to laboratory cultivation, implying an important role in infection ORFs identified by this technique are implied to have a significant role in infection establishment and/or maintenance In this technique random chromosomal fragments of target organism are cloned upstream of a promoter-less recombmase gene in a plasmid vector This construct is mtroduced mto the target organism which carries an antibiotic resistance gene flanked by resolvase sites Growth m the presence of the antibiotic removes from the population those fragments cloned mto the plasmid vector capable of supporting transcription of the recombmase gene and therefore have caused loss of antibiotic resistance The resistant pool is introduced mto a host and at various times after infection bacteria may be recovered and assessed for the presence of antibiotic resistance The chromosomal fragment carried by each antibiotic sensitive bacterium should carry a promoter or portion of a gene normally upregulated durmg infection Sequencmg upstream of the recombmase gene allows identification of the up regulated gene

RT-PCR may also be used to analyze gene expression patterns For RT PCR using the polynucleotides of the invention, messenger RNA is isolated from bacterial infected tissue, e g , 48 hour murine lung infections, and the amount of each mRNA species assessed by reverse transcription of the RNA sample primed with random hexanucleotides followed by PCR with gene specific primer pairs The determination of the presence and amount of a particular mRNA species by quantification of the resultant PCR product provides information on the bacterial genes which are transcribed m the infected tissue Analysis of gene transcription can be carried out at different times of infection to gain a detailed knowledge of gene regulation in bacterial pathogenesis allowing for a clearer understanding of which gene products represent targets for screens for antibactenals Because of the gene specific nature of the PCR primers employed it should be understood that the bacterial mRNA preparation need not be free of mammalian RNA This allows the investigator to carry out a simple and quick RNA preparation from infected tissue to obtain bacterial mRNA species which are very short lived m the bacterium ( the order of 2 mmute halflives) Optimally the bacterial mRNA is prepared from infected murine lung tissue by mechanical disruption in the presence of TRIzole (GIBCO-BRL) for very short periods of time, subsequent processing according to the manufacturers of TRIzole reagent and DNAase treatment to remove contaminating DNA Preferably the process is optimized by finding those conditions which give a maximum amount of Streptococcus pneumoniae 16S nbosomal RNA as detected by probing Northerns with a suitably labeled sequence specific oligonucleotide probe Typically a 5' dye labeled primer is used in each PCR primer pair m a PCR reaction which is terminated optimally between 8 and 25 cycles The PCR products are separated on 6% polyacrylamide gels with detection and quantification usmg GeneScanner (manufactured by ABI)

Gridding and Polynucleotide Subtraction Methods have been described for obtaining information about gene expression and identity using so called "high density DNA arrays" or grids See, e g , M Chee et al , Science, 274 610-614 ( 1996) and other references cited therein Such gπddmg assays have been employed to identify certain novel gene sequences, referred to as Expressed Sequence Tags (EST) (Adams et a , Science, 252 1651-1656 (1991)) A vanety of techmques have also been described for identifying particular gene sequences on the basis of their gene products For example, see International Patent Application No WO91/07087, published May 30, 1991 In addition, methods have been descnbed for the amplification of desired sequences For example, see International Patent Application No W091/17271, published November 14, 1991 The polynucleotides of the invention may be used as components of polynucleotide arrays, preferably high density arrays or grids These high density arrays are particularly useful for diagnostic and prognostic purposes For example, a set of spots each comprising a different gene, and further comprising a polynucleotide or polynucleotides of the mvention, may be used for probing, such as using hybridization or nucleic acid amplification, using a probes obtained or derived from a bodily sample, to determine the presence of a particular polynucleotide sequence or related sequence m an individual Such a presence may indicate the presence of a pathogen, particularly Streptococcus pneumoniae, and may be useful m diagnosing and/or prognosing disease or a course of disease A grid comprising a number of variants of the polynucleotide sequence of SEQ ID NO 1 or 3 are preferred Also preferred is a comprising a number of variants of a polynucleotide sequence encodmg the polypeptide sequence of SEQ ID NO 2 or 4 Antibodies

The polypeptides and polynucleotides of the mvention or vanants thereof, or cells expressmg the same can be used as immunogens to produce antibodies immunospecific for such polypeptides or polynucleotides respectively

In certam prefened embodiments of the mvention there are provided antibodies against deoD polypeptides or polynucleotides

Antibodies generated against the polypeptides or polynucleotides of the mvention can be obtamed by administering the polypeptides and or polynucleotides of the mvention, or epitope-beaπng fragments of either or both, analogues of either or both, or cells expressmg either or both, to an ammal, preferably a nonhuman, usmg routme protocols For preparation of monoclonal antibodies, any techmque known m the art that provides antibodies produced by contmuous cell line cultures can be used Examples mclude vanous techmques, such as those in Kohler, G and Milstem, C , Nature 256 495-497 (1975), Kozbor et al , Immunology Today 4 72 (1983), Cole et al , pg 77-96 m MONOCLONAL ANTIBODIES AND CANCER THERAPY. Alan R Liss, Inc (1985)

Techmques for the production of smgle cham antibodies (U S Patent No 4,946,778) can be adapted to produce smgle cham antibodies to polypeptides or polynucleotides of this mvention Also, transgemc mice, or other orgamsms such as other mammals, may be used to express humanized antibodies immunospecific to the polypeptides or polynucleotides of the mvention

Alternatively, phage display technology may be utilized to select antibody genes with binding activities towards a polypeptide of the invention either from repertoires of PCR amplified v-genes of lymphocytes from humans screened for possessmg anti-deoD or from naive libraries (McCafferty. et al , (1990), Nature 348, 552-554, Marks, et al , (1992) Biotechnology 10, 779- 783) The affinity of these antibodies can also be improved by, for example, chain shuffling (Clackson et al . (1991) Nature 352 628)

The above-descnbed antibodies may be employed to isolate or to identify clones expressmg the polypeptides or polynucleotides of the mvention to punfy the polypeptides or polynucleotides by. for example, affinity chromatography

Thus, among others, antibodies against deoD-polypeptide or deoD-polynucleotide may be employed to treat infections, particularly bactenal infections

Polypeptide variants include antigemcally, epitopically or lmmunologically equivalent variants form a particular aspect of this invention A polypeptide or polynucleotide of the invention, such as an antigemcally or lmmunologically equivalent derivative or a fusion protein of the polypeptide is used as an antigen to immunize a mouse or other animal such as a rat or chicken The fusion protein may provide stability to the polypeptide The antigen may be associated, for example by conjugation, with an lmmunogenic carrier protein for example bovme serum albumin, keyhole limpet haemocyamn or tetanus toxoid Alternatively, a multiple antigenic polypeptide comprising multiple copies of the polypeptide, or an antigemcally or lmmunologically equivalent polypeptide thereof may be sufficiently antigenic to improve lmmunogenicity so as to obviate the use of a carrier

Preferably, the antibody or variant thereof is modified to make it less lmmunogenic in the individual For example, if the individual is human the antibody may most preferably be "humanized," where the complimentanty determining region or regions of the hybπdoma-denved antibody has been transplanted mto a human monoclonal antibody, for example as described in ones et al (1986), Nature 321, 522-525 or Tempest et al , (1991) Biotechnology 9, 266-273

In accordance with an aspect of the mvention, there is provided the use of a polynucleotide of the mvention for therapeutic or prophylactic purposes, m particular genetic immunization Among the particularly preferred embodiments of the mvention are naturally occurnng alle c vanants of deoD polynucleotides and polypeptides encoded thereby

The use of a polynucleotide of the invention in genetic immunization will preferably employ a suitable delivery method such as direct injection of plasmid DNA mto muscles (Wolff et al , Hum Mol Genet (1992) 1 363. Manthorpe et al , Hum Gene Ther (1983) 4 419), delivery of DNA complexed with specific protein carriers (Wu et al , J Biol Chem (1989) 264 16985), coprecipitation of DNA with calcium phosphate (Benvemsty & Reshef, PNAS USA, (1986) 83 9551), encapsulation of DNA in various forms of hposomes (Kaneda et al , Science (1989) 243 375), particle bombardment (Tang et al , Nature (1992) 356 152, Eisenbraun et al , DNA Cell Biol (1993) 12 791) and in vivo infection using cloned retroviral vectors (Seeger et al , PNAS USA (1984) 81 5849)

