WO1998041234A1 - Novel compounds - Google Patents

Abstract

The invention provides ratA polypeptides and DNA (RNA) encoding ratA polypeptides and methods for producing such polypeptides by recombinant techniques. Also provided are methods for utilizing ratA polypeptides to screen for antibacterial compounds.

Description

NOVEL COMPOUNDS

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, in these and in other regards, the invention relates to novel polynucleotides and polypeptides of the rat family, hereinafter referred to as "ratA".

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 of Streptococcus pneumoniae infections has risen dramatically in the past 20 years. 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 has created a demand for both new anti-microbial agents and diagnostic tests for this organism.

The translation product of the ratA gene is one of at least two polypeptides that constitute an amidotransferase which catalyses the transfer of an amino group from a donor to Glu- tRNA(Gln) in order to form Gln-tRNA(Gln). The reaction is essential to bacteria.

Clearly, there is a need for factors, such as the novel compounds of the invention, having a present benfit of being useful to screen compounds for antibiotic activity. Such factors may also be used 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 which can play a role in preventing, ameliorating or correcting infections, dysfunctions or diseases.

The polypeptides of the invention have ammo acid sequence homology to a known amidase from Synechocystis sp protein. Each patent application to which this mvention claims pπonty is hereby incoφorated by reference in its entirety.

SUMMARY OF THE INVENTION

It is an object of the invention to provide polypeptides that have been identified as novel ratA polypeptides by homology between the amino acid sequence set out in Figure 2 [SEQ ID NO:2 or 4] and a known ammo acid sequence or sequences of other proteins such as amidase from Synechocystis sp protein.

It is a further object of the invention to provide polynucleotides that encode ratA polypeptides, particularly polynucleotides that encode the polypeptide herein designated ratA. In a particularly preferred embodiment of the invention the polynucleotide comprises a region encoding ratA polypeptides compnsing the sequence set out in Figure 1 [SEQ ID NO: 1 or 3], or a vanant thereof.

In another particularly preferred embodiment of the invention there is a novel ratA protein from Streptococcus pneumoniae compnsing the ammo acid sequence of Figure 2 [SEQ ID NO:2 or 4], or a vanant thereof.

In accordance with another aspect of the invention there is provided an isolated nucleic acid molecule encoding a mature polypeptide expressible by the Streptococcus pneumoniae 0100993 strain contained in NCIMB Deposit No. 40794. A further aspect of the invention there are provided isolated nucleic acid molecules encoding ratA, particularly Streptococcus pneumoniae ratA, including mRNAs, cDNAs, genomic DNAs. Further embodiments of the invention include biologically, diagnostically, prophylactically, clinically or therapeutocally useful vanants thereof, and compositions compnsing the same. In accordance with another aspect of the invention, there is provided the use of a polynucleotide of the invention for therapeutic or prophylactic puφoses, in particular genetic immunization. Among the particularly preferred embodiments of the invention are naturally occurπng allehc vanants of ratA and polypeptides encoded thereby. Another aspect of the invention there are provided novel polypeptides of Streptococcus pneumoniae referred to herein as ratA as well as biologically, diagnostically, prophylactically, clinically or therapeutically useful vanants thereof, and compositions compnsing the same. Among the particularly preferred embodiments of the invention are vanants of ratA polypeptide encoded by naturally occurπng alleles of the ratA gene.

In a preferred embodiment of the invention there are provided methods for producing the aforementioned ratA polypeptides.

In accordance with yet another aspect of the invention, there are provided inhibitors to such polypeptides, useful as antibactenal agents, including, for example, antibodies.

In accordance with certain preferred embodiments of the invention, there are provided products, compositions and methods for (I) assessing ratA expression, (u) treating disease, 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,, (in) assaying genetic vanation, (iv) and admimstenng a ratA polypeptide or polynucleotide to an organism to raise an lmmunological response against a bactena, especially a Streptococcus pneumoniae bactena.

In accordance with certain preferred embodiments of this and other aspects of the invention there are provided polynucleotides that hybndize to ratA polynucleotide sequences, particularly under stnngent conditions.

In certain preferred embodiments of the invention there are provided antibodies against ratA polypeptides.

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 of a polypeptide or polynucleotide of the invention compnsmg: contacting a polypeptide 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 or polynucleotide to assess the binding to or other interaction with the compound, such binding or interaction being associated with a second component capable of providing a detectable signal m response to the binding or interaction of the polypeptide or polynucleotide with the compound; and determining whether the compound binds to or otherwise interacts with and activates or inhibits an activity of the polypetide or polynucleotide by detecting the presence or absence of a signal generated from the binding or interaction of the compound with the polypeptide or polynucleotide. In accordance with yet another aspect of the invention, there are provided ratA agonists and antagonists, preferably bacteriostatic or bacteriocidalagonists and antagonists.

In a further aspect of the invention there are provided compositions comprising a ratA polynucleotide or a ratA polypeptide for administration to a cell or to a multicellular organism.

Various changes and modifications within the spirit and scope of the disclosed invention will become readily apparent to those skilled in the art from reading the following descriptions and from reading the other parts of the present disclosure.

BRIEF DESCRIPTION OF THE DRAWINGS

The following drawings depict certain embodiments of the invention. They are illustrative only and do not limit the invention otherwise disclosed herein.

Figure 1 shows the polynucleotide sequence of Streptococcus pneumoniae ratA [SEQ ID NO:l or 3].

Figure 2 shows the amino acid sequence of Streptococcus pneumoniae ratA [SEQ ID NO:2 or 4] deduced from the polynucleotide sequence of Figure 1. GLOSSARY

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

"Host cell" 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, including but not limited to those described in (Computational Molecular Biology, Lesk, A.M., ed., Oxford University Press, New York, 1988; Biocomputing: Informatics and Genome Projects, Smith, D.W., ed., Academic Press, New York, 1993; Computer Analysis of Sequence Data, Part I, Griffin, A.M., and Griffin, H.G., eds., Humana Press, New Jersey, 1994; Sequence Analysis in Molecular Biology, von Heinje, G., Academic Press, 1987; and Sequence Analysis Primer, Gribskov, M. and Devereux, J., eds., M Stockton Press, New York, 1991; and Carillo, H., and Lipman, D., SIAM J. Applied Math., 48: 1073 (1988). Methods to determine identity are designed to give the largest match between the sequences tested. Moreover, methods to determine identity are codified in publicly available computer programs. Computer program methods to determine identity between two sequences include, but are not limited to, the GCG program package (Devereux, J., et al., Nucleic Acids Research 12(1): 387 ( 1984)), BLASTP, BLASTN, and FASTA (Atschul, S.F. et al., J. Molec. Biol. 215: 403-410 (1990). The BLAST X program is publicly available from NCBI and other sources (BLAST Manual, Altschul, S., et al, NCBI NLM NIH Bethesda, MD 20894; Altschul, S., et al, J. Mol. Biol. 215: 403-410 (1990). The well known Smith Waterman algonthm may also be used to determine identity. Parameters for polypeptide sequence companson include the following:

1) Algorithm: Needleman and Wunsch, J. Mol Biol. 48: 443-453 (1970) Companson matrix: BLOSSUM62 from Hentikoff and Hentikoff, Proc. Natl. Acad. Sci. USA. 89:10915-10919 (1992) Gap Penalty: 12 Gap Length Penalty: 4

A program useful with these parameters is publicly available as the "gap" program from Genetics Computer Group, Madison WI. The aforementioned parameters are the default parameters for peptide compansons (along with no penalty for end gaps). Parameters for polynucleotide comparison include the following: 1) Algonthm: Needleman and Wunsch, J. Mol Biol. 48: 443-453 (1970) Companson matnx: matches = +10, mismatch = 0 Gap Penalty: 50 Gap Length Penalty: 3 Available as: The "gap" program from Genetics Computer Group, Madison WI. These are the default parameters for nucleic acid compansons.

A preferred meaning for "identity" for polynucleotides and polypeptides, as the case may be, are provided in (1) and (2) below.

