WO2003080654A2

WO2003080654A2 - Helicobacter flagellar, motility polypeptides

Info

Publication number: WO2003080654A2
Application number: PCT/CA2003/000430
Authority: WO
Inventors: Annie J. Aubry; Susan M. Logan
Original assignee: National Research Council Of Canada
Priority date: 2002-03-26
Filing date: 2003-03-26
Publication date: 2003-10-02
Also published as: WO2003080654A3; AU2003218551A8; WO2003080654B1; AU2003218551A1

Abstract

The invention relates to flagellar glycosylation processes of helicobacter having a polar flagellum or polar flagella, polar flagellar motilitypolypeptides, nucleic acid molecules encoding the polypeptides. The invention also relates to uses of flagellar glycosylation process changes which may be the result of mutational changes which can be tracked in associated polypeptides and nucleic acid molecules in detecting and treating disease as well as screening candidate pharmaceutical compounds.

Description

TITLE: Helicobacter Motility Polypeptides FIELD OF THE INVENTION

The invention relates to flagellar glycosylation processes of bacteria, including gram-negative bacteria, having a polar flagellum or polar flagella, polar flagellar motility polypeptides, nucleic acid molecules encoding the polypeptides. The invention also relates to uses of flagellar glycosylation process changes which may be the result of mutational changes which can be tracked in associated polypeptides and nucleic acid molecules in detecting and treating disease as well as screening candidate pharmaceutical compounds. BACKGROUND OF THE INVENTION Helicobacter pylori is a motile, Gram negative, microaerophilic, spiral shaped organism that colonizes the stomachs of at least half the worlds population. While most infected individuals are asymptomatic, a significant number develop a more serious pathology. H. pylori is the causative agent of chronic type B gastritis, a prerequisite for duodenal ulcers and more recently the organism has been associated with mucosa- associated lymphoid tissue (MALT) and with B-cell MALT lymphomas. Numerous bacterial factors have been suggested to play a role in the pathogenesis of H. pylori infection while only a few factors including urease, motility and vacuolating cytotoxin have been definitively characterised. Motility is a key factor in the adaptation of many bacterial pathogens which colonize mucosal surfaces and all gastric Helicobacter spp demonstrate highly efficient flagellar motility under conditions of elevated viscosity such as that found in the gastric lumen. Helicobacter cells possess a unipolar bundle of sheathed flagella. The complex filament is comprised of two flagellin subspecies, the more abundant FlaA protein which comprises the majority of the filament, and a second slightly larger protein, FlaB which appears to be exclusively located proximal to the hook within the assembled filament. The two flagellin structural genes are unlinked on the chromosome and the flaA ivaάflaB genes are preceded by different promoters (σ for flaA, σ⁵⁴ for flaB). Complex flagella assembled from two or more structural flagellin proteins are found in a diverse range of bacterial species including Caulobacter, Campylobacter, Aeromonas and Vibrio, and also within members of the Archaea. There is increasing evidence that protein glycosylation is involved in the flagellar assembly process for a number of these organisms, most notably for Caulobacter crescentus, Halobacter halobium and Campylobacter jejuni. The complex flagella of the gastrointestinal pathogen Campylobacter jejuni is heavily glycosylated with pseudaminic acid (Pse5Ac7Ac), a "sialic acid of a new type" along with related derivatives of this unique carbohydrate molecule(Thibault, P., Logan, S.M., Kelly, J. F., Brisson, J-R, Ewing, C.P., Trust, T.J., and Guerry, P. (2001) J. Biol. Chem. 276, 34862-34870). Studies on H. felis by Josenhans et al (1999) Mol. Micro. 33:350-362 provided the first evidence that flagellin of Helicobacter spp may also be glycosylated using periodic acid treatment and subsequent labeling of flagellin with digoxygenin-tagged hydrazine. More recently, Josenhans et α/.(2002) FEMS Microbiol. Lett. 210:165-72 proposed that the function of the HP0326 bicistronic operon was likely involved in glycosylation of flagellin from H. pylori although the functional basis of this proposal remains unclear.

Given the importance of motility in Helicobacter associated diseases, and because of our interest in the assembly of complex flagella of mucosal pathogens, we initiated this study to determine the genetic basis of the glycosylation process in Helicobacter pylori and to examine flagellin to determine the structural nature of the glycosyl moieties present. We describe here the identification of polysaccharide gene loci which are involved in the assembly of Helicobacter flagella and characterize the nature and degree of glycosylation found on H pylori flagellin. SUMMARY OF THE INVENTION

Applicants have identified genes involved in the synthesis and/or assembly of flagella of Helicobacter. Isogenic mutants of the genes result in a non-motile phenotype, no structural filament production, and a loss of ability to colonize the mouse stomach. The genes may encode enzymes involved in glycosylation of flagella. Identification of the gene loci involved and also characterization of the metabolic pathways leading to glycosylation allows us to use the disruptions of the gene loci, their products and the production and/or accumulation of metabolites to screen candidate antibacterial agents. Also, the demonstration that many of the glycosylation genes identified in Helicobacter pylori have homologues in other bacterial species will allow us to screen candidate compounds which may have more general antibacterial application.

Broadly stated the present invention contemplates an isolated polynucleotide encoding a Helicobacter motility polypeptide of the invention, including mRNAs, DNAs, cDNAs, genomic DNAs, PNAs, as well as antisense analogs and biologically, diagnostically, prophylactically, clinically or therapeutically useful variants or fragments thereof, and compositions comprising same.

In particular, the present invention contemplates an isolated polynucleotide comprising a sequence that comprises at least 18 nucleotides and hybridizes under stringent conditions to the complementary nucleic acid sequence of SEQ. ID. NO. 1, 3, 17 or 20 or a degenerate form thereof. In an embodiment the polynucleotide comprises a region encoding a polypeptide comprising a sequence set out in SEQ ID NO: 2, 4, 18, 19 or 21 that includes a full length polynucleotide or .a variant thereof. In a preferred embodiment the polynucleotide encodes a polynucleotide designated herein as HP0326(A or B) or HP0178. HP0114 and HP0840 are also contemplated.

The polynucleotides of the invention permit identification of untranslated nucleic acid sequences or regulatory sequences which specifically promote expression of genes operatively linked to the promoter regions. The invention therefore contemplates a polynucleotide encoding a regulatory sequence of a polynucleotide of the invention such as a promoter sequence, preferably a regulatory sequence of a Helicobacter motility gene.

The polynucleotides encoding a mature polypeptide of the invention may include only the coding sequence for the mature polypeptide; the coding sequence for the mature polypeptide and additional coding sequences (e.g. leader or secretory sequences, proprotein sequences); the coding sequence for the mature polypeptide (and optionally additional coding sequences) and non-coding sequence, such as introns or non-coding sequences 5' and/or 3' of the coding sequence of the mature polypeptide.

The polynucleotides of the invention may be inserted into an appropriate expression vector, and the vector may contain the necessary elements for the transcription and translation of an inserted coding sequence. Accordingly, recombinant expression vectors may be constructed which comprise a polynucleotide of the invention, and where appropriate one or more transcription and translation elements linked to the polynucleotide.

Vectors are contemplated within the scope of the invention which comprise regulatory sequences of the invention, as well as chimeric gene constructs wherein a regulatory sequence of the invention is operably linked to a polynucleotide sequence encoding a heterologous protein (i.e. a protein not naturally expressed in the host cell), and a transcription termination signal. A vector can be used to transform host cells to express a polypeptide of the invention, or a heterologous protein. Therefore, the invention further provides host cells containing a vector of the invention.

The invention also contemplates an isolated Helicobacter motility polypeptide encoded by a polynucleotide of the invention. In an embodiment, the invention provides a polypeptide comprising the amino acid sequence of SEQ ID NO: 2, 4, 18, 19 or 21 or a variant thereof. Further embodiments of the invention provide biologically, diagnostically, prophylactically, clinically or therapeutically useful variants thereof and compositions comprising a polypeptide of the invention. Among the embodiments of the invention are variants of a polypeptide of the invention encoded by naturally occurring alleles of a Helicobacter motility gene.

Polypeptides of the invention may be obtained as an isolate from natural cell sources, but they are preferably produced by recombinant procedures. In one aspect the invention provides a method for preparing a polypeptide of the invention utilizing an isolated polynucleotide of the invention. In an embodiment a method for preparing a

Helicobacter motility polypeptide is provided comprising:

(a) transferring a recombinant expression vector of the invention having a polynucleotide sequence encoding a Helicobacter motility polypeptide, into a host cell; (b) selecting transformed host cells from untransformed host cells;

(c) culturing a selected transformed host cell under conditions which allow expression of the Helicobacter motility polypeptide; and

(d) isolating the Helicobacter motility polypeptide.

The invention further broadly contemplates a recombinant Helicobacter motility polypeptide obtained using a method of the invention.

A polypeptide of the invention may be conjugated with other molecules, such as proteins, to prepare fusion proteins or chimeric proteins. This may be accomplished, for example, by the synthesis of N-terminal or C-terminal fusion proteins.

The invention further contemplates antibodies having specificity against an epitope of a polypeptide of the invention. Antibodies may be labeled with a detectable substance and used to detect polypeptides of the invention in biological samples, tissues, and cells. The invention also permits the construction of nucleotide probes which are unique to the polynucleotides of the invention or to polypeptides of the invention. Therefore, the invention also relates to a probe comprising a sequence encoding a polypeptide of the invention, or a part thereof. The probe may be labeled, for example, with a detectable substance and it may be used to select from a mixture of nucleotide sequences a polynucleotide of the invention including polynucleotides encoding a polypeptide which displays one or more of the properties of a polypeptide of the invention.

In accordance with an aspect of the invention there is provided a method of, and products for, diagnosing and monitoring diseases by determining the presence of polynucleotides and polypeptides of the invention.

Still further the invention provides a method for evaluating a test compound or agent for its ability to modulate the activity of a polypeptide or polynucleotide of the invention. For example a substance that inhibits or enhances the catalytic activity of a polypeptide of the invention may be evaluated. "Modulate" refers to a change or an alteration in the biological activity of a polypeptide of the invention. Modulation may be an increase or a decrease in activity, a change in characteristics, or any other change in the biological, functional, or immunological properties of the polypeptide.

In an embodiment, the invention provides a method for identifying a compound having anti-polar flagellum activity, which method comprises: (a) exposing a polypeptide of the invention to a test sample of said compound and detecting any interaction of said polypeptide and said test sample, interaction being taken as an indication of anti-polar flagellum activity; or

(b) exposing a polynucleotide of the invention to a test sample of said compound and detecting any interaction of said polynucleotide and said test sample, interaction being taken as an indication of anti-polar flagellum activity; or

(c) exposing a bacterium having a polar flagellum to a test sample of said compound and detecting abnormal flagellar form or behaviour, said abnormal form or behaviour being taken as an indication of anti-polar flagellum activity; or (d) exposing a bacterium having a polar flagellum to a test sample of said compound and detecting abnormal accumulation of flagellar sugars or intermediates thereof, said abnormal accumulation being taken as an indication of anti-polar flagellum activity; or (e) growing H. pylori bacteria in semisolid motility media in presence of said compound and detecting abnormal motility patterns, abnormal motility patterns being taken as an indication of antipolar flagellum activity. Compounds that modulate the biological activity of a polypeptide or polynucleotide of the invention may also be identified using the methods of the invention by comparing the pattern and level of expression of a polynucleotide or polypeptide of the invention in cells and organisms, in the presence, and in the absence of the compounds.

Methods are also contemplated that identify compounds or substances (e.g. polypeptides) which bind to regulatory sequences (e.g. promoter sequences, enhancer sequences, negative modulator sequences). The substances and compounds identified using the methods of the invention, antibodies, and antisense polynucleotides may be used to modulate the biological activity of a polypeptide or polynucleotide of the invention, and they may be used in the prevention and treatment of disease. In an aspect of the invention the substances and compounds are inhibitors of polypeptides of the invention that are useful as antibacterial agents.

In accordance with an aspect of the invention there are provided agonists and antagonists of a Helicobacter motility gene or polypeptide, preferably bacteriostatic or bacteriocidal agonists or antagonists.

Accordingly, the polynucleotides and polypeptides of the invention, antibodies and substances and compounds may be formulated into compositions for administration to a cell or to a multicellular organism. Therefore, the present invention also relates to a composition comprising one or more of a polynucleotide or polypeptide of the invention, antibody or a substance or compound identified using the methods of the invention, and a pharmaceutically acceptable carrier, excipient or diluent. A method for treating or preventing a disease is also provided comprising administering to a patient in need thereof, a composition of the invention. In accordance with certain embodiments of the invention, there are provided products, compositions and methods for assessing Helicobacter motility gene expression, treating disease, assaying genetic variation, and administering a polypeptide or polynucleotide of the invention to an organism to raise an immunological response against a bacteria.

In accordance with a further aspect of the invention, there are provided processes for utilizing polypeptides or polynucleotides of the invention, for in vitro purposes related to scientific research, synthesis of DNA, and manufacture of vectors.

In another embodiment of the invention there is provided a computer readable medium having stored thereon a member selected from the group consisting of: (a) a polynucleotide comprising the sequence of SEQ ID NO. 1, 3, 17 or 20; (b) a polypeptide comprising the sequence of SEQ ID NO. 2, 4, 18, 19 or 21; (c) a data set of polynucleotide sequences wherein at least one of said sequences comprises the sequence of SEQ ID NO. 1, 3, 17 or 20; (d) a data set of polypeptide sequences wherein at least one of said sequences comprises the sequence of SEQ ID NO. 2, 4, 18, 19 or 21; (e) a data set representing a polynucleotide sequence comprising the sequence of SEQ ID NO. 1, 3, 17 or 20; and (f) a data set representing a polynucleotide sequence encoding a polypeptide sequence comprising the sequence of SEQ ID NO. 2, 4, 18, 19 or 21.

A further embodiment of the invention provides a computer based method for performing homology identification, said method comprising the steps of providing a polynucleotide sequence comprising the sequence of SEQ ID NO. 1, 3, 17 or 20 in a computer readable medium; and comparing said polynucleotide sequence to at least one polynucleotide or polypeptide sequence to identify homology.

A further embodiment of the invention provides a computer based method for performing homology identification, said method comprising the steps of: providing a polypeptide sequence comprising the sequence of SEQ ID NO. 2, 4, 18, 19 or 21 in a computer readable medium; and comparing said polypeptide sequence to at least one polynucleotide or polypeptide sequence to identify homology.

A further embodiment of the invention provides a computer based method for polynucleotide assembly, said method comprising the steps of: (a) providing a first polynucleotide sequence comprising the sequence of SEQ ID NO. 1, 3, 17 or 20 in a computer readable medium; and (b) screening for at least one overlapping region between said first polynucleotide sequence and a second polynucleotide sequence.

A further embodiment of the invention provides a computer based method for performing homology identification, said method comprising the steps of: (a) providing a polynucleotide sequence comprising the sequence of SEQ ID NO. 1, 3, 17 or 20 in a computer readable medium; and (b) comparing said polynucleotide sequence to at least one polynucleotide or polypeptide sequence to identify homology.

A further embodiment of the invention provides a computer based method for performing homology identification, said method comprising the steps of: (a) providing a polypeptide sequence comprising the sequence of SEQ ID NO. 2, 4, 18, 19 or 21 in a computer readable medium; and (b) comparing said polypeptide sequence to at least one polynucleotide or polypeptide sequence to identify homology.

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

The invention will now be described in relation to the drawings in which:

Figure 1. Electrospray mass spectrum of intact flagellin from H. pylori 1061. The reconstructed molecular mass profile is shown in A, and indicates two peaks at Mr: 55362 and 55050 Da. Sequence coverage map of H. pylori 1061 FlaA (B) and FlaB (C) proteins.

The sites of O-linked Pse5Ac7Ac glycosylation are boxed. Numbers shown above boxes in (C) correspond to Pse5Ac7Ac units found on the identified tryptic peptide.

Figure 2. Motility of Helicobacter pylori 1061 (A) and M6 (B) strains and the respective isogenic mutants in motility agar. Figure 3. Transmission electron micrographs of negatively stained H. pylori.

A. H. pylori 1061; B. HP0178 isogenic mutant; C. HP0326A isogenic mutant; D. HP0326B isogenic mutant; E. HPOl 14 isogenic mutant. Scale bars represent lμm. Note the appearance of empty flagellar sheaths in panels B and D, and the presence of truncated sheaths at the cell poles in panels C and E.

