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WO2001000796A2 - Glycosyltransferases de helicobacter pylori utilisees comme nouvelle cible dans la prevention et le traitement des infections par h. pylori - Google Patents

Glycosyltransferases de helicobacter pylori utilisees comme nouvelle cible dans la prevention et le traitement des infections par h. pylori Download PDF

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Publication number
WO2001000796A2
WO2001000796A2 PCT/CA2000/000777 CA0000777W WO0100796A2 WO 2001000796 A2 WO2001000796 A2 WO 2001000796A2 CA 0000777 W CA0000777 W CA 0000777W WO 0100796 A2 WO0100796 A2 WO 0100796A2
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Prior art keywords
seq
glycosyltransferase
lps
leu
helicobacter
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WO2001000796A3 (fr
Inventor
Susan M. Logan
Warren Wakarchuk
Wayne Conlan
Mario A. Monteiro
Eleonora Altman
Koji Hiratsuka
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National Research Council of Canada
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National Research Council of Canada
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Priority to AU56684/00A priority Critical patent/AU5668400A/en
Priority to CA002377427A priority patent/CA2377427A1/fr
Publication of WO2001000796A2 publication Critical patent/WO2001000796A2/fr
Priority to US10/451,685 priority patent/US20040110261A1/en
Priority to AU2001268886A priority patent/AU2001268886A1/en
Priority to EP01947090A priority patent/EP1299527A2/fr
Priority to CA002417692A priority patent/CA2417692A1/fr
Priority to AU2001268885A priority patent/AU2001268885A1/en
Priority to PCT/CA2001/000970 priority patent/WO2002000888A1/fr
Priority to CA002417699A priority patent/CA2417699A1/fr
Priority to PCT/CA2001/000969 priority patent/WO2002000851A2/fr
Priority to EP01947091A priority patent/EP1299549A1/fr
Publication of WO2001000796A3 publication Critical patent/WO2001000796A3/fr
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/10—Transferases (2.)
    • C12N9/1048—Glycosyltransferases (2.4)
    • C12N9/1051—Hexosyltransferases (2.4.1)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/10—Transferases (2.)
    • C12N9/1048—Glycosyltransferases (2.4)
    • C12N9/1081—Glycosyltransferases (2.4) transferring other glycosyl groups (2.4.99)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P19/00—Preparation of compounds containing saccharide radicals
    • C12P19/44—Preparation of O-glycosides, e.g. glucosides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/51—Medicinal preparations containing antigens or antibodies comprising whole cells, viruses or DNA/RNA
    • A61K2039/52—Bacterial cells; Fungal cells; Protozoal cells
    • A61K2039/522—Bacterial cells; Fungal cells; Protozoal cells avirulent or attenuated
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies

Definitions

  • the invention relates to newly identified and isolated polynucleotides and polypeptides of bacterial origin, in particular to novel polynucleotides and polypeptides related to glycosyltransferases involved in biosynthesis of lipopolysaccharides of Helicobacter pylori.
  • Helicobacter pylori is a spiral, microaerophilic, Gram-negative bacterium infecting about 50% of the global human population, and is now recognised as the most common bacterial pathogen of humans worldwide. It is the causative agent of chronic active gastritis in all who harbour it, is responsible for the development of most gastro-duodenal ulcers, and is formally recognised as the carcinogen for certain gastric cancers (Blaser, Gastroenterology 102: 720-727 (1992); Parsonnet et al, N. Engl. J. Med. 325: 1127-1131 (1991 )). H.
  • LPS lipopolysaccharides
  • the sugars found in the O-chain vary among bacterial species, whereas the composition of the core polysaccharide is relatively constant.
  • Lipopolysaccharides are released from bacteria undergoing lysis and are toxic to animals and humans. They are often referred to as endotoxins.
  • H. pylori LPS unlike typical LPS, has low endotoxic properties.
  • Fresh clinical isolates usually display typical smooth type LPS (S-type). The O-chain polysaccharide structure of H.
  • pylori type strain (NCTC11637) LPS is composed of a type 2 ⁇ /-acetyllactosamine (LacNAc) chain of various lengths and this O-chain may be partially ⁇ -L- fucosylated or less commonly ⁇ -D-giucosylated or ⁇ -D-galactosylated and may be terminated at the nonreducing end by Lewis blood group epitopes which mimic human cell surface glycoconjugates and glycolipids.
  • Lewis blood group epitopes which mimic human cell surface glycoconjugates and glycolipids.
  • the Lewis antigens present on the O-chain polysaccharide might reduce the immunogenicity of this molecule during infection, or might trigger autoimmunity.
  • the ability to produce structurally defined truncated LPS molecules should help elucidate the biological role of LPS in H. pylori infection and immunity and possibly open a new approach to the treatment and prevention of H. pylori infections.
  • Known methods of prevention and treatment of H. pylori infections are either immunogenic or drug-based.
  • the immunogenic approach is mostly intended to provide an immunogenic protection against the bacterium by vaccinating the individual with a usually bacterium-derived immunogen, to elicit an immune response of the organism to future H. pylori infections.
  • immunogens antigens
  • derived from the LPS of H. pylori are known in this group of treatments (see, for example, WO 97/14782 and WO 87/07148).
  • H. pylori infections are treated with antibacterial drugs or combinations of such drugs, intended to eradicate the bacterial population in the infected individual.
  • antibacterial drugs or combinations of such drugs intended to eradicate the bacterial population in the infected individual.
