EP1100879A1 - Pneumococcal nrdg protein - Google Patents

Abstract

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

Description

' PNEUMOCOCCAL NRDG PROTEIN. '

FIELD OF THE INVENTION

This invention relates to newly identified polynucleotides and polypeptides. and their production and uses, as well as their variants, agonists and antagonists, and their uses. In particular, the invention relates to polynucleotides and polypeptides of the nrdG (anaerobic ribonucleotide triphosphate reductase) family, as well as their variants, hereinafter referred to as "nrdG." "nrdG polynucleotide(s)," and "nrdG polypeptide(s)" as the case may be.

BACKGROUND OF THE INVENTION

The Streptococci make up a medically important genera of microbes known to cause several types of disease in humans, including, for example, otitis media, conjunctivitis, pneumonia, bacteremia, meningitis. sinusitis, pleural empyema and endocarditis, and most particularly meningitis, such as for example infection of cerebrospinal fluid. Since its isolation more than 100 years ago, Streptococcus pneumoniae has been one of the more intensively studied microbes. For example, much of our early understanding that DNA is, in fact, the genetic material was predicated on the work of Griffith and of Avery, Macleod and McCarty using this microbe. Despite the vast amount of research with S. pneumoniae, many questions concerning the 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 oi Streptococcus pneumoniae infections has risen dramatically in the past few decades. This has been attributed to the emergence of multiply antibiotic resistant strains and an increasing population of people with weakened immune systems. It is no longer uncommon to isolate Streptococcus pneumoniae strains which are resistant to some or all of the standard antibiotics. This phenomenon has created an unmet medical need and demand for new anti-microbial agents, vaccines, drug screening methods, and diagnostic tests for this organism.

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

Clearly, there exists a need for polynucleotides and polypeptides. such as the nrdG embodiments of the invention, that have a present benefit of, among other things, being useful to screen compounds for antimicrobial activity Such factors are also useful to determine their role in pathogenesis of infection, dysfunction and disease There is also a need for identification and characterization of such factors and their antagonists and agonists to find ways to prevent, ameliorate or correct such infection, dysfunction and disease

SUMMARY OF THE INVENTION

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

Vanous changes and modifications within the spint and scope of the disclosed invention will become readily apparent to those skilled in the art from reading the following descnptions and from reading the other parts of the present disclosure

DESCRIPTION OF THE INVENTION

The invention relates to nrdG polypeptides and polynucleotides as descnbed in greater detail below In particular, the invention relates to polypeptides and polynucleotides of a nrdG of Streptococcus pneumoniae, which is related by amino acid sequence homology to nrdG polypeptide The invention relates especially to nrdG having the nucleotide and amino acid sequences set out in Table 1 as SEQ D NO 1 and SEQ ID NO 2 respectively Note that sequences recited in the Sequence Listing below as "DNA^" represent an exemplification of the invention, since those of ordmary skill will recogmze that such sequences can be usefully employed in polynucleotides in general, including ribopolynucleotides

TABLE 1 NrdG Polynucleotide and Polypeptide Sequences

(A) Streptococcus pneumoniae nrdG porynucleotide sequence [SEQ ID NO: 1].

5 ' -ATGAATAATCCAAAACCACAAGAATGGAAAAGCGAGGAACTTAGTCAAGG TCGTATCATTGACTACAAGGCCTTTAACTTTGTGGACGGCGAAGGCGTGC

GCAACTCTCTCTATGTATCAGGCTGTATGTTTCACTGCGAGGGATGTTAT

AATGTTGCGACTTGGTCTTTTAATGCTGGCATTCCCTATACAGCAGAATT

AGAAGAGCAGATTATGGCAGACCTTGCCCAACCCTATGTTCAAGGCTTGA

CTTTGCTGGGAGGGGAGCCTTTTCTCAATACTGGGATTCTCTTGCCACTT GTTAAGCGGATTCGGAAGGAATTGCCAGACAAGGACATCTGGTCCTGGAC

CGGCTACACTTGGGAAGAAATGATGTTGGAAACTCCAGATAAACTGGAAT

TCTTGTCACTGATTGACATTCTTGTCGATGGAAGATATGATCGAACTAAG

AGAAATCTTATGCTCCAGTTTCGAGGTTCATCTAACCAACGAATTATCGA

TGTGCAAAAATCGCTCAAAAGTGGGCAAGTAGTGATTTGGGACAAGCTCA ATGACGGAAAAGAAAGCTATGAACAGGTGAAGAGAGAATGA-3 '

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

NH₂-MNNPKPQE KSEELSQGRI IDYKAFNFVDGEGVRNSLYVSGCMFHCEGCY NVATWSFNAGIPYTAELEEQIMADLAQPYVQGLTLLGGEPFLNTGILLPL VKRIRKELPDKDI S TGYT EEMMLETPDKLEFLSLIDILVDGRYDRTK RNLMLQFRGSSNQRIIDVQKSLKSGQWIWDKLNDGKESYEQVKRE-COOH

Deposited materials A deposit containing a Streptococcus pneumoniae 0100993 strain has been deposited with the

National Collections of Industrial and Marine Bacteria Ltd. (herein "NCIMB"), 23 St. Machar Drive, Aberdeen

AB2 IRY, Scotland on 11 April 1996 and assigned deposit number 40794. The deposit was described as

Streptococcus pneumoniae 0100993 on deposit.

On 17 April 1996 a Streptococcus pneumoniae 0100993 DNA library in E. coli was similarly deposited with the NCIMB and assigned deposit number 40800. The Streptococcus pneumoniae strain deposit is referred to herein as "the deposited strain" or as "the DNA of the deposited strain."

The deposited strain contains a full length nrdG gene. The sequence of the polynucleotides contained in the deposited strain, as well as the amino acid sequence of any polypeptide encoded thereby, are controlling in the event of any conflict with any description of sequences herein. The deposit of the deposited strain has been made under the terms of the Budapest Treaty on the

International Recognition of the Deposit of Micro-organisms for Purposes of Patent Procedure. The deposited strain will be irrevocably and without restriction or condition released to the public upon the issuance of a patent. The deposited strain is provided merely as convenience to those of skill in the art and is not an admission that a deposit is required for enablement such as that required under 35 U.S. C. §112. A license may be required to make, use or sell the deposited strain, and compounds derived therefrom, and no such license is hereby granted.

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

Polypeptides

NrdG polypeptide of the invention is substantially phylogenetically related to other proteins of the nrdG (anaerobic ribonucleotide triphosphate reductase) family.

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

Among the particularly preferred embodiments of the invention are variants of nrdG polypeptide encoded by naturally occurring alleles of the nrdG 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 97-99% 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 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 amino acid sequence of SEQ ID NO:2, over the entire length of SEQ ID NO:2.

The polypeptides of the invention include a polypeptide of Table 1 [SEQ ID NO:2] (in particular the mature polypeptide) as well as polypeptides and fragments, particularly those which have the biological activity of nrdG, and also those which have at least 70% identity to a polypeptide of Table 1 [SEQ ID NO:l]or the relevant portion, preferably at least 80% identity to a polypeptide of Table 1 [SEQ LD NO:2and more preferably at least 90% identity to a polypeptide of Table 1 [SEQ ID NO:2] and still more preferably at least 95% identity to a polypeptide of Table 1 [SEQ ID NO:2] and also include portions of such polypeptides with such portion of the polypeptide generally containing at least 30 amino acids and more preferably at least 50 amino acids.

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

X-(R, )_m-(R₂)-(R₃)_n-Y wherein, at the amino terminus, X is hydrogen, a metal or any other moiety described herein for modified polypeptides, and at the carboxyl temiinus, Y is hydrogen, a metal or any other moiety described herein for modified polypeptides, Ri and R3 are any amino acid residue or modified amino acid residue, m is an integer between 1 and 1000 or zero, n is an integer between 1 and 1000 or zero, and R₂ is an amino acid sequence of the invention, particularly an amino acid sequence selected from Table 1 or modified forms thereof. In the formula above, R ^s oriented so that its amino terminal amino acid residue is at the left, covalently bound to Ri and its carboxy terminal amino acid residue is at the right, covalently bound to R3. Any stretch of amino acid residues denoted by either Ri or R3, where m and/or n is greater than 1, may be either a heteropolymer or a homopolymer, preferably a heteropolymer. Other preferred embodiments of the invention are provided where m is an integer between 1 and 50, 100 or 500, and n is an integer between 1 and 50, 100, or 500.

It is most preferred that a polypeptide of the invention is derived from Streptococcus pneumoniae, however, it may preferably be obtained from other organisms of the same taxonomic genus. A polypeptide of the invention may also be obtained, for example, from organisms of the same taxonomic family or order.

A fragment is a variant polypeptide having an amino acid sequence that is entirely the same as part but not all of any amino acid sequence of any polypeptide of the invention. As with nrdG polypeptides, fragments may be "free-standing," or comprised within a larger polypeptide of which they form a part or region, most preferably as a single continuous region in a single larger polypeptide. Preferred fragments include, for example, truncation polypeptides having a portion of an amino acid sequence of Table 1 [SEQ ID NO:2], or of variants thereof, such as a continuous series of residues that includes an amino- and/or carboxyl-terminal amino acid sequence. Degradation forms of the polypeptides of the invention produced by or in a host cell, particularly a Streptococcus pneumoniae, are also preferred. Further preferred are fragments characterized by structural or functional attributes such as fragments that comprise alpha-helix and alpha-hehx forming regions, beta-sheet and betø-sheet-forrning regions, turn and turn- forming regions, coil and coil-forming regions, hydrophilic regions, hydrophobic regions, alpha amphipathic regions, beta amphipathic regions, flexible regions, surface-forming regions, substrate binding region, and high antigenic index regions. Further preferred fragments include an isolated polypeptide comprising an amino acid sequence having at least 15, 20, 30, 40, 50 or 100 contiguous amino acids from the amino acid sequence of SEQ ID NO:2, or an isolated polypeptide comprising an amino acid sequence having at least 15, 20, 30, 40, 50 or 100 contiguous amino acids truncated or deleted from the amino acid sequence of SEQ ID NO:2.

Also preferred are biologically active fragments which are those fragments that mediate activities of nrdG, including those with a similar activity or an improved activity, or with a decreased undesirable activity. Also included are those fragments that are antigenic or immunogenic in an animal, especially in a human. Particularly preferred are fragments comprising 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 individual, particularly a human.

