WO1998037918A1

WO1998037918A1 - Staphylococcus 3-dehydroquinate synthase

Info

Publication number: WO1998037918A1
Application number: PCT/US1998/004483
Authority: WO
Inventors: James R. Brown; David J. Payne
Original assignee: Smithkline Beecham Corporation
Priority date: 1997-02-28
Filing date: 1998-02-27
Publication date: 1998-09-03
Also published as: EP0968293A1; JP2001516211A; EP0968293A4

Abstract

The invention provides 3-dehydroquinate synthase polypeptides and polynucleotides encoding 3-dehydroquinate synthase polypeptides and methods for producing such polypeptides by recombinant techniques. Also provided are methods for utilizing 3-dehydroquinate synthase polypeptides or polynucleotides to screen for antibacterial compounds.

Description

STAPHYLOCOCCUS 3-DEHYDROQUINATΕ SYNTHASE

FIELD OF THE INVENTION

This invention relates to newly identified polynucleotides and polypeptides, and their production and uses, as well as their vanants, agonists and antagonists, and their uses In particular, m these and in other regards, the invention relates to novel polynucleotides and polypeptides of the aro family, hereinafter referred to as "3-dehydroqumate synthase ".

BACKGROUND OF THE INVENTION

It is particularly preferred to employ Staphylococcal genes and gene products as targets for the development of antibiotics. The Staphylococci make up a medically important genera of microbes They are known to produce two types of disease, invasive and toxigemc Invasive infections are characterized generally by abscess formation effecting both skm surfaces and deep tissues. S. aureus is the second leading cause of bacteremia in cancer patients. Osteomyelitis, septic arthritis, septic thrombophlebitis and acute bacterial endocarditis are also relatively common There are at least three clinical conditions resulting from the toxigemc properties of Staphylococci. The manifestation of these diseases result from the actions of exotoxms as opposed to tissue invasion and bacteremia. These conditions include: Staphylococcal food poisoning, scalded skm syndrome and toxic shock syndrome.

The frequency of Staphylococcus aureus infections has risen dramatically m 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 Staphylococcus aureus 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.

3-dehydroquιnate synthase is a key enzyme in the choπsmate biosynthetic pathway This gene is named aro B in Bacillus subtilus and E.coli. 3-dehydroqumate synthase converts 3-deoxy-D-arabmo-heptulosonate 7-phosphate to 3-dehydroqumate (Biosynthesis of Aromatic Ammo Acids, D. Henner and C. Yanofsky (p.269). In Bacillus subtilus & other Gram positive bacteria, Eds Sonenshem, Hoch & Losick, ASM Washington, DC, 1993). Inhibition of this reaction will prevent the synthesis of aromatic amino acids, p-ammobenzoic acid (precursor for folate) and ubiqumone. These essential metabolites are in limiting concentrations in mammalian tissues and thus inhibition of this enzyme is a valid antibacterial strategy.

Clearly, there is a need for factors, such as the novel compounds of the invention, having a present benefit 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 m preventing, ameliorating or correcting infections, dysfunctions or diseases. The polypeptides of the invention have ammo acid sequence homology to a known

Bacillus subtilus aroB ( 3 -dehydroqumate synthase protein.

SUMMARY OF THE INVENTION

It is an object of the invention to provide polypeptides that have been identified as novel 3 -dehydroqumate synthase polypeptides by homology between the ammo acid sequences set out in Figure 2 [SEQ LD NO 2] and a known amino acid sequence or sequences of other proteins such as Bacillus subtilus aroB ( 3 -dehydroqumate synthase protein.

It is a further object of the invention to provide polynucleotides that encode 3- dehydroqumate synthase polypeptides, particularly polynucleotides that encode the polypeptide herein designated 3 -dehydroqumate synthase .

In a particularly preferred embodiment of the invention the polynucleotide comprises a region encoding 3-dehydroqumate synthase polypeptides compπsing a sequence set out in Figure 1 [SEQ ID NO: 1], or a variant thereof. In another particularly preferred embodiment of the invention there is a novel 3- dehydroquinate synthase protein from Staphylococcus aureus comprising an ammo acid sequence of Figure 2 [SEQ ID NO:2], or a variant thereof

In accordance with another aspect of the invention there is provided an isolated nucleic acid molecule encoding a mature polypeptide expressible by the Staphylococcus aureus WCUH 29 strain contained m NCIMB Deposit No. 40771.

A further aspect of the invention there are provided isolated nucleic acid molecules encoding 3 -dehydroqumate synthase , particularly Staphylococcus aureus 3 -dehydroqumate synthase , including mRNAs, cDNAs, genomic DNAs. Further embodiments of the invention include biologically, diagnostically, prophylactically, clinically or therapeutically useful vaπants thereof, and compositions comprising 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 purposes, in particular genetic immunization. Among the particularly preferred embodiments of the invention are naturally occurring allehc variants of 3 -dehydroqumate synthase and polypeptides encoded thereby

Another aspect of the invention there are provided novel polypeptides of Staphylococcus aureus referred to herein as 3 -dehydroqumate synthase as well as biologically, diagnostically, prophylactically, clinically or therapeutically useful vaπants thereof, and compositions comprising the same.

Among the particularly preferred embodiments of the invention are variants of 3- dehydroquinate synthase polypeptide encoded by naturally occurring alleles of the 3- dehydroqumate synthase gene. In a preferred embodiment of the invention there are provided methods for producing the aforementioned 3 -dehydroqumate synthase polypeptides.

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

In accordance with certain preferred embodiments of the invention, there are provided products, compositions and methods for assessing 3 -dehydroqumate synthase expression, treating disease, for example, disease, such as, infections of the upper respiratory tract (e.g., otitis media, bacteπal tracheitis, acute epiglottitis, thyroiditis), lower respiratory (e.g , empyema, lung abscess), cardiac (e.g., infective endocarditis), gastrointestinal (e.g., secretory diarrhoea, splenic absces, retropeπtoneal abscess), CNS (e.g., cerebral abscess), eye (e.g., blephaπtis, conjunctivitis, keratitis, endophthalmitis, preseptal and orbital celluhtis, darcryocystitis), kidney and urinary tract (e.g., epididymitis, mtrarenal and permephπc absces, toxic shock syndrome), skin (e.g., impetigo, follicuhtis, cutaneous abscesses, celluhtis, wound infection, bacteπal myositis) bone and joint (e.g., septic arthπtis, osteomyelitis), assaying genetic variation, and admmisteπng a 3-dehydroqumate synthase polypeptide or polynucleotide to an organism to raise an immunological response against a bacteπa, especially a Staphylococcus aureus bacteπa.

In accordance with certain preferred embodiments of this and other aspects of the invention there are provided polynucleotides that hybπdize to 3 -dehydroqumate synthase polynucleotide sequences, particularly under stπngent conditions In certain preferred embodiments of the invention there are provided antibodies against 3 -dehydroqumate synthase polypeptides

In accordance with yet another aspect of the invention, there are provided 3- dehydroqumate synthase agonists and antagonists, preferably bacteπostatic or bacteriocidal agonists and antagonists.

In a further aspect of the invention there are provided compositions comprising a 3- dehydroqumate synthase polynucleotide or a 3 -dehydroqumate synthase polypeptide for administration to a cell or to a multicellular organism

Vaπous changes and modifications within the spiπt 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 Staphylococcus aureus 3- dehydroqumate synthase [SEQ ID NO.1 ]

Figure 2 shows ammo acid sequence of Staphylococcus aureus 3 -dehydroqumate synthase [SEQ LD NOS:2 and 3] deduced from the polynucleotide sequence of Figure 1.

Asterisks (*) denote putative stop codons in the ORF. The continuous stretches of ammo acid sequence of four or more residues found between the asterisks make up SEQ ID NOS 4 through 23.