  • triple therapies in which patients are administered simultaneously two different antibiotics and an acid secretion inhibiting drug.
  • the efficacy of these therapies varies and is often adversely affected by the developing resistance to broad spectrum antibiotics used for this purpose and by side effects of antibiotic therapies, which frequently result in termination of the therapy before completely healing the infection.
  • H. pylori drugs modulating the activity of enzymes specific to the bacteria
  • An ideal anti-helicobacterial drug should be selective, meaning that the drug should inhibit H. pylori but not the bacterial population of the microfiora of the lower intestine.
  • the molecular target of the drug should be unique to H. pylori and/or should be related to its unique phenotypic characteristics, particularly those facilitating the colonization of bacterium's natural ecological niche (the human stomach). While improving the understanding of H. pylori pathogenesis, the present invention provides means for developing new anti-helicobacterial drugs possessing such desirable characteristics.
  • the present invention provides isolated and/or recombinant nucleic acids which encode certain glycosyltransferases of Helicobacter origin.
  • the invention also provides recombinant DNA constructs and vectors containing polynucleotide sequences encoding such glycosyltransferases or portions thereof. These nucleic acids and constructs may be used to produce recombinant glycosyltransferases of Helicobacter origin by expressing the polynucleotide sequences in suitable host cells.
  • the invention provides isolated polypeptides having the enzymatic activity of glycosyltransferases of Helicobacter origin. Such polypeptides are useful, among other things, for the identification of modulators, in particular inhibitors of their enzymatic activity, which inhibitors are potential antimicrobial agents. Using the isolated polypeptides of the present invention, potential inhibitors of these enzymes can be screened for antimicrobial or antibiotic effects, without culturing pathogenic strains of Helicobacter bacteria, such as H. pylori.
  • preferred glycosyltransferases of Helicobacter origin are glycosyltransferases of H. pylori involved in the biosynthesis of the bacterial lipopolysaccharide (LPS), in particular of LPS core or
  • the present invention provides novel antigens and vaccines used in immunization against Helicobacter bacteria, in particular H. pylori.
  • the novel antigens are derived from bacteria having deactivated gene of a glycosyltransferase involved in the biosynthesis of the bacterial lipopolysaccharide, in particular of LPS core or LPS O-chain. Purified or partially purified LPS isolated from such mutants is a preferred antigen.
  • Fig. 1 shows amino acid sequence alignment of glycosyltransferases from H. pylori, H. influenzae, H. somnus and N. meningitidis. Multiple sequence alignment was performed using the Clustal Alignment Programme (Higgins et al, Gene 73: 237-244 (1988)). Designations on the left side refer to the origin of the sequences; HP0826 of genebank AE000594 (Tomb et al, Nature 388:539-547 (1997)), Haemophilus influenzae lex2B, U05670 (Cope et al, Mol. Microbiol.
  • Fig. 2 shows a complete FAB-MS spectrum of the methylated intact LPS of 26695::HP0826kan strain.
  • Fig. 3 is a schematic showing the chemical structure of LPS from parent strains 26695 and SS1 and isogenic mutants of HP0826, HP0159 and HP0479.
  • Fig. 4 shows results of CZE-MS/MS analysis (+ion mode) of delipidated LPS from H. pylori 26695::0159 mutant. Tandem mass spectrum of precursor ions at m/z 902 (doubly protonated ions). Separation conditions: 10 mM ammonium acetate containing 5% methanol, pH 9.0, +25 kV. For MS/MS experiments, nitrogen as a collision gas, Ei aD : 70 eV (laboratory frame of reference).
  • Fig. 5 shows results of CZE-MS/MS (+ion mode) analysis of delipidated LPS from H. pylori 0479 mutants. Tandem mass spectrum of precursor ions at m/z 1612. Separation conditions: 10 mM ammonium acetate containing 5% methanol, pH 9.0, +25 kV. For MS/MS experiments, nitrogen as a collision gas, E
  • identity and similarity mean the degree of sequence relatedness between two or more polynucleotide or polypeptide sequences as determined by the match between strings of such sequences. "Identity” or “similarity” can be readily quantified by algorithms well known to those skilled in the art, implemented in a number of publicly available computer software packages, for example BLAST software package available from NCBI and other sources. The identity or similarity is usually expressed as a percentage of identity with respect to some reference sequence.
  • a polynucleotide having a sequence 95% identical to a reference nucleotide sequence 5% of the nucleotides of the reference sequence have been deleted or substituted with another nucleotide, or 5% of another nucleotides have been inserted into the reference sequence.
  • substitutions, insertions, and/or deletions may take place anywhere between 5' and 3' terminal positions, either interspersed individually among nucleotides of the reference sequence or in one or more contiguous groups within the reference sequence.
  • isolated as used herein means altered by the hand of man with respect to its natural state. For a substance occurring in nature, it means that this substance has been changed or removed from its natural environment, or both.
  • a polynucleotide or a polypeptide naturally present in a living organism is not isolated, but the same polynucleotide or polypeptide separated from its natural matrix and coexisting materials is isolated, as the term is employed herein.
  • polynucleotide or “nucleic acid” refers to any polyribonucleotide or polydeoxyribonucleotide, which may be unmodified or modified RNA or DNA, whether single- or double-stranded.
  • polypeptide or “protein” refers to any peptide or protein comprising at least two amino acid residues joined to each other by peptide bonds or modified peptide bonds.
  • variant means a polynucleotide or polypeptide that differs from a reference polynucleotide or polypeptide but retains its essential properties.