Fragments of the polypeptides of the invention may be employed for producing the corresponding full- length polypeptide by peptide synthesis; therefore, these variants may be employed as intermediates for producing the full-length polypeptides of the invention. In one embodiment, the nrdG protein has a glycyl radical as part of its polypeptide structure, the radical located at the position corresponding to that reported for E. coli nrdG by Sun et al., J Biol. Chem. 270: 2443-2446, 1995. To generate the glycyl radical, the gene product is acted upon by an activase such as that reported by Sun et al. in E. coli or one from Streptococcus pneumoniae.

In addition to the standard single and triple letter representations for amino 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 amino acids may appear at such a designated position in the polypeptide sequence.

Polynucleotides

It is an object of the invention to provide polynucleotides that encode nrdG polypeptides, particularly polynucleotides that encode the polypeptide herein designated nrdG.

In a particularly preferred embodiment of the invention the polynucleotide comprises a region encoding nrdG polypeptides comprising a sequence set out in Table 1 [SEQ LD NO: 1] which includes a full length gene, or a variant thereof. The Applicants believe that this full length gene is essential to the growth and/or survival of an organism which possesses it, such as Streptococcus pneumoniae . As a further aspect of the invention there are provided isolated nucleic acid molecules encoding and/or expressing nrdG polypeptides and polynucleotides, particularly Streptococcus pneumoniae nrdG polypeptides and polynucleotides, including, for example, unprocessed RNAs, ribozyme RNAs, niRNAs. cDNAs, genomic DNAs, B- and Z-DNAs. Further embodiments of the invention include biologically, diagnostically. prophylactically, clinically or therapeutically useful polynucleotides and polypeptides, and vanants thereof, and compositions compnsing the same

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

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

Using the information provided herein, such as a polynucleotide sequence set out in Table 1 [SEQ ID NO 1]. a polynucleotide of the invention encoding nrdG polypeptide may be obtained using standard cloning and screening methods, such as those for cloning and sequencing chromosomal DNA fragments from bactena using Streptococcus pneumoniae 0100993 cells as starting matenal, followed by obtaining a full length clone For example, to obtain a polynucleotide sequence of the invention, such as a polynucleotide sequence given in Table 1 [SEQ ID NO 1], typically a library of clones of chromosomal DNA of Streptococcus pneumoniae 0100993 in E coli or some other suitable host is probed with a radiolabeled oligonucleotide, preferably a 17-mer or longer, deπved from a partial sequence Clones carrying DNA identical to that of the probe can then be distinguished usmg stringent hybridization conditions By sequencmg the individual clones thus identified by hybridization with sequencing primers designed from the original polypeptide or polynucleotide sequence it is then possible to extend the polynucleotide sequence m both directions to determine a full length gene sequence Convemently, such sequencmg is performed, for example, usmg denatured double stranded DNA prepared from a plasmid clone Suitable techniques are descπbed by Maniatis, T , Fntsch, E F and Sambrook et al , MOLECULAR CLONING, A LABORATORY MANUAL, 2nd Ed . Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York (1989) (see in particular Screening By Hybridization 1 90 and Sequencmg Denatured Double-Stranded DNA Templates 13 70) Direct genomic DNA sequencing may also be performed to obtain a full length gene sequence Illustrative of the invention, each polynucleotide set out in Table 1 [SEQ O NO 1] was discovered in a DNA library denved from Streptococcus pneumoniae 0100993

Moreover, each DNA sequence set out in Table 1 [SEQ LD NO 1] contains an open reading frame encoding a protein having about the number of amino acid residues set forth in Table 1 [SEQ ED NO 2] with a deduced molecular weight that can be calculated usmg amino acid residue molecular weight values well known to those skilled in the art The polynucleotide of SEQ ED NO 1, between nucleotide number 1 and the stop codon which begins at nucleotide number 589 of SEQ ID NO 1 , encodes the polypeptide of SEQ ID NO 2 In a further aspect, the present invention provides for an isolated polynucleotide comprising or consisting of: (a) a polynucleotide sequence which has at least 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; (b) 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 100% exact, to the amino acid sequence of SEQ ID NO:2. over the entire length of SEQ ID NO:2.

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

The invention provides a polynucleotide sequence identical over its entire length to a coding sequence (open reading frame) in Table 1 [SEQ ID NO:l]. Also provided by the invention is a coding sequence for a mature polypeptide or a fragment thereof, by itself as well as a coding sequence for a mature polypeptide or a fragment in reading frame with another coding sequence, such as a sequence encoding a leader or secretory sequence, a pre-, or pro- or prepro-protein sequence. The polynucleotide of the invention may also contain at least one non-coding sequence, including for example, but not limited to at least one non-coding 5' and 3' sequence, such as the transcribed but non-translated sequences, termination signals (such as rho-dependent and rho-mdependentteirnination signals), ribosome binding sites, Kozak sequences, sequences that stabilize mRNA, introns, and polyadenylation signals. The polynucleotide sequence may also comprise additional coding sequence encoding additional amino acids. For example, a marker sequence that facilitates purification of the fused polypeptide can be encoded. In certain embodiments of the invention, the marker sequence is a hexa- histidine peptide, as provided in the pQE vector (Qiagen, Inc.) and described in Gentz et al, Proc. Natl. Acad. Sci., USA 86: 821-824 (1989), or an HA peptide tag (Wilson et al. , Cell 37: 767 (1984), both of which may be useful in purifying polypeptide sequence fused to them. Polynucleotides of the invention also include, but are not limited to, polynucleotides comprising a structural gene and its naturally associated sequences that control gene expression. A preferred embodiment of the invention is a polynucleotide of consisting of or comprising nucleotide 1 to the nucleotide immediately upstream of or including nucleotide 589 set forth in SEQ ID NO:l of Table 1, both of which encode the nrdG polypeptide. The invention also includes a polynucleotide consisting of or comprising a polynucleotide of the formula:

X-(Rι )_m-(R₂)-(R₃)_n-Y wherein, at the 5' end of the molecule, X is hydrogen, a metal or a modified nucleotide residue, or together with Y defines a covalent bond, and at the 3' end of the molecule, Y is hydrogen, a metal, or a modified nucleotide residue, or together with X defines the covalent bond, each occurrence of Ri and R3 is 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 R is a nucleic acid sequence or modified nucleic acid sequence of the invention, particularly a nucleic acid sequence selected from Table 1 or a modified nucleic acid sequence thereof. In the polynucleotide formula above, R is 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 R_j 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 shows a first strand to which the second strand is complementary. In another preferred embodiment m and/or n is an integer between 1 and 1000. Other preferred embodiments of the invention are provided where m is an integer between 1 and 50, 100 or 500, and n is an integer between 1 and 50, 100, or 500. It is most preferred that a polynucleotide of the invention is derived from Streptococcus pneumoniae, however, it may preferably be obtained from other organisms of the same taxonomic genus. A polynucleotide of the invention may also be obtained, for example, from organisms of the same taxonomic family or order.

The term "polynucleotide encoding a polypeptide" as used herein encompasses polynucleotides that include a sequence encoding a polypeptide of the invention, particularly a bacterial polypeptide and more particularly a polypeptide of the Streptococcus pneumoniae nrdG having an amino acid sequence set out in Table 1 [SEQ ID NO:2]. The term also encompasses polynucleotides that include a single continuous region or discontinuous regions encoding the polypeptide (for example, polynucleotides interrupted by integrated phage, an integrated insertion sequence, an integrated vector sequence, an integrated transposon sequence, or due to RNA editing or genomic DNA reorganization) together with additional regions, that also may contain coding and/or non-coding sequences .

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

Further particularly preferred embodiments are polynucleotides encoding nrdG vanants, that have the ammo acid sequence of nrdG polypeptide of Table 1 [SEQ ED NO 2] in which several, a few, 5 to 10, 1 to 5, 1 to 3, 2, 1 or no amino acid residues are substituted, modified, deleted and/or added, in any combination

Especially prefeπed among these are silent substitutions, additions and deletions, that do not alter the properties and activities of nrdG polypeptide

Further preferred embodiments of the invention are polynucleotides that are at least 70% identical over their entire length to a polynucleotide encoding nrdG polypeptide having an ammo acid sequence set out in Table 1 [SEQ ID NO 2], and polynucleotides that are complementary to such polynucleotides Alternatively, most highly preferred are polynucleotides that compπse a region that is at least 80% identical over its entire length to a polynucleotide encoding nrdG polypeptide and polynucleotides complementary thereto In this regard, polynucleotides at least 90% identical over their entire length to the same are particularly preferred, and among these particularly preferred polynucleotides, those with at least 95% are especially prefeπed Furthermore, those with at least 97% are highly prefeπed among those with at least 95%, and among these those with at least 98% and at least 99% are particularly highly prefeπed, with at least 99% being the more prefeπed

Prefeπed embodiments are polynucleotides encoding polypeptides that retain substantially the same biological function or activity as the mature polypeptide encoded by a DNA of Table 1 [SEQ ED NO 1] In accordance with certain prefeπed embodiments of this invention there are provided polynucleotides that hybπdize, particularly under stπngent conditions, to nrdG polynucleotide sequences, such as those polynucleotides in Table 1

The invention further relates to polynucleotides that hybπdize to the polynucleotide sequences provided herein In this regard, the invention especially relates to polynucleotides that hybπdize under stπngent conditions to the polynucleotides descπbed herein As herem used, the terms "stπngent conditions" and

"stπngent hybπdization conditions" mean hybπdization occurring only if there is at least 95% and preferably at least 97% identity between the sequences A specific example of strmgent hybridization conditions is overnight incubation at 42°C m a solution compnsmg 50% formarmde, 5x SSC (150mM NaCl, 15mM tπsodium 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 Clomng A Laboratory Manual, Second Edition, Cold Spring Harbor, N.Y., (1989), particularly Chapter 11 therein. Solution hybridization may also be used with the polynucleotide sequences provided by the invention.