(1) Polynucleotide embodiments further include an isolated polynucleotide compnsing a polynucleotide sequence having at least a 50, 60, 70, 80, 85, 90, 95, 97 or 100% identity to the reference sequence of SEQ ID NO: 1 or 3, wherein said polynucleotide sequence may be identical to the reference sequence of SEQ ID NO: 1 or 3 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 m the reference sequence or in one or more contiguous groups withm the reference sequence, and wherein said number of nucleotide alterations is determined by multiplying the total number of nucleotides m SEQ ID NO.1 or 3 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 3, or:

ι_n < x_n (^χ _n • y)>

wherein n_n is the number of nucleotide alterations, x_n is the total number of nucleotides in SEQ ID NO: l or 3, 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 pnor to subtracting it from x_n. Alterations of a polynucleotide sequence encoding the polypeptide of SEQ ID NO:2 or 4 may create nonsense, missense or frameshift mutations m 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 mvention may be identical to the reference sequence of SEQ ID NO:2 or 4, that is it may be 100% identical, or it may include up to a certain integer number of ammo acid alterations as compared to the reference sequence such that the percent identity is less than 100% identity. Such alterations are selected from the group consisting of at least one 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 withm the reference sequence. The number of nucleic acid alterations for a given percent identity is determined by multiplying the total number of amino acids in SEQ ID NO:2 or 4 by the integer defining the percent identity divided by 100 and then subtracting that product from said total number of ammo acids in SEQ ID NO:2 or 4, or:

n_n ≤ x_n - (x_n • y), wherein n_n is the number of amino acid alterations, x_n is the total number of ammo acids in SEQ ID NO:2 or 4, 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-mteger product of x_n and y is rounded down to the nearest integer pnor to subtracting it from x_n.

(2) Polypeptide embodiments further include an isolated polypeptide compnsing 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 or 4, wherein said polypeptide sequence may be identical to the reference sequence of SEQ ID NO:2 or 4 or may include up to a certain integer number of ammo acid alterations as compared to the reference sequence, wherein said alterations are selected from the group 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 ammo acids in SEQ ID NO:2 or 4 by the integer defining the percent identity divided by 100 and then subtracting that product from said total number of ammo acids in SEQ ID NO:2 or 4, or:

ι_a < x_a (^χ _a • y).

wherein n_a is the number of amino acid alterations, x_a is the total number of ammo acids in SEQ ID NO:2 or 4, 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-mteger product of x_a and y is rounded down to the nearest integer pnor to subtracting it from x_a.

By way of example, a polypeptide sequence of the present invention may be identical to the reference sequence of SEQ ID NO:2 or 4, 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 ammo acid deletion, substitution, including conservative and non-conservative substitution, or insertion, and wherein said alterations may occur at the ammo- or carboxy-termmal 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 ammo acids m SEQ ID NO:2 or 4 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 4, or:

ⁿa ≤ ^xa " (^xa • Y)>

wherein n_a is the number of amino acid alterations, x_a is the total number of amino acids in SEQ ID NO:2 or 4, 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. "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 onginal 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 matenals of its natural state is "isolated", as the term is employed herein. "Polynucleotιde(s)" generally refers to any polynbonucleotide or polydeoxnbonucleotide, which may be unmodified RNA or DNA or modified RNA or DNA. "Polynucleotide(s)" include, without limitation, single- and double-stranded DNA, DNA that is a mixture of single- and double-stranded regions or single-, double- and tπple-stranded regions, single- and double-stranded RNA, and RNA that is mixture of single- and double- stranded regions, hybnd molecules compnsing DNA and RNA that may be single-stranded or, more typically, double-stranded, or tπple-stranded regions, or a mixture of single- and double- stranded regions. In addition, "polynucleotide" as used herein refers to tnple-stranded regions compnsing RNA or DNA or both RNA and DNA. The strands in such regions may be from the same molecule or from different molecules. The regions may include all of one or more of the molecules, but more typically involve only a region of some of the molecules. One of the molecules of a tnple-hehcal region often is an ohgonucleotide. As used herein, the term "polynucleotide(s)" also includes DNAs or RNAs as descπbed 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 compnsing unusual bases, such as mosme, or modified bases, such as tπtylated bases, to name just two examples, are polynucleotides as the term is used herein. It will be appreciated that a great vanety of modifications have been made to DNA and RNA that serve many useful puφoses known to those of skill in the art. The term "polynucleotide(s)" as it is employed herein embraces such chemically, enzymatically or metabohcally modified forms of polynucleotides, as well as the chemical forms of DNA and RNA charactenstic of viruses and cells, including, for example, simple and complex cells. "Polynucleotide(s)" also embraces short polynucleotides often referred to as ohgonucleotide(s). "Polypeptιde(s)" refers to any peptide or protem compnsing two or more ammo acids joined to each other by peptide bonds or modified peptide bonds. "Polypeptide(s)" refers to both short chains, commonly referred to as peptides, oligopeptides and ohgomers and to longer chains generally referred to as proteins. Polypeptides may contain amino acids other than the 20 gene encoded ammo acids "Polypeptιde(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 descnbed in basic texts and m 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 ammo acid side-chams, and the ammo or carboxyl termini. Modifications include, for example, acetylation, acylation, ADP-nbosylatoon, amidation, covalent attachment of flavin, covalent attachment of a heme moiety, covalent attachment of a nucleotide or nucleotide denvative, covalent attachment of a lipid or lipid denvative, covalent attachment of phosphotidylinositol, cross-linkmg, cychzation, disulfide bond formation, demethylation, formation of covalent cross-links, formation of cysteine, formation of pyroglutamate, formylation, gamma-carboxylation, glycosylation, GPI anchor formation, hydroxylation, lodination, methylation, myπstoylation, oxidation, proteolytic processing, phosphorylation, prenylation, racemization, glycosylation, lipid attachment, sulfation, gamma-carboxylation of glutamic acid residues, hydroxylation and ADP-nbosylation, selenoylation, sulfation, transfer-RNA mediated addition of amino acids to proteins, such as arg ylation, 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 182:626-646 (1990) and Rattan et al., Protein Synthesis: Posttranslational Modifications and Aging, Ann. N.Y. Acad. Sci. 663: 48-62 (1992) each of which is incoφorated by reference herein in its entirety. 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.

"Vanant(s)" as the term is used herein, is a polynucleotide or polypeptide that differs from a reference polynucleotide or polypeptide respectively, but retains essential properties. A typical variant of a polynucleotide differs in nucleotide sequence from another, reference polynucleotide. Changes in the nucleotide sequence of the vanant may or may not alter the amino acid sequence of a polypeptide encoded by the reference polynucleotide. Nucleotide changes may result m amino acid substitutions, additions, deletions, fusions and truncations in the polypeptide encoded by the reference sequence, as discussed below. A typical vanant of a polypeptide differs in ammo acid sequence from another, reference polypeptide. Generally, differences are limited so that the sequences of the reference polypeptide and the variant are closely similar overall and, in many regions, identical. A vanant and reference polypeptide may differ in ammo acid sequence by one or more substitutions, additions, deletions m any combination. A substituted or inserted ammo acid residue may or may not be one encoded by the genetic code. A variant of a polynucleotide or polypeptide may be a naturally occurnng such as an allehc vanant, or it may be a vanant that is not known to occur naturally. Non-naturally occurnng vanants of polynucleotides and polypeptides may be made by mutagenesis techniques, by direct synthesis, and by other recombinant methods known to skilled artisans.

DESCRIPTION OF THE INVENTION

The invention relates to novel ratA polypeptides and polynucleotides as descnbed in greater detail below. In particular, the invention relates to polypeptides and polynucleotides of a novel ratA gene of Streptococcus pneumoniae, which is related by ammo acid sequence homology to amidase from Synechocystis sp polypeptide. The invention relates especially to ratA having the nucleotide and amino acid sequences set out in Figure 1 [SEQ ID NO:l or 3] and Figure 2 [SEQ ID NO: 2 or 4] respectively, and to the ratA nucleotide sequences of the DNA deposited m NCIMB Deposit No. 40794 and ammo acid sequences encoded thereby.

Deposited materials

A deposit containing a Streptococcus pneumoniae 0100993 strain has been deposited with the National Collections of Industrial and Manne Bactena Ltd. (NCIMB), 23 St.