  • a typical variant of a polynucleotide differs in nucleotide sequence from another, reference polynucleotide. Changes in the nucleotide sequence of the variant may or may not alter the amino acid sequence of a polypeptide encoded by the reference polynucleotide.
  • a typical variant of a polypeptide differs in amino acid sequence from another, reference polypeptide. These difference are usually limited and variants of a polypeptide are closely similar overall and identical in many regions.
  • a variant of a polynucleotide or polypeptide may be naturally occurring, such as an alleiic variant, or may be prepared by mutagenesis techniques, by direct synthesis, or by other recombinant methods well known to those skilled in the art.
  • a “fragment” can be considered as a variant of a polynucleotide or polypeptide, having the same nucleotide or amino acid sequence as part of the reference polynucleotide or peptide.
  • a fragment may be "free-standing” or comprised within a larger polynucleotide or polypeptide, normally as a single continuous region.
  • Nucleic acids referred to herein as "recombinant” are nucleic acids which have been produced by recombinant DNA methodology, including those nucleic acids that are generated by procedures which rely upon a method of artificial recombination, such as polymerase chain reaction (PCR) and/or cloning into a vector using restriction enzymes.
  • PCR polymerase chain reaction
  • the invention provides novel isolated polynucleotides and polypeptides, as described in greater detail below.
  • the invention provides isolated polynucleotides and polypeptides related to glycosyltransferases involved in the biosynthesis of bacterial lipopolysaccharides of bacteria of the genus Helicobacter, more particularly the lipopolysaccharides of the species Helicobacter pylori and various strains thereof.
  • the glucosyltransferases as those involved in the biosynthesis of the bacterial LPS, in particular of LPS core or LPS O-chain.
  • the invention provides isolated polynucleotides and polypeptides identical over their entire lengths to sequences set out in Table 1.
  • Preferred embodiments of the invention are polynucleotides coding for H. pylori glycosyltransferases involved in the biosynthesis of the core or O-chain regions of the bacterial lipopolysacchahde (LPS), in particular polynucleotides having sequences shown in Table 1 (SEQ ID NO: 1 , 3, 5, 7, 9, 11 , 13 and 15), polynucleotides closely related thereto, as well as fragments and variants thereof.
  • LPS bacterial lipopolysacchahde
  • Another preferred embodiments of the invention are polynucleotides that are at least 70% identical over their entire length to polynucleotides shown in Table 1 , preferably at least 80% identical, more preferably at least 90% identical, most preferably at least 95% identical, and polynucleotides that are complementary to such polynucleotides. Furthermore, those with at least 97% are highly preferred among those with at least 95%, and among these those with at least 98% and at least 99% are particularly highly preferred, with at least 99% being the most preferred.
  • polynucleotides showing substantial identity to the polynucleotides shown in Table 1 SEQ ID NO: 1 , 3, 5, 7, 9, 11 , 13 and 15
  • Polynucleotides shown in Table 1 correspond to open reading frames HP0826 (SEQ ID NO: 1 ), HP0159 (SEQ ID NO: 3), HP0479 (SEQ ID NO: 5) and HP1191 (SEQ ID NO:7) of the genomic DNA of H. pylori strain 26695, to open reading frames SS0826 (SEQ ID NO: 9), SS0159 (SEQ ID NO: 11 ) and SS0479 (SEQ ID NO: 13) of the genomic DNA of H. pylori strain SS1 , and to open reading frame PJ1-0479 (SEQ ID NO:15) of the genomic DNA of H. pylori strain PJ1.
  • ORFs HP0826, HP0159, HP0479 and HP1191 have been identified using the complete annotated genome sequence of H. pylori strain 26695 and BLAST analysis as potentially coding for glycosyltransferases. They have been proven, directly or indirectly, to encode a ⁇ -1 ,4-galactosyltransferase (HP0826), a ⁇ -1 ,6-glucosyltransferase (HP0159), a heptosyltransferase (HP0479), and an ADP-heptose-LPS heptosyltransferase II (HP1191 ), which are enzymes involved in the biosynthesis of the H. pylori lipopolysaccharide. ORFs identified by BLAST analysis have been cloned, expressed, and isolated using techniques well known to those skilled in the art, also discussed more in detail further in this disclosure.
  • the isolated polynucleotides of the present invention can be used in the production of polypeptides they encode.
  • a polynucleotide containing all or part of the coding sequence for a Helicobacter glycosyltransferase can be incorporated into various DNA constructs, such as expression cassettes, and vectors, such as recombinant plasmids, adapted for further manipulation of polypeptide sequences or for the production of the encoded polypeptide in suitable host cells, either eukaryotic, such as yeast or plant cells, or prokaryotic, such as bacteria, for example E. coli. This can be achieved using recombinant DNA techniques and methodologies well known to those skilled in the art.
  • the present invention further provides recombinant nucleic acids comprising polynucleotide sequences which encode glycosyltransferases involved in the biosynthesis of lipopolysaccharides of bacteria of the genus Helicobacter, more particularly of lipopolysaccharides of the species Helicobacter pylori and various strains thereof.
  • the invention provides recombinant nucleic acids comprising polynucleotides identical over their entire lengths to polynucleotides having sequences set out in Table 1 , as well as fragments and variants of such sequences.
  • fragments and variants preferred are those coding for polypeptides retaining the biological function or activity of the reference polypeptides.