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

As discussed elsewhere herein regarding polynucleotide assays of the invention, for instance, the polynucleotides of the invention, may be used as a hybridization probe for RNA, cDNA and genomic DNA to isolate full-length cDNAs and genomic clones encoding nrdG and to isolate cDNA and genomic clones of other genes that have a high identity, particularly high sequence identity, to the nrdG 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 prefeπed 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 nrdG gene may be isolated by screening using a DNA sequence provided in Table 1 [SEQ ID NO:l] 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 mvention may be employed, for example, as research reagents and mateπals for discovery of treatments of and diagnostics for diseases, particularly human diseases, as further discussed herein relatmg to polynucleotide assays

The polynucleotides of the invention that are oligonucleotides derived from a sequence of Table 1 [SEQ ID NOS 1 or 2] may be used in the processes herem as descπbed, but preferably for PCR, to determine whether or not the polynucleotides identified herem in whole or m part are transcπbed in bacteria in infected tissue It is recognized that such sequences will also have utility in diagnosis of the stage of infection and type of 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 intenor to the mature poh-peptide (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 prefeπed that these complementary polynucleotides are fully complementary to each polynucleotide with which they are complementary A precursor protein, having a mature form of the polypeptide fused to one or more prosequences may be an inactive form of the polypeptide When prosequences are removed such inactive precursors generally are activated Some or all of the prosequences may be removed before activation Generally, such precursors are called proproteins

In addition to the standard A, G, C, T/U representations for nucleotides, the term "N" may also be used m 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 prefeπed that N is not a nucleic acid that when taken in combination with adjacent nucleotide positions, when read in the correct read g frame, would have the effect of generating a premature termination codon in such reading frame In sum, a polynucleotide of the mvention may encode a mature protein, a mature protem plus a leader sequence (which may be refeπed to as a preprotein), a precursor of a mature protem having one or more prosequences that are not the leader sequences of a preprotein, or a preproprotem, which is a precursor to a proprotern. havmg a leader sequence and one or more prosequences, which generally are removed dunng processmg steps that produce active and mature forms of the polypeptide Vectors, Host Cells, Expression Systems

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

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

For recombinant production of the polypeptides of the mvention, host cells can be genetically engmeered to incorporate expression systems or portions thereof or polynucleotides of the mvention Introduction of a polynucleotide mto the host cell can be effected by methods descπbed m many standard laboratory manuals, such as Davis, et al , BASIC METHODS IN MOLECULAR BIOLOGY, (1986) and Sambrook, et al , MOLECULAR CLONING A LABORATORY MANUAL, 2nd Ed , Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N Y (1989), such as, calcium phosphate transfection, DEAE-dextran mediated transfection, transvection, micromjection, catiomc hpid-mediated transfection. electroporation, transduction, scrape loading, ballistic mtroduction and infection

Representative examples of appropnate hosts include bacteπal cells, such as cells of streptococci, staphylococci, enterococci E coli, streptomyces, cyanobactena, Bacillus subtihs, and Streptococcus pneumoniae, fungal cells, such as cells of a yeast, Kluveromyces, Saccharomyces, a basidiomycete, Candida albicans and Aspergillus, insect cells such as cells oϊ Drosophila S2 and Spodoptera Sf9, animal cells such as CHO, COS, HeLa, C127, 3T3, BHI , 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 clude, among others, chromosomal-, episomal- and virus-deπved vectors, for example, vectors denved from bactenal plasmids, from bacteπophage, from transposons, from yeast episomes, from insertion elements, from yeast chromosomal elements, from viruses such as baculoviruses, papova viruses, such as SV40, vaccinia viruses, adenovrruses, fowl pox viruses, pseudorabies viruses, picornavmises and retroviruses, and vectors denved from combinations thereof, such as those denved from plasmid and bacteπophage 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 vaπety of well-known and routme techniques, such as, for example, those set forth m Sambrook et al , MOLECULAR CLONING, A LABORATORY MANUAL, (supra)

In recombinant expression systems m eukaryotes, for secretion of a translated protem mto the lumen of the endoplasnuc reticulum, mto the peπplasmic space or mto the extracellular environment, appropnate secretion signals may be incorporated mto the expressed polypeptide These signals may be endogenous to the polypeptide or they may be heterologous signals Polypeptides of the mvention can be recovered and punfied from recombinant cell cultures by well- known methods including ammonium sulfate or ethanol precipitation, acid extraction, anion or cation exchange chromatography, phosphocellulose chromatography, hydrophobic mteraction chromatography, affinity chromatography, hydroxylapatite chromatography, and lectin chromatography Most preferably, high performance liquid chromatography is employed for purification Well known techniques for refolding protem may be employed to regenerate active conformation when the polypeptide is denatured dunng isolation and or puπfication

Diagnostic, Prognostic, Serotyping and Mutation Assays

This mvention is also related to the use of nrdG polynucleotides and polypeptides of the mvention for use as diagnostic reagents Detection of nrdG polynucleotides and/or polypeptides m a eukaryote, particularly a mammal, and especially a human, will provide a diagnostic method for diagnosis of disease, stagmg of disease or response of an infectious organism to drugs Eukaryotes, particularly mammals, and especially humans, particularly those infected or suspected to be infected with an orgamsm compnsmg the nrdG gene or protein, may be detected at the nucleic acid or ammo acid level by a vaπety of well known techniques as well as by methods provided herem Polypeptides and polynucleotides for prognosis, diagnosis or other analysis may be obtained from a putatively infected and/or infected mdividual's bodily mateπals 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 pπor 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 orgamsm present in an mdividual, may be made by an analysis of the genotype of a selected polynucleotide of the orgamsm Deletions and insertions can be detected by a change m size of the amplified product m compaπson to a genotype of a reference sequence selected from a related orgamsm, preferably a different species of the same genus or a different strain of the same species Point mutations can be identified by hybndizmg amplified DNA to labeled nrdG polynucleotide sequences Perfectly or sigmficantly matched sequences can be distinguished from imperfectly or more sigmficantly mismatched duplexes by DNase or RNase digestion, for DNA or RNA respectively, or by detecting differences m melting temperatures or renaturation kinetics Polynucleotide sequence differences may also be detected by alterations m 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 aπay of oligonucleotides probes compπsmg nrdG nucleotide sequence or fragments thereof can be constructed to conduct efficient screening of, for example, genetic mutations, serotype, taxonomic classification or identification Aπay technology methods are well known and have general applicability and can be used to address a vaπety of questions m molecular genetics including gene expression, genetic linkage, and genetic vanabihty (see, for example, Chee et al , Science, 274 610 (1996))

Thus in another aspect, the present mvention relates to a diagnostic kit which compnses (a) a polynucleotide of the present invention, preferably the nucleotide sequence of SEQ ID NO 1, or a fragment thereof , (b) a nucleotide sequence complementary to that of (a), (c) a polypeptide of the present mvention, preferably the polypeptide of SEQ ID NO 2 or a fragment thereof, or (d) an antibody to a polypeptide of the present mvention, preferably to the polypeptide of SEQ ID NO 2

It will be appreciated that in any such kit, (a), (b), (c) or (d) may compπse a substantial component Such a kit will be of use m diagnosing a disease or susceptibility to a Disease, among others This mvention also relates to the use of polynucleotides of the present mvention as diagnostic reagents Detection of a mutated form of a polynucleotide of the mvention, preferable, SEQ ED NO 1. 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 vaπety of techniques, such as those descπbed 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 mapping of relevant sequences to chromosomes according to the present mvention may be an important step m coπelating those sequences with pathogenic potential and/or an ecological niche of an organism and/or drug resistance of an organism, as well as the essentiality of the gene to the organism. Once a sequence has been mapped to a precise chromosomal location, the physical position of the sequence on the chromosome can be coπelated with genetic map data. Such data may be found on-line in 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 (coinheritance of physically adjacent genes) or mating studies, such as by conjugation.

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

Cells from an organism carrying mutations or polymorphisms (allelic variations) in a polynucleotide and/or polypeptide of the invention may also be detected at the polynucleotide or polypeptide level by a variety of techniques, to allow for serotyping, for example. For example, RT-PCR can be used to detect mutations in the RNA. It is particularly prefeπed to use RT-PCR in conjunction with automated detection systems, such as, for example, GeneScan. RNA, cDNA or genomic DNA may also be used for the same purpose, PCR. As an example, PCR primers complementary to a polynucleotide encoding nrdG polypeptide can be used to identify and analyze mutations. Examples of representative primers are shown below in Table 2.

Table 2 Primers for amplification of nrdG polynucleotides SEQ ID NO PRIMER SEQUENCE

3 5'-ATGAATAATCCAAAACCACA-3'

4 5'-TCATTCTCTCTTCACCTGTT-3'

The invention also includes primers of the formula:

X-(Rι)_m-(R₂)-(R₃)_n-Y wherein, 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, Ri and R3 are any nucleic acid residue or modified nucleotide residue, m is an integer between 1 and 20 or zero , n is an integer between 1 and

20 or zero, and R₂ is a primer sequence of the invention, particularly a primer sequence selected from Table 2. En the polynucleotide formula above R is oπented 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 prefeπed embodiment m and/or n is an integer between 1 and 10.

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

The mvention further provides a process for diagnosing disease, preferably bacterial infections, more preferably infections caused by Streptococcus pneumoniae, compπsmg determining from a sample derived from an individual, such as a bodily material, an mcreased level of expression of polynucleotide havmg a sequence of Table 1 [SEQ ID NO 1] Increased or decreased expression of a nrdG polynucleotide can be measured usmg any on of the methods well known m the art for the quantitation of polynucleotides, such as, for example, amplification, PCR, RT-PCR, RNase protection, Northern blotting, spectrometry and other hybridization methods In addition, a diagnostic assay m accordance with the mvention for detecting over-expression of nrdG 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 nrdG polypeptide, m a sample denved from a host, such as a bodily matenal, are well-known to those of skill m the art Such assay methods include 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 Bactenol 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 comparmg pre-infection and post infection profiles, genes up and down regulated dunng infection can be identified and the RT-PCR product sequenced and matched to ORF "unknowns " In Vivo Expression Technology (IVET) is described by Camilli et al Proc Nat 'I 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 m infection establishment and/or maintenance In this technique random chromosomal fragments of target organism are cloned upstream of a promoter-less recombmase gene m a plasmid vector This construct is introduced into the target orgamsm which carπes an antibiotic resistance gene flanked by resolvase sites Growth in the presence of the antibiotic removes from the population those fragments cloned mto the plasmid vector capable of supporting transcπption of the recombmase gene and therefore have caused loss of antibiotic resistance The resistant pool is mtroduced into a host and at vaπous 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 dunng mfection Sequencing 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 mvention, messenger RNA is isolated from bacteπal mfected tissue, e g , 48 hour murme lung infections, and the amount of each mRNA species assessed by reverse transcπption 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 bacteπal genes which are transcribed m the infected tissue Analysis of gene transcription can be carried out at different times of infection to gam a detailed knowledge of gene regulation in bactenal pathogenesis allowing for a clearer understanding of which gene products represent targets for screens for antibacteπals Because of the gene specific nature of the PCR prrmeis employed it should be understood that the bacterial mRNA preparation need not be free of mammalian RNA This allows the mvestigator to carry out a simple and quick RNA preparation from infected tissue to obtam bactenal mRNA species which are very short lived in the bactenum (m the order of 2 minute halflives) Optimally the bactenal mRNA is prepared from infected murme lung tissue by mechanical disruption m the presence of TRIzole (GIBCO-BRL) for very short peπods of time, subsequent processmg accordmg 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 oϊ Streptococcus pneumoniae 16S πbosomal RNA as detected by probmg Northerns with a suitably labeled sequence specific oligonucleotide probe Typically a 5' dye labeled primer is used in each PCR primer pair in 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 using 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, 274:6 0-614 (1996) and other references cited therein. Such gridding 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 variety of techniques have also been described for identifying particular gene sequences on the basis of their gene products. For example, s International Patent Application No. WO91/07087, published May 30, 1991. In addition, methods have been described 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 aπays, 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 invention, 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 in an individual. Such a presence may indicate the presence of a pathogen, particularly Streptococcus pneumoniae, and may be useful in 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 are preferred. Also preferred is a comprising a number of variants of a polynucleotide sequence encoding the polypeptide sequence of SEQ ID NO:2.