Machar Dnve, Aberdeen AB2 1RY, Scotland on 11 Apnl 1996 and assigned NCIMB

Deposit No. 40794. The Streptococcus pneumoniae strain deposit is referred to herein as "the deposited strain" or as "the DNA of the deposited strain."

The deposited matenal is a strain that contains the full length ratA DNA, referred to as "NCIMB 40794" upon depositThe sequence of the polynucleotides contained m the deposited matenal, as well as the amino acid sequence of the polypeptide encoded thereby, are controlling in the event of any conflict with any descnption of sequences herein.

The deposit 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 strain will be irrevocably and without restriction or condition released to the public upon the issuance of a patent. The deposit is provided merely as convenience to those of skill m the art and is not an admission that a deposit is required for enablement, such as that required under 35 U.S.C. §112.

A license may be required to make, use or sell the deposited matenals, and no such license is hereby granted.

Polypeptides

The polypeptides of the invention include the polypeptide of Figure 2 [SEQ ID NO:2 or 4] (in particular the mature polypeptide) as well as polypeptides and fragments, particularly those which have the biological activity of ratA, and also those which have at least 70% identity to the polypeptide of Figure 2 [SEQ ID NO:2 or 4] or the relevant portion, preferably at least 80% identity to the polypeptide of Figure 2 [SEQ ID NO:2 or 4], and more preferably at least 90% similanty (more preferably at least 90% identity) to the polypeptide of Figure 2 [SEQ ID NO:2 or 4] and still more preferably at least 95% similanty (still more preferably at least 95%> identity) to the polypeptide of Figure 2 [SEQ ID NO:2 or 4] 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 ammo acids.

A fragment is a vanant polypeptide having an amino acid sequence that entirely is the same as part but not all of the ammo acid sequence of the aforementioned polypeptides. As with ratA polypeptides fragments may be "free-standmg," or compπsed withm a larger polypeptide of which they form a part or region, most preferably as a single continuous region, a single larger polypeptide.

Preferred fragments include, for example, truncation polypeptides having a portion of the amino acid sequence of Figure 2 [SEQ ID NO:2 or 4], or of vanants thereof, such as a continuous senes of residues that includes the ammo terminus, or a continuous seπes of residues that includes the carboxyl terminus. Degradation forms of the polypeptides of the invention in a host cell, particularly a Streptococcus pneumoniae, are also preferred. Further preferred are fragments charactenzed by structural or functional attributes such as fragments that compnse alpha-helix and alpha-helix forming regions, beta-sheet and beta-sheet-forming regions, turn and tum-formmg 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.

Also preferred are biologically active fragments which are those fragments that mediate activities of ratA, 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 lmmunogemc in an animal, especially in a human. Particularly preferred are fragments compnsing 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. Vanants that are fragments of the polypeptides of the invention may be employed for producing the corresponding full-length polypeptide by peptide synthesis; therefore, these vanants may be employed as intermediates for producing the full-length polypeptides of the invention.

Polynucleotides Another aspect of the mvention relates to isolated polynucleotides that encode the ratA polypeptide having the deduced ammo acid sequence of Figure 2 [SEQ ID NO:2 or 4] and polynucleotides closely related thereto and vanants thereof.

Using the information provided herein, such as the polynucleotide sequence set out m Figure 1 [SEQ ED NO:l or 3], a polynucleotide of the invention encoding ratA 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 the sequence given in Figure 1 [SEQ ID NO:l or 3], typically a library of clones of chromosomal DNA of Streptococcus pneumoniae 0100993 m E.coli or some other suitable host is probed with a radiolabeled ohgonucleotide, preferably a 17-mer or longer, denved from a partial sequence. Clones carrying DNA identical to that of the probe can then be distinguished using stnngent conditions. By sequencing the individual clones thus identified with sequencing pnmers designed from the ongmal sequence it is then possible to extend the sequence in both directions to determine the full gene sequence. Conveniently, such sequencing is performed using denatured double stranded DNA prepared from a plasmid clone. Suitable techniques are descnbed by Mamatis, T., Fntsch, E.F. and Sambrook et al., MOLECULAR CLONING, A LABORATORY MANUAL, 2nd Ed.; Cold Spring Harbor Laboratory Press, Cold Spπng Harbor, New York (1989), which is incoφorated by reference herein m its entirety, (see in particular Screening By Hybndization 1.90 and Sequencing Denatured Double- Stranded DNA Templates 13.70). Illustrative of the invention, the polynucleotide set out in Figure 1 [SEQ ID NO:l or 3] was discovered in a DNA library denved from Streptococcus pneumoniae 0100993. The DNA sequence set out in Figure 1 [ SEQ ID NO:l or 3] contains an open readmg frame encoding a protein having about the number of ammo acid residues set forth in Figure 2 [SEQ ID NO:2 or 4] with a deduced molecular weight that can be calculated using amino acid residue molecular weight values well known in the art. ratA of the invention is structurally related to other proteins of the rat family, as shown by the results of sequencing the DNA encoding ratA of the deposited strain. The protein exhibits greatest homology to amidase from Synechocystis sp protein among known proteins. RatA of Figure 2 [SEQ ID NO:2 or 4] has about 50% identity over its entire length and about 70% similanty over its entire length with the amino acid sequence of amidase from Synechocystis sp polypeptide.

The invention provides a polynucleotide sequence identical over its entire length to the coding sequence in Figure 1 [SEQ ID NO:l or 3].Also provided by the invention is the codmg sequence for the mature polypeptide or a fragment thereof, by itself as well as the coding sequence for the mature polypeptide or a fragment in readmg frame with other codmg sequence, such as those encoding a leader or secretory sequence, a pre-, or pro- or prepro- protein sequence. The polynucleotide may also contain non-codmg sequences, including for example, but not limited to non-codmg 5' and 3' sequences, such as the transcnbed, non- translated sequences, termination signals, nbosome binding sites, sequences that stabilize mRNA, trons, polyadenylation signals, and additional coding sequence which encode 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-histidme peptide, as provided in the pQE vector (Qiagen, Inc.) and descnbed in Gentz et al., Proc. Nat/. Acad. Sci., USA 86: 821-824 (1989), or an HA tag (Wilson et al, Cell 37: 767 (1984), incoφorated by reference herein in its entirety. Polynucleotides of the invention also include, but are not limited to, polynucleotides compnsing a structural gene and its naturally associated sequences that control gene expression.

The term "polynucleotide encodmg a polypeptide" as used herein encompasses polynucleotides that include a sequence encoding a polypeptide of the invention, particularly a bactenal polypeptide and more particularly a polypeptide of the Streptococcus pneumoniae ratA having the amino acid sequence set out in Figure 2 [SEQ ID ΝO:2 or 4]. The term also encompasses polynucleotides that include a single continuous region or discontinuous regions encoding the polypeptide (for example, interrupted by integrated phage or an insertion sequence or editing) together with additional regions, that also may contain codmg and/or non-codmg sequences. The invention further relates to vanants of the polynucleotides descnbed herein that encode for vanants of the polypeptide having the deduced ammo acid sequence of Figure 2 [SEQ ID NO: 2 or 4] Vanants that are fragments of the polynucleotides of the invention may be used to synthesize full-length polynucleotides of the invention.

Further particularly preferred embodiments are polynucleotides encoding ratA vanants, that have the amino acid sequence of ratA polypeptide of Figure 2 [SEQ ID NO:2 or 4] m which several, a few, 5 to 10, 1 to 5, 1 to 3, 2, 1 or no ammo acid residues are substituted, deleted or added, in any combination. Especially preferred among these are silent substitutions, additions and deletions, that do not alter the properties and activities of ratA.

Further preferred embodiments of the invention are polynucleotides that are at least 70% identical over their entire length to a polynucleotide encodmg ratA polypeptide having the ammo acid sequence set out in Figure 2 [SEQ ID NO:2 or 4], and polynucleotides that are complementary to such polynucleotides. Alternatively, most highly preferred are polynucleotides that compnse a region that is at least 80% identical over its entire length to a polynucleotide encoding ratA polypeptide of the deposited strain 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 preferred. Furthermore, those with at least 97% are highly preferred among those with at least 95%, and among these those with at least 98% and at least 99% are particularly highly preferred, with at least 99% being the more preferred.