  • the isolated polynucleotides and fragments thereof can also be used as DNA diagnostic probes specific to H. pylori, for diagnostic or similar purposes. They may be used, for example, to check whether or not the polynucleotides according to the present invention are transcribed in bacteria of an infected tissue. They may be also useful in diagnosis of the stage of infection and determining the specific pathogen involved.
  • the isolated polynucleotides of the present invention may further be used as hybridization probes for RNA, cDNA and genomic DNA, for example to isolate cDNA or genomic clones of other genes that have a high sequence similarity to the polynucleotides of the present invention. Such probes will comprise at least 15 bases, preferably at least 30 bases, but may have even more than 50 bases.
  • Preferred isolated or recombinant polypeptides of the present invention are those showing the activity of glycosyltransferases involved in biosynthesis of the bacterial lipopolysaccharides of bacteria of the genus Helicobacter, more particularly lipopolysaccharides of the species Helicobacter pylori and various strains thereof.
  • polypeptides coded by polynucleotides having sequences shown in Table 1 SEQ ID NOs: 1 , 3, 5, 7, 9, 11 , 13 and 15
  • polypeptides coded by polynucleotides having sequences shown in Table 1 SEQ ID NOs: 1 , 3, 5, 7, 9, 11 , 13 and 15
  • polypeptides coded by polynucleotides having sequences shown in Table 1 SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14 and 16
  • polypeptides closely related thereto as well as fragments and variants thereof preferred are those having the same biological function or activity as the polypeptides appearing in Table 1.
  • Polypeptides having amino acid sequences shown in Table 1 correspond to those coded by open reading frames HP0826 (SEQ ID NO: 2), HP0159 (SEQ ID NO: 4), HP0479 (SEQ ID NO: 6) and HP1191 (SEQ ID NO:8) of the genomic DNA of H. pylori strain 26695, by open reading frames SS0826 (SEQ ID NO: 10), SS0159 (SEQ ID NO: 12) and SS0479 (SEQ ID NO: 14) of the genomic DNA of H. pylori strain SS1 , and by open reading frame PJ0479 of the genomic DNA of H. pylori strain PJ1.
  • these ORFs have been cloned and expressed in suitable host cells and their function has been determined in vitro using techniques well known to those skilled in the art and discussed more in detail further in this disclosure.
  • Polypeptides of the present invention can be produced as discussed above in connection with recombinant nucleic acids of the present invention. They can be recovered and purified from recombinant cell cultures by methods and techniques well known to those skilled in the art, including ammonium sulfate or ethanol precipitation, acid extraction, and various forms of chromatography, in particular ion exchange and high performance liquid chromatography (HPLC). Well known techniques for refolding protein may be employed to regenerate active conformation when the polypeptide is denaturated during isolation and/or purification.
  • the invention also relates to methods of screening compounds, to identify those which enhance (agonists) or block (antagonists) the action of polynucleotides or polypeptides of the present invention.
  • antagonists acting as bacteriostatic or bactericidal agents are of particular interest.
  • Potential antagonists include small organic molecules, peptides, polypeptides and antibodies that bind to a polynucleotide or polypeptide of the present invention and therefore inhibit its activity.
  • Polynucleotides and polypeptides of the present invention may be used to assess the binding of small molecule substrates and ligands from various sources, including cells, cell-free preparations, chemical libraries, and natural product mixtures.
  • the substrates and ligands may be natural substrates and ligands or may be structural or functional mimetics.
  • Polypeptides of the present invention are particularly useful for screening chemical compounds modulating the enzymatic activity of glycosyltransferases of Helicobacter origin involved in the biosynthesis of bacterial lipopolysaccharides, to identify those which enhance (agonists) or inhibit (antagonists or inhibitors) the action of Helicobacter glycosyltransferases, in particular compounds that are bacteriostatic and/or bactericidal.
  • the method of screening may involve high- throughput techniques and assays. In a typical assay, a synthetic reaction mix comprising a polypeptide of the present invention and a labelled substrate or ligand of such polypeptide is incubated in the absence and in the presence of a candidate substance, a potential agonist or antagonist of the enzyme under study.
  • Detection of the rate or level of production of the product from the substrate may be enhanced by using a suitable reporter system, such as a colorimetricaily labelled substrate which is converted into a colorimetricaily assayable product or a reporter gene responsive to changes in the enzymatic activity of the polypeptide.
  • a suitable reporter system such as a colorimetricaily labelled substrate which is converted into a colorimetricaily assayable product or a reporter gene responsive to changes in the enzymatic activity of the polypeptide.
  • polypeptides of the present invention showing enzymatic activity of Helicobacter glycosyltransferases are also useful for the enzymatic synthesis of bacterial lipopolysaccharides and fragments thereof. When included in suitable reaction mixtures, these polypeptides catalyze the transfer of mono- or oligosaccharide residues to a suitable acceptor. In a preferred embodiment, the polypeptides of the present invention are used for the preparation of various mimics, analogues and derivatives of Helicobacter lipopolysaccharides.
  • the invention provides novel mutants of Helicobacter bacteria, in particular mutants of H. pylori, having mutated (deactivated) genes of glycosyltransferases involved in the biosynthesis of bacterial lipopolysaccharides, in particular of the core or O-chain regions of LPS.
  • Structural analysis of LPS isolated from the mutants confirmed that O-chain synthesis has been affected by the mutations and revealed the exact structure of the truncated LPS molecules.
  • the mutant strains were also shown to have a reduced capacity of gastric colonization.
  • the mutant bacteria expressing the truncated LPS and the LPS isolated from such mutants are useful as sources of antigens to be used 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.