Figure 4. Analysis of flagellin expression in H. pylori 1061 and isogenic mutants. A. Whole cell lysate probed with rabbit polyclonal (JB3) to H. pylori flagellin. B. Sheared flagellar preparation probed with rabbit polyclonal (JB3) to H. pylori flagellin. C. Whole cell lysate probed with monoclonal antibody (Mab72c) to H. pylori flagellin. D. Sheared flagellar preparation probed with monoclonal antibody (Mab72c) to H. pylori flagellin. E. RT-PCR of flaA mRNA. Lane 1, H. pylori 1061, Lane 2, ΗP0178 isogenic mutant, Lane 3, HP0326A isogenic mutant, Lane 4, HP0326B isogenic mutant, Lane 5, HP0114 isogenic mutant. F. Whole cell lysates of isogenic mutants and isogenic mutants complemented with wild type copies of their respective genes and probed with rabbit polyclonal antibody JB3. Lane 1, 1061::HP0178, lane 2 1061::HP0178 with pHelHPO 178 lane 3, M6::HP0326A, lane 4, M6::HP0326A with pHel HP0326A/B, Lane 5, M6::HP0326B, lane 6 M6::HP0326B with pHel0326A/B. Figure 5. Identification of activated sugar-nucleo tides from cytosolic extracts of

H. pylori using CE/ESMS and precursor ion scanning. Precursors (negative ion mode) of m/z 322 (A), m/z 385 (B) from H. pylori strain 1061. Product ion scan (positive ion mode) of m/z 317 (C) arising from collisional activation in the ion source. Precursors (negative ion mode) of m/z 322 (D) and m/z 385 (E) from H. pylori isogenic mutant HP0326B. Product ion scan (positive ion mode) of m/z 229 (F) arising from collisional activation in the ion source. DETAILED DESCRIPTION OF THE INVENTION

In accordance with the present invention there may be employed conventional molecular biology, microbiology, and recombinant DNA techniques within the skill of the art. Such techniques are explained fully in the literature. See for example, Sambrook, Fritsch, & Maniatis, Molecular Cloning: A Laboratory Manual, Second Edition (1989) Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y); DNA Cloning: A Practical Approach, Volumes I and II (D.N. Glover ed. 1985); Oligonucleotide Synthesis (M.J. Gait ed. 1984); Nucleic Acid Hybridization B.D. Hames & SJ. Higgins eds. (1985); Transcription and Translation B.D. Hames & S.J. Higgins eds (1984); Animal Cell Culture R.I. Freshney, ed. (1986); Immobilized Cells and enzymes IRL Press, (1986); and B. Perbal, A Practical Guide to Molecular Cloning (1984). Glossary

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

The term "complementary" refers to the natural binding of polynucleotides under permissive salt and temperature conditions by base-pairing. For example, the sequence "A-G-T" binds to the complementary sequence "T-C-A". Complementarity between two single-stranded molecules may be "partial", in which only some of the nucleic acids bind, or it may be complete when total complementarity exists between the single stranded molecules. The term "consisting essentially of or "consisting of a polynucleotide sequence refers to the disclosed polynucleotide sequence, and also encompasses polynucleotide sequences which are identical except for a base change or substitution therein. As known to those skilled in the art, a limited number of base changes or substitutions may be made in a short oligonucleotide sequence resulting in a sequence maintaining substantial function (ranging, from approximately 50%) to greater than 100% of the activity) of the original unmodified sequence.

"Disease(s)" means a condition or disease caused by or related to infection by a bacteria that comprises a polypeptide or polynucleotide of the invention.

"Host cell" is a cell which has been transformed or transfected, or is capable of being transformed or transfected by an exogenous polynucleotide sequence.

"Identity," as known in the art, is a relationship between two or more polypeptide sequences or two or more polynucleotide sequences, as determined by comparing the sequences. "Identity" also refers to the degree of sequence relatedness between polypeptide or polynucleotide sequences as determined by the match between strings of such sequences. "Identity" may be calculated by conventional methods, including but not limited to those described in Computational Molecular Biology, Lesk, A. M., ed., Oxford University Press, New York, 1988; Biocomputing: Informatics and Genome Projects, Smith, D. W., ed., Academic Press, New York, 1993; Computer Analysis of Sequence Data, Part I, Griffin, A. M., and Griffin, H. G., eds., Humana Press, New Jersey, 1994; Sequence Analysis in Molecular Biology, von Heinje, G., Academic Press, 1987; and Sequence Analysis Primer, Gribskov, M. and Devereux, J., eds., M Stockton Press, New York, 1991; and Carillo, H., and Lipman, D., SIAM J. Applied Math., 48. 1073 (1988). Methods to determine identity are designed to give the highest match between the sequences tested. Methods to determine identity are codified in publicly available computer programs. Examples of 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 NTH Bethesda, Md. 20894; Altschul, S., et al., J. Mol. Biol. 215: 403-410 (1990). The Smith Waterman algorithm known in the art may also be used to determine identity.

Parameters for comparison of polypeptide sequences include the following: (1) Algorithm: Needleman and Wunsch, J. Mol Biol. 48: 443-453 (1970); (2) Comparison matrix: BLOSSUM62 from Hentikoff and Hentikoff, Proc. Natl. Acad. Sci. USA. 89:10915-10919 (1992); (3) Gap Penalty: 12; and (4) Gap Length Penalty: 4. A useful publicly available program with these parameters is the "gap" program from Genetics Computer Group, Madison Wis. The above-mentioned comparison parameters are the default parameters for peptide comparisons (along with no penalty for end gaps).

Parameters for comparison of polynucleotide sequences include the following: (1)

Algorithm: Needleman and Wunsch, J. Mol Biol. 48: 443-453 (1970); (2) Comparison matrix: matches=+10, mismatch=0; (3) Gap Penalty: 50; and (4) Gap Length Penalty: 3.

The "gap" program from Genetics Computer Group, Madison, Wis. is a publicly available program with these default parameters for nucleic acid comparisons.

A preferred meaning for "identity" for polynucleotides and polypeptides is as follows: (1) Polynucleotide embodiments may include an isolated polynucleotide comprising a polynucleotide sequence having at least 30, 40, 50, 60, 70, 80,

85, 90, 95, 97 or 100% identity to the sequence of SEQ ID NO:l, 3, 17 or

20, where the polynucleotide sequence may be identical to the sequence of

SEQ ID NO: 1, 3, 17 or 20 or may include up to a certain integer number of nucleotide alterations as compared to the sequence of SEQ ID NO: 1, 3, 17 or 20. The alterations may be selected from the group consisting of at least one nucleotide deletion, substitution, including transition and transversion, or insertion. The alterations may occur at the 5' or 3' terminal positions of the sequence of SEQ ID NO: 1, 3, 17 or 20 or anywhere between those terminal positions, interspersed either individually among the nucleotides in the sequence of SEQ ID NO: 1, 3, 17 or 20 or in one or more contiguous groups within this sequence. The number of nucleotide alterations can be determined by multiplying the total number of nucleotides in SEQ ID NO: 1, 3, 17 or 20 by the integer defining the percent identity divided by 100 and then subtracting that product from the total number of nucleotides in SEQ ID NO:l, 3, 17 or 20. (2) Polypeptide embodiments may include an isolated polypeptide comprising a polypeptide having at least a 30, 40. 50, 60, 70, 80, 85, 90, 95, 97 or 100% identity to a polypeptide sequence of SEQ ID NO: 2, 4, 18, 19 or 21 where the polypeptide sequence may be identical to the sequence of SEQ ID NO: 2, 4, 18, 19 or 21 or may include up to a certain integer number of amino acid alterations as compared to the sequence. The alterations may be selected from the group consisting of at least one amino acid deletion, substitution, including conservative and non-conservative substitution, or insertion, and where the alterations may occur at the amino- or carboxy- terminal positions of the reference polypeptide sequence or anywhere between those terminal positions, interspersed either individually among the amino acids in the reference sequence or in one or more contiguous groups within the reference sequence. The number of amino acid alterations is determined by multiplying the total number of amino acids in SEQ ID NO: 2, 4, 18, 19 or 21 by the integer defining the percent identity divided by 100 and then subtracting that product from said total number of amino acids in SEQ ID NO: 2, 4, 18, 19 or 21.

The term "isolated" refers to a polynucleotide or polypeptide changed and/or removed from its natural environment, purified or separated, or substantially free of cellular material or culture medium when produced by recombinant DNA techniques, or chemical reactants, or other chemicals when chemically synthesized. A polynucleotide or polypeptide that is introduced into an organism by transformation, genetic manipulation, or any other recombinant method is "isolated" even if it is still present in an organism, which may be living or non-living. Preferably, an isolated polynucleotide or polypeptide is at least 60% free, more preferably at least 75% free, and most preferably at least 90% free from other components with which they are naturally associated.

"Polynucleotide(s)" generally refers to any polyribonucleotide or polydeoxribonucleotide, which may be unmodified RNA or DNA or modified RNA or DNA, including mRNAs, DNAs, cDNAs and genomic DNA. "Polynucleotide(s)" include, without limitation, single- and double-stranded DNA, DNA that is a mixture of single- and double-stranded regions or single-, double- and triple-stranded regions, single- and double-stranded RNA, and RNA that is mixture of single- and double-stranded regions, hybrid molecules comprising DNA and RNA that may be single-stranded or, more typically, double-stranded, or triple-stranded regions, or a mixture of single- and double- stranded regions. The term also includes triple-stranded regions comprising RNA or DNA or both RNA and DNA. The strands in such triple-stranded regions may be from the same molecule or from different molecules. The regions may include all or one or more of the molecules, but typically involve only a region of some of the molecules. One of the molecules of a triple-helical region often is an oligonucleotide. As used herein, the term "polynucleotide(s)" also includes DNAs or RNAs as described herein that contain one or more modified bases. Thus, DNAs or RNAs with backbones modified for stability or for other reasons are within the meaning of the term "polynucleotide(s)". "Polynucleotide(s)" also includes DNAs or RNAs comprising unusual bases, such as inosine, or modified bases, such as tritylated bases, to name just two examples. 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 and the term "polynucleotide(s)" embraces such chemically, enzymatically or metabolically modified forms of polynucleotides, as well as the chemical forms of DNA and RNA characteristic of viruses and cells, including, for example, simple and complex cells. "Polynucleotide(s)" also includes short polynucleotides often referred to as oligonucleotide(s). The term "polynucleotides" and in particular DNA or RNA, refers only to the primary and secondary structure and it does not limit it to any particular tertiary forms. The term "polynucleotide encoding a polypeptide" encompasses polynucleotides that include a sequence encoding a polypeptide of the invention, particularly a bacteria polypeptide and more particularly a polypeptide of Helicobacter pylori having an amino acid sequence set out in SEQ ID NO: 2, 4, 18, 19 or 21. The term also contemplates polynucleotides that include a single continuous region or discontinuous regions encoding the polypeptide (e.g. interrupted by integrated phage or an insertion sequence or editing) together with additional regions, that also may contain coding and/or non-coding sequences.

"Polypeptide(s)" refers to any peptide or protein comprising two or more amino acids joined to each other by peptide bonds or modified peptide bonds. The term includes both short chains, commonly referred to as peptides, oligopeptides and oligomers and to longer chains generally referred to as proteins. Polypeptides may contain amino acids other than the 20 gene encoded amino acids. "Polypeptide(s)" as used herein includes those modified either by natural processes, such as processing and other post-translational modifications, but also by chemical modification techniques. Such modifications are well described in basic texts and research literature, and they are well known to those of skill in the art. The same type of modification may be present in the same or varying degree at several sites in a given polypeptide, and a given polypeptide may contain many types of modifications. Modifications may occur anywhere in a polypeptide, including the peptide backbone, the amino acid side-chains, and the amino or carboxyl termini. Examples of modifications include, acetylation, acylation, ADP-ribosylation, amidation, covalent attachment of flavin, covalent attachment of a heme moiety, covalent attachment of a nucleotide or nucleotide derivative, covalent attachment of a lipid or lipid derivative, covalent attachment of phosphotidylinositol, cross-linking, cyclization, disulfide bond formation, demethylation, formation of covalent cross-links, formation of cysteine, formation of pyroglutamate, formylation, gamma-carboxylation, glycosylation, GPI anchor formation, hydroxylation, iodination, methylation, myristoylation, oxidation, proteolytic processing, phosphorylation, prenylation, racemization, glycosylation, lipid attachment, sulfation, gamma-carboxylation of glutamic acid residues, hydroxylation and ADP-ribosylation, selenoylation, sulfation, transfer-RNA mediated addition of amino acids to proteins, such as arginylation, and ubiquitination. (See, for example, PROTEINS- STRUCTURE AND MOLECULAR PROPERTIES, 2^nd 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. These polypeptides may result from post-translational natural processes and may be made by entirely synthetic methods.

"Variant(s)" as used herein refers to a polynucleotide or polypeptide that differs from a reference polynucleotide or polypeptide respectively, but retains essential properties. A typical variant of a polynucleotide differs in nucleotide sequence from another polynucleotide. Changes in the nucleotide sequence of the variant may or may not alter the amino acid sequence of an encoded polypeptide. Nucleotide changes may result in amino acid substitutions, additions, deletions, fusions and truncations in the polypeptide encoded by the reference sequence. A typical variant of a polypeptide differs in amino acid sequence from another, reference polypeptide. Differences are generally limited so that the sequences of the reference polypeptide and the variant are very similar overall and, in many regions, identical. A variant may differ in amino acid sequence by one or more substitutions, additions, deletions in any combination. A substituted or inserted amino 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 variant such as an allelic variant, or it may be a variant that is not known to occur naturally. Mutagenesis techniques, direct synthesis, and other recombinant methods known to skilled artisans may be used to produce non-naturally occurring variants of polynucleotides and polypeptides.

A "ligand" refers to a compound or entity that associates with a polypeptide of the invention or part thereof, including acceptor molecules or analogues or parts thereof, and donor molecules or analogues or parts thereof. Polynucleotides

As hereinbefore mentioned, the invention provides isolated polynucleotides, (including a full length Helicobacter motility gene) that encode Helicobacter motility polypeptides, or fragments, variants, homologs thereof, and polynucleotides having substantial identity thereto, and variants thereof. Preferably, the polynucleotides encode polypeptides that retain substantially the same biological function or activity of a mature Helicobacter motility polypeptide. In an embodiment of the invention an isolated polynucleotide is contemplated which comprises:

(i) a polynucleotide encoding a polypeptide having substantial sequence identity, preferably at least 50%), more preferably at least 70% sequence identity, with an amino acid sequence of SEQ. ID. NO. 2, 4, 18, 19 or 21;

(ii) polynucleotides complementary to (i); (iii) polynucleotides differing from any of the polynucleotides of (i) or (ii) in codon sequences due to the degeneracy of the genetic code; (iv) a polynucleotide comprising at least 10, 15, or 18, preferably at least 20 nucleotides and capable of hybridizing under stringent conditions to a polynucleotide of SEQ. ID. NO. 1, 3, 17 or 20 or to a degenerate form thereof; (v) a polynucleotide encoding an allelic or species variation of a polypeptide comprising an amino acid sequence of SEQ. ID. NO. 2, 4, 18, 19 or 21; or (vi) a fragment, or allelic or species variation of (i), (ii) or (iii)

In a specific embodiment, the isolated polynucleotide comprises: (i) a polynucleotide having substantial sequence identity, preferably at least 50%), more preferably at least 70% sequence identity with a sequence of SEQ. ID. NO. 1, 3, 17 or 20; (ii) polynucleotides complementary to (i), preferably complementary to a full sequence of SEQ. ID. NO. 1, 3, 17 or 20; (iii) polynucleotides differing from any of the nucleic acids of (i) to (ii) in codon sequences due to the degeneracy of the genetic code; or (iv) a fragment, or allelic or species variation of (i), (ii) or (iii). In a preferred embodiment the isolated nucleic acid comprises a polynucleotide encoded by an amino acid sequence of SEQ. ID. NO. 2, 4, 18, 19 or 21 or comprises or consists essentially of a polynucleotide of SEQ. ID. NO. 1, 3, 17 or 20 wherein T can also be U. The DNA sequence set out in SEQ ID NO: 1, 3, 17 or 20 contains an open reading frame encoding a polypeptide comprising the amino acid residues set forth in SEQ ID NO: 2, 4, 18, 19 or 21, respectively, with a deduced molecular weight that can be calculated using amino acid residue molecular weight values well known in the art. Preferably, a polynucleotide of the present invention has substantial sequence identity using the preferred computer programs cited herein, for example at least 30%, 40%, 50%, 60%, 70%, 75%, 80%, 90%, more preferably at least 95%, 96%, 97%, 98%, or 99% sequence identity to a sequence of SEQ. ID. NO. 1, 3, 17 or 20. Isolated nucleic acid molecules encoding a polypeptide of the invention and having a sequence which differs from a polynucleotide of SEQ. ID. NO. 1, 3, 17 or 20 due to degeneracy in the genetic code are also within the scope of the invention. As one example, DNA sequence variations within a Helicobacter motility gene may result in silent mutations which do not affect the amino acid sequence. Variations in one or more nucleotides may exist among organisms within a genus due to natural allelic variation. Any and all such nucleic acid variations are within the scope of the invention. DNA sequence variations may also occur which lead to changes in the amino acid sequence of a polypeptide of the invention. These amino acid variations are also within the scope of the present invention. In addition, species variations i.e. variations in nucleotide sequence naturally occurring among different species, are within the scope of the invention.

The invention contemplates 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 sequences, including those encoding a leader or secretory sequence, a pre-, or pro- or prepro- protein sequence. A polynucleotide of the invention may also contain non-coding sequences, including, but not limited to non-coding 5' and 3' sequences, such as the transcribed, non-translated sequences, termination signals, ribosome binding sites, sequences that stabilize mRNA, introns, polyadenylation signals, and additional coding sequence which encode additional amino acids. The additional sequences may be a marker sequence that facilitates purification of the fused polypeptide, the sequences may play a role in processing of a polypeptide 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. Additional sequences may be at the amino or carboxyl-terminal end or interior to the mature polypeptide.

Polynucleotides of the invention also include, but are not limited to, polynucleotides comprising a structural gene and its naturally associated sequences that control gene expression.