Antibodies The polypeptides and polynucleotides of the invention or variants thereof, or cells expressing the same can be used as immunogens to produce antibodies immunospecific for such polypeptides or polynucleotides respectively.

In certain prefeπed embodiments of the invention there are provided antibodies against nrdG polypeptides or polynucleotides. Antibodies generated against the polypeptides or polynucleotides of the invention can be obtained by administering the polypeptides and/or polynucleotides of the invention, or epitope-bearing fragments of either or both, analogues of either or both, or cells expressing either or both, to an animal, preferably a nonhuman, using routine protocols. For preparation of monoclonal antibodies, any technique known in the art that provides antibodies produced by continuous cell line cultures can be used. Examples include various techniques, such as those in Kohler, G. and Milstein, C, Nature 256: 495-497 (1975); Kozbor et al, Immunology Today 4: 72 (1983); Cole et al, pg. 77-96 in MONOCLONAL ANTIBODIES AND CANCER THERAPY, Alan R. Liss, Inc. (1985). Techmques for the production of single chain antibodies (U.S. Patent No. 4,946,778) can be adapted to produce single chain antibodies to polypeptides or polynucleotides of this invention. Also, transgenic mice, or other organisms such as other mammals, may be used to express humanized antibodies immunospecific to the polypeptides or polynucleotides of the invention.

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 possessing anti-nrdG 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-described antibodies may be employed to isolate or to identify clones expressing the polypeptides or polynucleotides of the invention to purify the polypeptides or polynucleotides by, for example, affinity chromatography.

Thus, among others, antibodies against nrdG-polypeptide or nrdG-polynucleotide may be employed to treat infections, particularly bacterial infections. Polypeptide variants include antigenically, epitopically or immunologically equivalent variants form a particular aspect of this invention.

A polypeptide or polynucleotide of the invention, such as an antigenically or immunologically 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 immunogenic carrier protein for example bovine serum albumin, keyhole limpet haemocyanin or tetanus toxoid. Alternatively, a multiple antigenic polypeptide comprising multiple copies of the polypeptide, or an antigenically or immunologically equivalent polypeptide thereof may be sufficiently antigenic to improve immunogenicity so as to obviate the use of a carrier. Preferably, the antibody or variant thereof is modified to make it less immunogenic in the individual. For example, if the individual is human the antibody may most preferably be "humanized," where the complimentarity determimng region or regions of the hybridoma-derived antibody has been transplanted into a human monoclonal antibody, for example as described in Jones et al (1986), Nature 321, 522-525 or Tempest et al , (1991) Biotechnology 9, 266-273

In accordance with an aspect of the invention, there is provided the use of a polynucleotide of the invention for therapeutic or prophylactic purposes, in particular genetic immunization Among the particularly prefeπed embodiments of the mvention are naturally occurring allehc vanants of nrdG 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 into 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 protem earners (Wu et al , J Biol Chem (1989) 264 16985), coprecipitation of DNA with calcium phosphate (Benve sty & Reshef, PNAS USA, (1986) 83 9551), encapsulation of DNA m 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 usmg 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 binding of small molecule substrates and ligands in, for example, cells, cell-free preparations, chemical hbraπes, and natural product mixtures These substrates and ligands may be natural substrates and ligands or may be structural or functional mimetics See, e g , Coligan et al , Current Protocols n Immunology 1(2) Chapter 5 (1991) Polypeptides and polynucleotides of the present mvention are responsible for many biological functions, including many disease states, m particular the Diseases hereinbefore mentioned It is therefore desirable to devise screening methods to identify compounds which stimulate or which inhibit the function of the polypeptide or polynucleotide Accordingly, in a further aspect, the present mvention provides for a method of screening compounds to identify those which stimulate or which inhibit the function of a polypeptide oi polynucleotide of the mvention, as well as related polypeptides and polynucleotides In general, agonists or antagonists may be employed for therapeutic and prophylactic purposes for such Diseases as herembefore mentioned Compounds may be identified from a vaπety of sources, for example, cells, cell-free preparations, chemical hbraπes, and natural product mixtures Such agonists, antagonists or inhibitors so-identified may be natural or modified substrates, hgands. receptors, enzymes, etc , as the case may be, of nrdG polypeptides and polynucleotides, or may be structural or functional mimetics thereof (see Coligan et al , Current Protocols in Immunology 1(2) Chapter 5 (1991))

The screening methods may simply measure the binding of a candidate compound to the polypeptide or polynucleotide, or to cells or membranes bearing the polypeptide or polynucleotide, or a fusion protein of the polypeptide by means of a label directly or indirectly associated with the candidate compound. Alternatively, the screening method may involve competition with a labeled competitor. Further, these screening methods may test whether the candidate compound results in a signal generated by activation or inhibition of the polypeptide or polynucleotide, using detection systems appropriate to the cells comprising the polypeptide or polynucleotide. Inhibitors of activation are generally assayed in the presence of a known agonist and the effect on activation by the agonist by the presence 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, in 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 screening 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 nrdG polypeptide and/or polynucleotide activity in the mixture, and comparing the nrdG polypeptide and/or polynucleotide activity of the mixture to a standard. Fusion proteins, such as those made from Fc portion and nrdG polypeptide, as hereinbefore described, can also be used for high-throughput screening assays to identify antagonists of the polypeptide of the present invention, as well as of phylogenetically and and/or functionally related polypeptides (see D. Bennett et al., J Mol Recognition, 8:52-58 (1995); and K. Johanson et al., J Biol Chem, 270(16): 9459-9471 (1995)).

The polynucleotides, polypeptides and antibodies that 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 in cells. For example, an ELISA assay may be constructed for measuring secreted or cell associated levels of polypeptide using monoclonal and polyclonal antibodies by standard methods known in the art. This can be used to discover agents which may inhibit or enhance the production of polypeptide (also called antagonist or agonist, respectively) from suitably manipulated cells or tissues.

The invention also provides a method of screening compounds to identify those which enhance (agonist) or block (antagonist) the action of nrdG polypeptides or polynucleotides, particularly those compounds that are bacteristatic and/or bactericidal. The method of screening may involve high-throughput techniques. For example, to screen for agonists or antagonists, a synthetic reaction mix, a cellular compartment, such as a membrane, cell envelope or cell wall, or a preparation of any thereof, comprising nrdG polypeptide and a labeled substrate or ligand of such polypeptide is incubated in the absence or the presence of a candidate molecule that may be a nrdG agonist or antagonist. The ability of the candidate molecule to agonize or antagonize the nrdG polypeptide is reflected in decreased binding of the labeled ligand or decreased production of product from such substrate Molecules that bind gratuitously, i e , without mducmg the effects of nrdG polypeptide are most likely to be good antagonists Molecules that bmd well and. as the case may be. increase the rate of product production from substrate, 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 nrdG polynucleotide or polypeptide activity, and binding assays known m the art

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

The fluorescence polarization value for a fluorescently-tagged molecule depends on the rotational coπelation time or tumbling rate Protem complexes, such as formed by nrdG polypeptide associatmg with another nrdG polypeptide or other polypeptide, labeled to comprise a fluorescently- labeled molecule will have higher polarization values than a fluorescently labeled monomeπc protem It is prefeπed that this method be used to characteπze small molecules that disrupt polypeptide complexes Fluorescence energy transfer may also be used characteπze small molecules that interfere with the formation of nrdG polypeptide drmers, tπmers, tetramers or higher order structures, or structures formed by nrdG polypeptide bound to another polypeptide NrdG polypeptide can be labeled with both a donor and acceptor fluorophore Upon mixing of the two labeled species and excitation of the donor fluorophore, fluorescence energy transfer can be detected by observing fluorescence of the acceptor Compounds that block dimerization will inhibit fluorescence energy transfer

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

A scintillation proximity assay may be used to characterize the interaction between an association of nrdG polypeptide with another nrdG polypeptide or a different polypeptide. NrdG polypeptide can be coupled to a scintillation-filled bead. Addition of radio-labeled nrdG polypeptide results in binding where the radioactive source molecule is in close proximity to the scintillation fluid. Thus, signal is emitted upon nrdG polypeptide binding and compounds that prevent nrdG polypeptide self-association or an association of nrdG polypeptide and another polypeptide or small molecule will diminish 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- facilitated ion channels in suspended membrane bilayers and hence to a measurable change in the admittance (similar to impedence) 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 libraries. In other embodiments of the invention there are provided methods for identifying compounds which bind to or otherwise interact with and inhibit or activate an activity or expression of a polypeptide and/or polynucleotide of the invention comprising: contacting a polypeptide and/or polynucleotide of the invention with a compound to be screened under conditions to permit binding to or other interaction between the compound and the polypeptide and/or polynucleotide to assess the binding to or other interaction with the compound, such binding or interaction preferably being associated with a second component capable of providing a detectable signal in response to the binding or interaction of the polypeptide and/or polynucleotide with the compound; and deteimining whether the compound binds to or otherwise interacts with and activates or inhibits an activity or expression of the polypeptide and/or polynucleotide by detecting the presence or absence of a signal generated from the binding or interaction of the compound with the polypeptide and/or polynucleotide. Another example of an assay for nrdG agonists is a competitive assay that combines nrdG and a potential agonist with nrdG-binding molecules, recombinant nrdG binding molecules, natural substrates or ligands, or substrate or ligand mimetics, under appropriate conditions for a competitive inhibition assay. nrdG can be labeled, such as by radioactivity or a colorimetric compound, such that the number of nrdG molecules bound to a binding molecule or converted to product can be determined accurately to assess the effectiveness of the potential antagonist.