  • the antigenic preparation is administered orally to the host, but parenteral admistration 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.
  • Chemically modified LPS isolated from mutants expressing the truncated LPS is a preferred antigen for use in vaccines according to the present invention. It is isolated from the 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 LPS are preferred. The purity of an LPS preparation is expressed as the weight percentage of the total Helicobacter antigens present in the preparation. The purified LPS can be used as antigen either directly or after being conjugated to a suitable carrier protein.
  • LgtB and LgtE proteins of N. meningitidis have been shown to be galactosyltransferases involved in the transfer of galactose in a ⁇ -1 ,4 linkage in the terminal lacto-N-neotetraose structure.
  • LgtB is responsible for the addition of Gal to GlcNAc, an identical function to that described here for HP0826, while LgtE catalyses the addition of Gal to Glc (Wakarchuk er al, supra).
  • Synthetic oligonucleotide primers which contained BamHI restriction sites which flanked the 5' and 3' ends of HP0826, HP0619, and HP0805 respectively, were used in a PCR reactions containing chromosomal DNA of H. pylori 26695 or SS1 as a template. A single PCR product was obtained in each case and this was cloned into pUC19 to give plasmids pHP0826, pHP0805, and pHP0619. DNA sequencing was used to confirm the identity of the cloned PCR products from 26695 and SS1.
  • HP0159 displayed homology to the rfaJ, lipopolysaccharide 1 ,2-glucosyltransferase gene from a number of bacterial species
  • HP0479 and HP1191 displayed homology to waaC and waaF respectively, which are heptosyltransferase genes responsible for the addition of LD heptose to KDO in the core backbone.
  • ⁇ -1 ,4-galactosyltransferase activity was also present in parent H. pylori strains but not in the H. pylori HP0826 mutants.
  • the assays were followed by TLC analysis of the reaction mixtures as previously described (Gilbert et al, Eur. J. Biochem. 249: 187-194 (1997)).
  • a more sensitive capillary electrophoresis (CE) analysis of the reaction mixtures clearly demonstrated a loss of galactosyltransferase activity in the mutants.
  • the product of the enzymatic reaction had an identical CE mobility compared to a known FCHASE- aminophenyl- ⁇ -N-acetyllactosamine standard, and was subjected to NMR analysis to determine the linkage.
  • the 1 H and 13 C chemical shift data (Table 2) and 1 D NOE analysis were consistent with the linkage of the Gal being ⁇ -1 ,4 to the GlcNAc.
  • the product was also sensitive to ⁇ -galactosidase.
  • Their respective 13 C signals are at (132.5, 123.3, 121.5), (132.7, 121.5, 104.3) and (131.1 , 121.5, 104.3) ppm.
  • Functional analysis of rfaJ homologue HP0159
  • Enzyme activity was detected in extracts of E. coli pHP0159 using the synthetic acceptor molecule FCHASE aminophenyl- ⁇ -maltose or FCHASE aminophenyl- ⁇ - glucose and UDP-Glc as the donor. Activity was also present in parent H. pylori strains but not in H. pylori HP0159 mutants.
  • the assays were followed by TLC and CE analysis of the reaction mixtures as previously described (Gilbert er al, Eur. J. Biochem. 249: 187-194 (1997)).
  • the more sensitive capillary electrophoresis (CE) analysis of the reaction mixtures demonstrated a loss of glucosyltransferase activity in the mutants.
  • Complementation analysis was used to determine the function of the HP1191 from Helicobacter pylori strain 26695.
  • the H. pylori HP1191 gene was amplified by PCR (see Table 6 for primer sequences used) and cloned into pUC19 to obtain pHP1191.
  • WaaF mutant strain S. typhimurium 3789 was electroporated with this recombinant plasmid, and one of the resultant transformants selected for further study.
  • SDS-PAGE was used to analyze LPS molecules produced by the relevant S. typhimurium strains.
  • the LPS of the wild type strain formed the ladder like pattern indicative of the presence of the O antigen repeat unit whereas the LPS of the S.
  • H. pylori mutants carrying a disrupted HP0826 gene were constructed by alleiic exchange. Briefly, the HP0826 ORF cloned in pUC19 was disrupted by using reverse primers 5TACAGATCGCTTCATTGAGTTCT3" and
  • Km r colonies were isolated and passaged on the same medium. Individual colonies were selected and screened for the presence of a double cross over mutation in the chromosome of the kan mutant. To assess the insertion site of the disrupted gene PCR analysis was used, with chromosomal DNA from parent and mutant H. pylori strains as templates and the primer pair 5 ⁇ CACTGGCATCATACAAT3' and
  • H. pylori mutants carrying disrupted HP0159 and HP0479 genes was carried out in essentially the same manner as above.
  • LPS molecules of H. pylori strains 26695, SS1 ( M. A. Monteiro et al, Eur. J. Biochem. 267: 305-320 (2000) and type strain NCTC 11637 (Aspinall et al, supra) have been determined to carry O- chain regions that express Le x and Le y blood-group determinants. These Lewis-mimicking O chains were shown to be covalently connected to a core oligosaccharide. Sugar composition analysis (Table 4) of the intact LPSs of H.
  • pylon 26695::HP0826kan, SS1 ::HP0826kan and NCTC 11637::HP0826kan demonstrated a clear reduction in levels of those sugars known to form the O chain components, namely L-Fuc, D-Gal and D- GlcNAc, when compared to parent LPSs.