Also included in the invention are polynucleotides of the formula: X-(Rι)m -(Z)-(R₂)„ -Y

wherein, at the 5' end of the molecule, X is hydrogen or a metal or together with Y defines a covalent bond, and at the 3' end of the molecule, Y is hydrogen or a metal or together with X defines a covalent bond, each occurrence of Ri and R₂ is independently any nucleic acid residue, m is an integer between 1 and 3000 or zero, preferably between 1 and 1000, n is an integer between 1 and 3000 or zero, preferably between 1 and 1000, and Z is a polynucleotide sequence of the invention, particularly a sequence selected from SEQ ID NO: 1, 3, 17 or 20. Any stretch of nucleotide residues denoted by either R group, where m and/or n is greater than 1, may be either a heteropolymer or a homopolymer, preferably a heteropolymer. In an embodiment, X and Y together define a covalent bond and the polynucleotide of the above formula is a closed, circular polynucleotide, which can be a double-stranded polynucleotide wherein the formula shows a first strand to which the second strand is complementary.

Fragments of a polynucleotide of the invention, include fragments that are a stretch of at least about 10, 15, 18, 20, 40, 50, 100, or 150 nucleotides, more typically at least 50 to 100 nucleotides but less than 2 kb. It will further be appreciated that variant forms of the polynucleotides of the invention which arise by alternative splicing of an mRNA corresponding to a cDNA of the invention are encompassed by the invention. Polynucleotides that encode for variants of polypeptides of the invention are particularly contemplated that have an amino acid sequence of SEQ ID NO: 2, 4, 18, 19 or 21, in which several, a few, 5 to 10, 1 to 5, 1 to 3, 2, 1, or no amino acid residues are substituted. Preferred among these variants are silent substitutions, additions, and deletions that do not alter the properties and activities of the polypeptide.

Another aspect of the invention provides a polynucleotide which hybridizes under selective conditions, e.g. high stringency conditions, to a polynucleotide which comprises a sequence which encodes a polypeptide of the invention. Preferably the sequence encodes an amino acid sequence of SEQ. ID. NO. 2, 4, 18, 19 or 21 or part thereof and comprises at least 18 nucleotides. Selectivity of hybridization occurs with a certain degree of specificity rather than being random. Appropriate stringency conditions which promote DNA hybridization are known to those skilled in the art, or can be found in Current Protocols in Molecular Biology, John Wiley & Sons, N.Y. (1989), 6.3.1-6.3.6. For example, hybridization may occur at 30°C in 750 mM NaCl, 75mM trisodium citrate, and 1% SDS, preferably 37°C in 500mM NaCl, 500 mM trisodium citrate, 1% SDS, 35% formamide, and lOOμg/ml denatured salmon sperm DNA (ssDNA), and more preferably 42°C in 250 mM NaCl, 25 mM trisodium citrate, 1% SDS, 50% formamide, and 200 μg/ml ssDNA. Useful variations on these conditions will be readily apparent to those skilled in the art.

The stringency may be selected based on the conditions used in the wash step. Wash step stringency conditions may be defined by salt concentration and by temperature. Generally, wash stringency can be increased by decreasing salt concentration or by increasing temperature. By way of example, a stringent salt concentration for the wash step is preferably less than about 30 mM NaCl and 3mM trisodium citrate, and more preferably less than about 15 mM NaCl and 1.5 mM trisodium citrate. Stringent temperature conditions will generally include temperatures of a least about 25°C, more preferably at least about 68°C. In a preferred embodiment, the wash steps will be carried out at 42°C in 15 mM NaCl, 1.5mM trisodium citrate, and 0.1% SDS. In a more preferred embodiment the wash steps are carried out at 68°C in 15 mM NaCl, 1.5mM trisodium citrate, and 0.1% SDS. Variations on these conditions will be readily apparent to those skilled in the art. The polynucleotides of the inventions are preferably derived from Helicobacter pylori, however, they may be obtained from other bacterial species.

An isolated polynucleotide of the invention which comprises DNA can be isolated by preparing a labeled nucleic acid probe based on all or part of a nucleic acid sequence of SEQ. ID. NO. 1, 3, 17 or 20. The labeled nucleic acid probe is used to screen an appropriate DNA library (e.g. a cDNA or genomic DNA library). For example, a cDNA library can be used to isolate a cDNA encoding a polypeptide of the invention by screening the library with the labeled probe using standard techniques. Alternatively, a genomic DNA library can be similarly screened to isolate a genomic clone encompassing a Helicobacter motility gene. Polynucleotides isolated by screening of a cDNA or genomic DNA library can be sequenced by standard techniques.

An isolated polynucleotide of the invention which is DNA can also be isolated by selectively amplifying a polynucleotide of the invention. "Amplifying" or "amplification " refers to the production of additional copies of a nucleic acid sequence and is generally carried out using polymerase chain reaction (PCR) technologies well known in the art (Dieffenbach, C. W. and G. S. Dveksler (1995) PCR Primer, a Laboratory Manual, Cold Spring Harbor Press, Plainview, NN.). In particular, it is possible to design synthetic oligonucleotide primers from a nucleotide sequence of SEQ. ID. NO. 1 for use in PCR. A nucleic acid can be amplified from cDNA or genomic DNA using these oligonucleotide primers and standard PCR amplification techniques. The nucleic acid so amplified can be cloned into an appropriate vector and characterized by DNA sequence analysis. cDNA may be prepared from mRNA, by isolating total cellular mRNA by a variety of techniques, for example, by using the guamdimum-thiocyanate extraction procedure of Chirgwin et al., Biochemistry, 18, 5294-5299 (1979). cDNA is then synthesized from the mRNA using reverse transcriptase (for example, Moloney MLV reverse transcriptase available from Gibco/BRL, Bethesda, MD, or AMV reverse transcriptase available from Seikagaku America, Inc., St. Petersburg, FL). An isolated polynucleotide of the invention which is RNA can be isolated by cloning a cDNA encoding a polypeptide of the invention into an appropriate vector which allows for transcription of the cDNA to produce an RNA molecule which encodes the polypeptide. For example, a cDNA can be cloned downstream of a bacteriophage promoter, (e.g. a T7 promoter) in a vector, cDNA can be transcribed in vitro with T7 polymerase, and the resultant RNA can be isolated by conventional techniques.

A polynucleotide of the invention may be engineered using methods known in the art to alter the Helicobacter motility encoding sequence for a variety of purposes including modification of the cloning, processing, and/or expression of the gene product. Procedures such as DNA shuffling by random fragmentation and PCR reassembly of gene fragments and synthetic oligonucleotides may be used to engineer the nucleic acid molecules. Mutations may be introduced by oligonucleotide-mediated site-directed mutagenesis to create for example new restriction sites, change codon preference, or produce variants.

Polynucleotides of the invention may be chemically synthesized using standard techniques. Methods of chemically synthesizing polydeoxynucleotides are known, including but not limited to solid-phase synthesis which, like peptide synthesis, has been fully automated in commercially available DNA synthesizers (See e.g., Itakura et al. U.S. Patent No. 4,598,049; Caruthers et al. U.S. Patent No. 4,458,066; and Itakura U.S. Patent Nos. 4,401,796 and 4,373,071).

Determination of whether a particular polynucleotide is a Helicobacter motility gene or encodes a polypeptide of the invention can be accomplished by expressing the cDNA in an appropriate host cell by standard techniques, and testing the expressed protein in the methods described herein. A cDNA encoding a polypeptide of the invention can be sequenced by standard techniques, such as dideoxynucleotide chain termination or Maxam-Gilbert chemical sequencing, to determine the nucleic acid sequence and the predicted amino acid sequence of the encoded protein. The polynucleotides of the invention may be extended using a partial nucleotide sequence and various PCR-based methods known in the art to detect upstream sequences such as promoters and regulatory elements. For example, restriction-site PCR which uses universal and nested primers to amplify unknown sequences from genomic DNA within a cloning vector may be employed (See Sarkar, G, PCR Methods' Applic. 2:318-322, 1993). Inverse PCR which uses primers that extend in divergent directions to amplify unknown sequences from a circularized template may also be used. The template in inverse PCR is derived from restriction fragments adjacent to known sequences in human and yeast artificial chromosome DNA (See e.g. Lagerstrom, M., at al, PCR Methods Applic. 1:111- 119, 1991). Other methods for retrieving unknown sequences are known in the art (e.g. Parker, J.D. et al, Nucleic Acids Res. 19:305-306, 1991). In addition, PCR, nested primers, and PROMOTERFINDER libraries (Clontech, Palo Alto, California) may be used to walk genomic DNA.

It is preferable when screening for full-length cDNAs to use libraries that have been size-selected to include larger cDNAs. For situations in which an oligo d(T) library does not yield a full-length cDNA, it is preferable to use random-primed libraries which often include sequences containing the 5' regions of genes. Genomic libraries may be useful for extending the sequence into 5 'non-translated regulatory regions.

Commercially available capillary electrophoresis systems may be employed to analyse the size or confirm the sequence of PCR or sequencing products. The system may use flowable polymers for elecfrophoretic separation, four different nucleotide-specific, laser-stimulated fluorescent dyes, and a charge coupled device camera for detection of the emitted wavelengths. Commercially available software (e.g. GENOTYPER and SEQUENCE NAVIGATOR, Perkin-Elmer) may convert the output/light intensity to electrical signal, and the entire process from loading of samples, and computer analysis and electronic data display may be computer controlled. This procedure may be particularly useful for sequencing small DNA fragments which may be present in limited amounts in a particular sample.

In accordance with another aspect of the invention, the polynucleotides isolated using the methods described herein are mutant Helicobacter motility gene alleles. For example, the mutant alleles may be isolated from organisms either known or proposed to contribute to a disease. Mutant alleles and mutant allele products may be used in therapeutic and diagnostic methods described herein. For example, a cDNA of a mutant Helicobacter motility gene may be isolated using PCR as described herein, and the DNA sequence of the mutant allele may be compared to the normal allele to ascertain the mutation(s) responsible for the loss or alteration of function of the mutant gene product. A genomic library can also be constructed using DNA from an organism suspected of or known to carry a mutant allele, or a cDNA library can be constructed using RNA from organisms known to express the mutant allele. A polynucleotide encoding a normal Helicobacter motility gene or any suitable fragment thereof, may then be labeled and used as a probe to identify the corresponding mutant allele in such libraries. Clones containing mutant sequences can be purified and subjected to sequence analysis. In addition, an expression library can be constructed using cDNA from RNA isolated from organisms known or suspected to express a mutant Helicobacter motility allele. Gene products from putatively mutant organisms may be expressed and screened, for example using antibodies specific for a polypeptide as described herein. Library clones identified using the antibodies can be purified and subjected to sequence analysis. Antisense molecules and ribozymes are contemplated within the scope of the invention. "Antisense" refers to any composition containing nucleotide sequences which are complementary to a specific DNA or RNA sequence. Ribozymes are enzymatic RNA molecules that can be used to catalyze the specific cleavage of RNA. Antisense molecules and ribozymes may be prepared by any method known in the art for the synthesis of polynucleotides. These include techniques for chemically synthesizing oligonucleotides such as solid phase phosphoramidite chemical synthesis. Alternatively, RNA molecules may be generated by in vitro and in vivo transcription of DNA sequences encoding a polypeptide of the invention. Such DNA sequences may be incorporated into a wide variety of vectors with suitable RNA polymerase promoters such as T7 or SP6. Alternatively, these cDNA constructs that synthesize antisense RNA constitutively or inducibly can be introduced into organisms. RNA molecules may be modified to increase intracellular stability and half-life. Possible modifications include, but are not limited to, the addition of flanking sequences at the 5' and/or 3' ends of the molecule or the use of phosphorothioate or 2' O-methyl rather than phosphodiesterase linkages within the backbone of the molecule. This concept is inherent in the production of PNAs and can be extended in all of these molecules by the inclusion of nontraditional bases such as inosine, queosine, and wybutosine, as well as acetyl-, methyl-, thio-, and similarly modified forms of adenine, cytidine, guanine, thymine, and uridine which are not as easily recognized by endogenous endonucleases. Polypeptides

A polypeptide of the invention includes a polypeptide of SEQ.ID. NO: 2, 4, 18, 19 or 21. In addition to polypeptides comprising an amino acid sequence of SEQ.ID. NO. 2, 4, 18, 19 or 21 the polypeptides of the present invention include truncations or fragments, and variants, and homologs.

Truncated polypeptides may comprise peptides of between 3 and 70 amino acid residues, ranging in size from a tripeptide to a 50 mer polypeptide, preferably 30 to 50 amino acids. In one aspect of the invention, fragments of a polypeptide of the invention are provided having an amino acid sequence of at least five consecutive amino acids of SEQ.ID. NO. 2, 4, 18, 19 or 21 where no amino acid sequence of five or more, six or more, seven or more, or eight or more, consecutive amino acids present in the fragment is present in a polypeptide other than a Helicobacter motility polypeptide of the invention. In an embodiment of the invention the fragment is a stretch of amino acid residues of at least 12 to 20 contiguous amino acids from particular sequences such as the sequences of SEQ.ID. NO. 2, 4, 18, 19 or 21. The fragments may be immunogenic and preferably are not immunoreactive with antibodies that are immunoreactive to polypeptides other than a Helicobacter motility polypeptide of the invention. Particularly preferred are fragments that are antigenic or immunogenic in an animal, especially in a human.

A fragment may be characterized by structural or functional attributes such as fragments that comprise alpha-helix and alpha-helix forming regions, beta-sheet and beta- sheet-forming regions, turn and turn-forming regions, coil and coil-forming regions, hydrophilic regions, hydrophobic regions, alpha amphipathic regions, beta amphipathic regions, flexible regions, surface-forming regions, substrate binding regions, and high antigenic index regions. In a preferred embodiment, the invention provides biologically active fragments which are those fragments that mediate activities of a Helicobacter motility polypeptide, including those with a similar activity or an improved activity, or with a decreased undesirable activity. Particularly preferred are fragments comprising domains of enzymes that confer a function essential for viability of Helicobacter species or the ability to initiate, maintain, or cause disease in an individual, particularly a human.

Truncated polypeptides may have an amino group (-NH2), a hydrophobic group (for example, carbobenzoxyl, dansyl, or T-butyloxycarbonyl), an acetyl group, a 9- fluorenylmethoxy-carbonyl (PMOC) group, or a macromolecule including but not limited to lipid-fatty acid conjugates, polyethylene glycol, or carbohydrates at the amino terminal end. The truncated polypeptides may have a carboxyl group, an amido group, a T- butyloxycarbonyl group, or a macromolecule including but not limited to lipid-fatty acid conjugates, polyethylene glycol, or carbohydrates at the carboxy terminal end.

A truncated polypeptide or fragment 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, of a single larger polypeptide.

The polypeptides of the invention may also include variants of a Helicobacter motility polypeptide of the invention, and/or truncations thereof as described herein, which may include, but are not limited to a polypeptide of the invention containing one or more amino acid substitutions, insertions, and/or deletions. Amino acid substitutions may be of a conserved or non-conserved nature. Conserved amino acid substitutions involve replacing one or more amino acids of a Helicobacter motility polypeptide amino acid sequence with amino acids of similar charge, size, and/or hydrophobicity characteristics. When only conserved substitutions are made the resulting analog is preferably functionally equivalent to a Helicobacter motility polypeptide of the invention. Non-conserved substitutions involve replacing one or more amino acids of the Helicobacter motility polypeptide amino acid sequence with one or more amino acids which possess dissimilar charge, size, and/or hydrophobicity characteristics. One or more amino acid insertions may be introduced into a polypeptide of the invention. Amino acid insertions may consist of single amino acid residues or sequential amino acids ranging from 2 to 15 amino acids in length.

Deletions may consist^' of the removal of one or more amino acids, or discrete portions from the Helicobacter motility polypeptide amino acid sequence. The deleted amino acids may or may not be contiguous. The lower limit length of the resulting analog with a deletion mutation is about 10 amino acids, preferably 50 amino acids.

Allelic variants of a Helicobacter motility polypeptide at the protein level differ from one another by only one, or at most, a few amino acid substitutions. A species variation of a Helicobacter motility polypeptide is a variation which is naturally occurring among different species of an organism.

The polypeptides of the invention include homologs of a Helicobacter motility polypeptide and/or truncations thereof as described herein. Such Helicobacter motility polypeptide homologs include proteins whose amino acid sequences are comprised of the amino acid sequences of Helicobacter motility polypeptide regions from other species that hybridize under selective hybridization conditions (see discussion of selective and in particular stringent hybridization conditions herein) with a probe used to obtain a polypeptide. These homologs will generally have the same regions which are characteristic of a Helicobacter motility polypeptide. It is anticipated that a protein comprising an amino acid sequence which has at least 30%, 40%, 50%, 60%, 70%, 75%,

80%, 85%, or 90% identity, more preferably 95%, 96%, 97%, 98%, or 99% identity with an amino acid sequence of SEQ. ID. NO. 2, 4, 18, 19 or 21 will be a homolog of a polypeptide of the invention. A percent amino acid sequence homology or identity is calculated using the methods described herein, preferably the computer programs described herein.

The invention also contemplates isoforms of polypeptides of the invention. An isoform contains the same number and kinds of amino acids as a polypeptide of the invention, but the isoform has a different molecular structure. The isoforms contemplated by the present invention preferably have the same properties as a polypeptide of the invention as described herein.