Potential antagonists include, among others, small organic molecules, peptides, polypeptides and antibodies that bind to a polynucleotide and/or polypeptide of the invention and thereby inhibit or extinguish its activity or expression. Potential antagonists also may be small organic molecules, a peptide, a polypeptide such as a closely related protein or antibody that binds the same sites on a binding molecule, such as a binding molecule, without inducing nrdG-induced activities, thereby preventing the action or expression of nrdG polypeptides and/or polynucleotides by excluding nrdG polypeptides and/or polynucleotides from binding.

Potential antagonists include a small molecule that binds to and occupies the binding site of the polypeptide thereby preventing binding to cellular binding molecules, such that normal biological activity is prevented. Examples of small molecules include but are not limited to small organic molecules, peptides or peptide-like molecules. Other potential antagonists include antisense molecules (see Okano, J. Neurochem. 56: 560 (1991); OUGODEOXYNUCLEOTIDES AS ANTISENSE INHIBITORS OF GENE EXPRESSION, CRC Press, Boca Raton, FL (1988), for a description of these molecules). Prefeπed potential antagonists include compounds related to and variants of nrdG.

Other examples of potential polypeptide antagonists include antibodies or, in some cases, oligonucleotides or proteins which are closely related to the ligands, substrates, receptors, enzymes, etc., as the case may be, of the polypeptide, e.g., a fragment of the ligands, substrates, receptors, enzymes, etc.; or small molecules which bind to the polypeptide of the present invention but do not elicit a response, so that the activity of the polypeptide is prevented.

Certain of the polypeptides of the invention are biomimetics, functional mimetics of the natural nrdG polypeptide. These functional mimetics may be used for, among other things, antagonizing the activity of nrdG polypeptide or as a antigen or immunogen in a manner described elsewhere herein. Functional mimetics of the polypeptides of the invention include but are not limited to truncated polypeptides. For example, prefeπed functional mimetics include, a polypeptide comprising the polypeptide sequence set forth in SEQ ED NO:2 lacking 20, 30, 40, 50, 60, 70 or 80 amino- or carboxy-terminal amino acid residues, including fusion proteins comprising 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 prefeπed that these cassettes comprise 5' and 3' restriction sites to allow for a convenient means to ligate the cassettes together when desired. It is further prefeπed that these cassettes comprise gene expression signals known in the art or described elsewhere herein.

Thus, in another aspect, the present invention relates to a screening kit for identifying agonists, antagonists, ligands, 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 in a method for the structure-based design of an agonist, antagonist 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 site(s), binding site(s) or motif(s) of an agonist, antagonist or inhibitor; (c) synthesizing candidate compounds that are predicted to bind to or react with the deduced binding site(s), reactive site(s), and/or motif(s); and

(d) testing whether the candidate compounds are indeed agonists, antagonists or inhibitors. It will be further appreciated that this will normally be an iterative process, and this iterative process may be performed using automated and computer-controlled steps. In a further aspect, the present invention provides methods of treating abnormal conditions such as, for instance, a Disease, related to either an excess of, an under-expression of, an elevated activity of, or a decreased activity of nrdG polypeptide and/or polynucleotide.

If the expression and/or activity of the polypeptide and/or polynucleotide is in excess, several approaches are available. One approach comprises administering to an individual in need thereof an inhibitor compound (antagonist) as herein described, optionally in combination with a pharmaceutically acceptable carrier, in an amount effective to inhibit the function and/or expression of the polypeptide and/or polynucleotide, such as, for example, by blocking the binding of ligands, substrates, receptors, enzymes, etc., or by 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 nrdG 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 various subclasses (IgG, IgM, IgA, IgE). Preferred as an immunoglobulin is the constant part of the heavy chain of human IgG, particularly IgGl, where fusion takes place at the hinge 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 screening, diagnosis and therapy. A further aspect of the invention also relates to polynucleotides encoding such fusion proteins. Examples of fusion protein technology can be found in International Patent Application Nos. W094/29458 and W094/22914.

In still another approach, expression of the gene encoding endogenous nrdG polypeptide can be inhibited using expression blocking techniques. This blocking may be targeted against any step in gene expression, but is preferably targeted against transcription and/or translation. An examples of a known technique of this sort involve the use of antisense sequences, either internally generated or separately administered (see, for example, O'Connor, J Neurochem (1991) 56:560 in Oligodeoxynucleotides as Antisense Inhibitors of Gene Expression, CRC Press, Boca Raton, FL (1988)). Alternatively, oligonucleotides 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 screening of antibacterial drugs. Additionally, the polynucleotide sequences encoding the amino terminal regions of the encoded protein or Shine-Delgarno or other translation facilitating sequences of the respective mRNA can be used to construct antisense sequences to control the expression of the coding sequence of interest. The invention also provides the use of the polypeptide, polynucleotide, agonist or antagonist of the invention to interfere with the initial physical interaction between a pathogen or pathogens and a eukaryotic, preferably mammalian, host responsible for sequelae of infection. In particular, the molecules of the invention may be used: in the prevention of adhesion of bacteria, in particular gram positive and/or gram negative bacteria, to eukaryotic, preferably mammalian, extracellular matrix proteins on in-dwelling devices or to extracellular matrix proteins in wounds; to block bacterial adhesion between eukaryotic, preferably mammalian, extracellular matrix proteins and bacterial nrdG proteins that mediate tissue damage and/or; to block the normal progression of pathogenesis in infections initiated other than by the implantation of in-dwelling devices or by other surgical techniques. In accordance with yet another aspect of the mvention, there are provided nrdG agomsts and antagonists, preferably bacteπstatic or bacteπcidal agomsts and antagonists

The antagonists 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 causmg stomach cancer, ulcers, and gastritis (International Agency for Research on Cancer (1994) Schistosomes, Liver Flukes and Hehcobacter Pylori (International Agenc> 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 gastπc adenocarcmoma, classifymg the bacterium as a Group I (defimte) carcmogen Prefeπed antimicrobial compounds of the mvention (agonists and antagonists of nrdG polypeptides and/or polynucleotides) found using screens provided by the invention, or known m the art, particularly naπow-spectrum antibiotics, should be useful m the treatment of H pylori infection Such treatment should decrease the advent of H pylori -mduced cancers, such as gastrointestinal carcinoma Such treatment should also prevent, inhibit and/or cure gastπc ulcers and gastπtis Vaccines

There are provided by the mvention, products, compositions and methods for assessmg nrdG expression, treating disease, assaying genetic vanation, and administering a nrdG polypeptide and/or polynucleotide to an orgamsm to raise an immunological response against a bacteπa, especialK a Streptococcus pneumoniae bacteπa

Another aspect of the mvention relates to a method for inducing an immunological response m an mdividual, particularly a mammal which comprises inoculating the individual with nrdG polynucleotide and/or polypeptide, or a fragment or variant thereof, adequate to produce antibody and/ or T cell immune response to protect said mdividual from infection, particularly bacteπal mfection and most particularly Streptococcus pneumoniae infection Also provided are methods whereby such immunological response slows bacteπal replication Yet another aspect of the invention relates to a method of inducing immunological response m an mdividual which comprises delivering to such mdividual a nucleic acid vector, sequence or nbozyme to direct expression of nrdG polynucleotide and/or polypeptide, or a fragment or a vaπant thereof, for expressing nrdG polynucleotide and/or polypeptide, or a fragment or a vaπant thereof in vivo in order to mduce an immunological response, such as, to produce antibody and/ or T cell immune response, including, for example, cytokme-producmg T cells or cytotoxic T cells, to protect said mdividual, preferably a human, from disease, whether that disease is already established withm the mdividual or not One example of administering the gene is by accelerating it mto the desired cells as a coating on particles or otherwise Such nucleic acid vector may compπse DNA, RNA. a ribozyme, a modified nucleic acid, a DNA/RNA hybrid, a DNA-protem complex or an RNA-protein complex

A further aspect of the mvention relates to an immunological composition that when introduced into an individual, preferably a human, capable of havmg mduced within it an immunological response, induces an immunological response m such individual to a nrdG polynucleotide and/or polypeptide encoded therefrom, wherein the composition comprises a recombinant nrdG polynucleotide and/or polypeptide encoded therefrom and/or compnses DNA and/or RNA which encodes and expresses an antigen of said nrdG 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 arising from CTL or CD4+ T cells

A nrdG 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 protem and producmg a fused or modified protem which will have antigenic and/or immunogenic properties, and preferably protective properties Thus fused recombinant protem, preferably further comprises an antigenic co-protein, such as hpoprotein D from Hemophύus mfluenzae, Glutathione-S-transferase (GST) or beta-galactosidase, or any other relatively large co-protem which solubihzes the protem and facilitates production and purification thereof Moreover, the co-protem may act as an adjuvant in the sense of providing a generalized stimulation of the immune system of the orgamsm receivmg the protem The co-protem may be attached to either the ammo- or carboxy-termmus of the first protem

Provided by this invention are compositions, particularly vaccme compositions, and methods compnsmg the polypeptides and/or polynucleotides of the mvention and lmmunostimulatory DNA sequences, such as those described m Sato, Y et al Science 273 352 (1996) Also, provided by this mvention are methods usmg the described polynucleotide or particular fragments thereof, which have been shown to encode non-vaπable regions of bacteπal cell surface proteins, m polynucleotide constructs used m such genetic immunization experiments m 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, denved from the requisite organ of the animal successfully resistmg or clearing infection, for the development of prophylactic agents or therapeutic treatments of bacteπal infection, particularly Streptococcus pneumoniae infection, m 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 bacteπa to damaged tissue Examples of tissue damage include wounds m skin or connective tissue caused, for example, by mechanical, chemical, thermal or radiation damage or by implantation of indwelling devices, or wounds in the mucous membranes, such as the mouth, throat, mammary glands, urethra or vagina