Antagonists and Agonists - Assays and Molecules Polypeptides and polynucleotides of the mvention may also be used to assess the bmdmg of small molecule substrates and hgands m, for example, cells, cell-free preparations, chemical branes. and natural product mixtures These substrates and hgands may be natural substrates and hgands or may be structural or functional mimetics See, e g , Co gan et al , Current Protocols in Immunology 1(2) Chapter 5 (1991) Polypeptides and polynucleotides of the present mvention are responsible for many biological functions, mcludmg many disease states, m particular the Diseases herembefore 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, m a further aspect the present mvention 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 may be employed for therapeutic and prophylactic purposes for such Diseases as herembefore mentioned Compounds may be identified from a vanety of sources, for example, cells, cell-free preparations, chemical hbranes, and natural product mixtures Such agomsts, antagomsts or inhibitors so-identified may be natural or modified substrates, hgands, receptors, enzymes, etc , as the case may be, of deoD polypeptides and polynucleotides. or may be structural or functional mimetics thereof (see Cohgan et al , Current Protocols in Immunology 1(2) Chapter 5 (1991))

The screening methods may simply measure the bmdmg of a candidate compound to the polypeptide or polynucleotide, or to cells or membranes bearing the polypeptide or polynucleotide, or a fusion protem of the polypeptide by means of a label directly or indirectly associated with the candidate compound Alternatively, the screemng method may involve competition with a labeled competitor Further, these screening methods may test whether the candidate compound results in a signal generated by activation or inhibition of the polypeptide or polynucleotide. using detection systems appropriate to the cells comprising the polypeptide or polynucleotide Inhibitors of activation are generally assayed m the presence of a known agonist and the effect on activation by the agomst by the presence of the candidate compound is observed Constitutively active polypeptide and/or constitutively expressed polypeptides and polynucleotides may be employed in screening methods for inverse agonists or inhibitors, m the absence of an agonist or inhibitor, by testing whether the candidate compound results in inhibition of activation of the polypeptide or polynucleotide, as the case may be Further, the screemng methods may simply comprise the steps of mixing a candidate compound with a solution containing a polypeptide or polynucleotide of the present invention, to form a mixture, measuring deoD polypeptide and/or polynucleotide activity in the mixture, and comparing the deoD polypeptide and/or polynucleotide activity of the mixture to a standard Fusion protems, such as those made from Fc portion and deoD polypeptide, as hereinbefore described, can also be used for high-throughput screemng assays to identify antagonists of the polypeptide of the present invention, as ell as of phylogenetically and and/or functionally related polypeptides (see D Bennett et al . I Mol Recognition, 8 52-58 (1995), and K ohanson et al , I Biol Chem, 270(16) 9459-9471 (1995))

The polynucleotides, polypeptides and antibodies that bind to and/or interact with a polypeptide of the present invention may also be used to configure screening methods for detecting the effect of added compounds on the production of mRNA and or polypeptide m cells For example, an ELISA assay may be constructed for measunng secreted or cell associated levels of polypeptide using monoclonal and polyclonal antibodies by standard methods known in the art This can be used to discover agents 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

(agomst) or block (antagomst) the action of deoD polypeptides or polynucleotides, particularly those compounds that are bactenstatic and/or bactencidal The method of screening may volve high- throughput techmques 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 deoD polypeptide and a labeled substrate or hgand of such polypeptide is mcubated m the absence or the presence of a candidate molecule that may be a deoD agomst or antagomst The ability of the candidate molecule to agonize or antagonize the deoD 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 deoD 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 deoD 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 binding techniques known in the art These techniques include, but are not limited to, ligand bmdmg and crosshnking assays m which the polypeptide is labeled with a radioactive isotope (for instance, ^^1), chemically modified (for mstance. biotmylated), or fused to a peptide sequence suitable for detection or purification, and incubated with a source of the putative receptor (e g , cells, cell membranes, cell supernatants. tissue extracts, bodily materials) Other methods include biophysical techniques such as surface plasmon resonance and spectroscopy These screening methods may also be used to identify agonists and antagonists of the polypeptide which compete with the binding of the polypeptide to its receptor(s), if any Standard methods for conducting such assays are well understood m the art

The fluorescence polanzation value for a fluorescently-tagged molecule depends on the rotational correlation time or tumbling rate Protem complexes, such as formed by deoD polypeptide associating with another deoD 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 energy transfer may also be used characterize small molecules that interfere with the formation of deoD polypeptide dimers, tamers, tetramers or higher order structures, or structures formed by deoD polypeptide bound to another polypeptide DeoD polypeptide can be labeled with both a donor and acceptor fluorophore Upon mixing of the two labeled species and excitation of the donor fluorophore, fluorescence energy transfer can be detected by observing fluorescence of the acceptor Compounds that block dimeπzation will inhibit fluorescence energy transfer

Surface plasmon resonance can be used to momtor the effect of small molecules on deoD polypeptide self-association as well as an association of deoD polypeptide and another polypeptide or small molecule deoD 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 deoD polypeptide -coated surface and specific binding can be detected in realtime by monitoring the change in resonance angle caused by a change in local refractive index This technique can be used to characterize the effect of small molecules on kinetic rates and equilibrium bmdmg constants for deoD polypeptide self-association as well as an association of deoD polypeptide and another polypeptide or small molecule

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

ICS biosensors have been described by AMBRI (Australian Membrane Biotechnology Research Institute) They couple the self-association of macromolecules to the closing of gramacidin-facihtated ion channels in suspended membrane bilayers and hence to a measurable change m the admittance (similar to impedance) of the biosensor This approach is linear over six decades of admittance change and is ideally suited for large scale, high through-put screening of small molecule combinatorial hbranes

In other embodiments of the mvention there are provided methods for identifying compounds which bmd to or otherwise mteract with and inhibit or activate an activity or expression of a polypeptide and/or polynucleotide of the mvention compnsmg contacting a polypeptide and/or polynucleotide of the mvention with a compound to be screened under conditions to permit bmdmg to or other mteraction between the compound and the polypeptide and or polynucleotide to assess the bmdmg to or other mteraction with the compound, such bmdmg or mteraction preferably bemg associated with a second component capable of providmg a detectable signal in response to the bmdmg or mteraction of the polypeptide and/or polynucleotide with the compound, and determinmg whether the compound bmds to or otherwise mteracts 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 bmdmg or mteraction of the compound with the polypeptide and/or polynucleotide Another example of an assay for deoD agomsts is a competitive assay that combmes deoD and a potential agomst with deoD-binding molecules, recombinant deoD bmdmg molecules, natural substrates or hgands, or substrate or ligand mimetics, under appropnate conditions for a competitive inhibition assay deoD can be labeled, such as by radioactivity or a colonmetnc compound, such that the number of deoD molecules bound to a bmdmg molecule or converted to product can be determined accurately to assess the effectiveness of the potential antagomst

Potential antagomsts mclude. among others, small orgamc molecules, peptides, polypeptides and antibodies that bmd to a polynucleotide and/or polypeptide of the mvention and thereby inhibit or extinguish its activity or expression Potential antagomsts also may be small orgamc molecules, a peptide, a polypeptide such as a closely related protem or antibody that bmds the same sites on a bmdmg molecule, such as a bmdmg molecule, without mducmg deoD-mduced activities, thereby preventmg the action or expression of deoD polypeptides and/or polynucleotides by excluding deoD polypeptides and/or polynucleotides from bmdmg

Potential antagomsts mclude a small molecule that bmds to and occupies the bmdmg site of the polypeptide thereby preventmg bmdmg to cellular bmdmg molecules, such that normal biological activity is prevented Examples of small molecules mclude but are not limited to small orgamc molecules, peptides or peptide-hke molecules Other potential antagomsts mclude antisense molecules (see Okano, J Neurochem 56 560 (1991), OLIGODEOXYNUCLEOTIDES AS ANTISENSE INHIBITORS OF GENE EXPRESSION, CRC Press, Boca Raton, FL (1988), for a descnption of these molecules) Preferred potential antagomsts mclude compounds related to and vanants of deoD

Other examples of potential polypeptide antagomsts mclude antibodies or, m some cases, ohgonucleotides or proteins which are closely related to the hgands, substrates, receptors, enzymes, etc , as the case may be, of the polypeptide, e g , a fragment of the hgands, substrates, receptors, enzymes, etc , or small molecules which bmd to the polypeptide of the present mvention but do not elicit a response, so that the activity of the polypeptide is prevented

Certam of the polypeptides of the mvention are biomimetics, functional mimetics of the natural deoD polypeptide These functional mimetics may be used for, among other thmgs, antagonizing the activity of deoD polypeptide or as a antigen or immunogen m a manner descnbed elsewhere herem Functional mimetics of the polypeptides of the mvention mclude but are not limited to truncated polypeptides For example, prefened functional mimetics mclude, a polypeptide compnsmg the polypeptide sequence set forth m SEQ ID NO 2 lacking 20, 30, 40, 50, 60, 70 or 80 ammo- or carboxy-terminal ammo acid residues, mcludmg fusion proteins compnsmg one or more of these truncated sequences Polynucleotides encoding each of these functional mimetics may be used as expression cassettes to express each mimetic polypeptide It is prefened that these cassettes compnse 5' and 3' restnction sites to allow for a convement means to gate the cassettes together when desired It is further prefened that these cassettes compnse gene expression signals known m the art or descnbed elsewhere herem