The present invention also provides a polypeptide of the invention conjugated with a selected protein, or a marker, to produce fusion proteins or chimeric proteins. Also included in the invention are polypeptides of the formula:

X-(R₁)_m -(Z)-(R₂)_n -Y

wherein, at the amino terminus, X is hydrogen or a metal, and at the carboxy terminus Y is hydrogen or a metal, or together Y and X define a covalent bond, each occurrence of Ri and R is independently any amino acid residue, m is an integer between 1 and 1000 or zero, preferably between 1 and 1000, n is an integer between 1 and 3000 or zero, preferably between 1 and 1000, and Z is a polypeptide of the invention, particularly a sequence selected from SEQ ID NO: 2, 4, 18, 19 or 21. Any stretch of amino acid residues denoted by either R group, where m and/or n is greater than 1, may be either a heteropolymer or a homopolymer, preferably a heteropolymer. Where, in a preferred embodiment, X and Y together define a covalent bond, the polypeptide of the above formula is a closed, circular polypeptide. A polypeptide of the invention may be prepared using recombinant DNA methods.

Accordingly, polynucleotides of the present invention having a sequence which encodes a polypeptide of the invention may be incorporated in a known manner into an appropriate expression vector which ensures good expression of the polypeptide. Possible expression vectors include but are not limited to chromosomal, episomal and virus-derived vectors, so long as the vector is compatible with the host cell used. Representative examples of vectors are vectors derived from bacterial plasmids, from bacteriophage, 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 derived from combinations thereof, such as those derived from plasmid and bacteriophage genetic elements, such as cosmids and phagemids

The invention therefore contemplates a recombinant expression vector comprising a polynucleotide of the invention, and the necessary regulatory sequences for the transcription and translation of the inserted sequence. Suitable regulatory sequences may be derived from a variety of sources, including bacterial, fungal, viral, mammalian, or insect genes (For example, see the regulatory sequences described in Goeddel, Gene Expression Technology: Methods in Enzymology 185, Academic Press, San Diego, CA (1990). Selection of appropriate regulatory sequences is dependent on the host cell chosen as discussed below, and may be readily accomplished by one of ordinary skill in the art. The necessary regulatory sequences may be supplied by the native polypeptide and/or its flanking regions. The invention further provides a recombinant expression vector comprising a polynucleotide of the invention cloned into the expression vector in an antisense orientation. That is, the DNA molecule is linked to a regulatory sequence in a manner which allows for expression, by transcription of the DNA molecule, of an RNA molecule which is antisense to a polynucleotide sequence of SEQ. ID. NO. 1, 3, 17 or 20. Regulatory sequences linked to the antisense nucleic acid can be chosen which direct the continuous expression of the antisense RNA molecule in a variety of cell types, for instance a viral promoter and/or enhancer, or regulatory sequences can be chosen which direct tissue or cell type specific expression of antisense RNA.

The recombinant expression vectors of the invention may also contain a marker gene which facilitates the selection of host cells transformed or transfected with a recombinant molecule of the invention. Examples of marker genes are genes encoding a protein such as G418, dhfr, npt, als, pat and hygromycin which confer resistance to certain drugs, β-galactosidase, chloramphenicol acetyltransferase, firefly luciferase, trpB, hisD, herpes simplex virus thymidine kinase, adenine phosphoribosyl transferase, or an immunoglobulm or portion thereof such as the Fc portion of an immunoglobulm preferably IgG. Visible markers such as anthocyanins, beta-glucuronidase and its substrate GUS, and luciferase and its substrate luciferin, can be used to identify transformants, and also to quantify the amount of transient or stable protein expression attributable to a specific vector system (Rhodes, C. et al. (1995) Mol. Biol. 55:121-131). The markers can be introduced on a separate vector from the nucleic acid of interest.

The recombinant expression vectors may also contain genes that encode a fusion moiety which provides increased expression of the recombinant polypeptide; increased solubility of the recombinant polypeptide; and aid in the purification of the target recombinant polypeptide by acting as a ligand in affinity purification. For example, a proteolytic cleavage site may be added to the target recombinant polypeptide to allow separation of the recombinant polypeptide from the fusion moiety subsequent to purification of the fusion protein. Typical fusion expression vectors include pGEX (Amrad Corp., Melbourne, Australia), pMAL (New England Biolabs, Beverly, MA) and pRIT5 (Pharmacia, Piscataway, NJ) which fuse glutathione S-transferase (GST), maltose E binding protein, or protein A, respectively, to the recombinant protein.

The vectors may be introduced into host cells to produce a transformed or transfected host cell. The terms "transfected " and "fransfection" encompass the introduction of nucleic acid (e.g. a vector) into a cell by one of many standard techniques. A cell is "transformed" by a nucleic acid when the transfected nucleic acid effects a phenotypic change. Prokaryotic cells can be transfected or transformed with nucleic acid by, for example, electroporation or calcium-chloride mediated transformation. Nucleic acid can be introduced into mammalian cells via conventional techniques such as calcium phosphate or calcium chloride co-precipitation, DEAE-dextran-mediated fransfection, lipofectin, electroporation or microinjection. Suitable methods for transforming and fransfecting host cells can be found in Sambrook et al. (Molecular Cloning: A Laboratory Manual, 2nd Edition, Cold Spring Harbor Laboratory press (1989)), and other laboratory textbooks.

Suitable host cells include a wide variety of prokaryotic and eukaryotic host cells. For example, the proteins of the invention may be expressed in bacterial cells such as E. coli, insect cells (using baculovirus), yeast cells, or mammalian cells. Other suitable host cells can be found in Goeddel, Gene Expression Technology: Methods in Enzymology 185, Academic Press, San Diego, CA (1991).

Examples of appropriate host cells include bacterial cells, such as Streptococci, Staphylococci, Enterococci, E. coli, Helicobacter, Streptomyces, and Bacillus subtilis 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 host cell may also be chosen which modulates the expression of an inserted nucleic acid sequence, or modifies (e.g. glycosylation) and processes (e.g. cleaves) the polypeptide in a desired fashion. Host systems or cell lines may be selected which have specific and characteristic mechanisms for post-translational processing and modification of proteins. For long-term high-yield stable expression of the polypeptide, cell lines and host systems which stably express the gene product may be engineered. Host cells and in particular cell lines produced using the methods described herein may be particularly useful in screening and evaluating compounds that modulate the activity of a polypeptide of the invention.

Polypeptides of the invention may also be prepared by chemical synthesis using techniques well known in the chemistry of proteins such as solid phase synthesis (Merrifield, 1964, J. Am. Chem. Assoc. 85:2149-2154) or synthesis in homogenous solution (Houbenweyl, 1987, Methods of Organic Chemistry, ed. E. Wansch, Vol. 15 I and II, Thieme, Stuttgart). Protein synthesis may be performed using manual procedures or by automation. Automated synthesis may be carried out, for example, using an Applied Biosystems 431 A peptide synthesizer (Perkin Elmer). Various fragments of the polypeptides of the invention may be chemically synthesized separately and combined using chemical methods to produce the full length molecule.

N-terminal or C-terminal fusion polypeptides or chimeric polypeptides comprising a polypeptide of the invention conjugated with other molecules, (e.g. markers) may be prepared by fusing, through recombinant techniques, the N-terminal or C-terminal of a polypeptide of the invention, and the sequence of a selected molecule with a desired biological function (e.g. marker protein). The resultant fusion proteins contain a polypeptide of the invention fused to the selected molecule as described herein. Examples of molecules which may be used to prepare fusion proteins include immunoglobulins, glutathione-S-transferase (GST), protein A, hemagglutinin (HA), and truncated myc. Antibodies

Polypeptides of the invention, or cells expressing them can be used as an immunogen to produce antibodies immunospecific for such polypeptides. "Antibodies" as used herein includes monoclonal and polyclonal antibodies, chimeric, single chain, simianized antibodies and humanized antibodies, as well as Fab fragments, including the products of an Fab immunoglobulm expression library

In an embodiment of the invention, oligopeptides, peptides, or fragments used to induce antibodies to a polypeptide of the invention have an amino acid sequence consisting of at least 5 amino acids and more preferably at least 10 amino acids. The oligopeptides, etc. can be identical to a portion of the amino acid sequence of the natural protein, and they may contain the entire amino acid sequence of a small, naturally occurring molecule. Antibodies having specificity for a polypeptide of the invention may also be raised from fusion proteins created by expressing fusion proteins in bacteria as described herein.

Antibodies including monoclonal and polyclonal antibodies, fragments and chimeras, etc. may be prepared using methods known to those skilled in the art. Antibodies against polypeptides of the invention can be obtained by administering the polypeptides or epitope-bearing fragments, analogues or cells to an animal, preferably a nonhuman, using routine protocols. Monoclonal antibodies may be obtained by any technique known in the art that provides antibodies produced by continuous cell line cultures. (See for example, Kohler, G. and Milstein, C, Nature 256. 495-497 (1975); Kozbor et al, Imnmunology Today 4: 72 (1983); Cole et al., pg. 77-96 in MONOCLONAL ANTIBODIES AND CANCER THERAPY, Alan R. Liss, Inc. (1985).

Single chain antibodies to polypeptides of this invention can be prepared using methods known in the art (e.g. U.S. Pat. No. 4,946,778). Transgenic mice, or other organisms such as other mammals, may be used to express humanized antibodies. Phage display technology may also be utilized to select antibody genes with binding activities towards a polypeptide of the invention either from repertoires of PCR amplified v-genes of lymphocytes from humans screened for possessing anti-Helicobacter motility polypeptide activity or from naive libraries (McCafferty, J. et al., (1990), Nature 348, 552-554; Marks, J. et al., (1992) Biotechnology 10, 779-783). Chain shuffling can also be used to improve the affinity of these antibodies (Clackson, T. et al., (1991) Nature 352, 624-628). Applications

The polynucleotides, polypeptides, and antibodies of the invention may be used in the prognostic and diagnostic evaluation of disease. (See below). Methods for detecting polynucleotides and polypeptides of the invention, can be used to monitor disease in eukaryotes particularly mammals, and especially humans, particularly those infected or suspected to be infected with an organism comprising a Helicobacter motility gene or polypeptide of the invention, by detecting and localizing the polynucleotides and polypeptides. The applications of the present invention also include methods for the identification of agents (e.g. compounds) which modulate the biological activity of a polypeptide of the invention (See below). The compounds, antibodies, etc. may be used for the treatment of disease. (See below). Diagnostic and Prognostic Methods

A variety of methods can be employed for the diagnostic and prognostic evaluation of disease. Such methods may, for example, utilize polynucleotides of the invention, and fragments thereof, and antibodies of the invention. In particular, the polynucleotides and antibodies may be used, for example, for: (1) the detection of the presence of Helicobacter motility gene mutations, or the detection of either over- or under-expression of Helicobacter motility mRNA relative to a non-disorder state; and (2) the detection of either an over- or an under-abundance of a polypeptide of the invention relative to a non-disorder state or the presence of a modified (e.g., less than full length) polypeptide of the invention.

The methods described herein may be performed by utilizing pre-packaged diagnostic kits comprising at least one specific polynucleotide or antibody described herein, which may be conveniently used, e.g., in clinical settings, to screen and diagnose individuals and to screen and identify or monitor disease in individuals. Nucleic acid-based detection techniques and peptide detection techniques are described below. The samples that may be analyzed using the methods of the invention include those which are known or suspected to contain a polynucleotide or polypeptide of the invention. The methods may be performed on biological samples including but not limited to cells, lysates of cells which have been incubated in cell culture, genomic DNA (in solutions or bound to a solid support such as for Southern analysis), RNA (in solution or bound to a solid support such as for northern analysis), cDNA (in solution or bound to a solid support), an extract from cells or a tissue (e.g. bone, muscle, cartilage, skin), and biological fluids such as serum, urine, blood, and CSF. The samples may be derived from a patient or a culture. Methods for Detecting Polynucleotides

The invention provides a process for diagnosing disease, preferably bacterial infections, more preferably infections by Helicobacter pylori, comprising determining from a sample derived from an individual an increased level of expression of a polynucleotide of the invention. Increased or decreased expression of a polynucleotide of the invention can be measured using any of the methods well known in the art.

A polynucleotide of the invention may be used in southern or northern analysis, dot blot, or other membrane-based technologies; in PCR technologies; or in dipstick, pin, ELISA assays or microarrays utilizing fluids or tissues from patients to detect altered expression. Such qualitative or quantitative methods are well known in the art and some methods are described below.

The polynucleotides of the invention allow those skilled in the art to construct nucleotide probes for use in the detection of polynucleotides of the invention in biological materials. Suitable probes include polynucleotides based on nucleic acid sequences encoding at least 5 sequential amino acids from regions of a polynucleotide of the invention (see SEQ. ID. No. 1, 3, 17 or 20), preferably they comprise 15 to 30 nucleotides. A nucleotide probe may be labeled with a detectable substance such as a radioactive label which provides for an adequate signal and has sufficient half-life such as 32p. 3R, *- Q _or the like. Other detectable substances which may be used include antigens that are recognized by a specific labeled antibody, fluorescent compounds, enzymes, antibodies specific for a labeled antigen, and luminescent compounds. An appropriate label may be selected having regard to the rate of hybridization and binding of the probe to the nucleotide to be detected and the amount of nucleotide available for hybridization. Labeled probes may be hybridized to nucleic acids on solid supports such as nitrocellulose filters or nylon membranes as generally described in Sambrook et al, 1989, Molecular Cloning, A Laboratory Manual (2nd ed.). The nucleic acid probes may be used to detect Helicobacter motility genes, preferably in human cells. The nucleotide probes may also be useful for example in the diagnosis or prognosis of disease, and in monitoring the progression of a disease condition, or monitoring a therapeutic treatment.

The probe may be used in hybridization techniques to detect Helicobacter motility genes. The technique generally involves contacting and incubating a sample from a patient or other cellular source with a probe of the present invention under conditions favourable for the specific annealing of the probes to complementary sequences in the nucleic acids. After incubation, the non-annealed nucleic acids are removed, and the presence of nucleic acids that have hybridized to the probe if any are detected.

The detection of polynucleotides of the invention may involve the amplification of specific gene sequences using an amplification method such as PCR, followed by the analysis of the amplified molecules using techniques known to those skilled in the art. Suitable primers can be routinely designed by one of skill in the art. For example, primers may be designed using commercially available software, such as OLIGO 4.06 Primer Analysis software (National Biosciences, Plymouth Minn.) or another appropriate program, to be about 22 to 30 nucleotides in length, to have a GC content of about 50% or more, and to anneal to the template at temperatures of about 60°C to 72° C.

Genomic DNA may be used in hybridization or amplification assays of biological samples to detect abnormalities in cells involving a Helicobacter motility gene structure, including point mutations, insertions, and deletions. For example, direct sequencing, single stranded conformational polymorphism analyses, heteroduplex analysis, denaturing gradient gel electrophoresis, chemical mismatch cleavage, and oligonucleotide hybridization may be utilized. Mutations in the DNA sequence of a Helicobacter motility gene may be used to diagnose infection and to serotype and/or classify the infectious agent.

Genotyping techniques known to one skilled in the art can be used to type polymorphisms that are in close proximity to the mutations in a Helicobacter motility gene. The polymorphisms may be used to identify species of organisms that are likely to cause disease.

RT-PCR may be used to detect mutations in the RNA. In particular, RT-PCR may be used in conjunction with automated detection systems such as for example GeneScan.

The primers and probes may be used in the above described methods in situ i.e. directly on tissue sections (fixed and/or frozen) of patient tissue obtained from biopsies or resections.

Oligonucleotides derived from any of the polynucleotides of the invention may be used as targets in microarrays. "Microarray" refers to an array of distinct polynucleotides or oligonucleotides synthesized on a substrate, such as paper, nylon, or other type of membrane, filter, chip, glass slide, or any other suitable solid support. The microarrays can be used to monitor the expression level of large numbers of genes simultaneously (to produce a transcript image) and to identify genetic variants, mutations, and polymorphisms. This information can be useful in determining gene function, diagnosing disease, and in developing and monitoring the activity of therapeutic agents (Heller, R. et al. (1997) Proc. Natl. Acad, Sci. 94:2150-55). _. The polynucleotides of the present invention are useful for chromosome identification. The sequences can be specifically targeted to, and can hybridize with a particular location on an individual microbial chromosome, particularly a Helicobacter pylori chromosome. The mapping of relevant sequences to a chromosome is an important step in correlating those sequences with genes associated with microbial pathogenicity and disease, or to precise chromosomal regions critical to the growth, survival, and/or ecological niche of an organism. The physical position of the sequence on the chromosome can be correlated with genetic map data to define a genetic relationship between the gene and another gene or phenotype by, for example, linkage analysis.

Differences in the RNA or genomic sequence between microbes of different phenotypes may also be determined. A mutation or sequence observed in some or all of the organisms of a certain phenotype but not in organisms lacking that phenotype, will likely be the causative agent for the phenotype. Thus, chromosomal regions may be identified that confer pathogenicity, growth characteristics, survival characteristics, and/or ecological niche.

The polynucleotides of the invention may be used in differential screening and differential display methods known in the art. (e.g. see Chuang et al J. Bacteriol. 175: 2026, 1993). Genes are identified which are expressed in an organism by identifying mRNA present using randomly primed RT-PCR. Pre-infection and post-infection profiles are compared to identify genes up and down regulated during infection. Methods for Detecting Polypeptides

Antibodies specifically reactive with a polypeptide of the invention or derivatives thereof, such as enzyme conjugates or labeled derivatives, may be used to detect the polypeptides in various samples. They may be used as diagnostic or prognostic reagents and they may be used to detect abnormalities in the level of a polypeptide of the invention, or abnormalities in the structure of the polypeptides. Antibodies may also be used to screen potentially therapeutic compounds in vitro to determine their effects on a disease. In vitro immunoassays may also be used to assess or monitor the efficacy of particular therapies. The antibodies of the invention may also be used in vitro to determine the level of Helicobacter motility polypeptide expression in cells genetically engineered to produce a Helicobacter motility polypeptide.