Figure 3 shows the amino acid sequence of homolog to AroB, 3-dehydroquinate synthase Characters m bold represent the most significant homolog to aroB in B.subtilis which is 59%ιdentιcal and 77% similar. Underlined sequence represents the fragment of aro B from Staphylococcus already present in Genbank. Bold and underlined sequences represent a homolog 55% identical and 70% similar to B. subtilus aroB. 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 m the art, is a relationship between two or more polypeptide sequences or two or more polynucleotide sequences, as the case may be, as determined by compaπng 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, Bωcomputing Informatics and Genome Projects, Smith, D.W., ed., Academic Press, New York, 1993; Computer Analysis of Sequence Data, Part I, Griffin, A.M., and Griffin, H.G., eds., Humana Press, New Jersey, 1994; Sequence Analysis in Molecular Biology, von Hemje, G., Academic Press, 1987; and Sequence Analysis Primer, 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 m publicly available computer programs. Computer program methods to determine identity between two sequences include, but are not limited to, the GCG program package (Devereux, J., et al , Nucleic Acids Research 12(1) 387 (1984)), BLASTP, BLASTN, and FASTA (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 275: 403-410 (1990). The well known Smith Waterman algorithm may also be used to determine identity.

Parameters for polypeptide sequence comparison include the following. 1) Algorithm: Needleman and Wunsch, J. Mol Biol. 48: 443-453 (1970) Compaπson matrix. BLOSSUM62 from Hentikoff and Hentikoff, Proc. Natl Acad. Sci USA. 89:10915-10919 (1992) Gap Penalty. 12 Gap Length Penalty: 4 A program useful with these parameters is publicly available as the "gap" program from Genetics Computer Group, Madison WI The aforementioned parameters are the default parameters for peptide comparisons (along with no penalty for end gaps) Parameters for polynucleotide comparison include the following: 1) Algorithm: Needleman and Wunsch, J. Mol Biol 48- 443-453 (1970) Comparison matrix: matches = +10, mismatch = 0 Gap Penalty: 50 Gap Length Penalty: 3

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

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

(1) Polynucleotide embodiments further include an isolated polynucleotide comprising a polynucleotide sequence having at least a 50, 60, 70, 80, 85, 90, 95, 97 or 100% identity to the reference sequence of SEQ ID NO: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 m SEQ ID NO: 1 by the integer defining the percent identity divided by 100 and then subtracting that product from said total number of nucleotides in SEQ ID NO:l, or: ⁿn ≤ ^xn - (^xn ' y)> wherein n_n is the number of nucleotide alterations, x_n is the total number of nucleotides m

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-mteger 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 NOS:2 OR 3 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 NOS:2 OR 3, 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 within the reference sequence. The number of nucleic acid alterations for a given percent identity is determined by multiplying the total number of ammo acids in SEQ ID NOS:2 OR 3 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 NOS:2 OR 3, or: n_n < x_n - (x_n • y), wherein n_n is the number of ammo acid alterations, x_n is the total number of ammo acids in SEQ ID NOS:2 OR 3, 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 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 NOS:2 OR 3, 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 ammo acid deletion, substitution, including conservative and non-conservative substitution, or insertion, and wherein said alterations may occur at the ammo- or carboxy-terminal positions of the reference polypeptide sequence or anywhere between those terminal positions, interspersed either individually among the ammo acids the reference sequence or in one or more contiguous groups within the reference sequence, and wherein said number of ammo acid alterations is determined by multiplying the total number of ammo acids m SEQ ID NOS:2 OR 3 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 NOS:2 OR 3, or. n_a < x_a - (x_a • y), wherein n_a is the number of ammo acid alterations, x_a is the total number of ammo acids m SEQ ID NOS:2 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-mteger product of x_a and y is rounded down to the nearest integer prior to subtracting it from x_a

By way of example, a polypeptide sequence of the present invention may be identical to the reference sequence of SEQ ID NOS.2 OR 3, 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 ammo acid deletion, substitution, including conservative and non-conservative substitution, or insertion, and wherein said alterations may occur at the ammo- or carboxy-terminal positions of the reference polypeptide sequence or anywhere between those terminal positions, interspersed either individually among the amino acids in the reference sequence or in one or more contiguous groups within the reference sequence The number of ammo acid alterations for a given % identity is determined by multiplying the total number of amino acids in SEQ ID NOS:2 OR 3 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 NOS:2 0R 3, or n_a < x_a - (x_a • y), wherein n_a is the number of ammo acid alterations, x_a is the total number of ammo acids m SEQ ID NOS:2 OR 3, 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-mteger product of x_a and y is rounded down to the nearest integer prior to subtracting it from x_a

In preferred aspects of the present invention, the invention relates to nucleic acids encoding polypeptides with one of the above described degrees of apparent relatedness (e.g., identity or similarity) to one of SEQ ID NOS:4 or 5 or 6 or 7 or 8 or 9 or 10 or 11 or 12 or 13 or 14 or 15 or 16 or 17 or 18 or 19 or 20 or 21 or 22 or 23. All of the discussion below with reference to SEQ ID NOS.2 or 3 is equally applicable to any of SEQ ID NOS:4 through 23.

"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 oπgmal 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 mateπals of its natural state is "isolated", as the term is employed herein

"Polynucleotιde(s)" generally refers to any polyπbonucleotide or polydeoxπbonucleotide, which may be unmodified RNA or DNA or modified RNA or DNA "Polynucleotιde(s)" include, without limitation, single- and double-stranded DNA, DNA that is a mixture of single- and double-stranded regions or single-, double- and triple-stranded regions, single- and double-stranded RNA, and RNA that is mixture of single- and double- stranded regions, hybrid molecules comprising DNA and RNA that may be smgle-stranded or, more typically, double-stranded, or triple-stranded regions, or a mixture of single- and double- stranded regions. In addition, "polynucleotide" as used herein refers to tπple-stranded regions comprising RNA or DNA or both RNA and DNA. The strands in such regions may be from the same molecule or from different molecules. The regions may include all of one or more of the molecules, but more typically involve only a region of some of the molecules. One of the molecules of a tπple-hehcal region often is an oligonucleotide. As used herein, the term "polynucleotιde(s)" also includes DNAs or RNAs as described above that contain one or more modified bases. Thus, DNAs or RNAs with backbones modified for stability or for other reasons are "polynucleotιde(s)" as that term is intended herein. Moreover, DNAs or RNAs compπsmg unusual bases, such as inosine, 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 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 "polynucleotιde(s)" as it is employed herein embraces such chemically, enzymatically or metabo cally modified forms of polynucleotides, as well as the chemical forms of DNA and RNA characteπstic of viruses and cells, including, for example, simple and complex cells. "Polynucleotιde(s)" also embraces short polynucleotides often referred to as ohgonucleotιde(s).

"Polypeptιde(s)" refers to any peptide or protein compnsmg two or more ammo acids joined to each other by peptide bonds or modified peptide bonds. "Polypeptιde(s)" refers to both short chains, commonly referred to as peptides, ohgopeptides and o gomers and to longer chains generally referred to as proteins. Polypeptides may contain ammo 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 descπbed m 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 m the same or varying degree at several sites m a given polypeptide. Also, a given polypeptide may contain many types of modifications Modifications can occur anywhere in a polypeptide, including the peptide backbone, the ammo acid side-chams, and the amino or carboxyl termini. Modifications include, for example, acetylation, acylation, ADP-πbosylation, 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-hnkmg, cyc zation, disulfide bond formation, demethylation, formation of covalent cross-links, formation of cysteme, formation of pyroglutamate, formylation, gamma-carboxylation, glycosylation, GPI anchor formation, hydroxylation, lodmation, methylation, myπstoylation, oxidation, proteolytic processing, phosphorylation, prenylation, racemization, glycosylation, lipid attachment, sulfation, gamma-carboxylation of glutamic acid residues, hydroxylation and ADP-πbosylation, selenoylation, sulfation, transfer-RNA mediated addition of ammo acids to proteins, such as argmylation, and ubiquitmation. See, for instance, PROTEINS -

STRUCTURE AND MOLECULAR PROPERTIES, 2nd Ed, T. E. Creighton, W. H. Freeman and Company, New York (1993) and Wold, F, Posttranslational Protein Modifications:

Perspectives and Prospects, pgs. 1-12 in POSTTRANSLATIONAL COVALENT

MODIFICATION OF PROTEINS, B. C. Johnson, Ed., Academic Press, New York (1983), Seifter et al, Meth Enzymol 182:626-646 (1990) and Rattan et al. Protein Synthesis