Thus, in another aspect, the present invention relates to a screening kit for identifying agonists, antagonists, hgands, receptors, substrates, enzymes, etc for a polypeptide and/or polynucleotide of the present invention, or compounds which decrease or enhance the production of such polypeptides and/or polynucleotides , which comprises (a) a polypeptide and/or a polynucleotide of the present invention, (b) a recombinant cell expressing a polypeptide and/or polynucleotide of the present invention, (c) a cell membrane expressing a polypeptide and/or polynucleotide of the present invention, or (d) antibody to a polypeptide and or polynucleotide of the present invention, which polypeptide is preferably that of SEQ ID NO 2, and which polynucleotide is preferably that of SEQ ID NO 1

It will be appreciated that in any such kit, (a), (b), (c) or (d) may comprise a substantial component It will be readily appreciated by the skilled artisan that a polypeptide and/or polynucleotide of the present invention may also be used m a method for the structure-based design of an agonist, antagomst or inhibitor of the polypeptide and/or polynucleotide, by (a) determining in the first instance the three-dimensional structure of the polypeptide and/or polynucleotide. or complexes thereof, (b) deducing the three-dimensional structure for the likely reactive sιte(s). binding sιte(s) or motιf(s) of an agomst, antagomst or inhibitor, (c) synthesizing candidate compounds that are predicted to bind 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, antagonists or inhibitors It will be further appreciated that this will normally be an iterative process, and this iterative process may be performed usmg automated and computer-controlled steps

In a further aspect, the present 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 deoD 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 admmistermg to an mdividual m need thereof an inhibitor compound (antagomst) 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 blocking the bmdmg of hgands. substrates, receptors, enzymes, etc . or by inhibiting a second signal, and thereby alleviating the abnormal condition In another approach, soluble forms of the polypeptides still capable of 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 of the deoD polypeptide and/or polypeptide

In a further aspect, the present invention relates to genetically engineered soluble fusion proteins comprising a polypeptide of the present invention, or a fragment thereof, and various portions of the constant regions of heavy or light chains of immunoglobulins of vanous subclasses (IgG. IgM. IgA, IgE) Preferred as an lmmunoglobulm is the constant part of the heavy chain of human IgG, particularly IgGl, where fusion takes place at the lunge region In a particular embodiment, the Fc part can be removed simply by incorporation of a cleavage sequence which can be cleaved with blood clotting factor Xa Furthermore, this invention relates to processes for the preparation of these fusion proteins by genetic engineering, and to the use thereof for drug screemng, diagnosis and therapy A further aspect of the invention also relates to polynucleotides encoding such fusion proteins Examples of fusion protem technology can be found in International Patent Application Nos W094/29458 and W094/22914

In still another approach, expression of the gene encoding endogenous deoD polypeptide can be inhibited usmg expression blocking techniques This blocking may be targeted aga st 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 Ohgodeoxynucleotides 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 oligomers can be administered per se or the relevant oligomers 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 screemng of antibacterial drugs Additionally, the polynucleotide sequences encoding the amino 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 of the coding sequence of interest

The invention also provides the use of the polypeptide, polynucleotide, agonist or antagonist of the 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 m-dwelling devices or to extracellular matrix protems m wounds, to block bacterial adhesion between eukaryotic, preferably mammalian, extracellular matrix proteins and bacterial deoD protems that mediate tissue damage and/or, to block the normal progression of pathogenesis in infections 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 deoD 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

Hehcobacter pylori (herein "H pylori") 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) Sch stosomes, Liver Flukes and Hehcobacter Pylori (International Agency for Research on Cancer, Lyon, France, http //www uicc ch/ecp/ecp2904 htm) Moreover, the International Agency for Research on Cancer recently recognized a cause-and- effect relationship between H pylori and gastric adenocarcmoma, classifying the bacterium as a Group I (definite) carcmogen Preferred antimicrobial compounds of the invention (agonists and antagomsts of deoD 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 /τy/orz -induced cancers, such as gastrointestinal carcinoma Such treatment should also prevent, inhibit and/or cure gastric ulcers and gastntis Vaccines There are provided by the mvention, products, compositions and methods for assessmg deoD expression, treatmg disease, assaying genetic vanation. and administering a deoD polypeptide and or polynucleotide to an orgamsm to raise an immunological response against a bactena, especially a Streptococcus pneumoniae bactena

Another aspect of the invention relates to a method for inducing an immunological response in an individual, particularly a mammal which comprises inoculating the individual with deoD polynucleotide and/or polypeptide, or a fragment or variant thereof, adequate to produce antibody and or T cell immune response to protect said individual from infection, particularly bacterial infection and most particularly Streptococcus pneumoniae infection Also provided are methods whereby such immunological response slows bacterial replication Yet another aspect of the invention relates to a method of mducmg immunological response m an individual which comprises delivering to such individual a nucleic acid vector, sequence or ribozyme to direct expression of deoD polynucleotide and or polypeptide, or a fragment or a variant thereof, for expressing deoD polynucleotide and/or polypeptide, or a fragment or a variant thereof in vivo order to induce an immunological response, such as, to produce antibody and/ or T cell immune response, including, for example, cytokine-producing T cells or cytotoxic T cells, to protect said individual, preferably a human, from disease, whether that disease is already established within the individual or not One example of administering the gene is by accelerating it into the desired cells as a coating on particles or otherwise Such nucleic acid vector may comprise DNA, RNA, a ribozyme, a modified nucleic acid, a DNA/RNA hybrid, a DNA-protein complex or an RNA- protem complex A further aspect of the invention relates to an immunological composition that when introduced into an individual, preferably a human, capable of having induced within it an immunological response, induces an immunological response m such individual to a deoD polynucleotide and/or polypeptide encoded therefrom, wherein the composition comprises a recombinant deoD polynucleotide and/or polypeptide encoded therefrom and/or comprises DNA and/or RNA which encodes and expresses an antigen of said deoD polynucleotide, polypeptide encoded therefrom, or other polypeptide of the invention The immunological response may be used therapeutically or prophylactically and may take the form of antibody immunity and/or cellular immunity, such as cellular immunity ansmg from CTL or CD4+ T cells

A deoD polypeptide or a fragment thereof may be fused with co-protem or chemical moiety which may or may not by itself produce antibodies, but which is capable of stabilizing the first protein and producing a fused or modified protein which will have antigenic and/or lmmunogenic properties, and preferably protective properties Thus fused recombinant protem. preferably further comprises an antigenic co-protem, such as hpoprotem D from Hemophilus influenzae, Glutathione-S-transferase (GST) or beta-galactosidase, or any other relatively large co-protein which solubihzes the protem and facilitates production and purification thereof Moreover, the co-protem may act as an adjuvant in the sense of providmg a generalized stimulation of the immune system of the organism receiving the protein The co-protem may be attached to either the ammo- or carboxy-terminus of the first protem

Provided by this invention are compositions, particularly vaccine compositions, and methods comprising the polypeptides and/or polynucleotides of the invention and lmmunostimulatory DNA sequences, such as those described in Sato, Y et al Science 273 352 (1996)

Also, provided by this invention are methods using the described polynucleotide or particular fragments thereof, which have been shown to encode non-variable regions of bacterial cell surface proteins polynucleotide constructs used in such genetic immunization experiments in animal models of infection with Streptococcus pneumoniae Such experiments will be particularly useful for identifying protem epitopes able to provoke a prophylactic or therapeutic immune response It is believed that this approach will allow for the subsequent preparation of monoclonal antibodies of particular value, derived from the requisite organ of the animal successfully resisting or clearing infection, for the development of prophylactic agents or therapeutic treatments of bacterial infection, particularly Streptococcus pneumoniae infection, in mammals, particularly humans

A polypeptide of the invention may be used as an antigen for vaccination of a host to produce specific antibodies which protect against invasion of bacteria, for example by blocking adherence of bacteria to damaged tissue Examples of tissue damage include wounds m skin or connective tissue caused, for example, by mechamcal, chemical, thermal or radiation damage or by implantation of indwelling devices, or wounds m the mucous membranes, such as the mouth, throat, mammary glands, urethra or vagma