In an embodiment, the invention provides a diagnostic method for detecting over- expression of a polypeptide of the invention compared to normal control tissue samples. The method may be used to detect the presence of an infection. The antibodies may be used in any known immunoassays which rely on the binding interaction between an antigenic determinant of a polypeptide of the invention, and the antibodies. Examples of such assays are radioimmunoassays, enzyme immunoassays (e.g. ELISA), immunofluorescence, immunoprecipitation, latex agglutination, hemagglutination, and histochemical tests. The antibodies may be used to detect and quantify polypeptides of the invention in a sample in order to determine its role in particular cellular events or pathological states, and to diagnose and treat such pathological states.

Antigenic polypeptides of the invention or fragments thereof may be used in immunoassays to detect antibody levels and correlations can be made with diseases such as gastroduodenal disease and with duodenal ulcer in particular. Immunoassays based on well defined recombinant antigens can be developed. Antibodies to Helicobacter pylori motility polypeptides within biological samples such as blood or serum samples may be detected. The antibodies of the invention may be used in immuno-histochemical analyses, for example, at the cellular and sub-subcellular level, to detect a polypeptide of the invention, to localise it to particular cells and tissues, and to specific subcellular locations, and to quantitate the level of expression.

Cytochemical techniques known in the art for localizing antigens using light and electron microscopy may be used to detect a polypeptide of the invention. Generally, an antibody of the invention may be labeled and a polypeptide may be localised in tissues and cells based upon detection of the label.

Various methods of labeling polypeptides are known in the art and may be used to label antibodies and polypeptides of the invention. Examples of detectable substances include, but are not limited to, the following: radioisotopes (e.g., ³ H, ¹⁴ C, ³⁵S, ¹²⁵1, ¹³¹I), fluorescent labels (e.g., FITC, rhodamine, lanthanide phosphors), luminescent labels such as luminol; enzymatic labels (e.g., horseradish peroxidase, β-galactosidase, luciferase, alkaline phosphatase, acetylcholinesterase), biotinyl groups (which can be detected by marked avidin e.g., streptavidin containing a fluorescent marker or enzymatic activity that can be detected by optical or calorimetric methods), and predetermined polypeptide epitopes recognized by a secondary reporter (e.g., leucine zipper pair sequences, binding sites for secondary antibodies, metal binding domains, epitope tags). In some embodiments, labels are attached via spacer arms of various lengths to reduce potential steric hindrance. Antibodies may also be coupled to electron dense substances, such as fe ritiή or colloidal gold, which are readily visualised by electron microscopy.

An antibody or sample may be immobilized on a carrier or solid support which is capable of immobilizing cells, antibodies etc. For example, the carrier or support may be nitrocellulose, or glass, polyacrylamides, gabbros, and magnetite. The support material may have any possible configuration including spherical (e.g. bead), cylindrical (e.g. inside surface of a test tube or well, or the external surface of a rod), or flat (e.g. sheet, test strip). Indirect methods may also be employed in which the primary antigen-antibody reaction is amplified by the introduction of a second antibody, having specificity for the antibody reactive against a polypeptide of the invention. By way of example, if the antibody having specificity against a polypeptide of the invention is a rabbit IgG antibody, the second antibody may be goat anti-rabbit gamma-globulin labelled with a detectable substance as described herein. Where a radioactive label is used as a detectable substance, a polypeptide of the invention may be localized by radioautography. The results of radioautography may be quantitated by determining the density of particles in the radioautographs by various optical methods, or by counting the grains. Methods for Identifying or Evaluating Substances/Compounds The invention provides methods for identifying substances that . modulate the biological activity of a polypeptide of the invention including substances that interfere with, or enhance the activity of the polypeptide.

The substances and compounds identified using the methods of the invention include but are not limited to peptides such as soluble peptides including Ig-tailed fusion peptides, members of random peptide libraries and combinatorial chemistry-derived molecular libraries including libraries made of D- and/or L-configuration amino acids, phosphopeptides (including members of random or partially degenerate, directed phosphopeptide libraries), antibodies [e.g. polyclonal, monoclonal, humanized, antisense, oligosaccharides, anti-idiotypic, chimeric, single chain antibodies, fragments, (e.g. Fab, F(ab)2_> an Fab expression library fragments, and epitope-binding fragments thereof)], and small organic or inorganic molecules. The substance or compound may be an endogenous physiological compound or it may be a natural or synthetic compound. A substance of the invention may be a natural substrate or ligand (e.g. an acceptor or donor molecule) or a structural or functional mimetic. The substance may be a small molecule ligand in, for example, cells, cell-free preparations, chemical libraries, and natural product mixtures Substances which modulate a polypeptide of the invention can be identified based on their ability to associate with (or bind to) a polypeptide of the invention. Therefore, the invention also provides methods for identifying substances which associate with a polypeptide of the invention. Substances identified using the methods of the invention may be isolated, cloned and sequenced using conventional techniques. A substance that associates with a polypeptide of the invention may be an agonist or antagonist of the biological or immunological activity of the polypeptide.

The term "agonist", refers to a molecule that increases the amount of, or prolongs the duration of, or the activity of the polypeptide. The term "antagonist" refers to a molecule which decreases the biological or immunological activity of the polypeptide. Agonists and antagonists may include proteins, nucleic acids, carbohydrates, or any other molecules that associate with a polypeptide of the invention (including ligands or mimetics thereof).

Substances which can associate with a polypeptide of the invention may be identified by reacting the polypeptide with a test substance which potentially associates with the polypeptide, under conditions which permit the association, and removing and/or detecting polypeptide associated with the test substance. Substance-polypeptide complexes, free substance, or non-complexed polypeptide may be assayed, or the activity of the polypeptide may be assayed. Conditions which permit the formation of substance- polypeptide complexes may be selected having regard to factors such as the nature and amounts of the substance and the polypeptide.

The substance-polypeptide complex, free substance, or non-complexed polypeptide may be isolated by conventional isolation techniques, for example, salting out, chromatography, electrophoresis, gel filtration, fractionation, absorption, polyacrylamide gel electrophoresis, agglutination, or combinations thereof. To facilitate the assay of the components, antibody against a polypeptide of the invention or the substance, or labeled polypeptide, or a labeled substance may be utilized. The antibodies, polypeptide, or substances may be labeled with a detectable substance as described above. A polypeptide of the invention, or the substance used in the method of the invention may be insolubilized. For example, a polypeptide, or substance may be bound to a suitable carrier such as agarose, cellulose, dextran, Sephadex, Sepharose, carboxymethyl cellulose polystyrene, filter paper, ion-exchange resin, plastic film, plastic tube, glass beads, polyamine-methyl vinyl-ether-maleic acid copolymer, amino acid copolymer, ethylene-maleic acid copolymer, nylon, silk, etc. The carrier may be in the shape of, for example, a tube, test plate, beads, disc, sphere etc. The insolubilized polypeptide or substance may be prepared by reacting the material with a suitable insoluble carrier using known chemical or physical methods, for example, cyanogen bromide coupling.

The invention also contemplates a method for evaluating a compound for its ability to modulate the biological activity of a polypeptide of the invention, by assaying for an agonist or antagonist (i.e. enhancer or inhibitor) of the association of the polypeptide with a substance which associates with the polypeptide. The basic method for evaluating if a compound is an agonist or antagonist of the association of a polypeptide of the invention and a substance that associates with the polypeptide, is to prepare a reaction mixture containing the polypeptide and the substance under conditions which permit the formation of substance-polypeptide complexes, in the presence of a test compound. The test compound may be initially added to the mixture, or may be added subsequent to the addition of the polypeptide and substance. Control reaction mixtures without the test compound or with a placebo are also prepared. The formation of complexes is detected and the formation of complexes in the control reaction but not in the reaction mixture indicates that the test compound interferes with the interaction of the polypeptide and substance. The reactions may be carried out in the liquid phase or the polypeptide, substance, or test compound may be immobilized as described herein. In an embodiment of the invention, the substance is a natural substrate or ligand of a polypeptide of the invention, or a structural or functional mimetic thereof.

It will be understood that the agonists and antagonists i.e. inhibitors and enhancers that can be assayed using the methods of the invention may act on one or more of the binding sites on the polypeptide or substance including agonist binding sites, competitive antagonist binding sites, non-competitive antagonist binding sites or allosteric sites. The invention also makes it possible to screen for antagonists that inhibit the effects of an agonist of the interaction of a polypeptide of the invention with a substance which is capable of associating with or binding to the polypeptide. Thus, the invention may be used to assay for a compound that competes for the same binding site of a polypeptide of the invention.

In an embodiment, the invention provides a method of screening compounds to identify those which enhance (agonist) or block (antagonist) the action of polypeptides or polynucleotides of the invention, particularly those compounds that are bacteriostatic and/or bacteriocidal. The method of screening may involve high-throughput techniques. For example, to screen for agonists or antagonists, a synthetic reaction mix, a cellular compartment, such as a membrane, cell envelope or cell wall, or a preparation of any thereof, comprising a polypeptide of the invention and a labeled substrate or ligand of such polypeptide is incubated in the absence or the presence of a test compound that may be an agonist or antagonist. The ability of the test compound to agonize or antagonize the polypeptide is reflected in decreased binding of the labeled ligand or decreased production of product from such substrate. Molecules that bind gratuitously, i.e., without inducing the effects of a polypeptide of the invention are most likely to be good antagonists. Molecules that bind well . and increase the rate of product production from substrate are agonists. Detection of the rate or level of production of product from substrate may be enhanced by using a reporter system. Reporter systems that may be useful in this regard include but are not limited to calorimetric labeled substrate converted into product, a reporter gene that is responsive to changes in polynucleotide or polypeptide activity, and binding assays known in the art.

Another example of an assay for antagonists is a competitive assay that combines a polypeptide of the invention and a potential antagonist with molecules that bind a polypeptide of the invention, a recombinant binding molecule, natural substrate or ligand, or substrate or ligand mimetic, under appropriate conditions for a competitive inhibition assay. The polypeptide can be labeled, such as by radioactivity or a colorimetric compound, such that the number of polypeptides bound to a binding molecule or converted to product can be determined accurately to assess the effectiveness of the potential antagonist. Agents that modulate a polypeptide of the invention can also be identified based on their ability to interfere with or enhance the activity of a polypeptide of the invention.

The reagents suitable for applying the methods of the invention to evaluate compounds that modulate a polypeptide of the invention may be packaged into convenient kits providing the necessary materials packaged into suitable containers. The kits may also include suitable supports useful in performing the methods of the invention.

A substance that inhibits a polypeptide may be identified by treating a cell which expresses the polypeptide with a test substance, and analyzing the cell surface structures on the cell. Cell surface structures can be analyzed using the methods described herein. Cells that have not been treated with the substance or which do not express the polypeptide may be employed as controls.

Substances which inhibit transcription or translation of a Helicobacter motility gene may be identified by transfecting a cell with an expression vector comprising a recombinant molecule of the invention, including a reporter gene, in the presence of a test substance and comparing the level of expression of a Helicobacter motility gene, or the expression of the protein encoded by the reporter gene with a confrol cell transfected with the nucleic acid molecule in the absence of the substance. The method can be used to identify transcription and translation inhibitors of a Helicobacter motility gene. Compositions and Treatments The polynucleotides and polypeptides of the invention and substances or compounds identified by the methods described herein, antibodies, and antisense nucleic acid molecules of the invention may be used to treat diseases. Examples of diseases that may be treated include diseases associated with organisms that contain a polypeptide or polynucleotide of the present invention. In an embodiment the organisms are from the Helicobacter family, and are particularly Helicobacter pylori species.

Helicobacter pylori infects the stomachs of over one-third of the world's population causing stomach cancer, ulcers, and gastritis (International Agency for Research on Cancer (1994) Schistosomes, Liver Flukes and Helicobacter Pylori (International Agency for Research on Cancer, Lyon, France; http://www.uicc.ch/ecp/ecp2904.htm). There is also a recognized cause-and-effect relationship between H pylori and gastric adenocarcinoma, classifying the bacterium as a Group I (definite) carcinogen. Preferred agonists of the invention found using screens provided by the invention, particularly broad-spectrum antibiotics, will be useful in the treatment of H. pylori infection, and they should decrease the advent of H. pylori-induced cancers, such as gastrointestinal carcinoma. The agonists should also be useful in the treatment of gastric ulcers and gastritis. Accordingly, the proteins, substances, antibodies, and compounds etc. may be formulated into pharmaceutical compositions for administration to subjects in a biologically compatible form suitable for administration in vivo. By "biologically compatible form suitable for administration in vivo" is meant a form of the substance to be administered in which any toxic effects are outweighed by the therapeutic effects. The substances may be administered to living organisms including humans, and animals. Administration of a therapeutically active amount of the pharmaceutical compositions of the present invention is defined as an amount effective, at dosages and for periods of time necessary to achieve the desired result. For example, a therapeutically active amount of a substance may vary according to factors such as the disease state, age, sex, and weight of the individual, and the ability of antibody to elicit a desired response in the individual. Dosage regima may be adjusted to provide the optimum therapeutic response. For example, several divided doses may be administered daily or the dose may be proportionally reduced as indicated by the exigencies of the therapeutic situation.

The active substance may be administered in a convenient manner such as by injection (subcutaneous, intravenous, etc.), oral administration, inhalation, transdermal application, or rectal administration. Depending on the route of administration, the active substance may be coated in a material to protect the compound from the action of enzymes, acids and other natural conditions that may inactivate the compound.

The compositions described herein can be prepared by per se known methods for the preparation of pharmaceutically acceptable compositions which can be administered to subjects, such that an effective quantity of the active substance is combined in a mixture with a pharmaceutically acceptable vehicle. Suitable vehicles are described, for example, in Remington's Pharmaceutical Sciences (Remington's Pharmaceutical Sciences, Mack Publishing Company, Easton, Pa., USA 1985). On this basis, the compositions include, albeit not exclusively, solutions of the substances or compounds in association with one or more pharmaceutically acceptable vehicles or diluents, and contained in buffered solutions with a suitable pΗ and iso-osmotic with the physiological fluids. After pharmaceutical compositions have been prepared, they can be placed in an appropriate container and labeled for treatment of an indicated condition. For administration of a composition of the invention the labeling would include amount, frequency, and method of administration. The compositions, substances, compounds etc. may be indicated as therapeutic agents either alone or in conjunction with other therapeutic agents or other forms of treatment (e.g. chemotherapy or radiotherapy). By way of example, they can be used in combination with anti-proliferative agents, antimicrobial agents, immunostimulatory agents, or anti-inflammatories. In particular, they can be used in combination with anti- bacterial agents. They can be administered concurrently, separately, or sequentially with other therapeutic agents or therapies.

Polynucleotides of the invention or any fragment thereof, or antisense sequences may be used for therapeutic purposes. Antisense to a polynucleotide encoding a polypeptide of the invention may be used in situations to block the synthesis of the polypeptide. In particular, cells may be transformed with sequences complementary to polynucleotides of the invention. Thus, antisense sequences may be used to modulate activity of a polypeptide of the invention, or to achieve regulation of gene function. Sense or antisense oligomers or larger fragments, can be designed from various locations along the coding or regulatory regions of sequences encoding a polypeptide of the invention. Expression vectors may be derived from retroviruses, adenoviruses, herpes or vaccinia viruses or from various bacterial plasmids for delivery of nucleic acid sequences to the target organ, tissue, or cells. Vectors that express antisense nucleic acid sequences of Helicobacter motility genes can be constructed using techniques well known to those skilled in the art (see for example, Sambrook et al. (supra)). Genes encoding a Helicobacter motility polypeptide can be turned off by transforming a cell or tissue with expression vectors that express high levels of a polynucleotide of the invention. Such constructs may be used to introduce untranslatable sense or antisense sequences into a cell. Even if they do not integrate into the DNA, the vectors may continue to transcribe RNA molecules until all copies are disabled by endogenous nucleases.

Modification of gene expression may be achieved by designing antisense molecules, DNA, RNA, or Peptide nucleic acid (PNA), to the control regions of a Helicobacter motility gene i.e. the promoters, enhancers, and introns. Preferably the antisense molecules are oligonucleotides derived from the transcription initiation site (e.g. between positions -10 and +10 from the start site). Inhibition can also be achieved by using triple-helix base-pairing techniques. Triple helix pairing causes inhibition of the ability of the double helix to open sufficiently for the binding of polymerases, transcription factors, or regulatory molecules (see Gee J.E. et al (1994) In: Huber, B.E. and B.I. Carr, Molecular and Immunologic Approaches, Futura Publishing Co., Mt. Kisco, N.Y.). An antisense molecule may also be designed to block translation of mRNA by inhibiting binding of the transcript to the ribosomes. Ribozymes may be used to catalyze the specific cleavage of RNA. Ribozyme action involves sequence-specific hybridization of the ribozyme molecule to complementary target RNA, followed by endonucleolytic cleavage. For example, hammerhead motif ribozyme molecules may be engineered that can specifically and efficiently catalyze endonucleolytic cleavage of sequences encoding a polypeptide of the invention.

Specific ribosome cleavage sites within any RNA target may be initially identified by scanning the target molecule for ribozyme cleavage sites which include the following sequences: GUA, GUU, and GUC. Short RNA sequences of between 15 and 20 ribonucleotides corresponding to the region of the cleavage site of the target gene may be evaluated for secondary structural features which may render the oligonucleotide inoperable. The suitability of candidate targets may be evaluated by testing accessibility to hybridization with complementary oligonucleotides using ribonuclease protection assays.