Posttranslational Modifications and Aging, Ann N.Y. Acad. Sci. 663 ^• 48-62 (1992)

Polypeptides may be branched or cyclic, with or without branching. Cyclic, branched and branched circular polypeptides may result from post-translational natural processes and may be made by entirely synthetic methods, as well. "Vaπant(s)" as the term is used herein, is a polynucleotide or polypeptide that differs from a reference polynucleotide or polypeptide respectively, but retains essential properties. A typical variant of a polynucleotide differs m nucleotide sequence from another, reference polynucleotide. Changes m the nucleotide sequence of the variant may or may not alter the ammo acid sequence of a polypeptide encoded by the reference polynucleotide. Nucleotide changes may result in ammo acid substitutions, additions, deletions, fusions and truncations in the polypeptide encoded by the reference sequence, as discussed below. A typical vaπant of a polypeptide differs m 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, in many regions, identical. A variant and reference polypeptide may differ in ammo acid sequence by one or more substitutions, additions, deletions in 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 occurring such as an allehc variant, or it may be a variant that is not known to occur naturally. Non-naturally occurring variants of polynucleotides and polypeptides may be made by mutagenesis techniques, by direct synthesis, and by other recombinant methods known to skilled artisans. DESCRIPTION OF THE INVENTION

The invention relates to novel 3 -dehydroqumate synthase polypeptides and polynucleotides as descπbed in greater detail below In particular, the invention relates to polypeptides and polynucleotides of a novel 3 -dehydroqumate synthase gene of

Staphylococcus aureus, which is related by ammo acid sequence homology to Bacillus subtilus aroB ( 3 -dehydroqumate synthase polypeptide The invention relates especially to

3 -dehydroqumate synthase having the nucleotide and ammo acid sequences set out in Figure 1 [SEQ ID NO: 1] and Figure 2 [SEQ ID NO: 2] respectively, and to the 3 -dehydroqumate synthase nucleotide sequences of the DNA deposited in NCIMB Deposit No 40771 and amino acid sequences encoded thereby.

Deposited materials

A deposit containing a Staphylococcus aureus WCUH 29 strain has been deposited with the National Collections of Industrial and Marine Bacteria Ltd. (NCIMB), 23 St

Machar Drive, Aberdeen AB2 1RY, Scotland on 11 September 1995 and assigned NCIMB

Deposit No. 40771. The Staphylococcus aureus strain deposit is referred to herein as "the deposited strain" or as "the DNA of the deposited strain."

The deposited mateπal is a strain that contains the full length 3 -dehydroqumate synthase DNA, referred to as "NCIMB 40771" upon deposit. The sequence of the polynucleotides contained m the deposited mateπal, as well as the ammo acid sequence of the polypeptide encoded thereby, are controlling in the event of any conflict with any descπption 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 mateπals, and no such license is hereby granted. Polypeptides

The polypeptides of the invention include the polypeptides of Figure 2 [SEQ ID NOS:2 OR 3] (m particular the mature polypeptide) as well as polypeptides and fragments, particularly those which have the biological activity of 3 -dehydroqumate synthase , and also those which have at least 70% identity to the polypeptides of Figure 2 [SEQ ID NOS:2 OR 3] (see Figure 3) or the relevant portion, preferably at least 80% identity to a polypeptide of Figure 2 [SEQ ID NOS:2 OR 3], and more preferably at least 90% similarity (more preferably at least 90% identity) to a polypeptide of Figure 2 [SEQ ID NOS:2 OR 3] and still more preferably at least 95% similaπty (still more preferably at least 95% identity) to a polypeptide of Figure 2 [SEQ ID NOS:2 OR 3] and also include portions of such polypeptides with such portion of the polypeptide generally containing at least 30 ammo acids and more preferably at least 50 ammo acids. Putative stop codons the polynucleotide of Figure 1 are due to sequence errors.

Skilled artisans can readily determine the correct nucleotide sequence using the techmgs of thi invention, for example, using the sequences and organism disclosed herein. Thus, the ORFs depicted in Figure 2 deduced from the polynucleotide sequence of Figure 1 may be joined, each asterisk being replaced by the appropπate amino acid residue A fragment is a vaπant polypeptide having an ammo acid sequence that entirely is the same as part but not all of the ammo acid sequence of the aforementioned polypeptides As with 3 -dehydroqumate synthase 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, a single larger polypeptide. Preferred fragments include, for example, truncation polypeptides having a portion of an ammo acid sequence of Figure 2 [SEQ ID NOS:2 OR 3], or of vaπants thereof, such as a continuous seπes of residues that includes the ammo terminus, or a continuous series of residues that includes the carboxyl terminus. Degradation forms of the polypeptides of the invention m a host cell, particularly a Staphylococcus aureus, are also preferred. Further preferred are fragments characteπzed by structural or functional attπbutes such as fragments that compπse alpha-helix and alpha-helix forming regions, beta-sheet and beta-sheet-formmg regions, turn and turn-formmg regions, coil and coil-forming regions, hydrophihc 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 3 -dehydroqumate synthase , 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 compπsmg receptors or domains of enzymes that confer a function essential for viability of Staphylococcus aureus or the ability to initiate, or maintain cause disease m an individual, particularly a human

Variants that are 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.

Polynucleotides

Another aspect of the invention relates to isolated polynucleotides that encode the 3- dehydroqumate synthase polypeptide having a deduced ammo acid sequence of Figure 2 [SEQ ID NOS:2 OR 3] and polynucleotides closely related thereto and variants thereof.

Using the information provided herein, such as the polynucleotide sequence set out in

Figure 1 [SEQ ID NO:l], a polynucleotide of the invention encoding 3 -dehydroqumate synthase polypeptide may be obtained using standard cloning and screening methods, such as those for cloning and sequencing chromosomal DNA fragments from bacteπa using Staphylococcus aureus WCUH 29 cells as starting mateπal, 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], typically a library of clones of chromosomal

DNA of Staphylococcus aureus WCUH 29 in E coli or some other suitable host is probed with a radiolabeled oligonucleotide, preferably a 17-mer or longer, derived from a partial sequence. Clones carrying DNA identical to that of the probe can then be distinguished using stringent conditions. By sequencing the individual clones thus identified with sequencing primers designed from the original 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 described by Maniatis, T, Fritsch, E.F. and Sambrook et al,

MOLECULAR CLONING, A LABORATORY MANUAL, 2nd Ed.; Cold Spπng Harbor

Laboratory Press, Cold Spπng Harbor, New York (1989) (see m particular Screening By

Hybridization 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] was discovered in a DNA library deπved from Staphylococcus aureus WCUH 29.

The DNA sequence set out m Figure 1 [ SEQ ID NO:l] contains an open reading frame encoding a protein having about the number of ammo acid residues set forth in Figure 2 [SEQ ID NOS: 2 OR 3] once the putative stop codons are removed with a deduced molecular weight that can be calculated using ammo acid residue molecular weight values well known in the art. 3 -dehydroqumate synthase of the invention is structurally related to other proteins of the aro family, as shown by the results of sequencing the DNA encoding 3 -dehydroqumate synthase of the deposited strain. The protein exhibits greatest homology to Bacillus subtilus aroB (3 -dehydroqumate synthase protein among known proteins. 3 -dehydroqumate synthase of Figure 2 [SEQ ID NOS:2 OR 3] has about 59% identity over its entire length and about 77%o similaπty over its entire length with the ammo acid sequence of Bacillus subtilus aroB ( 3 -dehydroqumate synthase polypeptide.

The invention provides a polynucleotide sequence identical over its entire length to the coding sequence in Figure 1 [SEQ ID NO: 1]. Also provided by the invention is the coding 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 reading frame with other coding sequence, such as those encoding a leader or secretory sequence, a pre-, or pro- or prepro- protem 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 transcπbed, non- translated sequences, termination signals, πbosome binding sites, sequences that stabilize mRNA, mtrons, polyadenylation signals, and additional coding sequence which encode additional ammo 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 descπbed in Gentz et al, Proc. Natl Acad. Sci , USA 86- 821-824 (1989), or an HA tag (Wilson et al, Cell 37 767 (1984). Polynucleotides of the invention also include, but are not limited to, polynucleotides compπsing a structural gene and its naturally associated sequences that control gene expression.