The invention also includes a vaccme formulation which comprises an lmmunogenic recombmant polypeptide and/or polynucleotide of the invention together with a suitable carrier, such as a pharmaceutically acceptable carrier Since the polypeptides and polynucleotides may be broken down in the stomach, each is preferably administered parenterally, including, for example, administration that is subcutaneous, intramuscular, intravenous, or mtradermal Formulations suitable for parenteral administration include aqueous and non-aqueous sterile injection solutions which may contain anti-oxidants, buffers, bacteπstatic compounds and solutes which render the formulation isotonic with the bodily fluid, preferably the blood, of the individual, and aqueous and non-aqueous sterile suspensions which may include suspending agents or thickening agents The formulations may be presented in unit-dose or multi-dose containers, for example, sealed ampoules and vials and may be stored in a freeze-dned condition requiring only the addition of the sterile liquid carrier immediately prior to use The vaccme formulation may also include adjuvant systems for enhancing the lmmunogenicity of the formulation, such as oil-in water systems and other systems known m the art The dosage will depend on the specific activity of the vaccme and can be readily determined by routme experimentation

While the invention has been described with reference to certain deoD polypeptides and polynucleotides. it is to be understood that this covers fragments of the naturally occurring polypeptides and polynucleotides, and similar polypeptides and polynucleotides with additions, deletions or substitutions which do not substantially affect the lmmunogenic properties of the recombmant polypeptides or polynucleotides Compositions, kits and administration

In a further aspect of the mvention there are provided compositions compnsmg a deoD polynucleotide and or a deoD polypeptide for administration to a cell or to a multicellular orgamsm

The mvention also relates to compositions compnsmg a polynucleotide and/or a polypeptides discussed herem or their agomsts or antagomsts The polypeptides and polynucleotides of the mvention may be employed m combmation with a non-stenle or stenle earner or earners for use with cells, tissues or orgamsms, such as a pharmaceutical earner suitable for administration to an mdividual Such compositions compnse, for instance, a media additive or a therapeutically effective amount of a polypeptide and or polynucleotide of the mvention and a pharmaceutically acceptable earner or excipient Such earners may mclude, but are not lirmted to, salme, buffered salme, dextrose, water, glycerol. ethanol and combinations thereof The formulation should suit the mode of administration The mvention further relates to diagnostic and pharmaceutical packs and kits compnsmg one or more containers filled with one or more of the ingredients of the aforementioned compositions of the mvention Polypeptides, polynucleotides and other compounds of the mvention may be employed alone or m conjunction with other compounds, such as therapeutic compounds The pharmaceutical compositions may be administered m any effective, convement manner mcludmg, for instance, administration by topical, oral, anal, vagmal, mtravenous, mtrapentoneal. intramuscular, subcutaneous, mtranasal or mtradermal routes among others

In therapy or as a prophylactic, the active agent may be administered to an individual as an injectable composition, for example as a sterile aqueous dispersion, preferably isotonic Alternatively the composition may be formulated for topical application for example m the form of ointments, creams, lotions, eye omtments, eye drops, ear drops, mouthwash, impregnated dressings and sutures and aerosols, and may contain appropriate conventional additives, including, for example, preservatives, solvents to assist drug penetration, and emollients in ointments and creams Such topical formulations may also contain compatible conventional carriers, for example cream or ointment bases, and ethanol or oleyl alcohol for lotions Such carriers may constitute from about 1% to about 98% by weight of the formulation, more usually they will constitute up to about 80% by weight of the formulation

In a further aspect, the present mvention provides for pharmaceutical compositions compnsmg a therapeutically effective amount of a polypeptide and/or polynucleotide, such as the soluble form of a polypeptide and or polynucleotide of the present mvention, agomst or antagomst peptide or small molecule compound, m combmation with a pharmaceutically acceptable earner or excipient Such earners mclude. but are not limited to, salme, buffered salme, dextrose, water, glycerol, ethanol, and combmations thereof The mvention further relates to pharmaceutical packs and kits compnsmg one or more contamers filled with one or more of the mgredients of the aforementioned compositions of the mvention Polypeptides, polynucleotides and other compounds of the present mvention may be employed alone or m conjunction with other compounds, such as therapeutic compounds

The composition will be adapted to the route of administration, for instance by a systemic or an oral route Prefened forms of systemic administration mclude injection, typically by mtravenous injection Other injection routes, such as subcutaneous, intramuscular, or mtrapentoneal, can be used Alternative means for systemic administration mclude transmucosal and transdermal administration usmg penetrants such as bile salts or fusidic acids or other detergents In addition, if a polypeptide or other compounds of the present mvention can be formulated m an entenc or an encapsulated formulation, oral administration may also be possible Administration of these compounds may also be topical and or localized, m the form of salves, pastes, gels, and the like

For administration to mammals, and particularly humans, it is expected that the daily dosage level of the active agent will be from 0 01 mg/kg to 10 mg/kg, typically around 1 mg/kg The physician in any event will determine the actual dosage which will be most suitable for an individual and will vary with the age, weight and response of the particular individual The above dosages are exemplar}' of the average case There can, of course, be individual instances where higher or lower dosage ranges are merited, and such are withm the scope of this invention In-dwellmg devices include surgical implants, prosthetic devices and catheters, 1 e . devices that are introduced to the body of an individual and remain m position for an extended time Such devices include, for example, artificial joints, heart valves, pacemakers, vascular grafts, vascular catheters, cerebrospmal fluid shunts, urinary catheters, and continuous ambulatory peritoneal dialysis (CAPD) catheters The composition of the invention may be administered by injection to achieve a systemic effect against relevant bacteria shortly before insertion of an in-dwellmg device Treatment may be continued after surgery during the m-body time of the device In addition, the composition could also be used to broaden preoperative cover for any surgical techmque to prevent bacterial wound infections, especially Streptococcus pneumoniae wound infections Many orthopedic surgeons consider that humans with prosthetic jo ts should be considered for antibiotic prophylaxis before dental treatment that could produce a bacteremia Late deep infection is a senous complication sometimes leadmg to loss of the prosthetic jomt and is accompanied by significant morbidity and mortality It may therefore be possible to extend the use of the active agent as a replacement for prophylactic antibiotics m this situation In addition to the therapy described above, the compositions of this mvention may be used generally as a wound treatment agent to prevent adhesion of bacteria to matrix protems exposed in wound tissue and for prophylactic use in dental treatment as an alternative to, or in conjunction with, antibiotic prophylaxis

Alternatively, the composition of the invention may be used to bathe an indwelling device immediately before msertion The active agent will preferably be present at a concentration of 1 μ g/ml to lOmg/ml for bathing of wounds or indwelling devices

A vaccine composition is conveniently in mjectable form Conventional adjuvants may be employed to enhance the immune response A suitable unit dose for vaccmation is 0 5-5 microgram kg of antigen, and such dose is preferably administered 1 -3 times and with an interval of 1-3 weeks With the indicated dose range, no adverse toxicological effects will be observed with the compounds of the mvention which would preclude their administration to suitable individuals Sequence Databases, Sequences in a Tangible Medium, and Algorithms

Polynucleotide and polypeptide sequences form a valuable information resource with which to determine then: 2- and 3-dιmensιonal structures as well as to identify further sequences of similar homology These approaches are most easily facilitated by storing the sequence m a computer readable medium and then usmg the stored data m a known macromolecular structure program or to search a sequence database usmg well known searching tools, such as GCC

The polynucleotide and polypeptide sequences of the invention are particularly useful as components in databases useful for search analyses as well as m sequence analysis algorithms As used in this section entitled "Sequence Databases, Sequences in a Tangible Medium, and Algorithms," and in claims related to this section, the terms "polynucleotide of the mvention" and "polynucleotide sequence of the invention" mean any detectable chemical or physical characteristic of a polynucleotide of the invention that is or may be reduced to or stoied m a tangible medium, preferably a computer readable form For example, chromatographic scan data or peak data, photographic data or scan data therefrom, called bases, and mass spectrographic data As used m this section entitled Databases and Algorithms and m claims related thereto, the terms "polypeptide of the invention" and "polypeptide sequence of the invention" mean any detectable chemical or physical characteristic of a polypeptide of the invention that is or may be reduced to or stored m a tangible medium, preferably a computer readable form For example, chromatographic scan data or peak data, photographic data or scan data therefrom, and mass spectrographic data