Preferred embodiments are polynucleotides that encode polypeptides that retain substantially the same biological function or activity as the mature polypeptide encoded by the DNA of Figure 1 [SEQ ID NO: 1 or 3] .

The invention further relates to polynucleotides that hybridize to the herein above- described sequences. In this regard, the invention especially relates to polynucleotides that hybridize under stringent conditions to the herein above-described polynucleotides. As herein used, the terms "stringent conditions" and "stringent hybridization conditions" mean hybridization will occur only if there is at least 95% and preferably at least 97% identity between the sequences. An example of stringent hybridization conditions is overnight incubation at 42°C in a solution comprising: 50% formamide, 5xSSC (150mM NaCl, 15mM trisodium citrate), 50 mM sodium phosphate (pH7.6), 5x Denhardt's solution, 10% dextran sulfate, and 20 micrograms/ml denatured, sheared salmon sperm DNA, followed by washing the hybridization support in O.lx SSC at about 65°C. Hybridization and wash conditions are well known and exemplified in Sambrook, et al., Molecular Cloning: A Laboratory Manual, Second Edition, Cold Spring Harbor, N.Y., (1989), particularly Chapter 11 therein, the disclosure of which is hereby incoφorated in its entirety by reference. The invention also provides a polynucleotide consisting essentially of a polynucleotide sequence obtainable by screening an appropriate library containing the complete gene for a polynucleotide sequence set forth in SEQ ID NO: 1 or 3 under stringent hybridization conditions with a probe having the sequence of said polynucleotide sequence set forth in SEQ ID NO:l or 3 or a fragment thereof; and isolating said DNA sequence. Fragments useful for obtaining such a polynucleotide include, for example, probes and primers described elsewhere herein.

As discussed additionally herein regarding polynucleotide assays of the invention, for instance, polynucleotides of the invention as discussed above, may be used as a hybridization probe for RNA, cDNA and genomic DNA to isolate full-length cDNAs and genomic clones encoding ratA and to isolate cDNA and genomic clones of other genes that have a high sequence similarity to the ratA gene. Such probes generally will comprise at least 15 bases. Preferably, such probes will have at least 30 bases and may have at least 50 bases. Particularly preferred probes will have at least 30 bases and will have 50 bases or less. For example, the codmg region of the ratA gene may be isolated by screening usmg the DNA sequence provided m SEQ ED NO: 1 or 3 to synthesize an oligonucleotide probe. A labeled oligonucleotide having a sequence complementary to that of a gene of the invention is then used to screen a library of cDNA, genomic DNA or mRNA to determine which members of the library the probe hybndizes to.

The polynucleotides and polypeptides of the invention may be employed, for example, as research reagents and matenals for discovery of treatments of and diagnostics for disease, particularly human disease, as further discussed herein relating to polynucleotide assays. Polynucleotides of the invention that are ohgonucleotides denved from the sequences of SEQ ID NOS:l and or 2 may be used in the processes herein as descnbed, but preferably for PCR, to determine whether or not the polynucleotides identified herein m whole or in part are transcnbed in bactena 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 invention also provides polynucleotides that may encode a polypeptide that is the mature protein plus additional ammo or carboxyl-terminal ammo acids, or ammo acids interior to the mature polypeptide (when the mature form has more than one polypeptide chain, for instance). Such sequences may play a role in processing of a protein from precursor to a mature form, may allow protein transport, may lengthen or shorten protein half-life or may facilitate manipulation of a protein for assay or production, among other things. As generally is the case in vivo, the additional amino acids may be processed away from the mature protein by cellular enzymes.

A precursor protein, having the mature form of the polypeptide fused to one or more prosequences may be an inactive form of the polypeptide. When prosequences are removed such inactive precursors generally are activated. Some or all of the prosequences may be removed before activation. Generally, such precursors are called proprotems.

In sum, a polynucleotide of the invention may encode a mature protein, a mature protein plus a leader sequence (which may be referred to as a preprotem), a precursor of a mature protein having one or more prosequences that are not the leader sequences of a preprotem, or a preproprotem, which is a precursor to a proprotein, having a leader sequence and one or more prosequences, which generally are removed duπng processing steps that produce active and mature forms of the polypeptide. Vectors, host cells, expression

The invention also relates to vectors that compnse a polynucleotide or polynucleotides of the invention, host cells that are genetically engineered 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 invention.

For recombinant production, host cells can be genetically engineered to mcoφorate expression systems or portions thereof or polynucleotides of the invention. Introduction of a polynucleotide into the host cell can be effected by methods descnbed in many standard laboratory manuals, such as Davis et al., BASIC METHODS IN MOLECULAR BIOLOGY, (1986) and Sambrook et al., MOLECULAR CLONING. A LABORATORY MANUAL, 2nd Ed., Cold Spnng Harbor Laboratory Press, Cold Spnng Harbor, N.Y. (1989), such as, calcium phosphate transfection, DEAE-dextran mediated transfection, transvection, micromjection, cationic hpid-mediated transfection, electroporation, transduction, scrape loading, ballistic introduction and infection.

Representative examples of appropnate hosts include bacteπal cells, such as streptococci, staphylococci, enterococci E coh, streptomyces and Bacillus subtihs cells; fungal cells, such as yeast cells and Asperg lus cells; insect cells such as Drosoph a S2 and Spodoptera Sf9 cells; animal cells such as CHO, COS, HeLa, C127, 3T3, BHK, 293 and Bowes melanoma cells; and plant cells.

A great vanety of expression systems can be used to produce the polypeptides of the invention. Such vectors include, among others, chromosomal, episomal and virus-denved vectors, e g , vectors denved from bactenal plasmids, from bactenophage, from transposons, from yeast episomes, from insertion elements, from yeast chromosomal elements, from viruses such as baculovimses, papova viruses, such as SV40, vaccinia viruses, adenoviruses, fowl pox viruses, pseudorabies viruses and retroviruses, and vectors denved from combinations thereof, such as those denved from plasmid and bactenophage genetic elements, such as cosmids and phagemids. The expression system constructs may contain control regions that regulate as well as engender expression. Generally, any system or vector suitable to maintam, 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 into the expression system by any of a vanety of well-known and routine techniques, such as, for example, those set forth in Sambrook et al., MOLECULAR CLONING, A

LABORATORY MANUAL, (supra). For secretion of the translated protein into the lumen of the endoplasmic reticulum, into the penplasmic space or into the extracellular environment, appropnate secretion signals may be incoφorated into the expressed polypeptide. These signals may be endogenous to the polypeptide or they may be heterologous signals. Polypeptides of the invention can be recovered and purified from recombinant cell cultures by well-known methods including ammonium sulfate or ethanol precipitation, acid extraction, anion or cation exchange chromatography, phosphocellulose chromatography, hydrophobic interaction chromatography, affinity chromatography, hydroxylapatite chromatography, and lectin chromatography. Most preferably, high performance liquid chromatography is employed for punfication. Well known techniques for refolding protein may be employed to regenerate active conformation when the polypeptide is denatured duπng isolation and or punfication. Diagnostic Assays This invention is also related to the use of the ratA polynucleotides of the invention for use as diagnostic reagents. Detection of ratA in a eukaryote, particularly a mammal, and especially a human, will provide a diagnostic method for diagnosis of a disease. Eukaryotes (herein also "mdιvιdual(s)"), particularly mammals, and especially humans, infected with an organism compnsing the ratA gene may be detected at thenucleic acid level by a vanety of techniques. Nucleic acids for diagnosis may be obtained from an infected individual's cells and tissues, such as bone, blood, muscle, cartilage, and skm. Genomic DNA may be used directly for detection or may be amplified enzymatically by using PCR or other amplification technique pnor to analysis. RNA or cDNA may also be used in the same ways Using amplification, charactenzation of the species and strain of prokaryote present m an individual, may be made by an analysis of the genotype of the prokaryote gene. Deletions and insertions can be detected by a change in size of the amplified product in companson to the genotype of a reference sequence. Point mutations can be identified by hybndizmg amplified DNA to labeled ratA polynucleotide sequences Perfectly matched sequences can be distinguished from mismatched duplexes by RNase digestion or by differences in melting temperatures DNA sequence differences may also be detected by alterations in the electrophoretic mobility of the DNA fragments m gels, with or without denatuπng agents, or by direct DNA sequencing. See, e.g., Myers et al., Science, 230 1242 (1985), which is mcoφorated by reference herein in its entirety Sequence changes at specific locations also may be revealed by nuclease protection assays, such as RNase and SI protection or a chemical cleavage method. See, eg , Cotton et al., Proc Natl Acad. Set , USA, 85 4397-4401 (1985) which is incoφorated by reference herein m its entirety.