The term "polynucleotide encoding a polypeptide" as used herein encompasses polynucleotides that include a sequence encoding a polypeptide of the invention, particularly a bacteπal polypeptide and more particularly a polypeptide of the Staphylococcus aureus 3- dehydroquinate synthase having an ammo acid sequence set out in Figure 2 [SEQ ID NOS:2 OR 3]. 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 coding and/or non-codmg sequences.

The invention further relates to variants of the polynucleotides descπbed herein that encode for vaπants of the polypeptide having deduced ammo acid sequence of Figure 2 [SEQ ID NOS:2 OR 3]. Vaπants 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 3- dehydroqumate synthase variants, that have an amino acid sequence of 3 -dehydroqumate synthase polypeptide of Figure 2 [SEQ ID NOS:2 OR 3] in 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 3 -dehydroqumate synthase .

Further preferred embodiments of the invention are polynucleotides that are at least 70% identical over their entire length to a polynucleotide encoding 3 -dehydroqumate synthase polypeptide having an amino acid sequence set out in Figure 2 [SEQ ID NOS:2 OR 3], 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 3 -dehydroqumate synthase 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%o, 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:l].

The invention further relates to polynucleotides that hybridize to the herein above- descπbed sequences. In this regard, the invention especially relates to polynucleotides that hybridize under stπngent conditions to the herein above-described polynucleotides. As herein used, the terms "stπngent conditions" and "stπngent 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 m a solution comprising: 50% formamide, 5x SSC (150mM NaCl,

15mM tπsodium citrate), 50 mM sodium phosphate (pH 7.6), 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 m Sambrook, et al., Molecular Cloning: A Laboratory Manual, Second Edition, Cold Spring Harbor, N.Y, (1989), particularly

Chapter 11 therein.

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 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 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 3 -dehydroqumate synthase and to isolate cDNA and genomic clones of other genes that have a high sequence similaπty to the 3 -dehydroqumate synthase gene. Such probes generally will compπse 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 coding region of the 3 -dehydroqumate synthase gene may be isolated by screening using the known DNA sequence provided in SEQ ID NO: 1 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 hybπdizes to. The polynucleotides and polypeptides of the invention may be employed, for example, as research reagents and materials for discovery of treatments of and diagnostics for disease, particularly human disease, as further discussed herein relating to polynucleotide assays.

Polynucleotides of the invention that are ohgonucleotides derived from the sequences of SEQ ID NOS: 1 and/or 2 may be used in the processes herein as described, but preferably for PCR, to determine whether or not the polynucleotides identified herein in whole or in part are transcribed m bacteria m infected tissue. It is recognized that such sequences will also have utility in diagnosis of the stage of infection and type of infection the pathogen has attained.

The invention also provides polynucleotides that may encode a polypeptide that is the mature protein plus additional ammo or carboxyl-termmal ammo acids, or amino acids mteπor to the mature polypeptide (when the mature form has more than one polypeptide chain, for instance). Such sequences may play a role m 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 proproteins.

In sum, a polynucleotide of the invention may encode a mature protein, a mature protein plus a leader sequence (which may be referred to as a 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 compπse a polynucleotide or polynucleotides of the invention, host cells that are genetically engineered with vectors of the invention and the production of polypeptides of the invention by recombinant techniques.

Cell-free translation systems can also be employed to produce such proteins using RNAs deπved from the DNA constructs of the invention.

For recombinant production, host cells can be genetically engineered to incorporate expression systems or portions thereof or polynucleotides of the invention. Introduction of a polynucleotide into 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 Spπng Harbor Laboratory Press, Cold Spπng Harbor, N.Y. (1989), such as, calcium phosphate transfection, DEAE-dextran mediated transfection, transvection, micromjection, cationic lipid-mediated transfection, electroporation, fransduction, scrape loadmg, ballistic introduction and infection.

Representative examples of appropπate hosts include bacterial cells, such as streptococci, staphylococci, enterococci E coh, streptomyces and Bacillus subtihs cells; fungal cells, such as yeast cells and Aspergillus cells; insect cells such as Drosophila 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 variety of expression systems can be used to produce the polypeptides of the invention. Such vectors include, among others, chromosomal, episomal and virus-derived vectors, e g , vectors deπved from bacterial 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, adenoviruses, fowl pox viruses, pseudorabies viruses and retroviruses, and vectors deπved from combinations thereof, such as those deπved 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 in a host may be used for expression in this regard. The appropriate DNA sequence may be inserted into the expression system by any of a vaπety 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 peπplasmic space or into the extracellular environment, appropπate secretion signals may be incorporated 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 puπfied 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 lectm chromatography. Most preferably, high performance liquid chromatography is employed for puπfication. Well known techniques for refolding protein may be employed to regenerate active conformation when the polypeptide is denatured duπng isolation and or puπfication.

Diagnostic Assays This invention is also related to the use of the 3 -dehydroqumate synthase polynucleotides of the invention for use as diagnostic reagents. Detection of 3 -dehydroqumate synthase 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 compπsmg the 3- dehydroqumate synthase gene may be detected at the nucleic acid level by a vaπety of techniques.

Nucleic acids for diagnosis may be obtained from an infected individual's cells and tissues, such as bone, blood, muscle, cartilage, and skin Genomic DNA may be used directly for detection or may be amplified enzymatically by using PCR or other amplification technique prior to analysis RNA or cDNA may also be used in the same ways. Using amplification, characteπzation 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 m size of the amplified product m comparison to the genotype of a reference sequence. Point mutations can be identified by hybπdizmg amplified DNA to labeled 3 -dehydroqumate synthase 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 in gels, with or without denatuπng agents, or by direct DNA sequencing See, e.g , Myers et al. Science, 230 1242 (1985). 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, e g , Cotton et al, Proc Natl Acad Sci , USA, 85 4397-4401 (1985).

Cells carrying mutations or polymorphisms m the gene of the invention may also be detected at the DNA level by a vaπety of techniques, to allow for serotyp g, 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 purpose, PCR or RT-PCR. As an example, PCR pπmers complementary to a nucleic acid encoding 3 -dehydroqumate synthase can be used to identify and analyze mutations. These pπmers may also be used for amplifying 3- dehydroqumate synthase DNA isolated from a sample deπved 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 bacterial infections, more preferably infections by Staphylococcus aureus, and most preferably disease, such as, infections of the upper respiratory tract (e.g, otitis media, bacteπal tracheitis, acute epiglottitis, thyroiditis), lower respiratory (e.g, empyema, lung abscess), cardiac (e.g, infective endocarditis), gastrointestinal (e.g, secretory diarrhoea, splenic absces, retropeπtoneal abscess), CNS (e.g, cerebral abscess), eye (e.g, blephaπtis, conjunctivitis, keratitis, endophthalmitis, preseptal and orbital celluhtis, darcryocystitis), kidney and urinary tract (e.g, epididymitis, intrarenal and peπnephπc absces, toxic shock syndrome), skm (e.g, impetigo, follicuhtis, cutaneous abscesses, celluhtis, wound infection, bacteπal myositis) bone and joint (e.g, septic arthritis, osteomyelitis), comprising determining from a sample derived from an individual a increased level of expression of polynucleotide having the sequence of Figure 1 [SEQ ID NO' 1]. Increased or decreased expression of 3 -dehydroqumate synthase polynucleotide can be measured using 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 and other hybridization methods.

In addition, a diagnostic assay in accordance with the invention for detecting over- expression of 3 -dehydroqumate synthase 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 3 -dehydroqumate synthase protein, in a sample deπved from a host are well-known to those of skill in the art Such assay methods include radioimmunoassays, competitive-bmdmg assays, Western Blot analysis and ELISA assays. Antibodies The polypeptides of the invention or vaπants thereof, or cells expressing them can be used as an immunogen to produce antibodies lmmunospecific for such polypeptides. "Antibodies" as used herein includes monoclonal and polyclonal antibodies, chimeπc, single chain, simiamzed antibodies and humanized antibodies, as well as Fab fragments, including the products of an Fab lmmunolglobulm expression library. Antibodies generated against the polypeptides of the invention can be obtained by admimsteπng the polypeptides or epitope-beaπng 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 vaπous 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).