The invention also includes a vaccine formulation which comprises an immunogenic recombinant polypeptide and/or polynucleotide of the invention together with a suitable earner, such as a pharmaceutically acceptable carrier Smce the polypeptides and polynucleotides may be broken down in the stomach, each is preferably administered parenterally, mcludmg, for example, admmistration 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, bactenstatic compounds and solutes which render the formulation lsotomc with the bodily fluid, preferably the blood, of the individual, and aqueous and non-aqueous stenle suspensions which may include suspendmg agents or thickening agents The formulations may be presented in unit- dose or multi-dose contamers, for example, sealed ampoules and vials and may be stored in a freeze-dned condition requirmg only the addition of the stenle liquid earner immediately pπor to use The vaccine formulation may also include adjuvant systems for enhancing the lmmunogenicity of the formulation, such as oil-in water systems and other systems known in the art The dosage will depend on the specific activity of the vaccine and can be readily determined by routme experimentation

While the invention has been described with reference to certain nrdG 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 immunogenic properties of the recombinant polypeptides or polynucleotides

Compositions, kits and administration

In a further aspect of the mvention there are provided compositions compnsmg a nrdG polynucleotide and/or a nrdG polypeptide for admmistration 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 in combination with a non-steπle or stenle earner or earners for use with cells, tissues or organisms, such as a pharmaceutical earner suitable for admmistration to an mdividual Such compositions compnse, foi 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 limited to, saline, 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 in conjunction with other compounds, such as therapeutic compounds

The pharmaceutical compositions may be admmistered in any effective, convenient manner mcludmg, for instance, admmistration by topical, oral, anal, vaginal, mtravenous, intrapentoneal, intramuscular. subcutaneous, lntranasal or lntradermal routes among others

In therapy or as a prophylactic, the active agent may be admmistered to an mdividual as an injectable composition, for example as a sterile aqueous dispersion, preferably isotonic

Alternatively the composition may be formulated for topical application for example in the form of ointments, creams, lotions, eye ointments, eye drops, ear drops, mouthwash, impregnated dressmgs and sutures and aerosols, and may contain appropπate conventional additives, mcludmg, for example, preservatives, solvents to assist drug penetration, and emollients in ointments and creams Such topical formulations may also contam compatible conventional carriers, for example cream or omtment bases, and ethanol or oleyl alcohol for lotions Such earners 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, agonist or antagonist peptide or small molecule compound, m combination with a pharmaceutically acceptable earner or excipient Such earners mclude, but are not limited to, salme, buffered salme, dextrose, water, glycerol, ethanol, and combinations thereof The mvention further relates to 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 present mvention may be employed alone or in conjunction with other compounds, such as therapeutic compounds The composition will be adapted to the route of admimstration, for instance by a systemic or an oral route Prefeπed forms of systemic administration mclude injection, typically by mtravenous injection Other injection routes, such as subcutaneous, intramuscular, or intrapentoneal, can be used Alternative means for systemic admmistration mclude transmucosal and transdermal admmistration usmg penetrants such as bile salts or fusidic acids or other detergents. In addition, if a polypeptide or other compounds of the present invention can be formulated in an enteric or an encapsulated formulation, oral aclrninistration may also be possible. Administration of these compounds may also be topical and or localized, in 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 exemplary of the average case. There can, of course, be individual instances where higher or lower dosage ranges are merited, and such are within the scope of this invention.

In-dwelling devices include surgical implants, prosthetic devices and catheters, i.e.. devices that are introduced to the body of an individual and remain in position for an extended time. Such devices include, for example, artificial joints, heart valves, pacemakers, vascular grafts, vascular catheters, cerebrospinal fluid shunts, urinary catheters, 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-dwelling device. Treatment may be continued after surgery during the in-body time of the device. In addition, the composition could also be used to broaden perioperative cover for any surgical technique to prevent bacterial wound infections, especially Streptococcus pneumoniae wound infections.

Many orthopedic surgeons consider that humans with prosthetic joints should be considered for antibiotic prophylaxis before dental treatment that could produce a bacteremia. Late deep infection is a serious complication sometimes leading to loss of the prosthetic joint 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 in this situation.

In addition to the therapy described above, the compositions of this invention may be used generally as a wound treatment agent to prevent adhesion of bacteria to matrix proteins 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 insertion. The active agent will preferably be present at a concentration of 1 μg/ml to lOmg/ml for bathing of wounds or indwelling devices. A vaccine composition is conveniently in injectable form. Conventional adjuvants may be employed to enhance the immune response. A suitable unit dose for vaccination 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 invention 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 their 2- and 3 -dimensional structures as well as to identify further sequences of similar homology. These approaches are most easily facilitated by storing the sequence in a computer readable medium and then using the stored data in a known macromolecular structure program or to search a sequence database using 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 in 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 invention" 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 stored in 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 in this section entitled Databases and Algorithms and in 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 in 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 invention 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 comprising 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 the sequences comprises the sequence of a polypeptide sequence of the invention; a data set representing a polynucleotide sequence comprising the sequence of 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: a polynucleotide comprising the sequence of a polynucleotide sequence of the invention; a polypeptide comprising 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 analysis include, 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 sequencing chromatogram peak analysis.

A computer based method is provided for performing homology identification. This method comprises the steps of providing a first polynucleotide sequence comprising the sequence a polynucleotide of the invention in a computer readable medium; and comparing 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 comprising the steps of: providing a first polypeptide sequence comprising the sequence of a polypeptide of the invention in a computer readable medium; and comparing 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 comprising the steps of: providing a first polynucleotide sequence comprising the sequence of a polynucleotide of the invention in a computer readable medium; and screening 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 comprising 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 overlapping region between said first polypeptide sequence and at least one second polynucleotide or polypeptide sequence.

In another prefeπed embodiment of the invention 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: l; a polypeptide comprising the sequence of SEQ ID NO:2; a set of polynucleotide sequences wherein at least one of said sequences comprises the sequence of SEQ ID NO:l; a set of polypeptide sequences wherein at least one of said sequences comprises the sequence of SEQ ID NO:2; a data set representing a polynucleotide sequence comprising the sequence of SEQ ID NO:l; a data set representing a polynucleotide sequence encoding a polypeptide sequence comprising the sequence of SEQ ID NO:2; a polynucleotide comprising the sequence of SEQ ID NO:l; a polypeptide comprising the sequence of SEQ ID NO:2; a set of polynucleotide sequences wherein at least one of said sequences comprises the sequence of SEQ ID NO:l; a set of polypeptide sequences wherein at least one of said sequences comprises the sequence of SEQ ID NO:2; a data set representing a polynucleotide sequence comprising the sequence of SEQ ID NO: l; a data set representing a polynucleotide sequence encoding a polypeptide sequence comprising the sequence of SEQ ID NO:2. A further preferred 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 in a computer readable medium; and comparing 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 comprising the steps of: providing a polypeptide sequence comprising the sequence of SEQ ID NO:2 in a computer readable medium; and comparing 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:l 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: l 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 herein as being fully set forth. Any patent application to which this application claims priority is also incorporated by reference herein in its entirety in the manner described above for publications and references.

GLOSSARY

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

"Antibody(ies)" as used herein includes polyclonal and monoclonal antibodies, chimeric, single chain, and humanized antibodies, as well as Fab fragments, including the products of an Fab or other immunoglobulin expression library.

"Antigenically equivalent derivative(s)" as used herein encompasses a polypeptide, polynucleotide, or the equivalent of either which will be specifically recognized by certain antibodies which, when raised to the protein, polypeptide or polynucleotide according to the invention, interferes with the immediate physical interaction between pathogen and mammalian host. "Bispecific antibody(ies)" means an antibody comprising at least two antigen binding domains, each domain directed against a different epitope.

"Bodily material(s) means any material derived from an individual or from an organism infecting, infesting or inhabiting an individual, including but not limited to, cells, tissues and waste, such as, bone, blood, serum, cerebrospinal fluid, semen, saliva, muscle, cartilage, organ tissue, skin, urine, stool or autopsy materials..

"Disease(s)" means any disease caused by or related to infection by a bacteria, including , for example, otitis media, conjunctivitis, pneumonia, bacteremia, meningitis, sinusitis, pleural empyema and endocarditis, and most particularly meningitis, such as for example infection of cerebrospinal fluid.

"Fusion protein(s)" refers to a protein encoded by two, often unrelated, fused genes or fragments thereof. In one example, EP-A-0464 discloses fusion proteins comprising various portions of constant region of immunoglobulin molecules together with another human protein or part thereof. In many cases, employing an immunoglobulin Fc region as a part of a fusion protein is advantageous for use in therapy and diagnosis resulting in, 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 in the art, is a relationship between two or more polypeptide sequences or two or more polynucleotide sequences, as the case may be. as determined by comparing the sequences In the art, "identity" also means the degree of sequence relatedness between polypeptide or polynucleotide sequences, as the case may be, as determined by the match between strings of such sequences "Identity" can be readily calculated by known methods, mcludmg but not limited to those described in (Computational Molecular Biology, Lesk, A M , ed , Oxford University Press, New York, 1988, Biocomputing Informatics and Genome Projects, Smith, D W , ed , Academic Press, New York, 1993, Computer Analysis of Sequence Data, Part I, Griffin, A M , and Gπffin, H G , eds , Humana Press, New Jersey, 1994, Sequence Analysis in Molecular Biology, von Hemje, G , Academic Press, 1987, and Sequence Analysis Primer, Gπbskov, M and Devereux, J , eds , M Stockton Press, New York, 1991, and Caπllo, H , and Lipman, D , SIAM J Applied Math , 48 1073 (1988) Methods to determine identity are designed to give the largest match between the sequences tested Moreover, methods to determine identity are codified in publicly available computer programs Computer program methods to determine identity between two sequences include, but are not limited to, the GCG program package (Devereux, J , et al , Nucleic Acids Research 12(1) 387 (1984)), BLASTP, BLASTN, and FASTA (Altschul, S F et al , J Molec Biol 215 403-410 (1990) The BLAST X program is publicly available from NCBI and other sources (BLAST Manual, Altschul, S , et l , NCBI NLM NIH Bethesda, MD 20894, Altschul, S , et al , J Mol Biol 215 403-410 (1990) The well known Smith Waterman algoπthm may also be used to determine identity Parameters for polypeptide sequence comparison mclude the following Algoπthm Needleman and Wunsch, J Mol Biol 48 443-453 (1970)

Comparison matnx BLOSSUM62 from Henukoff 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 compansons (along with no penalty for end gaps)

Parameters for polynucleotide compaπson include the following Algorithm Needleman and Wunsch, J Mol Biol 48 443-453 (1970)

Companson matrix matches = +10, mismatch = 0 Gap Penalty 50 Gap Length Penalty 3 Available as: The "gap" program from Genetics Computer Group, Madison WI. These are the default parameters for nucleic acid comparisons.