Therapeutic efficacy and toxicity may be determined by standard pharmaceutical procedures in cell cultures or with experimental animals, such as by calculating the ED₅o (the dose therapeutically effective in 50% of the population) or LD₅* (the dose lethal to 50% of the population) statistics. The therapeutic index is the dose ratio of therapeutic to toxic effects and it can be expressed as the ED₅- LD₅₀ ratio. Pharmaceutical compositions which exhibit large therapeutic indices are preferred. Mutant Organisms The invention provides novel mutants of Helicobacter bacteria, in particular mutants of H. pylori, having mutated (deactivated) Helicobacter motility genes. In general, "mutated" refers to a sudden heritable change in the phenotype of an organism which can be spontaneous or induced by known mutagenic agents, including radiation and various chemicals.

Methods are known in the art that can be used to generate mutations to produce the mutant bacteria of the present invention. For example, the transposon, TnlO, can be used to produce chromosomal deletions in a wide variety of bacteria (Kleckner et al., J. Mol. Biol. 116:125-159, 1977; EPO Pub. No. 315,682; U.S. Pat. No. 5,387,744. Alternatively, methods may be used that involve introducing specific deletions in a Helicobacter motility gene in an organism. A specific deletion in the selected gene can be generated by either of two general methods. The first method generates a mutation in a gene isolated from a population of clones contained in a genomic DNA library using restriction enzymes and the second method generates the mutation in a gene of known sequence using PCR. Using the first method, the position of the gene on a vector is identified using transposon tagging and a restriction map of the recombinant DNA in the vector is generated. Information derived from the transposon tagging allows all or a portion of a gene to be excised from the vector using the known restriction enzyme sites.

The second method is based upon PCR. Divergent PCR primers are used to amplify the upstream and downstream regions flanking a specified segment of a Helicobacter motility DNA to be deleted from the gene, generating a PCR product consisting of the cloning vector and upstream and downstream flanking nucleotide sequences (Innes et al. Eds., PCR Protocols, 1990, Academic Press, New York). In a variation of this method, PCR products are produced representing portions of the gene or flanking sequence, which are then joined together in a cloning vector.

Mutagenesis of a Helicobacter motility gene may also be carried out by insertion of a marker into an insertion site in the gene. For example, a kanamycin resistance marker may be ligated into an insertion site created in a Helicobacter motility gene by reverse PCR.

The DNA containing the mutant gene can be introduced into the bacterial host by transformation using chemical means or electroporation, by recombinant phage infection, or by conjugation. In preferred embodiments the mutant gene is introduced into the chromosomes of the bacteria which can be accomplished using any of a number of methods well known in the art such as, for example, methods using temperature-sensitive replicons (Hamilton et al., J. Bacteriol. 171:4617-4622, 1989), linear transformation of recBC mutants (Jasin et al., J. Bacteriol. 159:783-786, 1984), or host restricted replicons known as sμicide vectors (Miller et al., J. Bacteriol. 170:2575-2583, 1988). The particular method used is coupled with an appropriate counter selection method such as, for example, by using PCR, nucleic acid hybridization, or an immunological method.

The invention also provides modified cell surface molecules from mutants of the invention. A modified molecule may be isolated from the mutant bacteria and at least partially purified using techniques well known to those skilled in the art. Preparations of at least 70%, particularly 80%, more particularly 90%, most particularly 95% pure molecules are preferred. The purity of a preparation is expressed as the weight percentage of the total Helicobacter antigens present in the preparation. A purified cell surface molecule can be used as antigen either directly or after being conjugated to a suitable carrier protein. Vaccines

Mutant bacteria and modified cell surface molecules isolated from such mutants are useful sources of antigens in vaccination against Helicobacter bacteria, in particular against H. pylori. Such vaccines are normally prepared from dead bacterial cells, using methods well known to those skilled in the art, and usually contain various auxiliary components, such as an appropriate adjuvant and a delivery system. A delivery system aiming at mucosal delivery is preferred. Preferably but not essentially, the antigenic preparation is administered orally to the host, but parenteral administration is also possible. Live vaccines based on H. pylori mutants may also be prepared, but would normally require an appropriate vector for mucosal delivery. Vaccines of the present invention are useful in preventing and reducing the number of H. pylori infections and indirectly in reducing the incidence of pathological conditions associated with such infections, in particular gastric cancer.

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 an antigen (e.g. modified cell surface molecule) adequate to produce antibody and/ or T cell immune response to protect said individual from infection, particularly bacterial infection and most particularly Helicobacter pylori infection. Also provided are methods whereby such immunological response slows bacterial replication. A further aspect of the invention relates to an immunological composition which, when introduced into an individual capable of having induced within it an immunological response, induces an immunological response in such individual to Helicobacter wherein the composition comprises a modified cell surface molecule. 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.

A modified cell surface molecule may be fused with a molecule which may not by itself produce antibodies, but is capable of stabilizing the modified cell surface molecule and producing an antigen which will have immunogenic and protective properties. Examples of such molecules are lipoprotein D from Hemophilus inβaenzae, glutathione-S- transferase (GST) or beta-galactosidase. Moreover, the molecule may act as an adjuvant in the sense of providing a generalized stimulation of the immune system.

The invention provides methods using a modified cell surface molecule in immunization experiments in animal models of infection with Helicobacter to identify 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 Helicobacter pylori infection, in mammals, particularly humans. A modified cell surface molecule may be used as an antigen for vaccination of a host to produce specific antibodies which protect against invasion of bacteria, for example by preventing colonization.

The invention also includes a vaccine formulation which comprises a modified cell surface molecule of the invention together with a suitable carrier. The formulation is preferably administered parenterally, including, for example, administration that is subcutaneous, intramuscular, intravenous, or intradermal. Formulations suitable for parenteral administration include aqueous and non-aqueous sterile injection solutions which may contain anti-oxidants, buffers, bacteriostats and solutes which render the formulation isotonic with the bodily fluid, preferably the blood, of the individual; and aqueous and non-aqueous sterile suspensions which may include suspending agents or thickening agents. The formulations may be presented in unit-dose or multi-dose containers, for example, sealed ampoules and vials and may be stored in a freeze-dried 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-in water systems and other systems known in the art. The dosage will depend on the specific activity of the vaccine and can be readily determined by routine experimentation.

The following non-limiting examples is illustrative of the present invention: Example 1 Studies on the structural, genetic and functional characterization of flagellin glycosylation in Helicobacter pylori

Mass spectrometry analyses of the complex polar flagella from Helicobacter pylori demonstrated that both FlaA and FlaB proteins are posttranslationally modified with pseudaminic acid (Pse5Ac7Ac, 5,7-diacetamido-3,5,7,9-tetradeoxy-L-glycero-L-manno- nonulosonic acid). Unlike Campylobacter, flagellar glycosylation in Helicobacter displays little heterogeneity in isoform or glycoform distribution although all glycosylation sites are located in the central core region of the protein monomer in a manner similar to that found in Campylobacter. Bioinformatic analysis has now revealed five genes (HP0840 (SEQ ID NO:20), HP0178 (SEQ ID NO:l), HP0326A, HP0326B (SEQ ID NO:17 for A/B), HP0114 (SEQ ID NO:3)) homologous to other prokaryote genes previously reported to be involved in motility, flagellar glycosylation or polysaccharide biosynthesis. Insertional mutagenesis of four of these homologs in Helicobacter (HP0178, HP0326A, HP0326B, HP0114) resulted in a non-motile phenotype, no structural flagella filament, and only minor amounts of flagellin protein detectable by western immunoblot. However, mRNA levels for the flagellin structural genes remained unaffected by each mutation. In view of the combined bioinformatic and structural evidence indicating a role for these gene products in glycan biosynthesis, subsequent investigations focused on the functional characterization of the respective gene products. A novel approach was devised to identify biosynthetic sugar-nucleotide precursors from intracellular metabolic pools of parent and isogenic mutants using CE-ESMS and precursor ion scanning. HP0326A, HP0326B and HP0178 gene products are directly involved in the biosynthesis of the nucleotide activated form of Pse, CMP-Pse. Mass spectral analyses of the cytosolic extract from the HP0326A and HP0326B isogenic mutants revealed the accumulation of a mono and a diacetamido trideoxyhexose UDP sugar nucleotide precursor. METHODS.

Bacterial strains and culture conditions. H. pylori 26695 (Tomb et al, 2000, op. cit.) used for the initial cloning was obtained from R. A. Aim, Astra Boston, J99 was obtained from D. Taylor, University of Alberta Edmonton, SSI was obtained from A. Lee, NSW, Australia, PJ2 is a fresh clinical isolate from Dr. W. Conlan, IBS, NRC, the highly motile strain H. pylori 1061 from Dr. P. Hoffman, Dalhousie University, and M6 used in complementation experiments with pH-EL shuttle vector from Dr. K. Eaton, Ohio State University, Ohio. Helicobacter strains were grown at 37°C on antibiotic supplemented trypticase soy agar plates containing 7% horse blood (GSS agar) in a microaerophilic environment for 48h.

Flagellin purification. Flagellins were purified as described previously (Kostrzynska et al, 1991) to the point of pH 2.0 disassociation-neufral pH reassociation. The protein extract was centrifuged using a Centricon YM-30 membrane filter (Millipore, Bedford) with a molecular weight cut-off of 30 kDa to remove other protein contaminants such as urease, neutrophil activating protein and elongation factor.

Cloning and insertional mutagenesis of HP0840, HP0178, and HP0326A, B and HP0114. PCR primers were designed to amplify each ORF using H. pylori 26695 sequence data. The product was cloned into pUC19 and plasmid DNA purified and sequenced. Briefly, each clone was disrupted by using reverse primers which were internal to each gene, in a PCR reaction and which resulted in deletion of 10-20bp within the ORF. This was followed by ligation of a Kan cassette (Labigne et al, 1988 J. Bacteriol. 170:1704-1708) to the gel purified product to make plasmids pAAHP0840kan, pAAHP0178kan, pAA0326A, pAA0326B and pAAOl 14. The mutated allele was returned to Helicobacter strains by natural transformation according to the method of Haas βt al ((1993) Mol. Microbiol 8:753-760).

Complementation Studies. Wild type copies of HP0178, HP0114 and HP0326A and B were obtained by PCR of genomic 26695 DNA using the following primers (HP0178: IF 5' CAAACACCCATTACTCTTAAATCATGCCAA3' (SEQ ID NO:5), 1R 5 'CCTACAATGAGCGTTCTATATCAGCGCT3 ' (SEQ ID NO:6),

HP0114: IF 5' CGGGATCCAATTCAAAGGGGCGTTAGCCC 3' (SEQ ID NO:7), 1R 5' GGAATTCTTACCATTCTTTTAAAGCCATTTTGATCGCT3' (SEQ ID NO:8), HP0326A/B: IF 5' CGGGATCCATGAGAGCGATCGCTATTGTTTTAGCCAGA3' (SEQ ID , NO:9), 1R 5'

GGGGTACCTCAAATCTCTAAAAACTCCCTTAATGCACCCT3' (SEQ ID NO:10)). The cloned genes were subsequently transferred to the pHEL shuttle vector (Heuermann et al, 1998 Mol. Gen. Genet. 257:519-528) and used to naturally transform the respective isogenic mutants in either M6 or 1061. Transformants were selected on chloramphenicol and kanamycin and initially stabbed onto motility agar. SDS-PAGE of whole cell lysates PCR PCR's were carried out by using a Perkin Elmer thermocycler and using PWO polymerase (Roche Molecular Biochemicals, Laval, Qc, Canada). DNA sequence analysis. PCR products and plasmid DNA's were sequenced using terminator chemistry and Taq cycle sequencing kits (Perkin Elmer Applied Biosystems) and analysed on an Applied Biosystems 373 DNA sequencer. Custom primers were synthesized on an Applied Biosystems 394 DNA/RNA synthesizer.

Motility Testing. Helicobacter cells were tested for motility by spotting cultures onto plates of Brucella medium with 0.4% agar and 10%FBS, or the same medium supplemented with lOmg/ml kanamycin, or lOmg/ml Kan and 4mg/ml chloramphenicol.

Electron Microscopy. A grid covered with a carbon-coated parlodion film was floated onto a 20μl sample drop and left for approximately 2 min for adsorption of the sample to the grid. The grid was then removed from the drop and floated on a drop of 1% ammonium molybdate and left for approximately 2 minutes. Excess stain was removed by touching the edge of the grid to a piece of Whatman No.l filter paper. All samples for electron microscopy were examined in a Zeiss EM902 transmission electron microscope (Carl Zeiss, Thornwood, NY, 10594, USA) operating at 80 kV with the energy loss spectrometer in place. SDS-PAGE and Western blot analysis. Sodium dodecylsulfate-polyacrylamide gel electrophoresis (SDS-PAGE) was performed with a mini slab gel apparatus (Biorad) according to the method of Laemmli ((1970) Nature 227:680-685). Samples solubilised in sample buffer were stacked in 4.5% acrylamide and separated in 12.5%> acrylamide. For Western blot experiments, the proteins were transferred to nitrocellulose membranes by the method of Towbin et al ((1979) PNAS 74:4350-4354). The blots were incubated with either a monoclonal antibody specific for Helicobacter flagellin (Mab 72c) or rabbit polyclonal to purified flagellin (JB3) at 1:10,000 dilution and the bound antibodies visualised with alkaline phosphatase conjugated goat anti-mouse antibody. LPS morphology was determined by a modification of the procedure of Hitchcock and Brown ((1983) J. Bacteriol. 154:269-277). Bacterial cells were boiled in SDS-PAGE solubilization buffer for 10 minutes and then digested with an equal volume of proteinase K (lmg/ml) at 60°C for 2 hours. Samples were then loaded onto SDS-PAGE gels. After electrophoresis, gels were stained for LPS by the silver staining procedure of Tsai and Frasch ((1982) Anal Biochem. 119:115-119).

RT-PCR. RNA was extracted from broth grown cells using an RNeasy kit (Qiagen Inc, CA). All RNA samples were treated with Rnase-free DNase (Qiagen I-nc, CA) to remove contaminating DNA. RNA was quantified and 30ng used in each RT-PCR reaction using a Sensiscript RT Kit and the two tube RT-PCR protocol (Qiagen Inc, CA). The flagellin specific primers used were flaA2-R,

5ΑGAAGCCGAAACGACATTGATGCTCTT3' (SEQ ID NO: 11), and flaA2-F 5'GATAAAATCGGTCAGGTTCGTATCGCT3' (SEQ ID NO:12)which amplified a 570bp fragment from the flaA mRNA and flaB2-R,

5'TAACCGCATGCTGTGTCCCACAAATCCCT3' (SEQ ID NO:13) and flaB2-F 5'GAATTTCAAATTGGCGCGTATTCTAACAC3' (SEQ ID NO:14) which amplified a 598bp fragment from the flaB mRNA . Control RT- PCR reactions without Sensiscript enzyme were performed with each RNA sample to confirm that the product observed on agarose gels was RNA specific.

Mouse colonisation. Specific pathogen free female CD1 mice were purchased from Charles River Laboratories, Montreal when they were 6-8 weeks old. Mice were inoculated with bacteria harvested from 24h broth culture. Aliquots of 0.2ml containing approx 10⁸ bacteria resuspended in PBS, were given by gavage directly into the gastric lumen using a 20g gavage needle. Two inocula were given over a 48 hour period. No attempt was made to neutralize gastric acidity prior to inoculation. To recover viable bacteria from the stomach, at week 4 following inoculation, mice were killed by CO₂ asphyxiation and their stomachs removed whole. Stomachs were cut open along the greater curvature and the exposed luminal surface was gently irrigated with 20ml of PBS. This step effectively dimimshed the numbers of ubiquitous contaminating bacteria that otherwise overgrow on GSS agar and mask the presence of the slower growing H. pylori organisms. The washed stomach tissue was then homogenised and serial dilutions plated on GSS agar. H. pylori colonies were counted following 3-6 days incubation.

Extraction of sugar-nucleotides from cytosolic bacterial extracts. Sugar-nucleotides were isolated from the cells using the method of Fritsch et al (1996) J. Chomatogr.A 727:223-230. Briefly, cells were lysed by sonication in ammonium bicarbonate (50 mM; pΗ 8.0) and cellular debris was removed by centrifugation (100, 000 g for 45 min). Ice- cold ethanol was added to the lysates to a final concentration of 60% and the insoluble material removed by a second centrifugation (100, 000 g for 45 min). The soluble fraction was then evaporated to dryness on a SpeedVac preconcentrator, reconstituted in deionised water and filtered through a 10, 000 Da cut-off cellulose membrane (Millipore, Bedford, MA, USA). Extraction of the cellular pool of sugar-nucleotides was achieved by subjecting the filtrate to Isoelute PE-AX anion-exchange cartridges (Chromatographic Specialties, Brockville, ON, Canada) and eluted with ammonium acetate (0.1M). The volatile salt was removed by freeze-drying twice and the extracts reconstituted in deionised water for CE-ESMS analysis.