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, transgenic 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 antι-3-dehydroquιnate synthase 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-descπbed antibodies may be employed to isolate or to identify clones expressing the polypeptides to purify the polypeptides by affinity chromatography.

Thus, among others, antibodies against 3 -dehydroqumate synthase - polypeptide may be employed to treat infections, particularly bacteπal infections and especially disease, such as, infections of the upper respiratory tract (e.g, otitis media, bacterial tracheitis, acute epiglottitis, thyroiditis), lower respiratory (e.g, empyema, lung abscess), cardiac (e.g, infective endocarditis), gastrointestinal (e.g, secretory diarrhoea, splenic absces, retropeπtoneal abscess), CNS (e.g, cerebral abscess), eye (e.g, blephaπtis, conjunctivitis, keratitis, endophthalmitis, preseptal and orbital celluhtis, darcryocystitis), kidney and uπnary tract (e.g, epididymitis, mtrarenal and peπnephπc absces, toxic shock syndrome), skm (e.g, impetigo, follicuhtis, cutaneous abscesses, celluhtis, wound infection, bacteπal myositis) bone and joint (e.g, septic arthritis, osteomyelitis).

Polypeptide vaπants include antigemcally, epitopically or immunologically equivalent variants that form a particular aspect of this invention The term "antigemcally equivalent derivative" 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 derivative" as used herein encompasses a peptide or its equivalent 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

The polypeptide, such as an antigemcally or immunologically equivalent derivative 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 lmmunogemc carrier protein for example bovme serum albumin (BSA) or keyhole limpet haemocyanm (KLH). Alternatively a multiple antigenic peptide comprising multiple copies of the protein or polypeptide, or an antigemcally or immunologically equivalent polypeptide thereof may be sufficiently antigenic to improve lmmunogenicity so as to obviate the use of a carrier.

Preferably, the antibody or variant thereof is modified to make it less lmmunogemc in the individual. For example, if the individual is human the antibody may most preferably be "humanized"; where the comphmentaπty determining regιon(s) of the hybπdoma-deπved antibody has been transplanted into a human monoclonal antibody , for example as described 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, Manthorpe et al. Hum. Gene Ther 1963:4, 419), delivery of DNA complexed with specific protein carriers (Wu et al , J Biol Chem 1989:264,16985), coprecipitation of DNA with calcium phosphate (Benvenisty & Reshef, PNAS, 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 using cloned retroviral vectors (Seeger et al, PNAS 1984:81,5849).

Antagonists and agonists - assays and molecules

Each of the polynucleotide sequences provided herein, particularly the DNA sequences, may be used in the discovery and development of antibacterial compounds. For example, the encoded protein, upon expression, can be used as a target for the screening of antibacterial drugs. Additionally, the DNA sequences encoding the ammo terminal regions of the encoded protein or Shme-Delgarno or other translation facilitating sequences of the respective mRNA can be used to construct antisense sequences to control the expression of the coding sequence of interest.

The invention also provides a method of screening compounds to identify those which enhance (agonist) or block (antagonist) the action of 3 -dehydroqumate synthase polypeptides or polynucleotides, particularly those compounds that are bacteπostatic and/or bacteπocidal. The method of screening may involve high-throughput techniques, including, for example, multiwell formats or multi-sample detection formats known to skilled artisans 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, compπsmg 3 -dehydroqumate synthase polypeptide and a labeled substrate or hgand of such polypeptide is incubated in the absence or the presence of a candidate molecule that may be a 3 -dehydroqumate synthase agonist or antagonist. Synthetic reaction mixes useful m this aspect of the invention may compπse, among other things, puπfied or recombinantly expressed 3 -dehydroqumate synthase polypeptide or polynucleotide. The ability of the candidate molecule to agonize or antagonize the 3 -dehydroqumate synthase polypeptide or polynucleotide is reflected in decreased binding of the labeled hgand to such polypeptide or polynucleotide, or decreased production of product from such substrate by such polypeptide, or such polynucleotide, as with catalytic RNAs. Molecules that bind gratuitously, i.e., without inducing the effects of 3 -dehydroqumate synthase polypeptide are most likely to be good antagonists Molecules that bind well and increase the rate of product production from substrate are most likely to be good 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 m this regard include but are not limited to coloπmetπc labeled substrate to be converted into product, a reporter gene that is responsive to changes in 3 -dehydroqumate synthase , radiolabeled substrate, among others.

Another example of an assay for 3 -dehydroqumate synthase antagonists is a competitive assay that combines 3 -dehydroqumate synthase polypeptide or polynucleotide and a potential antagonist with 3 -dehydroqumate synthase -binding molecules (molecules that bind such polynucleotide or polypeptide), recombinant 3 -dehydroqumate synthase -binding molecules, natural substrates or hgands, or substrate or hgand mimetics, under appropπate conditions for a competitive inhibition assay 3 -dehydroqumate synthase can be labeled, such as by radiolabehng or a coloπmetπc compound, such that the number of 3- dehydroquinate synthase molecules bound to a 3 -dehydroqumate synthase -binding molecule or converted to product can be determined accurately to assess the effectiveness of the potential antagonist. Using the disclosures herein, skilled artisans may design and perform many types of competitive assay. Potential antagonists include small organic molecules, peptides, peptide mimetics, polypeptides and antibodies that bind to a polynucleotide or polypeptide of the invention and preferably thereby inhibit or extinguish its activity. Potential antagonists also may be small organic molecules, a peptide, a peptide mimetic, a polypeptide, such as a closely related protein or antibody that binds the same sites on a 3 -dehydroqumate synthase -binding molecule without inducing 3 -dehydroqumate synthase -induced activities, thereby preventing the action of 3 -dehydroqumate synthase , such as by excluding 3 -dehydroqumate synthase from binding

Potential antagonists include a small molecule that binds to and occupies the binding site of 3 -dehydroqumate synthase polypeptide or polynucleotide 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- hke molecules, peptide mimetics Other potential antagonists include antisense molecules (see Okano, J Neurochem 56 560 (1991); OLIGODEOXYNUCLEOTIDES AS ANTISENSE INHIBITORS OF GENE EXPRESSION, CRC Press, Boca Raton, FL (1988), for a description of these molecules) Preferred potential antagonists include compounds related to and variants of 3 -dehydroqumate synthase , and compounds structurally related to known inhibitors of Bacillus subtilus aroB ( 3 -dehydroqumate synthase

Polypeptides of the invention may also be used to assess the binding of small molecule substrates and hgands m, for example, cells, cell-free preparations, chemical hbraπes, and natural product mixtures. These substrates and hgands may be natural substrates and hgands or may be structural or functional mimetics. See, e g , Coligan et al , Current

Protocols in Immunology 1(2) Chapter 5 (1991)

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 invention may be used: in the prevention of adhesion of bacteria, m particular bacteria, especially a gram positive bacteria, to mammalian extracellular matrix proteins on indwelling devices or to extracellular matrix proteins in wounds; to block 3 -dehydroqumate synthase protem-mediated mammalian cell invasion by, for example, initiating phosphorylation of mammalian tyrosine kmases (Rosenshme et al , Infect Immun 60:2211 (1992); to block bacterial adhesion between mammalian extracellular matrix proteins and bacterial 3 -dehydroqumate synthase 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 bacteπal growth or other metabolic function, especially of Staphylococcus aureus, as well as to treat or inhibit disease, such as, infections of the upper respiratory tract (e.g, otitis media, bacteπal tracheitis, acute epiglottitis, thyroiditis), lower respiratory (e.g, empyema, lung abscess), cardiac (e.g, infective endocarditis), gastrointestinal (e.g, secretory diarrhoea, splenic absces, retropeπtoneal abscess), CNS (e.g, cerebral abscess), eye (e.g, blephaπtis, conjunctivitis, keratitis, endophthalmitis, preseptal and orbital celluhtis, darcryocystitis), kidney and uπnary tract (e.g, epididymitis, mtrarenal and permephπc absces, toxic shock syndrome), skin (e.g, impetigo, follicuhtis, cutaneous abscesses, celluhtis, wound infection, bacteπal myositis) bone and joint (e.g, septic arthritis, osteomyelitis), among other diseases. Vaccines

Another aspect of the invention relates to a method for inducing an immunological response in an individual, particularly a mammal which comprises inoculating the individual with 3-dehydroqumate synthase , or a fragment or variant thereof, adequate to produce antibody and/ or T cell immune response to protect said individual from infection, particularly bacterial infection and most particularly Staphylococcus aureus infection. Also provided are methods whereby such immunological response slows bacterial replication. Yet another aspect of the invention relates to a method of inducing immunological response m an individual which comprises delivering to such individual a nucleic acid vector to direct expression of 3 -dehydroqumate synthase , or a fragment or a variant thereof, for expressing 3 -dehydroqumate synthase , or a fragment or a variant thereof in vivo in order to induce 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 individual from disease, whether that disease is already established withm the individual or not. One way of administering the gene is by accelerating it into the desired cells as a coating on particles or otherwise.