  • Methylation linkage analysis performed on the intact H. pylori mutant LPSs from each strain showed the presence of terminal and 3-substituted Fuc, terminal, 3-, and 6-(except in SS1 strain) substituted Glc, terminal, 3- and 4-substituted Gal, 2- (only in 26695), 3-(only in 26695), 6-(only in 26695), 7- and 2,7-substituted DD- Hep, 2- and 3,7-substituted LD-Hep, and terminal and 3-substituted GlcNAc units. All sugars were present in the pyranose conformation.
  • A-type primary glycosyl oxonium ions containing Lewis blood-group related Fuc and GlcNAc residues were observed at m/z 434 [Fuc, GlcNAc] , 508 [GlcNAc, Hep] + , and 682 [Fuc, GlcNAc, Hep] + .
  • the ion m/z 434 stood for a disaccharide composed of Fuc and GlcNAc and ion m/z 508 characterized a possible connection between the O-chain related GlcNAc and a heptose from the core region.
  • the ion m/z 682 [Fuc, GlcNAc, Hep] represented a moiety containing GlcNAc and Fuc residues of the O-chain region and a single heptose unit from the core region which bridges the O-chain and the core oligosaccharide. Since no terminal Hep unit was detected, these m/z 508 and 682 ions must originate from cleavage at the heptose glycosidic bond and represent a partial O-chain repeating unit [Fuc, GlcNAc, Hep] + .
  • Methylation analysis of the intact LPS from each strain showed the presence of terminal and 3-substituted L-Fuc, terminal and 4-substituted D-Glc, terminal, 3- and 4-substituted D-Gal, terminal, 2-, 6-, 7- and 2,7-substituted DD-Hep, terminal, 2- and 3-substituted LD-Hep and terminal, 3-substituted and 4-substituted D- GlcNAc. All sugars were present in the pyranose form.
  • the primary ion m/z 668 and its corresponding secondary ion m/z 228 pointed to the presence of the type 1 linear B blood group [Gal(1-3)Gal(1-3)GlcNAc] antigen, a blood group antigen found in the LPS of 26695 and SS1 (Monteiro et al, Eur. J. Biochem. 267:305-320 (2000)).
  • LPS from 26695::HP0159kan was treated with 0.1 M sodium acetate buffer, pH 4.2 (2 h, 100°C) and following the removal of lipid A by low speed centrifugation, subjected to the gel filtration chromatography on a Bio-Gel P-2 column, followed by capillary electrophoresis-electrospray mass spectrometry (CE-ESMS) as described previously (Thibault and Richards, Meth. Mol. Biol. 145: 327-343
  • FAB-MS analysis in the positive mode of the permethylated LPS from each strain indicated the presence of primary glycosyl oxonium ions at m/z 260 [GlcNAc] + and m/z 434 [Fuc,GlcNAc] + and secondary glycosyl oxonium ions at m/z 228 (260-32) [GlcNAc] + and m/z 402 (434-32) [Fuc,GlcNAc] + .
  • the parent SS1 cells produce considerable amounts of S type LPS displaying Lewis Y epitopes while cells in which HP0826 has been inactivated produce a faster migrating, rough type LPS molecule no longer displaying Lewis epitopes.
  • groups of mice were gavaged with either wild-type or mutated H. pylori SS1. Representative mice from each group were killed 6 or 12 weeks later and the stomach burdens of H. pylori, and level of Helicobacter- specific circulating immunoglobulin G determined.
  • pylori SS1 bacteria unable to produce S-type LPS are significantly impaired in their ability to initially colonise the murine stomach.
  • mice colonization experiments for the parent (SS1 ) strain of H. pylori and their mutant strains SS0826, SS0159 and SS0479 are summarized in Table 5.
  • mice colonization data Numbers in the table show levels of colonization of mice stomachs (as log 10 CFU/stomach +/- standard deviation) after the indicated number of weeks (WK) of infection. ND: not determined BDL: less than 500 bacteria
  • Exp 1 Individual mice inoculated by gavage on D1 , D3, D6 with 0.2ml of broth grown cells suspended in PBS at cell concentration of ⁇ 1 x 10 10 /ml.
  • Exp 2 Individual mice inoculated by gavage on D1 + D3 with 0.2ml of broth grown cells suspended in PBS at cell concentration of ⁇ 2 x 10 10 /ml.
  • mice Individual mice inoculated by gavage on D1 and D3 with 0.2ml of broth grown cells suspended in PBS at cell concentration of
  • SS0479 strain (/-/. pylori strain SS1 having disrupted gene HP0479) is the least capable of colonization.
  • H. pylori strain 26695 (Tomb et al, supra) used for the initial cloning was obtained from R. A. Aim, Astra, Boston.
  • H. pylori strain SS1 was obtained from A. Lee.
  • H. pylori reference strain ATCC43504 and H. pylori serogroup 0:3 isolate were from J. Penner.
  • PJ1 was a fresh clinical isolate of H. pylori.
  • Helicobacter strains were grown on at 37°C on antibiotic supplemented (Lee et al, supra) trypticase soy agar plates containing 7% horse blood (GSS agar) in a microaerophilic environment for 48h (Kan 20 ⁇ g/ml).
  • H. pylori cells harvested from 48h trypticase soy agar/horse blood plates and incubated for 36h in a Trigas (Nuaire, Madison, MN) incubator (85% N 2 , 10%CO 2 , 5%0 2 ) on a shaking platform.