The mvention provides a computer readable medium having stored thereon polypeptide sequences of the invention and/or polynucleotide sequences of the invention For example, a computer readable medium is provided comprising and having stored thereon a member selected from the group consisting of a polynucleotide comprising the sequence of a polynucleotide of the invention, a polypeptide compnsmg the sequence of a polypeptide sequence of the mvention, a set of polynucleotide sequences wherein at least one of the sequences comprises the sequence of a polynucleotide sequence of the invention, a set of polypeptide sequences wherem at least one of the sequences comprises the sequence of a polypeptide sequence of the invention, a data set representmg a polynucleotide sequence comprising the sequence of polynucleotide sequence of the invention, a data set representing a polynucleotide sequence encodmg a polypeptide sequence comprising the sequence of a polypeptide sequence of the invention, a polynucleotide compnsmg the sequence of a polynucleotide sequence of the invention, a polypeptide compnsmg the sequence of a polypeptide sequence of the invention, a set of polynucleotide sequences wherein at least one of the sequences comprises the sequence of a polynucleotide sequence of the invention, a set of polypeptide sequences wherein at least one of said sequences comprises the sequence of a polypeptide sequence of the invention, a data set representing a polynucleotide sequence comprising the sequence of a polynucleotide sequence of the invention, a data set representing a polynucleotide sequence encoding a polypeptide sequence comprising the sequence of a polypeptide sequence of the invention The computer readable medium can be any composition of matter used to store information or data, including, for example, commercially available floppy disks, tapes, chips, hard drives, compact disks, and video disks Also provided by the invention are methods for the analysis of character sequences or strings, particularly genetic sequences or encoded genetic sequences Preferred methods of sequence ana sis mclude, for example methods of sequence homology analysis, such as identity and similarity analysis, RNA structure analysis, sequence assembly, cladistic analysis, sequence motif analysis, open reading frame determination, nucleic acid base calling, nucleic acid base trimming, and sequencmg chromatogram peak analysis

A computer based method is provided for performing homology identification This method comprises the steps of providing a first polynucleotide sequence compnsmg the sequence a polynucleotide of the invention in a computer readable medium, and comparmg said first polynucleotide sequence to at least one second polynucleotide or polypeptide sequence to identify homology

A computer based method is also provided for performing homology identification, said method compnsmg the steps of providing a first polypeptide sequence comprising the sequence of a polypeptide of the invention in a computer readable medium, and comparmg said first polypeptide sequence to at least one second polynucleotide or polypeptide sequence to identify homology

A computer based method is still further provided for polynucleotide assembly, said method compnsmg the steps of providing a first polynucleotide sequence comprising the sequence of a polynucleotide of the invention m a computer readable medium, and screemng for at least one overlapping region between said first polynucleotide sequence and at least one second polynucleotide or polypeptide sequence

A computer based method is still further provided for polynucleotide assembly, said method compnsmg the steps of providing a first polypeptide sequence comprising a polypeptide of the invention in a computer readable medium, and screening for at least one overlappmg region between said first polypeptide sequence and at least one second polynucleotide or polypeptide sequence

Preferred embodiments of the assemble methods of the invention use the assembly method set forth m United States Patent Number 5,618,672

In another prefened embodiment of the mvention there is provided a computer readable medium having stored thereon a member selected from the group consisting of a polynucleotide comprising the sequence of SEQ ID NO 1 or 3. a polypeptide comprising the sequence of SEQ ID NO 2 or 4. a set of polynucleotide sequences wherein at least one of said sequences comprises the sequence of SEQ ID NO 1 or 3, a set of polypeptide sequences wherein at least one of said sequences comprises the sequence of SEQ ID NO 2 or 4, a data set representing a polynucleotide sequence comprising the sequence of SEQ ID NO 1 or 3, a data set representing a polynucleotide sequence encoding a polypeptide sequence comprising the sequence of SEQ ID NO 2 or 4, a polynucleotide comprising the sequence of SEQ ID NO 1 or 3, a polypeptide comprising the sequence of SEQ ID NO 2 or 4, a set of polynucleotide sequences wherem at least one of said sequences comprises the sequence of SEQ ID NO 1 or 3, a set of polypeptide sequences where at least one of said sequences comprises the sequence of SEQ ID NO 2 or 4, a data set representing a polynucleotide sequence comprising the sequence of SEQ ID NO 1 or 3, a data set representing a polynucleotide sequence encoding a polypeptide sequence compnsmg the sequence of SEQ ID NO 2 or 4 A further preferred embodiment of the invention provides a computer based method for performing homology identification, said method comprising the steps of providmg a polynucleotide sequence comprising the sequence of SEQ ID NO 1 or 3 in a computer readable medium, and comparmg said polynucleotide sequence to at least one polynucleotide or polypeptide sequence to identify homology A still further preferred embodiment of the invention provides a computer based method for performing homology identification, said method compnsmg the steps of providing a polypeptide sequence comprising the sequence of SEQ ID NO 2 or 4 in a computer readable medium, and comparmg said polypeptide sequence to at least one polynucleotide or polypeptide sequence to identify homology A further embodiment of the invention provides a computer based method for polynucleotide assembly, said method comprising the steps of providing a first polynucleotide sequence comprising the sequence of SEQ ID NO 1 or 3 in a computer readable medium, and screening for at least one overlapping region between said first polynucleotide sequence and a second polynucleotide sequence

A further embodiment of the invention provides a computer based method for performing homology identification, said method comprising the steps of providing a polynucleotide sequence comprising the sequence of SEQ ID NO 1 or 3 in a computer readable medium, and comparing said polynucleotide sequence to at least one polynucleotide or polypeptide sequence to identify homology

All publications and references, including but not limited to patents and patent applications, cited in this specification are herein incorporated by reference in 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 facihtate understanding of certam terms used frequently herem

"Antιbody(ιes)" as used herein includes polyclonal and monoclonal antibodies, chimenc, single chain, and humanized antibodies, as well as Fab fragments, including the products of an Fab or other lmmunoglobulm expression library

"Antigemcally equivalent deπvatιve(s)" as used herem encompasses a polypeptide, polynucleotide. or the equivalent of either which will be specifically recognized by certam antibodies which, when raised to the protem, polypeptide or polynucleotide accordmg to the invention, interferes with the immediate physical interaction between pathogen and mammalian host

"Bispecific antιbody(ιes)" means an antibody comprising at least two antigen bmdmg domains, each domain directed against a different epitope

"Bodily mateπal(s) means any matenal denved from an mdividual or from an orgamsm infecting, infesting or inhabiting an mdividual. mcludmg but not limited to, cells, tissues and waste, such as, bone, blood, serum, cerebrospmal fluid, semen, saliva, muscle, cartilage, organ tissue, skm, urine, stool or autopsy matenals "Dιsease(s)" means any disease caused by or related to infection by a bactena, mcludmg , 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 "Fusion proteιn(s)" refers to a protein encoded by two, often unrelated, fused genes or fragments thereof In one example, EP-A-0464 discloses fusion protems comprising various portions of constant region of lmmunoglobulm molecules together with another human protein or part thereof In many cases, employing an lmmunoglobulm Fc region as a part of a fusion protein is advantageous for use m therapy and diagnosis resulting m, for example, improved pharmacokinetic properties [see. e g , EP-A 0232262] On the other hand, for some uses it would be desirable to be able to delete the Fc part after the fusion protein has been expressed, detected and purified

"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 pohnucleotide sequences, as the case may be, as determmed by comparmg the sequences In the art, "identity" also means the degree of sequence relatedness between polypeptide or polynucleotide sequences, as the case may be, as determined by the match between strings of such sequences "Identity" can be readily calculated by known methods, including but not limited to those described in (Computational Molecular Biology, Lesk, A M , ed , Oxford University Press, New York, 1988, Biocomputing Informatics and Genome Projects, Smith, D W , ed , Academic Press, New York, 1993, Computer Analysis of Sequence Data, Part I, Gnffin, A M . and Gnffin, H G , eds , Humana Press, New ersey, 1994, Sequence Analysis in Molecular Biology, von Heinje, G , Academic Press, 1987, and Sequence Analysis Primer, Gnbskov, M and Devereux. , eds , M Stockton Press, New York, 1991, and Canllo, H , and Lipman, D , SIAM J Apphed Math , 48 1073 (1988) Methods to determme identity are designed to give the largest match between the sequences tested Moreover, methods to determine identity are codified in publicly available computer programs Computer program methods to determme identity between two sequences include, but are not limited to, the GCG program package (Devereux, , 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 B ol 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 Algonthm Needleman and Wunsch, I 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 mclude an isolated polynucleotide comprising a polynucleotide sequence havmg at least a 50, 60, 70, 80, 85, 90, 95, 97 or 100% identity to the reference sequence of SEQ ID NO 1, wherem said polynucleotide sequence may be identical to the reference sequence of SEQ ID NO 1 or may include up to a certam mteger number of nucleotide alterations as compared to the reference sequence, wherem said alterations are selected from the group consisting of at least one nucleotide deletion, substitution, including transition and transversion, or msertion, and wherem said alterations may occur at the 5' or 3' terminal positions of the reference nucleotide sequence or anywhere between those terminal positions, mterspersed either individually among the nucleotides in the reference sequence or in one or more contiguous groups within the reference sequence, and wherem said number of nucleotide alterations is determined by multiplying the total number of nucleotides m SEQ ID NO 1 by the integer defining the percent identity divided by 100 and then subtracting that product from said total number of nucleotides m SEQ ID NO 1, or

n_n < x_n - (x_n • y),

wherem n_n is the number of nucleotide alterations, x_n is the total number of nucleotides in SEQ ID NO 1, y is 0 50 for 50%, 0 60 for 60%, 0 70 for 70%, 0 80 for 80%, 0 85 for 85%, 0 90 for 90%, 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-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 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 wherem 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 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 1 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