Cells carrying mutations or polymoφhisms in the gene of the invention may also be detected at the DNA level by a vanety of techniques, to allow for serotypmg, for example. For example, RT-PCR can be used to detect mutations It is particularly preferred to used RT- PCR in conjunction with automated detection systems, such as, for example, GeneScan. RNA or cDNA may also be used for the same puφose, PCR or RT-PCR. As an example, PCR pπmers complementary to a nucleic acid encoding ratA can be used to identify and analyze mutations. These pnmers may also be used for amplifying ratA DNA isolated from a sample denved from an individual. The invention further provides these pπmers with 1, 2, 3 or 4 nucleotides removed from the 5' and/or the 3' end. The pπmers may be used to amplify the gene isolated from an infected individual such that the gene may then be subject to vaπous techniques for elucidation of the DNA sequence. In this way, mutations in the DNA sequence may be detected and used to diagnose infection and to serotype and/or classify the infectious agent.

The invention further provides a process for diagnosing, disease, preferably bacteπal infections, more preferably infections by Streptococcus pneumoniae, and most preferably otitis media, conjunctivitis, pneumonia, bacteremia, meningitis, sinusitis, pleural empyema and endocarditis, and most particularly meningitis, such as for example infection of cerebrospinal fluid,, compnsing determining from a sample denved from an individual a increased level of expression of polynucleotide having the sequence of Figure 1 [SEQ ID NO:l or 3]. Increased or decreased expression of ratA polynucleotide can be measured using any on of the methods well known m the art for the quantation of polynucleotides, such as, for example, amplification, PCR, RT-PCR, RNase protection, Northern blotting and other hybndization methods.

In addition, a diagnostic assay in accordance with the invention for detectmg over- expression of ratA protein compared to normal control tissue samples may be used to detect the presence of an infection, for example. Assay techniques that can be used to determine levels of a ratA protein, in a sample denved from a host are well-known to those of skill m the art. Such assay methods include radioimmunoassays, competitive-bmdmg assays, Western Blot analysis and ELISA assays Antibodies

The polypeptides of the invention or vanants thereof, or cells expressing them can be used as an lmmunogen to produce antibodies lmmunospecific for such polypeptides. "Antibodies" as used herein includes monoclonal and polyclonal antibodies, chimeπc, smgle chain, simiamzed antibodies and humanized antibodies, as well as Fab fragments, mcludmg the products of an Fab lmmunolglobulm expression library.

Antibodies generated agamst the polypeptides of the invention can be obtained by admmistenng the polypeptides or epitope-beanng fragments, analogues or cells 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 vanous techniques, such as those m Kohler, G. and Milstem, C, Nature 256: 495-497 (1975); Kozbor et al, Immunology Today 4. 72 (1983); Cole et al., pg. 77-96 MONOCLONAL ANTIBODIES AND CANCER THERAPY, Alan R. Liss, Inc. (1985) which is incoφorated by reference herein m its entirety.

Techniques for the production of single chain antibodies (U.S. Patent No. 4,946,778) can be adapted to produce single chain antibodies to polypeptides of this invention. Also, transgemc mice, or other organisms such as other mammals, may be used to express humanized antibodies.

Alternatively phage display technology may be utilized to select antibody genes with binding activities towards the polypeptide either from repertoires of PCR amplified v- genes of lymphocytes from humans screened for possessing anti-ratA or from naive libraries (McCafferty, J. et al., (1990), Nature 348, 552-554; Marks, J. et al., (1992) Biotechnology 10, 779-783). The affinity of these antibodies can also be improved by chain shuffling (Clackson, T. et al., (1991) Nature 352, 624-628).

If two antigen binding domains are present each domain may be directed against a different epitope - termed 'bispecific' antibodies.

The above-descnbed antibodies may be employed to isolate or to identify clones expressing the polypeptides to punfy the polypeptides by affinity chromatography.

Thus, among others, antibodies against ratA- polypeptide may be employed to treat infections, particularly bactenal infections and especially otitis media, conjunctivitis, pneumonia, bacteremia, meningitis, sinusitis, pleural empyema and endocarditis, and most particularly meningitis, such as for example infection of cerebrospinal fluid,. Polypeptide vanants include antigemcally, epitopically or immunologically equivalent vanants that form a particular aspect of this invention. The term "antigemcally equivalent denvative" as used herein encompasses a polypeptide or its equivalent which will be specifically recognized by certain antibodies which, when raised to the protein or polypeptide according to the invention, interfere with the immediate physical interaction between pathogen and mammalian host. The term "immunologically equivalent denvative" as used herein encompasses a peptide or its equivalent which when used in a suitable formulation to raise antibodies m a vertebrate, the antibodies act to interfere with the immediate physical interaction between pathogen and mammalian host. The polypeptide, such as an antigemcally or immunologically equivalent denvative or a fusion protein thereof is used as an antigen to immunize a mouse or other animal such as a rat or chicken. The fusion protein may provide stability to the polypeptide. The antigen may be associated, for example by conjugation, with an lmmunogenic earner protein for example bovme serum albumin (BSA) or keyhole limpet haemocyamn (KLH). Alternatively a multiple antigenic peptide compnsing multiple copies of the protein or polypeptide, or an antigemcally or immunologically equivalent polypeptide thereof may be sufficiently antigenic to improve lmmunogemcity so as to obviate the use of a earner.

Preferably, the antibody or variant thereof is modified to make it less lmmunogenic in the individual. For example, if the individual is human the antibody may most preferably be "humanized"; where the comphmentanty determining regιon(s) of the hybndoma-deπved antibody has been transplanted into a human monoclonal antibody , for example as descnbed in Jones, P. et al. (1986), Nature 321, 522-525 or Tempest et al.,(1991) Biotechnology 9, 266-273.

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, Manthoφe et al., Hum. Gene Ther. 1963:4, 419), delivery of DNA complexed with specific protein earners (Wu et al., J Biol Chem 1989:264,16985), coprecipitation of DNA with calcium phosphate (Benvemsty & Reshef, PNAS, 1986:83,9551), encapsulation of DNA m vanous forms of hposomes (Kaneda et al, Science 1989:243,375), particle bombardment (Tang et al., Nature 1992, 356:152, Eisenbraun et al., DNA Cell Biol 1993, 12:791) and in vivo infection using cloned retroviral vectors (Seeger et al., PNAS 1984:81,5849).

Antagonists and agonists - assays and molecules

Polypeptides of the invention may also be used to assess the binding of small molecule substrates and ligands in, for example, cells, cell-free preparations, chemical hbranes, and natural product mixtures. These substrates and ligands may be natural substrates and ligands or may be structural or functional mimetics. See, e.g , Cohgan et al , Current Protocols in Immunology 1(2)- Chapter 5 (1991). The invention also provides a method of screening compounds to identify those which enhance (agonist) or block (antagonist) the action of ratA polypeptides or polynucleotides, particularly those compounds that are bacteπostatic and/or bactenocidal. The method of screening may involve high-throughput techniques. For example, to screen for agonists or antagoists, a synthetic reaction mix, a cellular compartment, such as a membrane, cell envelope or cell wall, or a preparation of any thereof, compnsing ratA polypeptide and a labeled substrate or ligand of such polypeptide is mcubated in the absence or the presence of a candidate molecule that may be a ratA agonist or antagonist. The ability of the candidate molecule to agonize or antagonize the ratA polypeptide is reflected m decreased binding of the labeled ligand or decreased production of product from such substrate. Molecules that bind gratuitously, i.e., without inducing the effects of ratA polypeptide are most likely to be good antagonists. Molecules that bind well and increase the rate of product production from substrate are agonists. Detection of the rate or level of production of product from substrate may be enhanced by using a reporter system. Reporter systems that may be useful in this regard include but are not limited to coloπmetπc labeled substrate converted into product, a reporter gene that is responsive to changes in ratA polynucleotide or polypeptide activity, and binding assays known in the art.