  • Esche chia coli strain DH5 ⁇ was used as host for plasmid cloning experiments and was grown on L-agar plates at 37°C supplemented with ampicillin (50 ⁇ gml "1 ) and/or kanamycin (20 ⁇ gml "1 ) ⁇ -1 ,4-galactosyltransferase activity
  • Glycosyltransferase assays were performed essentially as described previously (Gilbert et al., supra). Cells were scraped from a 3 day old plate culture of H. pylori, the cells were stored frozen at -20°C. Cell extracts were made by mixing the cell pellet with 2 volumes of glass beads, and grinding with a ground glass pestle in the microcentrifuge tube. The paste was extracted twice with 50 ⁇ l of 50 mM MOPS-NaOH buffer pH 7.0.
  • Reactions contained 0.5 mM FCHASE- aminophenyl- ⁇ -GlcNAc, 10 mM MnCI 2 , 0.5 mM UDP-Gal, 50 mM MOPS-NaOH pH 7.0, and 10 ⁇ l of cell extract in a final volume of 20 ⁇ l.
  • FCHASE- aminophenyl- ⁇ -GlcNAc 10 mM MnCI 2
  • UDP-Gal 0.5 mM UDP-Gal
  • 50 mM MOPS-NaOH pH 7.0 50 mM MOPS-NaOH pH 7.0
  • DNA sequencing of PCR products was performed using an Applied Biosystems (model 370A) automated DNA sequencer using the manufacturers cycle sequencing kit. All standard methods of DNA manipulation were performed according to the protocols of Sambrook et al, Molecular Cloning: A Laboratory Manual, 2 nd edn. Cold Spring Harbor, New York: Cold Spring Harbor Laboratory Press (1989). DNA probes for Southern blotting were labelled with DIG-11-dUTP using DIG-High Prime (Boehringer Mannheim, Montreal, Canada) and detection of hybridized probe with DIG Luminescent Detection Kit (Boehringer Mannheim Montreal, Canada). Primers used for the PCR gene amplification and for mutant constructs are shown in Table 6. Table 6. Primer sequences for PCR amplification of HP0826, HP0159, HP0479 and HP1191 genes and for construction of respective mutant strains .
  • HP0479-mutF1 CAAAACCGCCAGGAGTTG
  • Electrophoresis and Western blotting SDS-PAGE was performed with a mini-slab gel apparatus (Biorad) by the method of Laemmli (Nature 227: 680-685 (1970)).
  • LPS samples were prepared from whole cells according to a previously described method (Logan et al, Infect. Immun. 45: 210-216 (1984)), equivalent amounts loaded in each lane and stained according to Tsai er al (Anal. Biochem. 119: 115-119 (1982)) or transferred to nitrocellulose for immunological detection as previously described (Logan et al, supra).
  • Anti Lewis monoclonal antibodies (Signet Laboratories Inc, Dedham, MA) were used at 1 :500 dilution.
  • the LPSs were isolated by the hot phenol-water extraction procedure (Westphal er al, Meth. Carbohydr. Chem. 5: 83-91 (1965)).
  • the LPSs were purified by gel- permeation-chromatography on a column of Bio-Gel P-2 (1 m x 1cm) with water as eluent. In all cases, only one carbohydrate positive fraction was obtained which eluted in the high M r range (Dubois et al, Anal. Chem. 28: 350-356 (1956)). These intact H. pylori LPSs then were used for chemical analyses.
  • Alditol acetate derivatives were analyzed by gas-liquid-chromatography mass-spectrometry (GLC-MS) using a Hewlett-Packard chromatograph equipped with a 30 m DB-17 capillary column [210°C (30 min) to 240°C at 2°C/min] and MS in the electron impact (El) mode was recorded using a Varian Saturn II mass spectrometer.
  • Methylation linkage analysis was carried out by the NaOH/DMSO/CH 3 l procedure (Ciucanu er al, Carbohydr. Res.
  • a fraction of the methylated sample was used for positive ion fast atom bombardment-mass spectrometry (FAB-MS) which was performed on a Jeol JMS-AX505H mass spectrometer with glycerol(1 ) : thioglycerol(3) as the matrix.
  • a 6 kV Xenon beam was used to produce pseudo molecular ions which were then accelerated to 3kV and their mass analyzed.
  • Product ion scan (B/E) and precursor ion scan (B 2 /E) were preformed on metastable ions created in the first free field with a source pressure of 5x10 "5 torr.
  • the interpretations of positive ion mass spectra of the permethylated LPS derivatives were as previously described by Dell et al (Carbohydr. Res. 200: 59-67 (1990).
  • Samples were analyzed on a crystal Model 310 CE instrument (ATI Unicam, Boston, MA, USA) coupled to an API 3000 mass spectrometer (Perkin- Elmer/Sciex, Concord, Canada) via a microlonspray interface.
  • a sheath solution (isopropanol-methanol, 2:1 ) was delivered at a flow rate of 1 ⁇ L/min to a low dead volume tee (250 ⁇ m i.d., Chromatographic Specialties, Brockville, Canada). All aqueous solutions were filtered through a 0.45- ⁇ m filter (Millipore, Bedford, MA, USA) before use.
  • An electrospray stainless steel needle (27 gauge) was butted against the low dead volume tee and enabled the delivery of the sheath solution to the end of the capillary column.
  • the separation were obtained on about 90 cm length bare fused-siiica capillary using 10 mM ammonium acetate/ammonium hydroxide in deionized waster, pH 9.0, containing 5% methanol.
  • a voltage of 25 kV was typically applied at the injection.