Such nucleic acid vector may comprise DNA, RNA, a modified nucleic acid, or a DNA/RNA hybrid. A further aspect of the invention relates to an immunological composition which, when introduced into an individual capable or having induced withm it an immunological response, induces an immunological response m such individual to a 3 -dehydroqumate synthase or protein coded therefrom, wherein the composition comprises a recombinant 3- dehydroquinate synthase or protein coded therefrom comprising DNA which codes for and expresses an antigen of said 3 -dehydroqumate synthase or protein coded therefrom. The immunological response may be used therapeutically or prophylactically and may take the form of antibody immunity or cellular immunity such as that arising from CTL or CD4+ T cells. A3 -dehydroqumate synthase polypeptide or a fragment thereof may be fused with co-protem which may not by itself produce antibodies, but is capable of stabilizing the first protein and producing a fused protein which will have lmmunogemc and protective properties Thus fused recombinant protein, preferably further comprises an antigenic co- protein, such as lipoprotem D from Hemophilus influenzae, Glutathione-S-transferase (GST) or beta-galactosidase, relatively large co-protems which solubihze the protein and facilitate production and purification thereof Moreover, the co-protein may act as an adjuvant m the sense of providing a generalized stimulation of the immune system. The co- protein may be attached to either the ammo or carboxy terminus of the first protein. Provided by this invention are compositions, particularly vaccine compositions, and methods comprising the polypeptides or polynucleotides of the invention and immunostimulatory DNA sequences, such as those described in Sato, Y. et al. Science 273 352 (1996).

Also, provided by this invention are methods using the described polynucleotide or particular fragments thereof which have been shown to encode non- variable regions of bacterial cell surface proteins in DNA constructs used m such genetic immunization experiments in animal models of infection with Staphylococcus aureus will be particularly useful for identifying protein 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 clearing infection for the development of prophylactic agents or therapeutic treatments of bacterial infection, particularly Staphylococcus aureus infection, in 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 bacteπa, for example by blocking adherence of bacteria to damaged tissue. Examples of tissue damage include wounds m 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 vagma The invention also includes a vaccine formulation which comprises an lmmunogemc recombinant protein of the invention together with a suitable carrier. Since the protein may be broken down m the stomach, it is preferably administered parenterally, including, for example, administration that is subcutaneous, intramuscular, intravenous, or mtradermal. Formulations suitable for parenteral administration include aqueous and non- aqueous sterile injection solutions which may contain anti-oxidants, buffers, bacteπostats and solutes which render the formulation isotonic with the bodily fluid, preferably the blood, of the individual, and aqueous and non-aqueous sterile suspensions which may include suspending agents or thickening agents. The formulations may be presented m unit-dose or multi-dose containers, for example, sealed ampules and vials and may be stored in a freeze-dπed condition requiring only the addition of the sterile liquid carrier immediately prior to use. The vaccine formulation may also include adjuvant systems for enhancing the immunogenicity of the formulation, such as oil-m water systems and other systems known m the art. The dosage will depend on the specific activity of the vaccine and can be readily determined by routine experimentation.

While the invention has been descπbed with reference to certain 3 -dehydroqumate synthase protein, it is to be understood that this covers fragments of the naturally occurring protein and similar proteins with additions, deletions or substitutions which do not substantially affect the lmmunogemc properties of the recombinant protein Compositions, kits and administration

The invention also relates to compositions compπsing the polynucleotide or the polypeptides discussed above or their agonists or antagonists The polypeptides of the invention may be employed in combination with a non-steπle or steπle earner or carriers for use with cells, tissues or organisms, such as a pharmaceutical carrier 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 saline, 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 compπsing 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, mtrapentoneal, intramuscular, subcutaneous, mtranasal or mtradermal routes among others

In therapy or as a prophylactic, the active agent may be administered to an individual as an mjectable composition, for example as a steπle 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 dressings and sutures and aerosols, and may contain appropriate conventional additives, including, for example, preservatives, solvents to assist drug penetration, and emollients in ointments and creams. Such topical formulations may also contain compatible conventional 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 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-dwellmg 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, cerebrospmal 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 m-dwellmg device. Treatment may be continued after surgery during the m-body time of the device. In addition, the composition could also be used to broaden peπoperative cover for any surgical technique to prevent bacterial wound infections, especially Staphylococcus aureus 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 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 mjectable 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.

Each reference cited herein is hereby incorporated by reference m its entirety Moreover, each patent application to which this application claims pπoπty is hereby incorporated by reference its entirety. EXAMPLES

The examples below are earned 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 SEQ ID NO:l was obtained from a library of clones of chromosomal DNA of Staphylococcus aureus in E coh. The sequencing data from two or more clones containing overlapping Staphylococcus aureus DNAs was used to construct the contiguous DNA sequence in SEQ ID NO: 1. Libraries may be prepared by routine methods, for example. Methods 1 and 2 below.

Total cellular DNA is isolated from Staphylococcus aureus WCUH 29 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 l lkbp in size are rendered blunt by treatment with exonuclease and DNA polymerase, and EcoRI linkers added. Fragments are hgated into the vector Lambda ZapII that has been cut with EcoRI, the library packaged by standard procedures and E.coli 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 hgated to the DNA and the fragments then hgated into the vector Lambda ZapII that have been cut with EcoRI, the library packaged by standard procedures, and E.coli infected with the packaged library. The library is amplified by standard procedures.

In the Sequence Listing below, "X" or "Xaa" can stand for an ammo acid or the ammo acid position for a stop codon.

SEQUENCE LISTING

(1) GENERAL INFORMATION

(1) APPLICANT: SmithKlme Beecham Corporation

(11) TITLE OF THE INVENTION: Novel Compounds

(m) NUMBER OF SEQUENCES: 22

(iv) CORRESPONDENCE ADDRESS:

(A) ADDRESSEE: Dechert, Price & Rhoads

(B) STREET: 4000 Bell Atlantic Tower, 1717 Arch Stre

(C) CITY: Philadelphia

(D) STATE: PA I F ) COUNTRY: USA

(E) ZIP: 19103-2793

(v) COMPUTER READABLE FORM:

(A) MEDIUM TYPE: Diskette

(B) COMPUTER: IBM Compatible

(C) OPERATING SYSTEM: DOS

(D) SOFTWARE: FastSEQ for Windows Version 2.0

(vi) CURRENT APPLICATION DATA:

(A) APPLICATION NUMBER:

(B) FILING DATE:

(C) CLASSIFICATION:

(vn) PRIOR APPLICATION DATA:

(A) APPLICATION NUMBER: 60/039,209

(B) FILING DATE: 28-FEB-1997

(vm) ATTORNEY/AGENT INFORMATION-

(A) NAME: Falk, Stephen T

(B) REGISTRATION NUMBER: 36,795

(C) REFERENCE/DOCKET NUMBER: GM50015 ( ix ) TELECOMMUNICAT ION INFORMATION :

(A) TELEPHONE : 215 - 994 - 2488

( B ) TELEFAX : 215- 994 - 2222

(C) TELEX:

(2) INFORMATION FOR SEQ ID NO : 1 :

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 881 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: double

(D) TOPOLOGY: linear

(xi) SEQUENCE DESCRIPTION: SEQ ID NO : 1 :