ⁿn ≤ ^xn " (^xn • y),

wherem 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 70 for 70%, 0 80 for 80%, 0 85 for 85% 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 include an isolated polypeptide compnsmg a polypeptide havmg at least a 50.60, 70, 80, 85, 90, 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 ammo acid alterations as compared to the reference sequence, wherem said alterations are selected from the group consisting of at least one ammo acid deletion, substitution, including conservative and non- conservative substitution, or insertion, and wherem said alterations may occur at the amino- or carboxy-termmal positions of the reference polypeptide sequence or anywhere between those terminal positions, interspersed either individually among the ammo acids in the reference sequence or in one or more contiguous groups withm the reference sequence, and wherem 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 subtractmg that product from said total number of ammo acids m SEQ ID NO 2, or

ⁿa ≤ ^xa ^" (^xa ^# 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 0 50 for 50%, 0 60 for 60%, 0 70 for 70%, 0 80 for 80%, 0 85 for 85%, 0 90 for 90%, 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 prior to subtractmg it from x_a

By way of example, a polypeptide sequence of the 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 mteger number of ammo 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 ammo acid deletion, substitution, including conservative and non- conservative substitution, or msertion, and wherem 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 amino acids in the reference sequence or m one or more contiguous groups within the reference sequence The number of ammo acid alterations for a given % identity 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 amino acids m SEQ ID NO 2. or

n_a < x_a - (x_a • y),

wherem 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. for instance 0 70 for 70%, 0 80 for 80%, 0 85 for 85% etc , 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 prior to subtracting it from x_a

"lmmunologically equivalent deπvative(s)" as used herein encompasses a polypeptide, polynucleotide. or the equivalent of either which when used in a suitable formulation to raise antibodies in a vertebrate, the antibodies act to interfere with the immediate physical interaction between pathogen and mammalian host "Immunospecific" means that charactenstic of an antibody whereby it possesses substantially greater affinity for the polypeptides of the mvention or the polynucleotides of the mvention than its affinity for other related polypeptides or polynucleotides respectively, particularly those polypeptides and polynucleotides m the pnor art

"Indιvιdual(s)" means a multicellular eukaryote, mcludmg. 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 coexisting 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 by any other recombinant method is "isolated" even if it is still present m said orgamsm, which orgamsm may be Irving or non-living

"Organιsm(s)" means a (I) prokaryote, mcludmg but not limited to, a member of the genus Streptococcus, Staphylococcus, Bordetella, Corynebactenum, Mycobacterium, Neisseria, Haemophilus, Actinomycetes, Streptomycetes, Nocardia, Enterobacter, Yersima, Fancisella, Pasturella, Moraxella, Acinetobacter, Erysψelothrix, Branhamella, Actinobacillus, Streptobacillus, Listena, Calymmatobacterium, Brucella, Bacillus, Clostridium, Treponema, Escherichia, Salmonella, Kleibsiella, Vibrio, Proteus, Erw nia, Borrelia, Leptospira, Spirillum, Campylobacter, Shigella, Legionella, Pseudomonas, Aeromonas, Rickettsia, Chlamydia, Borrelia and Mycoplasma. and further including, but not limited to, a member of the species or group, Group A Streptococcus, Group B Streptococcus, Group C Streptococcus, Group D Streptococcus, Group G Streptococcus, Streptococcus pneumoniae, Streptococcus pyogenes, Streptococcus agalactiae, Streptococcus faecalis, Streptococcus faecium, Streptococcus durans, Neisseria gonorrheae, Neisseria meningitidis, Staphylococcus aureus, Staphylococcus epidermidis, Corynebacterium dipthenae, Gardnerella vaginalis, Mycobacterium tuberculosis, Mycobacterium bovis, Mycobacterium ulcerans, Mycobacterium leprae, Actinomyctes israelii, Listena monocytogenes, Bordetella pertusis, Bordatella parapertusis, Bordetella bronchiseptica, Escherichia coh, Shigella dysenteriae, Haemophilus influenzae, Haemophilus aegyptius, Haemophilus parainfluenzae, Haemophilus ducreyi, Bordetella, Salmonella typhi, Citrobacter freundu, Proteus mirabihs, Proteus vulgaris, Yersima pestis, Kleibsiella pneumoniae, Serratia marcessens, Serratia hquefaciens, Vibrio cholera, Shigella dysentern, Shigella flexnen, Pseudomonas aeruginosa, Franscisella tularensis, Brucella abortis, Bacillus anthracis, Bacillus cereus, Clostridium perfnngens, Clostridium tetani, Clostridium botulmum, Treponema pallidum, Rickettsia rickettsii and Chlamydia trachomitis, (ii) an archaeon, including but not limited to Archaebacter, and (iii) a unicellular or filamentous eukaryote, including but not limited to, a protozoan, a fungus, a member of the genus Saccharomyces, Kluveromyces, or Candida, and a member of the species Saccharomyces 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 term "polynucleotide(s)" also includes DNAs or RNAs as described above that contain one or more modified bases Thus, DNAs or RNAs with backbones modified for stability or for other reasons are "polynucleotιde(s)" as that term is mtended herem Moreover, DNAs or RNAs compnsmg unusual bases, such as mosme. 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 "polynucleotide(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 "Polynucleotide(s)" also embraces short polynucleotides often refened to as ohgonucleotide(s) "Polypeptιde(s)" refers to any peptide or protem compnsmg two or more ammo acids jomed to each other by peptide bonds or modified peptide bonds "Polypeptide(s)" refers to both short chains, commonly refened to as peptides, oligopeptides and oligomers and to longer chains generally refened to as proteins Polypeptides may contain ammo acids other than the 20 gene encoded ammo acids "Polypeptide(s)" mclude those modified either by natural processes, such as processmg and other post- translational modifications, but also by chemical modification techmques Such modifications are well descnbed m basic texts and m more detailed monographs, as well as m a voluminous research literature, and they are well known to those of skill in the art It will be appreciated that the same type of modification may be present m the same or varying degree at several sites m a given polypeptide Also, a given polypeptide may contain many types of modifications Modifications can occur anywhere m a polypeptide, mcludmg the peptide backbone, the ammo acid side-chains, and the ammo or carboxyl termini Modifications mclude, for example, acetylation, acylation, ADP-nbosylation, amidation, covalent attachment of flavm, covalent attachment of a heme moiety, covalent attachment of a nucleotide or nucleotide denvative, covalent attachment of a lipid or hpid 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 processmg, phosphorylation, prenylation, racemization, glycosylation, hpid 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 argmvlation, and ubiquitination See, for instance, PROTEINS - STRUCTURE AND MOLECULAR PROPERTIES, 2nd Ed , T E Creighton, W H Freeman and Company, New York (1993) and Wold, F , Posttranslational Protem Modifications Perspectives and Prospects, pgs 1-12 m POSTTRANSLATIONAL COVALENT MODIFICATION OF PROTEINS, B C Johnson, Ed , Academic Press. New York (1983), Seifter et al , Meth Enzymol 182 626-646 (1990) and Rattan et al , Protein Synthesis Posttranslational Modifications and Aging, Ann N Y Acad Sci 663 48-62 (1992) Polypeptides may be branched or cyclic, with or without branching Cyclic, branched and branched circular polypeptides may result from post-translational natural processes and may be made by entirely synthetic methods, as well

"Recombmant 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 "Subtraction set" is one or more, but preferably less than 100. polynucleotides comprising at least one polynucleotide of the invention