A preferred meaning for "identity" for polynucleotides and polypeptides, as the case may be, are provided in (1) and (2) below. (1) Polynucleotide embodiments further include an isolated polynucleotide comprising a polynucleotide sequence having at least a 50, 60, 70, 80, 85, 90, 95, 97 or 100% identity to the reference sequence of SEQ ID NO:l, wherein said polynucleotide sequence may be identical to the reference sequence of SEQ ID NO: 1 or may include up to a certain integer number of nucleotide alterations as compared to the reference sequence, wherein said alterations are selected from the group consisting of at least one nucleotide deletion, substitution, including transition and transversion, or insertion, and wherein said alterations may occur at the 5' or 3' terminal positions of the reference nucleotide sequence or anywhere between those terminal positions, interspersed either individually among the nucleotides in the reference sequence or in one or more contiguous groups within the reference sequence, and wherein said number of nucleotide alterations is determined by multiplying the total number of nucleotides in SEQ ID NO: 1 by the integer defining the percent identity divided by 100 and then subtracting that product from said total number of nucleotides in SEQ ID NO:l, or:

n_n < x_n - (x_n • y),

wherein n_n is the number of nucleotide alterations, x_n is the total number of nucleotides in SEQ ID NO:l, y is 0.50 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 wherein any non-integer product of x_n and y is rounded down to the nearest integer prior to subtracting it from x_n. Alterations of a polynucleotide sequence encoding the polypeptide of SEQ ID NO: 2 may create nonsense, missense or frameshift mutations in this coding sequence and thereby alter the polypeptide encoded by the polynucleotide following such alterations.

By way of example, a polynucleotide sequence 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 consisting of at least one nucleic acid deletion, substitution, including transition and transversion, or insertion, and wherein said alterations may occur at the 5' or 3' terminal positions 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 ^{# v})>

wherein n_n is the number of nucleic acid alterations, x_n is the total number of nucleic acids in SEQ ID NO:l, y is, for instance 0.70 for 70%, 0.80 for 80%, 0.85 for 85% etc., • is the symbol for the multiplication operator, and wherein any non-integer product of x_n and y is rounded down to the nearest integer prior to subtracting it from x_n.

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

n_a < x_a - (x_a • y),

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

n_a < x_a - (x_a • y),

wherein n_a is the number of amino acid alterations, x_a is the total number of amino acids in SEQ ID NO:2, y is, for instance 0.70 for 70%, 0.80 for 80%, 0.85 for 85% etc., and • is the symbol for the multiplication operator, and wherein any non-integer product of x_a and y is rounded down to the nearest integer prior to subtracting it from x_a.

"Immunologically equivalent derivative(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 characteristic of an antibody whereby it possesses substantially greater affinity for the polypeptides of the invention or the polynucleotides of the invention than its affinity for other related polypeptides or polynucleotides respectively, particularly those polypeptides and polynucleotides in the prior art.

"Individual(s)" means a multicellular eukaryote, including, but not limited to a metazoan, a mammal, an ovid, a bovid, a simian, a primate, and a human.

"Isolated" means altered "by the hand of man" from its natural state, i.e., if it occurs in nature, it has been changed or removed from its original environment, or both. For example, a polynucleotide or a polypeptide naturally present in a living organism is not "isolated," but the same polynucleotide or polypeptide separated from the coexisting materials of its natural state is "isolated", as the term is employed herein. Moreover, a polynucleotide or polypeptide that is introduced into an organism by transformation, genetic manipulation or by any other recombinant method is "isolated" even if it is still present m said orgamsm. which orgamsm may be living or non-living

"Organιsm(s)" means a (1) prokaryote, mcludmg but not limited to, a member of the genus Streptococcus, Staphylococcus, Bordetella, Corynebactenum, Mycobacterium, Neissena, Haemophύus, Actinomycetes, Streptomycetes, Nocardia, Enterobacter, Yersinia, Fancisella, Pasturella, Moraxella, Acinetobacter, Erysipelothnx, Branhamella, Actinobacillus, Streptobacillus, Listena, Calymmatobactenum, Brucella, Bacillus, Clostndium, Treponema, Escherichia, Salmonella, Kleώsiella, Vibrio Proteus, Erwinia, Borreha, Leptospira, Spirillum, Campylobacter, Shigella, Legionella, Pseudomonas, Aeromonas, Rickettsia, Chlamydia, Borreha and Mycoplasma, and further mcludmg, but not limited to, a member of the species or group, Group A Streptococcus, Group B Streptococcus, Group C Streptococcus, Group D Streptococcus Group G Streptococcus, Streptococcus pneumoniae, Streptococcus pyogenes, Streptococcus agalactiae, Streptococcus faecahs, Streptococcus faecium, Streptococcus durans, Neissena gonorrheae, Neissena meningitidis, Staphylococcus aureus, Staphylococcus epidermidis, Corynebactenum dψthenae, Gardnerella vaginalis. Mycobactenum tuberculosis, Mycobactenum bovis, Mycobactenum ulcerans, Mycobactenum leprae, Act nomyctes israeln, Listena monocytogenes, Bordetella pertusis, Bordatella parapertusis, Bordetella bronchiseptica, Eschenchia coli, Shigella dysentenae, Haemophύus influenzae, Haemoph us aegyptius, Haemophilus parainfluenzae, Haemophύus ducreyi, Bordetella, Salmonella typhi, Citrobacter freundn, Proteus mirabil s, Proteus vulgans, Yersinia pestis, Kleibsiella pneumoniae, Serratia marcessens, Serratia liquefaciens, Vibno cholera, Shigella dysenteni, Shigella flexnen, Pseudomonas aeruginosa, Franscisella tularensis, Brucella abortis, Bacillus anthracis, Bacillus cereus, Clostndium perffingens, Clostndium tetani, Clostndium botuhnum, Treponema palhdum, Rickettsia nckettsn and Chlamydia trachomitis, (u) an archaeon, mcludmg but not limited to Archaebacter, and (in) a unicellular or filamentous eukaryote. mcludmg but not limited to, a protozoan, a fungus, a member of the genus Saccharomyces, Kluveromyces, or Candida, and a member of the species Saccharomyces cenvseae, Kluveromyces lactis, or Candida albicans

"Polynucleotide(s)" generally refers to any polynbonucleotide or polydeoxynbonucleotide, which may be unmodified RNA oi DNA or modified RNA or DNA "Polynucleotide(s)" mclude, without limitation, smgle- and double-stranded DNA, DNA that is a mixture of smgle- and double-stranded regions or single-, double- and tnple-stranded regions, smgle- and double-stranded RNA, and RNA that is mixture of single- and double-stranded regions, hybπd molecules compnsmg DNA and RNA that may be smgle-stranded or, more typically, double-stranded, or tnple-stranded regions, or a mixture of smgle- and double-stranded regions In addition, "polynucleotide" as used herem refers to tnple-stranded regions compnsmg RNA or DNA or both RNA and DNA The strands m such regions may be from the same molecule or from different molecules The regions may 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 "polynucleotide(s)" as that term is intended herein. Moreover, DNAs or RNAs comprising unusual bases, such as inosine, or modified bases, such as tritylated bases, to name just two examples, are polynucleotides as the term is used herein. It will be appreciated that a great variety of modifications have been made to DNA and RNA that serve many useful purposes known to those of skill in the art. The term "polynucleotide(s)" as it is employed herein embraces such chemically, enzymatically or metabolically modified forms of polynucleotides, as well as the chemical forms of DNA and RNA characteristic of viruses and cells, including, for example, simple and complex cells. "Polynucleotide(s)" also embraces short polynucleotides often refeπed to as oligonucleotide(s).

"Polypeptide(s)" refers to any peptide or protein comprising two or more amino acids joined to each other by peptide bonds or modified peptide bonds. "Polypeptide(s)" refers to both short chains, commonly refeπed to as peptides, oligopeptides and oligomers and to longer chains generally refeπed to as proteins. Polypeptides may contain amino acids other than the 20 gene encoded amino acids. "Polypeptide(s)" include those modified either by natural processes, such as processing and other post-translational modifications, but also by chemical modification techniques. Such modifications are well described in basic texts and in more detailed monographs, as well as in a voluminous research literature, and they are well known to those of skill in the art. It will be appreciated that the same type of modification may be present in the same or varying degree at several sites in a given polypeptide. Also, a given polypeptide may contain many types of modifications. Modifications can occur anywhere in a polypeptide, including the peptide backbone, the amino acid side-chains, and the amino or carboxyl termini. Modifications include, for example, acetylation, acylation, ADP- ribosylation, amidation, covalent attachment of flavin, covalent attachment of a heme moiety, covalent attachment of a nucleotide or nucleotide derivative, covalent attachment of a lipid or lipid derivative, covalent attachment of phosphotidyhnositol, cross-linking, cychzation, disulfide bond formation, demethylation, formation of covalent cross-links, formation of cysteine, formation of pyroglutamate, formylation, gamma- carboxylation, GPI anchor formation, hydroxylation, iodination, methylation, myristoylation, oxidation, proteolytic processing, phosphorylation, prenylation, racemization, glycosylation, lipid attachment, sulfation, gamma-caiboxylation of glutamic acid residues, hydroxylation and ADP-ribosylation, selenoylation, sulfation, transfer-RNA mediated addition of amino acids to proteins, such as arginylation, and ubiquitination. See, for instance, PROTEINS - STRUCTURE AND MOLECULAR PROPERTIES, 2nd Ed., T. E. Creighton, W. H. Freeman and Company, New York (1993) and Wold, F., Posttranslational Protein Modifications: Perspectives and Prospects, pgs. 1-12 in POSTTRANSLATIONAL COVALENT MODIFICATION OF PROTEINS, B. C. Johnson, Ed Academic Press, New York (1983), Seifter et al , Meth Enzymol 182626-646 (1990) and Rattan et al , Protem 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 introduced 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 compπsing at least one polynucleotide of the mvention