CCCAAACAAC CTATCCTTCA AACAATTATC CAATATTTGT TGAACATGGT GCAATTGACC 60

ATATTAGCAC GTATATTGAT CAGTTTGATC AAAGTTTTAT ATTAATTGAC GAGCATGTAA 120

ATCAATATTT TGCTGATAAA TTTGATGATA TTTTATCATA TGAAAATGTG CATAAAGTCA 180

TTATTCCAGC TGGTGAAAAG ACGAAAACAT TTGAGCAATA TCAAGAAACA TTAGAATACA 240

TTTTGTCACA TCATGTAACG AGCGTAATAC AGCGATTATA GCTGTTGGTG GTGGTGCGAC 300

AGGAGATTTT GCAGGATTTG TAGCAGCAAC ACTATTAAGA GGTGTCCATT TTATACAAGT 360

TCCTACAACG ATTTTGGCGC ATGATTCTAG TGTTGGCGGT AAAGTGGGTA TTAACTCAAA 420

ACAAGGTAAA AACCTTATCG GTGCATTTTA TCGTCCAACT GCTGTGATTT ATGATTTAGA 480

CTTTTTATAG ACGTTACCAT TTGAGCAAAT ATTAAGTGGC TATGCAGAAG TTTATAAGCA 540

TGCGTTATTG AATGGTGAAT CAACGACGCA AGAAATCGAA CAGCACTTTA AAGATAGAGA 600

GATATTACAG TCATTAAATG GTATGGATAA ATATATTGCT AAAGGTATTG AAACGAAGCT 660

GGATATTGTT GTTGCAGATG AAAAAGAACA AGGTGTACGT AAATTTTTAA ATTTAGGTCA 720

TACATTTGGT CATGCCGTGG AGTATAACCA CAAAATTGCA CACGGTCATG CCGTAATGAT 780 AGGCATAATA TATCAATTTA TTGTTGCGAA TATATTGTTC AATTCTAATC ACGATATCCA 340

ACATTATATT AATTATTTAA CGAAATTAGG TTATCCTTTA G 381

(2) INFORMATION FOR SEQ ID NO : 2 :

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 293 amino acids

(B) TYPE: amino acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

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

Gin Thr Thr Tyr Pro Ser Asn Asn Tyr Pro lie Phe Val Glu His Gly

1 5 10 15

Ala lie Asp His lie Ser Thr Tyr lie Asp Gin Phe Asp Gin Ser Phe

20 25 30 lie Leu lie Asp Glu His Val Asn Gin Tyr Phe Ala Asp Lys Phe Asp

35 40 45

Asp lie Leu Ser Tyr Glu Asn Val His Lys Val lie lie Pro Ala Gly

50 55 60

Glu Lys Thr Lys Thr Phe Glu Gin Tyr Gin Glu Thr Leu Glu Tyr lie 65 70 75 80

Leu Ser His His Val Thr Ser Val lie Gin Arg Leu Xaa Leu Leu Val

85 90 95

Val Val Arg Gin Glu He Leu Gin Asp Leu Xaa Gin Gin His Tyr Xaa

100 105 110

Glu Val Ser He Leu Tyr Lys Phe Leu Gin Arg Phe Trp Arg Met He

115 120 125

Leu Val Leu Ala Val Lys Trp Val Leu Thr Gin Asn Lys Val Lys Thr

130 135 140

Leu Ser Val His Phe He Val Gin Leu Leu Xaa Phe Met He Xaa Thr 145 150 155 160

Phe Tyr Arg Arg Tyr His Leu Ser Lys Tyr Xaa Val Ala Met Gin Lys

165 170 175

Phe He Ser Met Arg Tyr Xaa Met Val Asn Gin Arg Arg Lys Lys Ser

180 185 190

Asn Ser Thr Leu Lys He Glu Arg Tyr Tyr Ser His Xaa Met Val Trp

195 200 205

He Asn He Leu Leu Lys Val Leu Lys Arg Ser Trp He Leu Leu Leu 210 215 220 Gin Met Lys Lys Asn Lys Val Tyr Val Asn Phe Xaa He Xaa Val He 225 230 235 240

His Leu Val Met Pro Trp Ser He Thr Thr Lys Leu His Thr Val Met

245 250 255

Pro Xaa Xaa Xaa Ala Xaa Tyr He Asn Leu Leu Leu Arg He Tyr Cys

260 265 270

Ser He Leu He Thr He Ser Asn He He Leu He He Xaa Arg Asn

275 280 285

Xaa Val He Leu Xaa 290

(2) INFORMATION FOR SEQ ID NO: 3:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 293 amino acids

(B) TYPE: amino acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

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

Pro Asn Asn Leu Ser Phe Lys Gin Leu Ser Asn He Cys Xaa Thr Trp

1 5 10 15

Cys Asn Xaa Pro Tyr Xaa His Val Tyr Xaa Ser Val Xaa Ser Lys Phe

20 25 30

Tyr He Asn Xaa Arg Ala Cys Lys Ser He Phe Cys Xaa Xaa He Xaa

35 40 45

Xaa Tyr Phe He He Xaa Lys Cys Ala Xaa Ser His Tyr Ser Ser Trp

50 55 60

Xaa Lys Asp Glu Asn He Xaa Ala He Ser Arg Asn He Arg He His 65 70 75 80

Phe Val Thr Ser Cys Asn Glu Arg Asn Thr Ala He He Ala Val Gly

85 90 95

Gly Gly Ala Thr Gly Asp Phe Ala Gly Phe Val Ala Ala Thr Leu Leu

100 105 110

Arg Gly Val His Phe He Gin Val Pro Thr Thr He Leu Ala His Asp

115 120 125

Ser Ser Val Gly Gly Lys Val Gly He Asn Ser Lys Gin Gly Lys Asn

130 135 140

Leu He Gly Ala Phe Tyr Arg Pro Thr Ala Val He Tyr Asp Leu Asp 145 150 155 160

Phe Leu Xaa Thr Leu Pro Phe Glu Gin He Leu Ser Gly Tyr Ala Glu 165 170 175 Val Tyr Lys His Ala Leu Leu Asn Gly Glu Ser Thr Thr Gin Glu He

180 185 190

Glu Gin His Phe Lys Asp Arg Glu He Leu Gin Ser Leu Asn Gly Met

195 200 205

Asp Lys Tyr He Ala Lys Gly He Glu Thr Lys Leu Asp He Val Val

210 215 220

Ala Asp Glu Lys Glu Gin Gly Val Arg Lys Phe Leu Asn Leu Gly His 225 230 235 240

Thr Phe Gly His Ala Val Glu Tyr Asn His Lys He Ala His Gly His

245 250 255

Ala Val Met He Gly He He Tyr Gin Phe He Val Ala Asn He Leu

260 265 270

Phe Asn Ser Asn His Asp He Gin His Tyr He Asn Tyr Leu Thr Lys

275 280 285

Leu Gly Tyr Pro Leu 290

(2) INFORMATION FOR SEQ ID NO : 4 :

(l) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 93 ammo acids

(B) TYPE: amino acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

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

Gin Thr Thr Tyr Pro Ser Asn Asn Tyr Pro He Phe Val Glu His Gly

1 5 10 15

Ala He Asp His He Ser Thr Tyr He Asp Gin Phe Asp Gin Ser Phe

20 25 30

He Leu He Asp Glu His Val Asn Gin Tyr Phe Ala Asp Lys Phe Asp

35 40 45

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

50 55 60

Glu Lys Thr Lys Thr Phe Glu Gin Tyr Gin Glu Thr Leu Glu Tyr He

65 70 75 80

Leu Ser His His Val Thr Ser Val He Gin Arg Leu Xaa 85 90

(2) INFORMATION FOR SEQ ID NO : 5 :

(l) SEQUENCE CHARACTERISTICS: (A) LENGTH: 14 ammo acids

(B) TYPE: ammo acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(xi) SEQUENCE DESCRIPTION: SEQ ID NO : 5 :

Leu Leu Val Val Val Arg Gin Glu He Leu Gin Asp Leu Xaa 1 5 10

(2) INFORMATION FOR SEQ ID NO : 6 :

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 5 ammo acids

(B) TYPE: ammo acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 6:

1 5

(2) INFORMATION FOR SEQ ID NO : 7 :