"Vaπant(s)" as the term is used herein, is a polynucleotide or polypeptide that differs from a reference polynucleotide or polypeptide respectively, but retains essential properties A typical variant of a polynucleotide differs in nucleotide sequence from another, reference polynucleotide Changes in the nucleotide sequence of the variant may or may not alter the ammo acid sequence of a polypeptide encoded by the reference polynucleotide Nucleotide changes may result in amino acid substitutions, additions, deletions, fusion protems 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, m 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 ammo acid residue may or may not be one encoded by the genetic code The present mvention also mcludes mclude vanants of each of the polypeptides of the mvention, that is polypeptides that vary from the referents by conservative ammo acid substitutions, whereby a residue is substituted by another with like 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 variant of a polynucleotide or polypeptide may be a naturally occurring such as an allehc variant, or it may be a variant that is not known to occur naturally Non-naturally occurring 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 techmques. which are well known and routme 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 having a DNA sequence given in Table 1 [SEQ ID NO 1 or 3] was obtained from a library of clones of chromosomal DNA of Streptococcus pneumoniae m E coh The sequencing data from two or more clones containing 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 m order to size- fractionate according to standard procedures DNA fragments of up to 1 lkbp in size are rendered blunt by treatment with exonuclease and DNA polymerase, and EcoRI linkers added Fragments are ligated into the vector Lambda ZapII that has been cut with EcoRI, the library packaged b> 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 series of fragments for clomng mto library vectors (e g , Rsal, Pall, Alul, Bshl235I), and such fragments are size-fractionated according to standard procedures EcoRI linkers are ligated to the DNA and the fragments then ligated mto 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 Example 2

The S.pneumoniae deoD gene is expressed during infection in a respiratory tract infection model. The determination of expression during infection of a gene from Streptococcus pneumoniae Excised lungs from a 48 hour respiratory tract infection of Streptococcus pneumoniae 0100993 m the mouse is efficiently disrupted and processed m the presence of chaotropic agents and RNAase inhibitor to provide a mixture of animal and bacterial RNA The optimal conditions for disruption and processing to give stable preparations and high yields of bacterial RNA are followed by the use of hybridisation to a radiolabelled oligonucleotide specific to Streptococcus pneumoniae 16S RNA on Northern blots The RNAase free, DNAase free, DNA and protem free preparations of RNA obtained are suitable for Reverse Transcription PCR (RT-PCR) using unique primer pairs designed from the sequence of each gene of Streptococcus pneumoniae 0100993

a) Isolation of tissue infected with Streptococcus pneumoniae 0100993 from a mouse ammal model of infection (lungs) Streptococcus pneumoniae 0100993 is seeded onto TSA (Tryptic Soy Agar. BBL) plates containing 5% horse blood and allowed to grow overnight at 37°C in a C02 incubator Bacterial growth is scraped mto 5 ml of phosphate-buffered salme (PBS) and adjusted to an A600 ~ 0 6 (4 x 106/ml) Mice (male CBA/J-1 mice, approximately 20g) were anaesthetized with isoflurane and 50 microhters of the prepared bacterial inoculum is delivered by mtranasal instillation Animals are allowed to recover and observed twice daily for signs of moribundancy Forty-eight hours after infection the animals are euthanized by carbon dioxide overdose and their torsos swabbed with ethanol and then RNAZap The torso is then opened, and the lungs are aseptically removed Half of each pair of lungs is placed in a cryovial and immediately frozen in liquid nitrogen, the other half is used for bacterial enumeration after homogenization of the tissue m 1 ml of PBS

b) Isolation of Streptococcus pneumoniae 0100993 RNA from infected tissue samples Infected tissue samples, in 2-ml cryo-strorage tubes, are removed from -80°C storage mto a dry ice ethanol bath In a microbiological safety cabinet the samples are disrupted up to eight at a time while the remammg samples are kept frozen in the dry ice ethanol bath To disrupt the bacteria withm the tissue sample, 50-100 mg of the tissue is transfered to a FastRNA tube containing a silica/ceramic matrix (BIO 101) Immediately, 1 ml of extraction reagents (FastRNA reagents. BIO101) are added to give a sample to reagent volume ratio of approximately 1 to 20 The tubes are shaken in a reciprocating shaker (FastPrep FP120, BIO101) at 6000 rpm for 20-120 sec The crude RNA preparation is extracted with chloroform/isoamyl alcohol, and precipitated with DEPC-treated Isopropanol Precipitation Solution (BIO 101) RNA preparations are stored in this isopropanol solution at -80°C if necessary The RNA is pelleted (12,000g for 10 mm ). washed with 75% ethanol (v/v m DEPC- treated water), air-dried for 5-10 mm, and resuspended in 0 1 ml of DEPC-treated water, followed by 5-10 minutes at 55 °C Finally, after at least 1 minute on ice, 200 units of Rnasin (Promega) is added

RNA preparations are stored at -80 °C for up to one month For longer term storage the RNA precipitate can be stored at the wash stage of the protocol in 75% ethanol for at least one year at -20 °C

Quality of the RNA isolated is assessed by running samples on 1 % agarose gels 1 x TBE gels stained with ethidium bromide are used to visualise total RNA yields To demonstrate the isolation of bacterial RNA from the infected tissue 1 x MOPS, 2 2M formaldehyde gels are run and vacuum blotted to Hybond-N (Amersham) The blot is then hybridised with a 32P-labelled ohgonucletide probe specific to 16S rRNA of Streptococcus pneumoniae The size of the hybridising band is compared to that of control RNA isolated from m vitro grown Streptococcus pneumomae 0100993 in the Northern blot Correct sized bacterial 16S rRNA bands can be detected in total RNA samples which show degradation of the mammalian RNA when visualised on TBE gels

c) The removal of DNA from Streptococcus pneumoniae-deπved RNA

DNA was removed from 50 microgram samples of RNA by a 30 minute treatment at 37°C with 20 units of RNAase-free DNAasel (GenHunter) m the buffer supplied in a final volume of 57 microhters

The DNAase was inactivated and removed by treatment with TRIzol LS Reagent (Gibco BRL, Life Technologies) according to the manufacturers protocol DNAase treated RNA was resuspended in 100 microhtres of DEPC treated water with the addition of Rnasin as described before

d) The preparation of cDNA from RNA samples derived from infected tissue 3 microgram samples of DNAase treated RNA are reverse transcribed using a Superscript Preamplification System for First Strand cDNA Synthesis kit (Gibco BRL, Life Technologies) according to the manufacturers instructions 150 nanogram of random hexamers is used to prime each reaction Controls without the addition of SuperScnptll reverse transcriptase are also run Both +/-RT samples are treated with RNaseH before proceedmg to the PCR reaction

e) The use of PCR to determine the presence of a bacterial cDNA species PCR reactions are set up on ice m 0 2ml tubes by adding the following components 43 microhtres PCR Master Mix (Advanced Biotechnologies Ltd ), 1 microhtre PCR primers (optimally 18-25 basepairs in length and designed to possess similar annealing temperatures), each primer at lOmM initial concentration, and 5 microhtres cDNA

PCR reactions are run on a Perkm Elmer GeneAmp PCR System 9600 as follows 2 minutes at 94 °C, then 50 cycles of 30 seconds each at 94 °C, 50 °C and 72 °C followed by 7 minutes at 72 °C and then a hold temperature of 20 °C (the number of cycles is optimally 30-50 to determine the appearance or lack of a PCR product and optimally 8-30 cycles if an estimation of the starting quantity of cDNA from the RT reaction is to be made). 10 microhtre ahquots are then run out on 1% 1 x TBE gels stained with ethidium bromide, with PCR product, if present, sizes estimated by companson to a 100 bp DNA Ladder (Gibco BRL, Life Technologies) Alternatively if the PCR products are conveniently labelled by the use of a labelled PCR primer (e g labelled at the 5'end with a dye) a smtable aliquot of the PCR product is run out on a polyacrylamide sequencing gel and its presence and quantity detected usmg a suitable gel scanning system (e g ABI PnsmTM 377 Sequencer using GeneScanTM software as supplied by Perkm Elmer)

RT/PCR controls may include +/- reverse transcriptase reactions, 16S rRNA pnmers or DNA specific primer pairs designed to produce PCR products from non-transcribed Streptococcus pneumoniae 0100993 genomic sequences To test the efficiency of the primer pairs they are used in DNA PCR with Streptococcus pneumomae 0100993 total DNA PCR reactions are set up and run as described above using approx 1 microgram of DNA m place of the cDNA

Primer pairs which fail to give the predicted sized product in either DNA PCR or RT/PCR are PCR failures and as such are umnformative Of those which give the correct size product with DNA PCR two classes are distinguished in RT/PCR 1 Genes which are not transcribed m vivo reproducibly fail to give a product in RT/PCR, and 2 Genes which are transcribed in vivo reproducibly give the correct size product in RT/PCR and show a stronger signal m the +RT samples than the signal (if at all present) in -RT controls