Mass specfrometry. The flagellins were analysed by ESMS on a Micromass Q- TOF 2 (Manchester, UK) to determine the molecular mass of the intact glycoprotein. H. pylori flagellin was dissolved in water (2 % formic acid) and infused into the MS at a flow rate of 0.5 μL/min. For the identification of glycosylation sites, the flagellin was digested by trypsin (Promega, Madison) and analyzed using CapLC (Waters, Millford, USA) coupled to ESMS on the Q-TOF 2 instrument. Peptide separation was achieved using a linear gradient of 10-40 % acetonitrile (0.2%> formic acid) in 50 min and from 50.1 min to 60 min iri 80% acetonitrile (0.2%> formic acid) on home-made Jupiter C18 column 7 cm x 150 μm i.d., 5 μm particle size (Phenomenex, Torrance, CA). All MS/MS spectra were obtained using data-dependent experiments with Ar as a collision gas. Second-generation product ion spectra were obtained by increasing the cone voltage from 35 V to 80 V to form fragment ions in the orifice/skimmer region, while the desired precursor ion was selected by the quadrupole. The precise identification of the glycosylation site was achieved using β-elimination with ammonium hydroxide to leave a modified Ser/Thr residue that could be located using tandem mass specfrometry (Rademaker et al, 1996 Anal. Biochem. 257, 149-160). The tryptic digest was subjected to alkaline hydrolysis using 1 n L NΗ4OΗ for 18 h at 50 °C, dried down, redissolved in water and analyzed by LC/ESMS. Tryptic peptides showing the characteristic mass shift following β-elimination were subjected to MS/MS analyses to locate the position of the modified residue.

The separation and identification of sugar-nucleotides biosynthetic substrates was performed on a Crystal CE 310 system coupled to a PE/Sciex API 3000 triple-quadrupole or a Q-Star mass spectrometer (PE/Sciex, Concord ON, Canada) via a sheath flow interface (sheath buffer: isopropano methanol, 2:1 v/v). Electrophoretic separations were performed on an uncoated fused silica of 90 cm x 50 μm i.d (Polymicro technologies, Tucson AZ). The separation buffers were 30 mM morpholine/formate, pH 9.0 containing 5% (v:v) methanol (for negative ion detection) and 10 mM ammonium acetate pH 9.0 (for positive ion detection). A separation voltage of 30 kV was typically applied at the injection end of the capillary. The outlet of the capillary was tapered to ca. 15 μm i.d. using a laser puller (Sutter Instruments, Novato, CA).

Potential sugar-nucleotide precursors present in the cell lysates were identified using precursor ion scanning on the API 3000 (negative ion mode) for specific nucleotide fragment ions of CMP (m/z 322), UDP (m/z 323, 385, 403), GDP (m/z 362, 442, 424) and ADP (m/z 408, 346, 426). Suspected precursor ions identified were fragmented in-source by raising the orifice/skimmer voltage from 30 to 100 V to form positive oxonium ions. Product ion spectra were obtained for each characteristic oxoniums ions using nitrogen as collision gas at energies of 60 eV (laboratory frame of reference). Accession numbers. The H. pylori 1061 flaA and flaB DNA sequences described here (the coded polypeptides are SEQ ID NO:15 and SEQ ID NO:16) has been deposited in Genbank as AYl 55231 and AYl 55232. RESULTS Structural analysis of Helicobacter pylori flagellin. The flaA and flaB flagellin structural genes from strain 1061 were amplified by

PCR and subjected to DNA sequence analysis to determine the amino acid sequence of the translated proteins. The predicted aa sequence for flaA (1061) was identical to that of 26695 while the predicted aa sequence of the 1061 flaB gene had two amino acid substitutions at position 154 (T for an A) and 181 (E for an A) when compared to the predicted 26695 flaB sequence (GenbankAccession # AYl 55231 and AYl 55232). Flagella were purified from H pylori 1061 cells following the method of Kostrzynska et al (1991) using pH 2.0 disassociation. Electrospray mass specfrometry analyses of purified flagellin from H. pylori 1061 (Fig 1A) showed two well defined components at Mr:55049 and 55365 Da corresponding to the molecular mass of the monomeric FlaA protein (Mr: 53153.4 Da) with additional modifications accounting for 1896 and 2212 Da respectively (see below). These mass spectral analyses also indicated relatively little heterogeneity in isoform or glycoform distribution of the H. pylori flagellin (peak width 40Da) in contrast to those obtained from C. jejuni flagellin (Thibault, P., Logan, S.M., Kelly, J. F., Brisson, J-R, Ewing, C.P., Trust, T.J., and Guerry, P. (2001) J. Biol. Chem. 276, 34862-34870) where extensive heterogeneity in glycosylation profile resulted in a relatively large peak width extending to 700 Da. It is noteworthy that two other components of significantly lower intensity (< 10%> FlaA) were observed at 56683 Da and 56999 Da which were later shown to correspond to FlaB with modifications accounting for an additional 2794Da and 3160 Da respectively (see below).

Tandem mass specfrometry experiments performed on the multiply charged ions of H. pylori flagellin revealed an abundant fragment ion at m/z 317. Second generation product ion spectrum of m/z 317 showed a fragmentation pattern identical to that of pseudaminic acid (Pse5Ac7Ac), an unusual O-linked monosaccharide previously identified in C.jejuni and C. coli flagellins (Thibault et al, 2001, op.cit.). No evidence for additional carbohydrate residues was observed from these tandem mass specfrometry experiments in contrast to Campylobacter flagellins which displayed acetamidino and hydroxyproprionyl substituents on Pse5Ac7Ac (Thibault et al, 2001, op.cit.). Results from these combined analyses suggested that H. pylori FlaA protein is modified with a total of 7 O-linked Pse5Ac7Ac residues (7 x 316.1 Da = 2212.7 Da) whereas FlaB contains up to 10 Pse5Ac7Ac residues (10x316.1Da = 3161Da).

In order to precisely assign the location of glycosylation sites on H. pylori FlaA protein, cLC/ESMS experiments were performed on tryptic peptides from purified flagellin with and without alkaline hydrolysis. Suspected tryptic glycopeptides comprising the extra 316 Da residue were first subjected to tandem mass specfrometry to confirm the assignment. The flagellin tryptic digest was then subjected to base-catalyzed hydrolysis in the presence of NΗ OΗ to identify the site of O-linked attachment (Thibault et al. 2001 (op.cit.), Rademaker et al. 1996(op.cit.)). Upon alkaline hydrolysis, O-linked Ser and Thr residues yield modified amino acids of neutral mass of 86 and 100 Da that can be identified by the corresponding mass shift in the product ion spectrum of the β-eliminated products. By using this approach it was possible to map. all 7 sites of modifications on H. pylori flagellin (Fig IB). It is noteworthy that all glycosylation sites are located in the central core region consistent with that observed previously in Campylobacter flagellins. Similar analyses were also performed on FlaB present in this sample at a much lower concentration. While precise location of the glycosylation sites was very challenging in view of the overwhelming abundance of FlaA and the limited amount of flagellin sample available, it was possible to identify a total of 10 modification sites, all containing O- linked Pse5Ac7Ac in the central core region of the FlaB molecule (Fig 1C). Genetic analysis of 26695 and J99 genomes. Analysis of the genome sequences of H. pylori 26695 and J99 identified a number of ORF's with homology to genes involved in motility, flagellar glycosylation or polysaccharide biosynthesis in a number of other bacterial species, and for which there is substantial evidence of flagellar glycosylation. Five ORFs selected for further investigation were ΗP0840 (flaAl, GlcNac epimerase/dehydratase), HP0178 ( neuB/spore coat polysaccharide biosynthesis protein E), HP0326A (neuA, N acetylneuraminic acid synthetase), HP0326B (flmD, glycosyltransferase), and HP0114 (hypothetical protein, Cj 1318 family) which show homology to genes of Campylobacter and Caulobacter which have been implicated in the glycosylation process. With the exception of HP0840 which has been functionally characterized (Creuzenet et al, 2000 J. Biol.Chem. 275:34873- 34880) the annotations assigned to HP0326A, HP0326B, HP0178 and HP0114 have no functional basis. The similarity scores of all five predicted proteins are shown in Table 1.

The HP0326 ORF of 26695 has been shown by Josenhans et al (2002, op.cit.) to encode two overlapping ORF's, HP0326A and HP0326B and we have now expanded this observation to other Helicobacter isolates. Sequencing of PCR products spanning the HP0326 gene in the four strains examined in this study confirmed that this was indeed a common feature. This was in contrast to the single continuous ORF reported by Tomb, J- F., White, O. Kervalage, A. R., Clayton, R. A., Sutton, G. G, et al (1997) Nature 388:539-547. Generation of isogenic mutants of H. pylori. The five genes were amplified by PCR from genomic DNA of 26695 using primers specific for the 5' and 3' regions of each gene. The purified product was ligated into pUC19 and confirmed by DNA sequencing. PCR was used to determine the distribution of the five genes in 4 other Helicobacter isolates. In all strains examined a single PCR product was obtained for each gene as would be expected based on the whole genome microarray analysis of Salama et al (2000 PNAS 97:14668-14673) where each of these ORFs were present in 15 clinical helicobacter isolates tested. Insertionally inactivated copies of each gene were constructed by inverse PCR of the cloned genes in pUC19 and ligation of a kanamycin cassette in a non-polar orientation within each ORF. The orientation of the kanamycin cassette was confirmed by DNA sequencing. Transcription of genes lying downstream of each inactivated ORF should not be affected by insertion of the kan cassette in a non-polar orientation (also see below). Isogenic mutants of H pylori 1061, M6, SSI and PJ2 were made in ORFS HP0178, 0326A, 0326B, 0114 by natural transformation as previously described by Haas et al (1993, op.cit.). Plasmids pAA0178Kan, pAA0326AKan, pAA0326BKan pAA0114Kan and pAA0840Kan were used in transformation experiments and the successful inactivation of each gene confirmed by PCR. Repeated attempts to construct an isogenic mutant in HP0840 ORF in H pylori 1061, as well as in a number of other readily transformable H. pylori isolates, was unsuccessful although the HP0840 gene was shown to be present by PCR. This led us to conclude that insertional inactivation of this gene maybe a lethal event in H. pylori. Phenotypic characterisation of H pylori HP0178, HP0326A, HP0326B and HP0114 mutants. All mutant strains grew well and were not affected in viability or growth characteristics. The motility of H. pylori HP0178, HP0114 and HP0326A and B mutants in strains 1061 and M6 was determined by swarming on soft agar plates. In all strains where the gene had been insertionally inactivated, cells were completely non-motile as demonstrated by the small, sharply delineated colonies typical of ήon-motile cells on motility agar (Fig 2). In contrast, the parental strain did produce the diffuse spreading growth pattern characteristic of motile bacteria. Complementation of an H. pylori 1061 HP0178 isogenic mutant with the pHel shuttle vector (Heuermann and Haas, 1998, op.cit.) containing DNA spanning HP0180- HP0178 genes restored motility and flagellin production (Fig 4F, lane 2). Isogenic mutants HP0326A and HP0326B of H. pylori M6 were complemented with a pHel plasmid containing DNA spanning both open reading frames. Motility and flagellin production was restored in both mutants (Fig 4F, lane 4,6). To date we have been unable to complement the HP0114 mutation using the pHel shuttle vector containing a wild type copy of HP0114 and this may be due to the lack of promoter activity. Subsequent experiments to clone a larger fragment of DNA encompassing HP0115, HP0114 and 200bp of DNA upstream from HP0115 gene which includes a putative sigma 54 promoter in E. coli have been unsuccessful. However, insertional mutagenesis of the HP0114 gene has been performed in a number of strains of Helicobacter and in each case all transformants examined (>10 colonies/transformation) displayed the non motile phenotype providing convincing evidence that the phenotype we observed was due to the inactivation of HP0114 gene and not to some unrelated distant event which would likely not occur at such a high frequency. While a polar effect cannot be ruled out, the gene lying immediately downstream of HP0114, HP0113 is transcribed from the opposite strand. Electron microscopic examination of H. pylori.

All mutants generated in H. pylori 1061 were examined by transmission electron microscopy using negative staining and compared with the parental strain. No flagella could be detected in the mutant strains while the characteristic multiple polar, sheathed flagella were abundant on parental cells. While neither broth nor plate grown cells of the isogenic mutants were able to produce the typical polar tufts of flagella, examination of mutants revealed many cells that displayed empty truncated "sheath-like" structures at the polar tips of cells. Occasionally cells were observed with full-length sheaths lacking flagellar filaments. These observations demonstrate that normal flagellar assembly was severely affected as a result of any one of the four mutations (Fig 3) SDS-PAGE and Western blot analysis .

The presence of FlaA protein, the major component of the flagellar filament was determined by immunoblotting with a rabbit polyclonal antisera (JB3) made to purified flagellin and with a monoclonal antibody (Mab72c) directed towards H. pylori flagellin (Kostrzynska et al, 1991). Whole cell lysates and sheared flagellin preparations were probed following SDS-PAGE and the results are presented in Figure 4. Immunoblotting of whole cell lysates and sheared flagella from the parent strain, 1061, clearly demonstrated the presence of FlaA FlaB proteins as indicated by the positive immunoblot reaction with both polyclonal (Fig 4, Panel A, B lane 1) and monoclonal antibodies (Fig 4 Panel C, D, lane 1) while in cell lysates and flagella preparations of HP0178, HP0326A, HP0326B, and HPOl 14 isogenic mutants, flagellin protein production was clearly affected (Fig 4, panel A, B, C, D, lanes 2-5). No corresponding flagellin protein could be detected in either the whole cell lysates or sheared flagellin preparations of HP0326A, HP0326B, HPOl 78, and HPOl 14 isogenic mutants with either antisera. When immunoblots of whole cell lysates of the isogenic mutants were probed with either polyclonal or monoclonal antibody and were developed for a prolonged period, a weak reaction was obtained to either one or two proteins of slightly lower apparent MW (Fig 4, A, C lanes 2-5) which may correspond to non-glycosylated flagellin monomer or a truncated protein product. Western immunoblot analysis of sheared flagella preparations from the HPOl 14 isogenic mutant probed with polyclonal antisera also displayed a weakly reactive protein of higher molecular weight than either parental FlaA or FlaB protein (Fig 4B, lane 5). The identity of this protein is currently under investigation. Interestingly, the rabbit polyclonal antisera to purified Helicobacter flagellin protein also reacted strongly with a protein of approx 75000 Da in whole cell lysates from wildtype 1061 and the isogenic mutant HPOl 14, but this reactivity was absent in whole cell lysates of HP0326A, HP0326B and HP0178. We are currently in the process of characterizing this protein to determine its identity and whether it is also glycosylated (Fig 4A, lane 1,5). In summary, inactivation of any one of these four genes has resulted in severely reduced levels of flagellin protein as detected with either a monoclonal or polyclonal antisera made to purified flagellin. Reactivity of 1061 and the isogenic mutant HPOl 14 whole cell lysates to the flagellin polyclonal antibody has identified a second protein which may either be glycosylated or involved in the glycosylation process.

To determine if inactivation of any of these genes has also resulted in a change in LPS structure, proteinase K digested whole cell lysates were examined by SDS-PAGE and silver stain. No changes in the migration pattern of LPS from each isogenic mutant when compared to the parent strain was apparent upon growth in broth or on solid media.

RT-PCR analysis of flagellin mRNA. As the level of flagellin protein produced by each isogenic mutant was shown by western blotting to be substantially reduced, we next examined the levels of mRNA for the major flagellar structural protein, FlaA. As can be seen in Fig 4E, the levels of mRNA specific for flaA from each of the isogenic mutants appears to be identical to that from the 1061 parent cells suggesting that inactivation of these genes has had no effect on flaA transcription. Similar results were obtained for flaB mRNA transcription. Mouse colonisation studies.

The role of two of these polysaccharide genes in colonisation was then investigated using a clinical isolate of Helicobacter pylori PJ2 and the mouse adapted strain SSI, both of which are capable of colonising the stomachs of mice. At week 4 post challenge, both PJ2 and SSI parent strains had colonized the mouse stomach at typical levels. In comparison, colonization of mice by either the HP0178 or HP0326B isogenic mutants was severely affected and in the majority of mice in these experimental groups below the detectable limit. The results are presented in Table 2. It appears therefore that the ability of H. pylori cells to colonise the mouse stomach is severely affected in both the HP0326B and HPO 178 isogenic mutants.

Biosynthetic precursor accumulation.