Another example of an assay for ratA antagonists is a competitive assay that combines ratA and a potential antagonist with ratA-bindmg molecules, recombinant ratA bmdmg molecules, natural substrates or ligands, or substrate or ligand mimetics, under appropnate conditions for a competitive inhibition assay. RatA can be labeled, such as by radioactivity or a colonmetnc compound, such that the number of ratA molecules bound to a bmdmg molecule or converted to product can be determined accurately to assess the effectiveness of the potential antagonist. Potential antagonists include small organic molecules, peptides, polypeptides and antibodies that bind to a polynucleotide or polypeptide of the invention and thereby inhibit or extinguish its activity. Potential antagonists also may be small organic molecules, a peptide, a polypeptide such as a closely related protem or antibody that binds the same sites on a binding molecule, such as a binding molecule, without inducing ratA-mduced activities, thereby preventing the action of ratA by excluding ratA 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); OUGODEOXYNUCLEOΗDES AS ANTISENSE INHIBITORS OF GENE EXPRESSION, CRC Press, Boca Raton, FL (1988), for a descnption of these molecules). Preferred potential antagonists include compounds related to and vanants of ratA. Each of the DNA sequences provided herein may be used in the discovery and development of antibactenal compounds. The encoded protem, upon expression, can be used as a target for the screening of antibactenal drugs. Additionally, the DNA 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 or inhibitor of the invention to interfere with the initial physical interaction between a pathogen and mammalian host responsible for sequelae of infection. In particular the molecules of the mvention may be used: in the prevention of adhesion of bactena, m particular gram positive bacteria, to mammalian extracellular matnx proteins on in-dwellmg devices or to extracellular matnx proteins in wounds; to block ratA protem-mediated mammalian cell invasion by, for example, initiating phosphorylation of mammalian tyrosme kinases (Rosenshine et al, Infect. Immun. (50:2211 (1992); to block bacteπal adhesion between mammalian extracellular matnx proteins and bactenal ratA proteins that mediate tissue damage and; to block the normal progression of pathogenesis m infections initiated other than by the implantation of in-dwelling devices or by other surgical techniques.

The antagonists and agonists of the invention may be employed, for instance, to inhibit and treat otitis media, conjunctivitis, pneumonia, bacteremia, meningitis, sinusitis, pleural empyema and endocarditis, and most particularly meningitis, such as for example infection of cerebrospinal fluid,. Vaccines

Another aspect of the invention relates to a method for inducing an lmmunological response m an individual, particularly a mammal which compnses inoculating the individual with ratA, or a fragment or vanant thereof, adequate to produce antibody and/ or T cell immune response to protect said individual from infection, particularly bactenal infection and most particularly Streptococcus pneumoniae infection. Also provided are methods whereby such lmmunological response slows bactenal replication. Yet another aspect of the mvention relates to a method of inducing lmmunological response in an individual which compnses dehvenng to such individual a nucleic acid vector to direct expression of ratA, or a fragment or a vanant thereof, for expressing ratA, or a fragment or a vanant thereof in vivo in order to induce an lmmunological response, such as, to produce antibody and/ or T cell immune response, including, for example, cytokme-producing T cells or cytotoxic T cells, to protect said individual from disease, whether that disease is already established within the individual or not. One way of admmistenng the gene is by accelerating it into the desired cells as a coating on particles or otherwise. Such nucleic acid vector may compnse DNA, RNA, a modified nucleic acid, or a DNA/RNA hybnd.

A further aspect of the invention relates to an lmmunological composition which, when introduced into an individual capable or having induced within it an lmmunological response, induces an lmmunological response m such individual to a ratA or protein coded therefrom, wherein the composition compnses a recombinant ratA or protem coded therefrom compnsing DNA which codes for and expresses an antigen of said ratA or protem coded therefrom. The lmmunological response may be used therapeutically or prophylactically and may take the form of antibody immunity or cellular immunity such as that aπsing from CTL or CD4+ T cells.

A ratA polypeptide or a fragment thereof may be fused with co-protein which may not by itself produce antibodies, but is capable of stabilizing the first protein and producing a fused protein which will have lmmunogenic and protective properties. Thus fused recombinant protein, preferably further compnses an antigenic co-prote , such as lipoprotem D from Hemophilus influenzae, Glutathione-S-transferase (GST) or beta- galactosidase, relatively large co-protems which solubihse the protein and facilitate production and punfication thereof. Moreover, the co-protein may act as an adjuvant in the sense of providing a generalized stimulation of the immune system. The co-protem may be attached to either the ammo or carboxy terminus of the first protem. Provided by this invention are compositions, particularly vaccine compositions, and methods compnsing the polypeptides or polynucleotides of the invention and lmmunostimulatory DNA sequences, such as those descnbed m Sato, Y. et al. Science 273 : 352 (1996).

Also, provided by this invention are methods using the descnbed polynucleotide or particular fragments thereof which have been shown to encode non-vanable regions of bacterial cell surface proteins in DNA constructs used in such genetic immunization expeπments in animal models of infection with Streptococcus pneumoniae 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 from the requisite organ of the animal successfully resisting or cleanng infection for the development of prophylactic agents or therapeutic treatments of bacteπal infection, particularly Streptococcus pneumoniae infection, m mammals, particularly humans The polypeptide may be used as an antigen for vaccination of a host to produce specific antibodies which protect against invasion of bactena, for example by blocking adherence of bactena to damaged tissue. Examples of tissue damage include wounds in skin or connective tissue caused, e.g., by mechanical, chemical or thermal damage or by implantation of indwelling devices, or wounds in the mucous membranes, such as the mouth, mammary glands, urethra or vagina.

The invention also includes a vaccine formulation which compnses an lmmunogenic recombinant protem of the invention together with a suitable earner. Since the protein may be broken down in the stomach, it is preferably administered parenterally, including, for example, administration that is subcutaneous, intramuscular, intravenous, or lntradermal. Formulations suitable for parenteral administration include aqueous and non- aqueous sterile injection solutions which may contain anti-oxidants, buffers, bactenostats and solutes which render the formulation mstomc with the bodily fluid, preferably the blood, of the individual; and aqueous and non-aqueous sterile suspensions which may include suspending agents or thickening agents The formulations may be presented m unit-dose or multi-dose containers, for example, sealed ampules and vials and may be stored m a freeze-dπed condition requiπng only the addition of the stenle liquid earner immediately prior to use The vaccine formulation may also include adjuvant systems for enhancing the lmmunogemcity 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 routine expenmentation.

While the invention has been descnbed with reference to certain ratA protein, it is to be understood that this covers fragments of the naturally occurnng protem and similar proteins with additions, deletions or substitutions which do not substantially affect the lmmunogenic properties of the recombinant protein. Compositions, kits and administration

The invention also relates to compositions compnsing the polynucleotide or the polypeptides discussed above or their agonists or antagonists. The polypeptides of the invention may be employed m combination with a non-stenle or stenle earner or earners for use with cells, tissues or organisms, such as a pharmaceutical earner suitable for administration to a subject. Such compositions compπse, for instance, a media additive or a therapeutically effective amount of a polypeptide of the invention and a pharmaceutically acceptable earner or excipient. Such earners may include, but are not limited to, saline, buffered salme, dextrose, water, glycerol, ethanol and combinations thereof. The formulation should suit the mode of administration. The invention further relates to diagnostic and pharmaceutical packs and kits compnsing one or more containers filled with one or more of the ingredients of the aforementioned compositions of the invention.