Example 3

The deoD gene when mutated causes attenuation in S.pneumoniae infection models. A S pneumoniae deoD mutant was generated as described below When tested m a respiratory tract infection as described below, the mutant was found to be 2 logs attenuated compared to an infection with the wild-type parent strain When tested in an otitis media infection as described below, the mutant was found to be 0 5 logs attenuated compared to an mfection with the wild- type parent stram

a) Procedure for generating S pneumoniae alle c replacement mutants

A DNA construct is generated by PCR, consisting of 500bp chromosomal DNA fragments flanking an erythromycin resistance gene The chromosomal DNA sequences are usually the 500bp precedmg and following the gene of interest The allehc replacement cassette is mtroduced into S pneumomae R6 or S pneumoniae 100993 by transformation Competent cells are prepared according to published protocols DNA is introduced into the cells by incubation of 500ng of allehc replacement cassette with 106 cells at 30°C for 30 minutes The cells are transferred to 37°C for 90 minutes to allow expression of the erythromycin resistance gene Cells are plated in agar containing lug erythromycin per ml Followmg incubation at 37°C for 36 hours, any observed colonies are picked and grown overnight in Todd-Hewitt broth supplemented with 0 5% yeast extract Typically, in positive control experiments carried out m parallel which target a non-essential gene, 102-103 transformants containing the appropriate allehc replacement are obtained If erythromycin resistant colonies are only observed m transformation experiments using S pneumoniae R6, DNA from these cells are used to transform S pneumoniae 100993 The transformation procedure is identical to that for S pneumoniae R6 except that a competence stimulating heptadecapeptide (Havarstem et al , (1995) P N A S 92, 11140-11144) is added at a concentration of lug/ml in the initial transformation mix Mutants are selected by their ability to grow m agar containing lug erythromycin per ml

If no transformants are obtained in three separate transformation experiments, then the target gene is considered as being essential in vitro However, if colonies are obtained chromosomal DNA is prepared from these cells and examined using diagnostic PCR Ohgonucleotides designed to hybridize to sequences withm the allehc replacement cassette are used in conjunction with DNA primers hybridizing to chromosomal sequences outside the cassette to generate DNA products amplified by PCR of characteristic size This chromosomal DNA is also subject to Southern analysis in order to verify that the appropriate chromosomal DNA rearrangement has occurred

In order to demonstrate that the mutation is stably maintained, the defective strain is grown for many generations m the absence of selective pressure and then assayed for its ability to grow in the absence and presence of erythromycin

b) Procedures for respiratory tract infection with Streptococcus pneumoniae

Bacteria for infection are prepared by inoculation of tryptic soy agar plates containing 5% sheep blood from frozen stocks and overnight growth at 37°C m 5% C02 Bacteria are recovered from the plates, resuspended in phosphate-buffered sal e (PBS) and adjusted to A600-0 8 - 1 0 (approximately 107 - 108 cfu/ml) Ammals (male CBA/J mice, 14 - 16g) are anaesthetized with lsoflurane (3%), and 50ul of the prepared bacterial inoculum is administered by intranasal instillation usmg a pipetman No pam is inflicted on the animals at any point during this procedure, and no other anaesthetic is used To simulate the state in lmmunocompromised patients the immune system of the ammals may be suppressed by agents such as cyclophosphamide (150mg/kg I p on day -4 followed by lOOmg/kg on day -1) or by irradiation (700 rads) However, immunosuppression is not necessary for S pneumoniae infection m this model Animals are allowed to recover and given food and water ad libitum Animals are observed three times daily and those unlikely to survive the challenge (1 e . exhibiting cyanosis, hypothermia, staring coat or are moribund) are killed by C02 overdose Surviving animals are killed at 12 - 48 hours post-infection by carbon dioxide overdose, and lungs are aseptically removed, homogenized in 1ml of PBS. and enumerated for viable bacteria

c) Procedures for model of otitis media in Mongolian gerbils using Streptococcus pneumoniae Animals (male Mongolian gerbils. 40 - 60g) are anaesthetized with isoflurane (3%), and the area around the left ear bulla is prepared by swabbing with ethanol 40ul (~4 log 10 cfu) of S pneumoniae (isolate 0100993 or an isogemc mutant) is injected through the bone of the left bulla, and the animals are allowed to recover under observation Animals are given food and water ad libitum, and killed 96 hours post infection by carbon dioxide overdose The tympanic membrane is then examined and middle ear aspirates are obtained by injecting 250ul of phosphate-buffered saline into the middle ear cavity and withdrawing the fluid contained therein Aspirates are then serially diluted and evaluated for viable bacteria

Claims

What is claimed is:

1 An isolated polypeptide comprising an ammo acid sequence which has at least 70% identity to the amino acid sequence of SEQ ID NO 2 over the entire length of SEQ ID NO 2

2 An isolated polypeptide as claimed m claim 1 in which the amino acid sequence has at least 95% identity

3 The polypeptide as claimed in claim 1 comprising the ammo acid sequence of SEQ ID NO 2

4 The isolated polypeptide of SEQ ID NO 2

5 An isolated polynucleotide compnsmg a nucleotide sequence encoding a polypeptide that has at least 70% identity to the ammo acid sequence of SEQ ID NO 2, over the entire length of SEQ ID NO 2. or a nucleotide sequence complementary to said isolated polynucleotide

6 An isolated polynucleotide compnsmg a nucleotide sequence that has at least 70% identity to a nucleotide sequence encodmg a polypeptide of SEQ ID NO 2, over the entire codmg region, or a nucleotide sequence complementary to said isolated polynucleotide

7 An isolated polynucleotide which comprises a nucleotide sequence which has at least 70% identity to that of SEQ ID NO 1 over the entire length of SEQ ID NO 1 , or a nucleotide sequence complementary to said isolated polynucleotide

8 The isolated polynucleotide as claimed m any one of claims 5 to 7 in which the identity is at least 95%

9 An isolated polynucleotide selected from

(a) a polynucleotide compnsmg a nucleotide sequence encodmg the polypeptide of SEQ ID NO 2,

(b) the polynucleotide of SEQ ID NO 1, and

(c) a polynucleotide obtainable by screemng an appropnate library under stnngent hybndization conditions with a labeled probe having the sequence of SEQ ID NO 1 or a fragment thereof, or a nucleotide sequence complementary to said isolated polynucleotide

10 An expression system compnsmg a polynucleotide capable of producing a polypeptide of claim

I when said expression system is present in a compatible host cell

11 A host cell comprising the expression system of claim 15 or a membrane thereof expressing the polypeptide of claim 1

12 A process for producing a polypeptide of claim 1 comprising culturing a host cell of claim

I I under conditions sufficient for the production of said polypeptide and recovering the polypeptide from the culture medium

13 An antibody immunospecific for the polypeptide of claim 1

14 A method for screemng 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) measurmg 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 bearing the polypeptide) or a fusion protein thereof in the presence of a labeled competitor,

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

(d) mixing a candidate compound with a solution containing 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 in cells, using for instance, an ELISA assay

15 An agomst or antagonist to the polypeptide of claims 1 to 4 16 A compound which is

(a) an agonist or antagonist to the polypeptide of claims 1 to 4,

(b) isolated pohnucleotide of claims 5 to 9. or

(c) a nucleic acid molecule that modulates the expression of the nucleotide sequence encoding the polypeptide of claim 1. for use in therapy

17 A process for diagnosing a disease or a susceptibility to a disease in an individual related to expression or activity of the polypeptide of clami 1 m an mdividual compnsmg

(a) determining the presence or absence of a mutation in the nucleotide sequence encodmg said polypeptide in the genome of said individual, and/or

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

18 An isolated polynucleotide selected form the group consistmg of

(a) an isolated polynucleotide comprising a nucleotide sequence which has at least 70% identity to SEQ ID NO 3 over the entire length of SEQ ID NO 3,

(b) an isolated polynucleotide comprising a nucleotide sequence which has at least 70% identity to SEQ ID NO 1 over the entire length of SEQ ID NO 3,

(e) an isolated polynucleotide comprising the polynucleotide of SEQ ID NO 3,

(d) the polynucleotide of SEQ ID NO 3, or

(e) an isolated polynucleotide comprising a nucleotide sequence encoding a polypeptide which has at least 70% identity to the ammo acid sequence of SEQ ID NO 4, over the entire length of SEQ ID NO 4

19 A polypeptide selected from the group consistmg of

(a) a polypeptide which comprises an ammo acid sequence which has at least 70% identity to that of SEQ ID NO 4 over the entire length of SEQ ID NO 4,

(b) a polypeptide in which the ammo acid sequence has at least 70% identity to the ammo acid sequence of SEQ ID NO 4 over the entire length of SEQ ID NO 4, (c) a polypeptide which comprises the ammo acid of SEQ ID NO 4,

(d) a polypeptide which is the polypeptide of SEQ ID NO 4,

(e) a polypeptide which is encoded by a polynucleotide comprising the sequence contained m SEQ ID NO 3