The genetic analysis suggests that the gene products are involved in glycan biosynthesis. In view of the combined evidence ' from this analysis and the occurrence of Pse5Ac7Ac in H pylori 1061 flagellin, subsequent investigations focused on the identification of individual genes involved in the biosynthetic pathway of this unusual carbohydrate residue and its transfer to flagellin. Post translational modifications of the flagellin protein likely require the transfer of the Pse5Ac7Ac from its corresponding activated sugar-nucleotide via reactions catalysed by glycosyltransferases. Typically intracellular UDP nucleotides are associated with sugars such as GlcNAc, GalNAc, Glc and Gal whereas GDP and CMP form biosynthetic intermediates with Man and Neu5Ac, respectively. It is thus expected that Pse5Ac7Ac and its biosynthetic precursors would be present as activated nucleotide sugar molecules in the cytosolic pools of H pylori. The inhibition of the sugar-nucleotide transfer to the protein through insertional inactivation of genes involved either in this process or in the biosynthetic pathway is anticipated to result in an accumulation of enzymatic precursors that can be detected in the cytosol. By determining the concentration of these precursors in the wild type and isogenic mutants, inference can thus be made on the functional role of individual glycosylation genes. A novel approach was devised to identify such biosynthetic sugar- nucleotide precursors from intracellular metabolic pools of parent H pylori 1061 cells and isogenic mutants using capillary electrophoresis-electrospray mass specfrometry (CE-ESMS) and tandem mass specfrometry. Preliminary experiments performed on sugar nucleotide standards CMP-Neu5Ac, UDP-Glc, UDP-GalNAc, GDP-Man, GDP-Fuc, ADP-Glc indicated that dissociation of the anionic precxursors typically yield fragment ions specific to the nucleotide backbone (m/z 322 for CMP, m/z 323,385,403 for UDP, m/z 362, 424, 442, for GDP and m/z 346, 408, 426 for ADP). These fragment ions can be used to enable specific detection of sugar nucleotides using precursor ion scanning where biosynthetic precursor anions are identified based on the observation of those characteristic nucleotide fragment ions. Figure 5 illustrates the results obtained for the identification of cytosolic sugar nucleotides from the cell lysates of H. pylori 1061 and isogenic mutants. The precursor ion scan of m/z 322 from the parent strain of H. pylori 1061 (Figure 5 A) contained a number of cytidine related ions including hydrate fragment ions of CDP (m/z 420) and CTP (m/z 518) together with an ion at m/z 638 consistent with CMP-Pse5Ac7Ac. In contrast, no detectable signal was obtained for the precxxrsor ion scan of UDP (Fig 5B), GDP or ADP. Confirmation of the CMP-Pse5Ac7Ac assignment was achieved from the product ion scan (positive ion mode) of the suspected precursor with front-end collision induced dissociation (CID). Upon fragmentation, the sugar nucleotide dissociates to form an oxonium ion at m/z 317 that can be subsequently analysed by tandem mass specfrometry. The product ion spectrum of the resulting fragment (Fig 5C) was consistent with that obtain previously for Pse5Ac7Ac (Thibault et al, 2001, op.cit.). It is noteworthy that the presence of CMP-Pse5 Ac7Ac in the CMP precursor ion scan was also detected for the isogenic mutant ΗP0114 where the level of this metabolite remained constant, suggesting that this gene is not directly involved in the biosynthesis of CMP- Pse5Ac7c.

In contrast, no ion was detected in the precursor ion scan for CMP in HP0326B (Fig 5D), HP0326A and HP0178 isogenic mutants of H. pylori 1061, indicating that these gene products are directly related to the biosynthesis and/or activation of this novel carbohydrate. The precursor ion scanning (negative ion mode) for UDP-activated nucleotide sugars from the cytosolic extract of both the ΗP0326A and HP0326B isogenic mutants revealed the presence of two parent ions at m/z 589 and 631 (Fig 5E). However, in the case of the HPOl 78 isogenic mutant, no apparent build-up of any novel UDP nucleotide-sugar was detected suggesting that either this enzyme is not involved in steps requiring sugar-nucleotide precursors or that the rapid utilisation of precursor molecules resulted in concentration of biosynthetic substrates below the detection limits of the present technique (< 100 ng/mL).

The dissociation of biosynthetic precursors identified in Fig 5E with front-end collision-induced dissociation (CID) gave rise to oxonium ions at m/z 187 and 229 which are consistent with UDP-activated mono- and diacetamido trideoxyhexose, respectively. The product ion scan of m/z 229 is presented in Fig 5F, and shows consecutive losses of ketene (CH₂CO), water, and ammonia supporting this UDP-sugar substrate to comprise a diacetamido trideoxyhexose residue.

To confirm that the phenotype observed for HP0326B could be restored by the presence of a wild type copy of the gene in trans, precursor ion scan was performed on cell lysates of the HP0326B isogenic mutant transformed with the pHEL shuttle vector containing HP0326A and B genes. Precursor ion scans clearly showed the production of CMP-Pse5Ac7Ac, along with reduced levels of both UDP-activated mono- and diacetamido trideoxyhexose.

It can.be concluded from this analysis that HPOl 14 is not directly involved in the biosynthesis of CMP-Pse, while inactivation of HP0326A, B and 0178 led to an inability of cells to make CMP-Pse and in the case of HP0326A and B, increased levels of two UDP- activated trideoxyhexose precursor molecules was observed providing further evidence of a role in the biosynthetic pathway. Discussion

This work has identified four polysaccharide biosynthetic gene loci which are involved in the synthesis and/or assembly of Helicobacter flagella. Structural characterization of the flagellin proteins has identified 7 sites of glycosylation on FlaA and 10 sites on FlaB where pseudaminic acid is O-linked at serine or threonine in the central region of the molecule. As was the case for Campylobacter flagellin, glycosylation appears to be restricted to the central domain of the flagellin monomer a surface exposed region in the assembled filament. However, a major difference in the modification profile of H. pylori flagellin when compared to that found on Campylobacter flagellin is the lack of heterogeneity in the degree of modification as evidenced by the sharp intact mass profile indicating a total of seven sites. Moreover, only a single sugar species, Pse5Ac7Ac, was present on H. pylori flagellin in contrast to the numerous related derivatives found on Campylobacter flagellin (Thibault et al, 2001 (op.cit.), Logan et al, 2002, Mol. Micro. 46:587-597). This observation correlates well with the "glycosylation related" gene content of the genomes of Campylobacter and Helicobacter. While the Campylobacter genome contains 4 distinct carbohydrate biosynthetic loci with over 100 annotated carbohydrate biosynthesis genes, the two H. pylori genomes have only a very limited number of genes (<30) which show homology to either the larger pool of flagellar glycosylation genes found in the Campylobacter genome or to LPS/capsule biosynthesis genes. The relevance of this in terms of the pathogenesis of each organism remains to be established, but may be reflected in the particular mucosal environment in which each organism resides (stomach vs. intestine) and/or in the presence of a sheath covering the flagellar filament of H. pylori. As with Campylobacter, the glycosylation of Helicobacter flagellin may occur as a consequence of the local hydrophobicity surroimding selected serine/threonine residues. This report now expands the process of glycosylation of bacterial flagellins with the novel sugar Pse5Ac7Ac, to a second bacterial species which also has a polar complex flagella. Additional studies are required to establish if the presence of this charged acidic polysaccharide confers some novel property to the filament or if it is indeed an integral part of the assembly process. The observation by RT-PCR in this study that flagellin mRNA levels are imaffected in the isogenic mutants indicates that the products of these genes act at the posttranslational level and do not regulate flagellin expression at the transcriptional level.

This study provides a highly sensitive, novel method to study sugar biosynthetic pathways in the bacterial cell through CE-ESMS and precursor ion scanning. Functional characterization of flagellar glycosylation gene products has been accomplished by comparing isogenic mutants with wildtype cells. It appears that HP0326A, HP0326B and HPOl 78 proteins are directly involved in the biosynthesis of the nucleotide-activated form of Pse5Ac7Ac, CMP-Pse5Ac7Ac. HPOl 78 is annotated as an N-acetyl neuraminic acid synthase, which in E. coli Kl capsular biosynthesis is responsible for the condensation of PEP with the C6 sugar, N-acetylmannosamine to form N-acetylneuraminic acid (Vann et al, 1997 Glycobiology 7:697-701). Pse5Ac7Ac biosynthesis may occur via a similar condensation reaction of a diacetamido trideoxyhexose sugar with a C3, PEP like substrate. In this present study we screened only for nucleotide activated sugars in the metabolome and so we did not detect an increase in the respective C6 substrate of this condensation reaction. The inability of this strain to produce CMP-Pse5Ac7Ac clearly indicates a role for this gene in the biosynthetic pathway. We are currently developing alternate metabolome screening methods which may facilitate the identification of substrate molecules for this particular gene product.

Insertional inactivation of HP0326A and HP0326B resulted in accumulation of such a precursor (diacetamido trideoxyhexose sugar) activated with UDP indicating that both gene products are clearly involved in the pathway of CMP-Pse5Ac7Ac biosynthesis. HP0326A shows homology to a number of proteins annotated as CMP sialic acid synthetases and the loss of CMP-Pse as observed here is consistent with the expected gene function. Inability to activate Pse with CMP may lead to an accumulation of biosynthetic precursors through a form of feedback regulation. The HP0326B protein has no functional homolog in Campylobacter, and shows only limited homology to a protein from Clostridium which has been annotated as a glycosyltransferase and resides in a flagellar biosynthetic operon (see table 1). Results from this study indicate that HP0326B is involved in the flagellar glycosylation process as inactivation of this gene results in undetectable levels of CMP-Pse and in the accumulation of biosynthetic precursors.

The final flagellar glycosylation gene targets HPOl 14 does not appear to be involved in the biosynthesis of CMP-Pse5Ac7Ac but clearly plays a role in flagellar assembly in Helicobacter. While CMP-Pse5Ac7Ac production was unaffected in this mutant we are now investigating the possibility that this protein is involved in the transfer of Pse5Ac7Ac from CMP-Pse5Ac7Ac to the flagellin monomer or at a later stage in flagellin assembly following glycosylation of the protein. Although we are unclear of the role of HPOl 14 in the glycosylation process, we believe that the H. pylori system will be invaluable for defining the role of not only this Helicobacter gene but also the family of homologs found in both Campylobacter (Cj 1318 MAF family) and the related family of ORFs found in the Clostridium genome sequence. In Helicobacter, a second ORF HP0465 which shows limited homology to HPOl 14 is present in the genome and we are currently examining the role of this gene in flagellar assembly. In comparison to Campylobacter, the system of flagellar glycosylation in Helicobacter is relatively simple and so provides a very useful system to conduct functional studies. Sheathed or unsheathed polar flagella are common to a number of bacterial species including Vibrio, Pseudomonas, Campylobacter, Aeromonas and Caulobacter and substantial genetic and/or biochemical evidence for glycosylation of polar flagella from these species now exists. While the hierarchy of flagellar gene regulation and assembly has been extensively studied for the peritrichously flagellated E. coli and S. typhimurium, only recently have the genes and gene organization of polar motility systems of these other Gram negative bacteria received attention. It remains to be established what the biological role of glycosylation is for polar flagella but it seems clear from this study as well as from a number of other studies that glycosylation may be a unique feature of polar motility systems and appears to play a key role in the assembly process and may indeed contribute to the unique biological properties of these particular flagella.

Recent discoveries of derivatives of pseudaminic acid as components of cell surface glycopolymers or homologs of genes known to be involved in glycopolymer synthesis have been documented for a diverse number of bacterial species i.e. Legionella, Pseudomonas, Campylobacter. This suggests that these new higher sugars are more common to bacteria than previously believed and may provide novel targets for intervention strategies. To date the biological significance of these unique carbohydrate moieties either as a component of LPS/capsules or as a constituent of a glycoprotein remains elusive but their existence in cell surface associated molecules suggests they are likely to contribute significantly in host interactions. In confrast to sialic acids, little is known regarding the biosynthesis of 5,7-diamino-3,5,7,9-tefradeoxynon-2-ulosonic acids, and Helicobacter pylori may provide a useful model system to determine the biosynthetic pathways and to develop functional assays for the enzymes involved in these glycosylation processes. Helicobacter pylori is a prevalent human pathogen and in its most severe manifestations is responsible for a number of gastric diseases including peptic ulcer and gastric adenocarcinoma. Motility of Helicobacter pylori is essential for colonization and pathogenesis and is one of only a few well defined virulence factors for this organism. Structural examination of wild type H. pylori flagellin by mass spectroscopy has identified unusual O-linked monosaccharide residues on at least 9 sites. This novel biosynthetic process offers new targets for therapeutic intervention. Table 1 Homology of predicted H. pylori proteins.

26695 "/..identity/ Homolog Annotation (accession no.)

Gene (length similarity in aa) (length in aa)

Hp0840 64/76 (334) C.jejuni CJ1293 sugar nucleotide epimerase dehydratase

(333aa) (AL139078) 59/72 (363) C. crescentus FlniA flagellin modification protein (AE005697) 54/68 (336) F. nucleatum FN1689 UDP-N-acetylglucosamine 4,6 dehydratase,

UDP-4-dehydro-6-deoxy-2-acetamido-d- glucose 4 reductase (AE010474).

45/64 (333) M. janneschii M J 1061 capsular polysaccharide biosynthesis protein

D homolog. (U67549)

Hp0178 49/69 (343) Cjejunl CJ1317 NeuNAc synthetase/flagellar (B81275)

(340 aa) 45/65 (350) C. acetyrobatylicum CAC2187 sialic acid synthase (AAI 80145)

42/63 (352) A. punclata NeuB LPS biosynthesis/flagellar modification

(AAD45660)

40/56 (356) C.crescentus CC2868, flagellar locus putative NeuNAc synthetase (AAK24832)

44/61 (354) S. melilott capsule locus putative polysaccharide biosynthesis protein

(CAB62155)

34/52 (338) L. pneumophila LPS locus N-acetylneuraminic condensing enzyme

(CAB65212)

33/53 (339) S. agalactiae capsule locus Putative n-acetyl neuraminic acid synthetase

(AA 43615)

35/55 (334) M.jannaschii MJ1065 Spore coat polysaccaride biosynthesis protein

Hp0326A 39/57 (232) C. cytidylyltransferase

(208 aa) 37/53 (228) A. cytidylyltransferase

31/52 (286) S. meliloti capsule locus, rkpN putative polysaccharide biosynthesis protein

(AJ245666)

33/54 (229) P. aeruginosa 0 antigen locus OrflO (AF498418)

HP0326B 23/39 (339) B. subtills, SpsG spore coat polysaccharide biosynthesis

(290aa) (Z99123) 20/39 (484) M.jannaschii MJ1062 (U67549) Protein G, spore coat polysaccharide biosynthesis

28/46 (339) C. acetobutylic m CAC21.86 glycosyltransferase(AAK80l44)

(Flagellar locus) 32/50 (371) Synechocystis sp. (strain PCC 6803) Undecaprenyl-PP-MURNAC- PENTAPEPTIDE-UDP GLCNAC-GLCNAC transferase

27/47 (292) A. punctata flagellin modification FlmD (AF126256) locus

HP0114 32/51 (610) C.jejuni hypothetical protein family Unknown (AL139078)

(628aa) (CJ1318 family) 23/41 (439) C. acetobutylicum uncharacterized Unknown (AE007717, AE007720, conserved family AE007719) Table 2 Colonization of CDl mice with Helicobacter isogenic mutants.

*number of mice in group with >2.00 logioCFU.

Claims

WE CLAIM:

1. An isolated polynucleotide which encodes a Helicobacter motility polypeptide or immunogenic fragment thereof comprising: (a) a sequence encoding a polypeptide having substantial sequence identity, with an amino acid sequence of SEQ. ID. NO. 2, 4, 18, 19 or 21;

(b) a polynucleotide complementary to (a);

(c) a polynucleotide differing from a polynucleotide of (a) or (b) due to degeneracy of the genetic code; or (d) an insertional mutant, or allelic or species variation of (a), (b) or (c).

2. An isolated polynucleotide as claimed in claim 1 which comprises:

(a) a polynucleotide having substantial sequence identity with a sequence of SEQ. ID. NO. 1, 3, 17 or 20; (b) a polynucleotide complementary to (a);

(c) a polynucleotide differing from a polynucleotides of (a) to (b) due to degeneracy of the genetic code; or

(d) an insertional mutant, or allelic or species variation of (a), (b) or (c).

3. A vector comprising a polynucleotide of claim 1 or 2.

4. A transformed host cell comprising a polynucleotide of claim 1 or 2.

5. An isolated Helicobacter motility polypeptide comprising an amino acid sequence of SEQ. ID. NO. 2, 4, 18, 19 or 21.

6. An isolated polypeptide having at least 70% amino acid sequence identity to an amino acid sequence of SEQ. ID. NO. 2, 4, 18, 19 or 21.

7. A method for preparing a Helicobacter motility polypeptide comprising an amino acid sequence of SEQ. ID. NO. 2, 4, 18, 19 or 21 comprising culturing a transformed host cell according to claim 4 to allow expression of the polypeptide and isolating the polypeptide.

8. An antibody having specificity against an epitope of a polypeptide as claimed in claim 5 or 6.

9. An antibody as claimed in claim 8 labelled with a detectable substance for detecting the polypeptide in a biological sample, tissue or cell.

10. A nucleotide probe comprising a polynucleotide of at least 10 nucleotides and capable of hybridizing under stringent conditions to a polynucleotide of SEQ. ID. NO. 1, 3, 17 or 20 or to a complement or genetically degenerate form thereof.

11. A method of diagnosing or monitoring a disease by determining the presence of (a) a polynucleotide as claimed in claim 1 or 2, or (b) a polypeptide as claimed in claim 5 or 6.

12. A method for identifying a compound having anti-polar flagellum activity, which method comprises: (a) exposing a polypeptide as claimed in claim 5 or 6 to a test sample of said compound and detecting any interaction of said polypeptide and said test sample, interaction being taken as an indication of anti-polar flagellum activity; or

(b) exposing a polynucleotide as claimed in claim 1 or 2 to a test sample of said compound and detecting any interaction of said polynucleotide and said test sample, interaction being taken as an indication of anti-polar flagellum activity; or

(c) exposing a bacterium having a polar flagellum to a test sample of said compound and detecting abnormal flagellar form or behaviour, said abnormal form or behaviour being taken as an indication of anti-polar flagellum activity; or (d) exposing a bacterium having a polar flagellum to a test sample of said compound and detecting abnormal accumulation of flagellar sugars or intermediates thereof, said abnormal accumulation being taken as an indication of anti-polar flagellum activity; or

(e) growing H. pylori bacterium in semisolid motility media in presence of said compound and detecting abnormal motility patterns, said abnormal motility patterns being taken as an indication of antipolar flagellum activity.