"Varιant(s)" as the term is used herem, is a polynucleotide or polypeptide that differs from a reference polynucleotide or polypeptide respectively, but retains essential properties A typical variant of a polynucleotide differs m nucleotide sequence from another, reference polynucleotide Changes in the nucleotide sequence of the vaπant may or may not alter the ammo acid sequence of a polypeptide encoded by the reference polynucleotide Nucleotide changes may result m ammo acid substitutions, additions, deletions, fusion protems and truncations m the polypeptide encoded by the reference sequence, as discussed below A typical vaπant of a polypeptide differs in ammo acid sequence from another, reference polypeptide Generally, differences are limited so that the sequences of the reference polypeptide and the vaπant are closely similar overall and, m many regions, identical A vanant and reference polypeptide may differ m ammo acid sequence by one or more substitutions, additions, deletions m any combmation 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 prefeπed are vanants m which several, 5-10, 1-5, 1-3, 1-2 or 1 ammo acids are substituted, deleted, or added m any combination A vanant of a polynucleotide or polypeptide may be a naturally occurring such as an allehc vanant, or it may be a vanant that is not known to occur naturally Non-naturally occurnng variants of polynucleotides and polypeptides may be made by mutagenesis techmques, by direct synthesis, and by other recombmant methods known to skilled artisans EXAMPLES The examples below are earned out usmg standard techmques, which are well known and routine to those of skill m the art, except where otherwise descnbed m detail The examples are illustrative, but do not limit the mvention

Example 1 Strain selection, Library Production and Sequencing The polynucleotide havmg a DNA sequence given in Table 1 [SEQ ID NO 1] was obtained from a library of clones of chromosomal DNA of Streptococcus pneumoniae m E coli The sequencmg data from two or more clones containing overlappmg Streptococcus pneumoniae DNAs was used to construct the contiguous DNA sequence in SEQ ID NO 1 Libranes may be prepared by routme methods, for example Methods 1 and 2 below

Total cellular DNA is isolated from Streptococcus pneumoniae 0100993 accordmg to standard procedures and size-fractionated by either of two methods Method 1

Total cellular DNA is mechanically sheared by passage through a needle in order to size- fractionate according to standard procedures DNA fragments of up to l lkbp in size are rendered blunt by treatment with exonuclease and DNA polymerase, and EcoRI linkers added Fragments are hgated mto the vector Lambda ZapII that has been cut with EcoRI, the library packaged by standard procedures and E coli infected with the packaged library The library is amplified by standard procedures Method 2 Total cellular DNA is partially hydrolyzed with a one or a combmation of restπction enzymes appropπate to generate a series of fragments for cloning mto library vectors (e g , Rsal, Pall, Alul, Bshl235I), and such fragments are size-fractionated accordmg to standard procedures EcoRI linkers are hgated to the DNA and the fragments then hgated mto the vector Lambda ZapII that have been cut with EcoRI, the library packaged by standard procedures, and E coli mfected with the packaged library The library is amplified by standard procedures

Claims

What is claimed is:

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

(i) an isolated polypeptide comprising an amino acid having at least:

(a) 70% identity;

(b) 80% identity;

(c) 90% identity; or

(d) 95% identity to the amino acid sequence of SEQ ID NO:2 over the entire length of SEQ ID NO:2; (ii) an isolated polypeptide comprising the amino acid sequence of SEQ ID NO:2, (iii) an isolated polypeptide which is the amino acid sequence of SEQ ID NO:2, and (iv) a polypeptide which is encoded by a recombinant polynucleotide comprising the polynucleotide sequence of SEQ ID NO: 1.

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

(i) an isolated polynucleotide comprising a polynucleotide sequence encoding a polypeptide that has at least

(a) 70% identity;

(b) 80% identity;

(c) 90% identity; or

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

(a) 70% identity

(b) 80% identity;

(c) 90% identity; or

(d) 95% identity; over its entire length to a polynucleotide sequence encoding the polypeptide of SEQ ED NO:2; (iii) an isolated polynucleotide comprising a nucleotide sequence which has at least:

(a) 70% identity;

(b) 80% identity;

(c) 90% identity; or

(d) 95% identity; to that of SEQ ID NO: 1 over the entire length of SEQ ID NO: 1 ;

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

NO:2;

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

(vi) an isolated polynucleotide obtainable by screening an appropriate library under stringent hybridization conditions with a probe having the sequence of SEQ ID NO: 1 or a fragment thereof; (vii) an isolated polynucleotide encoding a mature polypeptide expressed by the nrdG gene contained in the Streptococcus pneumoniae; and

(viii) a polynucleotide sequence complementary to said isolated polynucleotide of (i), (ii), (iii), (iv), (v), (vi) or (vii).

3. An antibody antigenic to or immunospecific for the polypeptide of claim 1.

4. A method for the treatment of an individual:

(i) in need of enhanced activity or expression of the polypeptide of claim 1 comprising the step of:

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

(b) providing to the individual an isolated polynucleotide comprising a polynucleotide sequence encoding said polypeptide in a form so as to effect production of said polypeptide activity in vivo; or

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

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

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

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

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

(a) determining the presence or absence of a mutation in the nucleotide sequence encoding said polypeptide in the genome of said individual; or (b) analyzing for the presence or amount of said polypeptide expression in a sample derived from said individual.

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

(a) measuring the binding of a candidate compound to the polypeptide or to the cells or membranes bearing the polypeptide or a fusion protein thereof by means of a label directly or indirectly associated with the candidate compound;

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

(c) testing whether the candidate compound results in a signal generated by activation or inhibition of the polypeptide, using 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;

(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, or

(f) (1) contacting a composition comprising the polypeptide with the compound to be screened under conditions to permit interaction between the compound and the polypeptide to assess the interaction of a compound, such interaction being associated with a second component capable of providing a detectable signal in response to the interaction of the polypeptide with the compound; and

(2) determining whether the compound interacts with and activates or inhibits an activity of the polypeptide by detecting the presence or absence of a signal generated from the interaction of the compound with the polypeptide.

7. An agonist or an antagonist of the activity or expression polypeptide of claim 1.

8. An expression system comprising a polynucleotide capable of producing a polypeptide of claim 1 when said expression system is present in a compatible host cell.

9. A host cell comprising the expression system of claim 8 or a membrane thereof expressing a polypeptide selected from the group consisting of:

(i) an isolated polypeptide comprising an amino acid sequence selected from the group having at least:

(a) 70% identity;

(b) 80% identity;

(c) 90% identity; or

(d) 95% identity to the amino acid sequence of SEQ ID NO:2 over the entire length of SEQ ID NO:2; (ii) an isolated polypeptide comprising the amino acid sequence of SEQ ID NO:2; (iii) an isolated polypeptide which is the amino acid sequence of SEQ ID NO:2, and (iv) a polypeptide which is encoded by a recombinant polynucleotide comprising the polynucleotide sequence of SEQ ID NO:l.

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

(i) an isolated polypeptide comprising an amino acid sequence selected from the group having at least:

(a) 70% identity;

(b) 80% identity;

(c) 90% identity; or

(d) 95% identity to the amino acid sequence of SEQ ID NO:2 over the entire length of SEQ ID NO:2; (ii) an isolated polypeptide comprising the amino acid sequence of SEQ ID NO:2; (iii) an isolated polypeptide which is the amino acid sequence of SEQ ID NO:2, and (iv) a polypeptide which is encoded by a recombinant polynucleotide comprising the polynucleotide sequence of SEQ ID NO:l, comprising the step of culturing a host cell of claim 9 under conditions sufficient for the production of said polypeptide.

11. A process for producing a host cell comprising the expression system of claim 8 or a membrane thereof expressing a polypeptide selected from the group consisting of:

(i) an isolated polypeptide comprising an amino acid sequence selected from the group having at least: (a) 70% identity;

(b) 80% identity;

(c) 90% identity; or

(d) 95% identity to the amino acid sequence of SEQ ID NO:2 over the entire length of SEQ ID NO:2;

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

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

(iv) a polypeptide which is encoded by a recombinant polynucleotide comprising the polynucleotide sequence of SEQ ID NO:l, said process comprising the step of transforming or transfecting a cell with an expression system comprising a polynucleotide capable of producing said polypeptide of (i), (ii), (iii) or (iv) when said expression system is present in a compatible host cell such the host cell, under appropriate culture conditions, produces said polypeptide of (i), (ii), (iii) or (iv).

12. A host cell produced by the process of claim 11 or a membrane thereof expressing a polypeptide selected from the group consisting of:

(i) an isolated polypeptide comprising an amino acid sequence selected from the group having at least:

(a) 70% identity;

(b) 80% identity;

(c) 90% identity; or

(d) 95% identity to the amino acid sequence of SEQ ID NO:2 over the entire length of SEQ ID NO:2; (ii) an isolated polypeptide comprising the amino acid sequence of SEQ ID NO:2; (iii) an isolated polypeptide which is the amino acid sequence of SEQ ID NO:2, and (iv) a polypeptide which is encoded by a recombinant polynucleotide comprising the polynucleotide sequence of SEQ ID NO: 1.

13. 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 ; a polypeptide comprising the sequence of SEQ ID NO:2; a set of polynucleotide sequences wherein at least one of said sequences comprises the sequence of SEQ ID NO: l; a set of polypeptide sequences wherein at least one of said sequences comprises the sequence ofSEQ ID NO:2; a data set representing a polynucleotide sequence comprising the sequence of SEQ ID NO:l; a data set representing a polynucleotide sequence encoding a polypeptide sequence comprising the sequence of SEQ ID NO:2; a polynucleotide comprising the sequence of SEQ ID NO: l; a polypeptide comprising the sequence of SEQ ID NO:2; a set of polynucleotide sequences wherein at least one of said sequences comprises the sequence of SEQ ID NO:l; a set of polypeptide sequences wherein at least one of said sequences comprises the sequence of SEQ ID NO:2; a data set representing a polynucleotide sequence comprising the sequence of SEQ ID NO:l; a data set representing a polynucleotide sequence encoding a polypeptide sequence comprising the sequence of SEQ ID NO:2.

14. 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: l in a computer readable medium; and comparing said polynucleotide sequence to at least one polynucleotide or polypeptide sequence to identify homology.

15. 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:l in a computer readable medium; and screening for at least one overlapping region between said first polynucleotide sequence and a second polynucleotide sequence.