Polypeptides and other compounds of the invention may be employed alone or m conjunction with other compounds, such as therapeutic compounds. The pharmaceutical compositions may be administered in any effective, convenient manner including, for instance, administration by topical, oral, anal, vaginal, intravenous, intrapentoneal, intramuscular, subcutaneous, mtranasal or mtradermal routes among others.

In therapy or as a prophylactic, the active agent may be administered to an individual as an mjectable composition, for example as a stenle aqueous dispersion, preferably lsotomc.

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 dressings and sutures and aerosols, and may contain appropnate conventional additives, including, for example, preservatives, solvents to assist drug penetration, and emollients m ointments and creams. Such topical formulations may also contain compatible conventional earners, for example cream or ointment 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. 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 m 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 mented, and such are within the scope of this mvention.

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 orthopaedic 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 mvention 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 lμg/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 0.5-5μg/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. EXAMPLES

The examples below are carried out using standard techniques, which are well known and routine to those of skill in the art, except where otherwise described in detail. The examples are illustrative, but do not limit the invention. Example 1 Strain selection, Library Production and Sequencing

The polynucleotide having the DNA sequence given in SEQ ID NO: 1 or 3 was obtained from a library of clones of chromosomal DNA of Streptococcus pneumoniae in E. coli. The sequencing data from two or more clones containing overlapping Streptococcus pneumoniae DNAs was used to construct the contiguous DNA sequence in SEQ ID NO:l or 3. Libraries may be prepared by routine methods, for example: Methods 1 and 2 below. Total cellular DNA is isolated from Streptococcus pneumoniae 0100993 according to standard procedures and size-fractionated by either of two methods. Method 1

Total cellular DNA is mechanically sheared by passage through a needle in order to size-fractionate according to standard procedures. DNA fragments of up to 1 lkbp in size are rendered blunt by treatment with exonuclease and DNA polymerase, and EcoRI linkers added. Fragments are ligated into the vector Lambda ZapII that has been cut with EcoRI, the library packaged by standard procedures and E.coli infected with the packaged library. The library is amplified by standard procedures. Method 2 Total cellular DNA is partially hydrolyzed with a one or a combination of restriction enzymes appropriate to generate a series of fragments for cloning into library vectors (e.g., Rsal, Pall, Alul, Bshl235I), and such fragments are size-fractionated according to standard procedures. EcoRI linkers are ligated to the DNA and the fragments then ligated into the vector Lambda ZapII that have been cut with EcoRI, the library packaged by standard procedures, and E.coli infected with the packaged library. The library is amplified by standard procedures.

SEQUENCE LISTING

(1) GENERAL INFORMATION:

(i) APPLICANT: Smit Kline Beecham Corporation

(ii) TITLE OF INVENTION: Novel Compounds

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(vii) PRIOR APPLICATION DATA:

(A) APPLICATION NUMBER: 60/041,130

(B) FILING DATE: 20-MAR-1997

(A) APPLICATION NUMBER: 60/041,131

(B) FILING DATE: 20-MAR-1997

(viii) ATTORNEY/AGENT INFORMATION:

(A) NAME: Falk, Stephen T

(B) REGISTRATION NUMBER: 36,795

(C) REFERENCE/DOCKET NUMBER: GM50023 (ix) TELECOMMUNICATION INFORMATION:

(A) TELEPHONE: 215-994-2488

(B) TELEFAX: 215-994-2222

(C) TELEX:

(2) INFORMATION FOR SEQ ID NO:l;

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 1464 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS : double

(D) TOPOLOGY: linear

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:l:

ATGACTTTTA ACAATAAAAC TATTGAAGAG TTGCACAATC TCCTTGTCTC TAAGGAAATT 60

TCTGCAACAG AATTGACCCA AGCAACACTT GAAAATATCA AGTCTCGTGA GGAAGCCATC 120

AATTCATTTG TCACCATCGC TGAGGAGCAA GCTCTTGTTC AAGCTAAAGC CATTGATGAA 180

GCTGGAATTG ATGCTGACAA TGTCCTTTCA GGAATTCCAC TTGCTGTTAA GGATAACATC 240

TCTACAGACG GTATTCTCAC AACTGCTGCC TCAAAAATGC TCTACAACTA TGAGCCAATC 300

TTTGATGCGA CAGCTGTTGC CAATGCAAAA ACCAAAGGCA TGATTGTCGT TGGAAAGACC 360

AACATGGACG AATTTGCTAT GGGTGGTTCA GGTGAAACTT CACACTACGG AGCAACTAAA 420

AACGCTTGGG ACCACAGCAA GGTTCCTGGT GGGTCATCAA GTGGTTCTGC CGCAGCTGTA 480

GCCTCAGGAC AAGTTCGCTT GTCACTTGGT TCTGATACTG GTGGTTCCAT CCGCCAACCT 540

GCTGCCTTCA ACGGAATCGT TGGTCTCAAA CCAACCTACG GAACAGTTTC ACGTTTCGGT 600

CTCATTGCCT TTGGTAGCTC ATTAGACCAG ATTGGACCTT TTGCTCCTAC TGTTAAGGAA 660

AATGCCCTCT TGCTCAACGC TATTGCCAGC GAAGATGCTA AAGACTCTAC TTCTGCTCCT 720

GTCCGCATCG CCGACTTTAC TTCAAAAATC GGCCAAGACA TCAAGGGTAT GAAAATCGCT 780

TTGCCTAAGG AATACCTCGG TGAAGGAATT AACCCAGAGG TTAAGGAAAC CATTCTAAAT 840

GCCGCTAAAC ACTTTGAAAA ATTGGGTGCT ATTGTCGAAG AAGTCAGCCT TCCTCACTCT 900

AAATACGGAG TTGCCGTATA CTACATCATC GCTTCATCAG AAGCTTCATC AAACTTGCAA 960

CGCTTCGACG GTATCCGTTA CGGCTATCGC GCAGAAGATG CAACCAACCT TGATGAAATC 1020

TATGTAAACA GCCGAAGCCA AGGTTTTGGT GAAGAAGTGA AGCGCCGTAT CATGCTGGGT 1080

ACTTTCAGTC TTTCATCAGG TTACTACGAT GCCTACTATA AGAAGGCTGG ACAGGTCCGT 1140

ACACTTATCA TTCAAGATTT CGAAAAAGTC TTCGCGGATT ACGATTTGAT TTTGGGTCCA 1200

ACTGCTCCAA GTGTTGCCTA TGACTTGGAT TCTCTCAACC ATGACCCAGT TGCCATGTAC 1260

TTAGCCGACC TATTGACCAT ACCTGTAAAC TTGGCAGGAC TGCCTGGAAT TTCGATTCCT 1320

GCTGGATTCT CTCAAGGTCT ACCTGTCGGA CTCCAATTGA TTGGTCCCAA GTACTCTGAG 1380

GAAACCATTT ACCAAGCTGC TGCTGCTTTT GAAGCAACAA CAGACTACCA CAAACAACAA 1440

CCCGTGATTT TTGGAGGTGA CAAC 1464 (2) INFORMATION FOR SEQ ID NO : 2 :

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 488 amino acids

(B) TYPE: amino acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 2:

Met Thr Phe Asn Asn Lys Thr He Glu Glu Leu His Asn Leu Leu Val

1 5 10 15

Ser Lys Glu He Ser Ala Thr Glu Leu Thr Gin Ala Thr Leu Glu Asn

20 25 30

He Lys Ser Arg Glu Glu Ala He Asn Ser Phe Val Thr He Ala Glu

35 40 45

Glu Gin Ala Leu Val Gin Ala Lys Ala He Asp Glu Ala Gly He Asp

50 55 60

Ala Asp Asn Val Leu Ser Gly He Pro Leu Ala Val Lys Asp Asn He 65 70 75 80

Ser Thr Asp Gly He Leu Thr Thr Ala Ala Ser Lys Met Leu Tyr Asn

85 90 95

Tyr Glu Pro He Phe Asp Ala Thr Ala Val Ala Asn Ala Lys Thr Lys

100 105 110

Gly Met He Val Val Gly Lys Thr Asn Met Asp Glu Phe Ala Met Gly

115 120 125

Gly Ser Gly Glu Thr Ser His Tyr Gly Ala Thr Lys Asn Ala Trp Asp

130 135 140

His Ser Lys Val Pro Gly Gly Ser Ser Ser Gly Ser Ala Ala Ala Val 145 150 155 160

Ala Ser Gly Gin Val Arg Leu Ser Leu Gly Ser Asp Thr Gly Gly Ser

165 170 175

He Arg Gin Pro Ala Ala Phe Asn Gly He Val Gly Leu Lys Pro Thr

180 185 190

Tyr Gly Thr Val Ser Arg Phe Gly Leu He Ala Phe Gly Ser Ser Leu

195 200 205

Asp Gin He Gly Pro Phe Ala Pro Thr Val Lys Glu Asn Ala Leu Leu

210 215 220

Leu Asn Ala He Ala Ser Glu Asp Ala Lys Asp Ser Thr Ser Ala Pro 225 230 235 240

Val Arg He Ala Asp Phe Thr Ser Lys He Gly Gin Asp He Lys Gly

245 250 255

Met Lys He Ala Leu Pro Lys Glu Tyr Leu Gly Glu Gly He Asn Pro 260 265 270

Glu Val Lys Glu Thr He Leu Asn Ala Ala Lys His Phe Glu Lys Leu

275 280 285

Gly Ala He Val Glu Glu Val Ser Leu Pro His Ser Lys Tyr Gly Val

290 295 300

Ala Val Tyr Tyr He He Ala Ser Ser Glu Ala Ser Ser Asn Leu Gin 305 310 315 320

Arg Phe Asp Gly He Arg Tyr Gly Tyr Arg Ala Glu Asp Ala Thr Asn

325 330 335

Leu Asp Glu He Tyr Val Asn Ser Arg Ser Gin Gly Phe Gly Glu Glu

340 345 350

Val Lys Arg Arg He Met Leu Gly Thr Phe Ser Leu Ser Ser Gly Tyr

355 360 365

Tyr Asp Ala Tyr Tyr Lys Lys Ala Gly Gin Val Arg Thr Leu He He

370 375 380

Gin Asp Phe Glu Lys Val Phe Ala Asp Tyr Asp Leu He Leu Gly Pro 385 390 395 400

Thr Ala Pro Ser Val Ala Tyr Asp Leu Asp Ser Leu Asn His Asp Pro

405 410 415

Val Ala Met Tyr Leu Ala Asp Leu Leu Thr He Pro Val Asn Leu Ala

420 425 430

Gly Leu Pro Gly He Ser He Pro Ala Gly Phe Ser Gin Gly Leu Pro

435 440 445

Val Gly Leu Gin Leu He Gly Pro Lys Tyr Ser Glu Glu Thr He Tyr

450 455 460

Gin Ala Ala Ala Ala Phe Glu Ala Thr Thr Asp Tyr His Lys Gin Gin 465 470 475 480

Pro Val He Phe Gly Gly Asp Asn 485

(2) INFORMATION FOR SEQ ID NO: 3:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 70 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: double

(D) TOPOLOGY: linear

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 3:

TTTGCAACAG AATTGACCCA AGCAACACTT GAAAATATCA AGTCTCGTGA GGAAGCCATC 60 AATTCATTTG 70 (2) INFORMATION FOR SEQ ID NO: 4:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 70 amino acids

(B) TYPE: amino acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 4:

Phe Ala Thr Glu Leu Thr Gin Ala Thr Leu Glu Asn He Lys Ser Arg

1 5 10 15

Glu Glu Ala He Asn Ser Phe Val Thr He Ala Glu Glu Gin Ala Leu

20 25 30

Val Gin Ala Lys Ala He Asp Glu Ala Gly He Asp Ala Asp Asn Val

35 40 45

Leu Ser Gly He Pro Leu Ala Val Lys Asp Asn He Ser Thr Asp Gly

50 55 60

He Leu Thr Thr Ala Ala 65 70

Claims

What is claimed is:

1. An isolated polynucleotide comprising a polynucleotide sequence selected from the group consisting of:

(a) a polynucleotide having at least a 70% identity to a polynucleotide encoding a polypeptide comprising amino acids of SEQ ID NO:2 or 4;

(b) a polynucleotide which is complementary to the polynucleotide of (a); and

(c) a polynucleotide comprising at least 15 sequential bases of the polynucleotide of (a) or (b).

2. The polynucleotide of Claim 1 wherein the polynucleotide is DNA.

3. The polynucleotide of Claim 1 wherein the polynucleotide is RNA.

4. The polynucleotide of Claim 2 comprising the nucleotide sequence set forth in SEQ ID NO: l or 3.

5. The polynucleotide of Claim 2 comprising nucleotide sequence set forth in SEQ ID NO: l or 3.

6. The polynucleotide of Claim 2 which encodes a polypeptide comprising amino acid sequence of SEQ ED NO:2 or 4.

7. An isolated polynucleotide comprising a member selected from the group consisting of:

(a) a polynucleotide having at least a 70% identity to a polynucleotide encoding the same mature polypeptide expressed by the ratA gene contained in NCEMB Deposit No. 40794;

(b) a polynucleotide complementary to the polynucleotide of (a); and

(c) a polynucleotide comprising at least 15 bases of the polynucleotide of (a) or (b).

8. A vector comprising the DNA of Claim 2.

9. A host cell comprising the vector of Claim 8.

10. A process for producing a polypeptide comprising: expressing from the host cell of Claim 9 a polypeptide encoded by said DNA.

11. A process for producing a cell which expresses a polypeptide comprising transforming or transfecting the cell with the vector of Claim 8 such that the cell expresses the polypeptide encoded by the cDNA contained in the vector.

12. A process for producing a ratA polypeptide or fragment comprising culturing a host of claim 9 under conditions sufficient for the production of said polypeptide or fragment.

13. A polypeptide comprising an amino acid sequence which is at least 70% identical to amino acid sequence of SEQ ID NO:2 or 4.

14. A polypeptide comprising an amino acid sequence as set forth in SEQ ID NO:2 or 4.

15. An antibody against the polypeptide of claim 13.

16. An antagonist which inhibits the activity of the polypeptide of claim 13.

17. A method for the treatment of an individual having need of ratA comprising: administering to the individual a therapeutically effective amount of the polypeptide of claim 13.

18. The method of Claim 16 wherein said therapeutically effective amount of the polypeptide is administered by providing to the individual DNA encoding said polypeptide and expressing said polypeptide in vivo.

19. A method for the treatment of an individual having need to inhibit ratA polypeptide comprising: administering to the individual a therapeutically effective amount of the antagonist of Claim 16.

20. A process for diagnosing a disease related to expression of the polypeptide of claim 13 comprising: determining a nucleic acid sequence encoding said polypeptide.

21. A diagnostic process comprising: analyzing for the presence of the polypeptide of claim 13 in a sample derived from a host.

22. A method for identifying compounds which interact with and inhibit or activate an activity of the polypeptide of claim 13 comprising: 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 determining whether the compound interacts with and activates or inhibits an activity of the polypetide by detecting the presence or absence of a signal generated from the interaction of the compound with the polypeptide.

23. A method for inducing an immunological response in a mammal which comprises inoculating the mammal with ratA, or a fragment or variant thereof, adequate to produce antibody and/ or T cell immune response to protect said animal from disease.

24. A method of inducing immunological response in a mammal which comprises delivering a nucleic acid vector to direct expression of ratA fragment or a variant thereof, for expressing ratA, or a fragment or a variant thereof in vivo in order to induce an immunological response to produce antibody and/ or T cell immune response to protect said animal from disease.

25. An immunological composition comprising a DNA which codes for and expresses a ratA polynucleotide or protein coded therefrom which, when introduced into a mammal, induces an immunological response in the mammal to a given ratA polynucleotide or protein coded therefrom.

26. A polynucleotide consisting essentially of a DNA sequence obtainable by screening an appropriate library containing the complete gene for a polynucleotide sequence set forth in SEQ ID NO: 1 or 3 under stringent hybridization conditions with a probe having the sequence of said polynucleotide sequence set forth in SEQ ID NO:l or 3 or a fragment thereof; and isolating said DNA sequence.