(l) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 43 ammo acids

(B) TYPE: amino acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(xi) SEQUENCE DESCRIPTION: SEQ ID NO : 7 :

Glu Val Ser He Leu Tyr Lys Phe Leu Gin Arg Phe Trp Arg Met He

1 5 10 15

Leu Val Leu Ala Val Lys Trp Val Leu Thr Gin Asn Lys Val Lys Thr

20 25 30

Leu Ser Val His Phe He Val Gin Leu Leu Xaa 35 40

(2) INFORMATION FOR SEQ ID NO : 8 : (l) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 12 ammo acids

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(xi ) SEQUENCE DESCRIPTION: SEQ ID NO : 8 :

Thr Phe Tyr Arg Arg Tyr His Leu Ser Lys Tyr Xaa 1 5 10

(2) INFORMATION FOR SEQ ID NO: 9:

(l) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 12 ammo acids

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 9:

Val Ala Met Gin Lys Phe He Ser Met Arg Tyr Xaa 1 5 10

(2) INFORMATION FOR SEQ ID NO:10:

(l) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 22 ammo acids

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 10:

Met Val Asn Gin Arg Arg Lys Lys Ser Asn Ser Thr Leu Lys He Glu

1 5 10 15

20

(2) INFORMATION FOR SEQ ID NO: 11: (i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 31 amino acids

(B) TYPE: amino acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 11:

Met Val Trp He Asn He Leu Leu Lys Val Leu Lys Arg Ser Trp He

1 5 10 15

Leu Leu Leu Gin Met Lys Lys Asn Lys Val Tyr Val Asn Phe Xaa 20 25 30

(2) INFORMATION FOR SEQ ID NO: 12:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 20 amino acids

(B) TYPE: amino acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 12:

Val He His Leu Val Met Pro Trp Ser He Thr Thr Lys Leu His Thr

1 5 10 15

Val Met Pro Xaa 20

(2) INFORMATION FOR SEQ ID NO: 13:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 24 amino acids

(B) TYPE: amino acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(xi ) SEQUENCE DESCRIPTION : SEQ ID NO : 13 :

Tyr He Asn Leu Leu Leu Arg He Tyr Cys Ser He Leu He Thr He 1 5 10 15 Ser Asn He He Leu He He Xaa 20

(2) INFORMATION FOR SEQ ID NO: 14:

(l) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 14 ammo acids

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 14:

Pro Asn Asn Leu Ser Phe Lys Gin Leu Ser Asn He Cys Xaa 1 5 10

(2) INFORMATION FOR SEQ ID NO: 15:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 5 ammo acids

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:15:

Thr Trp Cys Asn Xaa

1 5

(2) INFORMATION FOR SEQ ID NO: 16:

(l) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 7 ammo acids

(B) TYPE: ammo acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 16:

Ser Lys Phe Tyr He Asn Xaa 1 5 (2) INFORMATION FOR SEQ ID NO: 17:

(l) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 9 ammo acids

(B) TYPE: ammo acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 17:

Arg Ala Cys Lys Ser He Phe Cys Xaa 1 5

(2) INFORMATION FOR SEQ ID NO: 18:

(l) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 5 ammo acids

(B) TYPE: ammo acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: I

Tyr Phe He He Xaa 1 5

(2) INFORMATION FOR SEQ ID NO: 19:

(l) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 7 ammo acids

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 19:

Ser His Tyr Ser Ser Trp Xaa 1 5

(2) INFORMATION FOR SEQ ID NO: 20: ( l ) SEQUENCE CHARACTERISTICS :

(A) LENGTH : 6 ammo acids

( C ) STRANDEDNESS : s ingle

( D ) TOPOLOGY : linear

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 20:

Lys Asp Glu Asn He Xaa 1 5

(2) INFORMATION FOR SEQ ID NO: 21:

(l) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 92 ammo acids

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 21:

Ala He Ser Arg Asn He Arg He His Phe Val Thr Ser Cys Asn Glu

1 5 10 15

Arg Asn Thr Ala He He Ala Val Gly Gly Gly Ala Thr Gly Asp Phe

20 25 30

Ala Gly Phe Val Ala Ala Thr Leu Leu Arg Gly Val His Phe He Gin

35 40 45

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

50 55 60

Gly He Asn Ser Lys Gin Gly Lys Asn Leu He Gly Ala Phe Tyr Arg

65 70 75 80

Pro Thr Ala Val He Tyr Asp Leu Asp Phe Leu Xaa 85 90

(2) INFORMATION FOR SEQ ID NO: 22:

(l) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 130 ammo acids

(B) TYPE: ammo acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 22:

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

1 5 10 15

His Ala Leu Leu Asn Gly Glu Ser Thr Thr Gin Glu He Glu Gin His

20 25 30

Phe Lys Asp Arg Glu He Leu Gin Ser Leu Asn Gly Met Asp Lys Tyr

35 40 45

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

50 55 60

Lys Glu Gin Gly Val Arg Lys Phe Leu Asn Leu Gly His Thr Phe Gly 65 70 75 80

His Ala Val Glu Tyr Asn His Lys He Ala His Gly His Ala Val Met

85 90 95

He Gly He He Tyr Gin Phe He Val Ala Asn He Leu Phe Asn Ser

100 105 110

Asn His Asp He Gin His Tyr He Asn Tyr Leu Thr Lys Leu Gly Tyr

115 120 125

Pro Leu 130

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 ammo acids of SEQ ID NOS:2 OR 3;

(b) a polynucleotide which is complementary to the polynucleotide of (a),

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

(d) a polynucleotide having at least a 70% identity to a polynucleotide encoding a polypeptide comp╧Çsing ammo acids of SEQ ID NOS:4 or 5 or 6 or 7 or 8 or 9 or 10 or 11 or 12 or 13 or 14 or 15 or 16 or 17 or 18 or 19 or 20 or 21 or 22 or 23.

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 set forth in SEQ ID

NO:l.

5. The polynucleotide of Claim 2 compnsmg the nucleotide set forth in SEQ ID

NO:l.

6. The polynucleotide of Claim 2 which encodes a polypeptide compnsmg a polypeptide of SEQ ID NOS:2 OR 3.

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 3 -dehydroqumate synthase gene contained in NCIMB Deposit No. 40771;

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

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

8. A vector comprising the DNA of Claim 2.

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

10. A process for producing a polypeptide compnsmg: 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 compnsmg transforming or transfectmg 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 3 -dehydroqumate synthase polypeptide or fragment compnsmg culturmg a host of claim 9 under conditions sufficient for the production of said polypeptide or fragment.

13. A polypeptide comprising an ammo acid sequence which is at least 70% identical to a polypeptide of SEQ ID NOS:2 OR 3

14. A polypeptide comp╧Çsing an amino acid sequence as set forth in SEQ ID NOS:2 0R 3

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 3 -dehydroqumate synthase comp╧Çsing: administering to the individual a therapeutically effective amount of the polypeptide of claim 13.

18. The method of Claim 17 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 3- dehydroqumate synthase polypeptide comprising: admmiste╧Çng 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 denved from a host.

22. A method for identifying compounds which bind to and inhibit an activity of the polypeptide of claim 13 compnsmg: contacting a compound under conditions to permit binding of the compound to the polypeptide to a composition comprising the polypeptide and a component capable of providing a detectable signal m response to the binding of the compound to the polypeptide; and determining whether the compound inhibits an activity of the polypeptide by detecting the presence 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 3-dehydroqu╬╣nate synthase , 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 3 -dehydroqumate synthase fragment or a va╧Çant thereof, for expressing 3 -dehydroqumate synthase , or a fragment or a va╧Çant 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 3 -dehydroqumate synthase polynucleotide or protein coded therefrom which, when introduced into a mammal, induces an immunological response m the mammal to a given 3 -dehydroqumate synthase polynucleotide or protein coded therefrom.

26 A polynucleotide consisting essentially of a DNA sequence obtainable by screening an approp╧Çate library containing the complete gene for a polynucleotide sequence set forth in SEQ ID N0:1 under stringent hybridization conditions with a probe having the sequence of said polynucleotide sequence set forth SEQ ID NO: l or a fragment thereof; and isolating said DNA sequence.