  • the outlet of the capillary was tapered to ca. 15 ⁇ m i.d. using a laser puller (Sutter Instruments, Novato, CA, USA). Mass spectra were acquired with dwell times of 3.0 ms per step of 1 m/z unit in full- mass-scan mode.
  • mice were purchased from Charles Rivers Laboratories, Montreal when they were 6-8 weeks old. Mice were maintained and used in accordance with the recommendations of the Canadian Council on Animal Care, Guide to the Care and Use of Experimental Animals (1993). Mice were inoculated with bacteria harvested from 36h broth culture. Aliquots of 0.2 ml, containing approximately 10 8 bacteria resuspended in PBS were given by gavage directly into the gastric lumen using a 20g gavage needle. Three inocula were given over a period of 6 days. No attempt was made to neutralize gastric acidity prior to inoculation. To recover viable bacteria from the stomach, mice were killed by C0 2 asphyxiation, and their stomachs removed whole.
  • Stomachs were cut open along the greater curvature, and the exposed lumenal surface was gently irrigated with 10 ml of sterile PBS, delivered via a syringe fitted with a 20g gavage needle, to dislodge the loosely adherent stomach contents. This step effectively diminished the small numbers of ubiquitous contaminating bacteria that otherwise overgrow on GSS agar to thereby 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.
  • Sera for antibody determinations were prepared from clotted blood obtained from a lateral tail vein during the course of an experiment or by cardiac puncture at the time of necropsy. Sera were screened for the presence of specific IgG isotype anti- H. pylori antibodies by ELISA essentially by the method of Engvall et al (J. Immunol. 109: 129-135 (1972)). Briefly, microtitre plates (Dynatech Immunolon II) were coated with 100 ⁇ l antigen (50 ⁇ g protein/ml in 0.05M carbonate buffer pH 9.8) and incubated overnight at 4°C. Antigen was prepared by resuspending plate grown H.
  • Titres were determined from plots of absorbance at 410 nm versus dilution and were defined as the reciprocal of the dilution giving an A» ⁇ o equivalent to 0.25. Standard negative and positive control sera identified by a preliminary ELISA of candidate samples were included on each plate. Titres were analysed statistically by Mann Whitney Rank Sum Test and were considered to be significantly different to comparative samples when p values ⁇ 0.05 were obtained.

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Abstract

L'invention concerne de nouveaux polynucléotides isolés codant pour les glycosyltransférases participant à la biosynthèse du lipopolysaccharide de Helicobacter pylori ainsi que des structures d'ADN et des vecteurs recombinants contenant des séquences polynucléotidiques codant pour ces glycosyltransférases. Ces structures d'acides nucléiques et vecteurs peuvent être utilisés dans la préparation des glycosyltransférases pour lesquelles elles codent au moyen de l'expression des séquences polynucléotidiques codantes dans des cellules cibles appropriés. L'invention concerne aussi des polypeptides isolés possédant l'activité enzymatique des glycosyltransférases de Helicobacter. Ces polypeptides sont particulièrement utiles pour cribler les modulateurs de leur activité enzymatique, notamment les inhibiteurs enzymatiques possédant une éventuelle activité antibactérienne.
PCT/CA2000/000777 1999-06-28 2000-06-28 Glycosyltransferases de helicobacter pylori utilisees comme nouvelle cible dans la prevention et le traitement des infections par h. pylori Ceased WO2001000796A2 (fr)

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AU56684/00A AU5668400A (en) 1999-06-28 2000-06-28 Glycosyltransferases of helicobacter pylori as a new target in prevention and treatment of h. pylori infections
CA002377427A CA2377427A1 (fr) 1999-06-28 2000-06-28 Glycosyltransferases de helicobacter pylori utilisees comme nouvelle cible dans la prevention et le traitement des infections par h. pylori
EP01947091A EP1299549A1 (fr) 2000-06-28 2001-06-28 Polypeptides de l'heptosyltransferase d'helicobacter pylori
US10/451,685 US20040110261A1 (en) 2000-06-28 2001-06-28 Helicobacter dd-heptosyltransferase
AU2001268886A AU2001268886A1 (en) 2000-06-28 2001-06-28 Helicobacter pylori heptosyl transferase polypeptides
EP01947090A EP1299527A2 (fr) 2000-06-28 2001-06-28 Helicobacter dd-heptosyltransferase
CA002417692A CA2417692A1 (fr) 2000-06-28 2001-06-28 Helicobacter dd-heptosyltransferase
AU2001268885A AU2001268885A1 (en) 2000-06-28 2001-06-28 Helicobacter dd-heptosyltransferase
PCT/CA2001/000970 WO2002000888A1 (fr) 2000-06-28 2001-06-28 Polypeptides de l'heptosyltransferase d'helicobacter pylori
CA002417699A CA2417699A1 (fr) 2000-06-28 2001-06-28 Polypeptides de l'heptosyltransferase d'helicobacter pylori
PCT/CA2001/000969 WO2002000851A2 (fr) 2000-06-28 2001-06-28 Helicobacter dd-heptosyltransferase

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Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2002000888A1 (fr) * 2000-06-28 2002-01-03 National Research Council Of Canada Polypeptides de l'heptosyltransferase d'helicobacter pylori
WO2002000851A3 (fr) * 2000-06-28 2002-03-28 Ca Nat Research Council Helicobacter dd-heptosyltransferase
WO2002007763A3 (fr) * 2000-07-12 2002-06-20 Chiron Spa Helicobacter pylori avec biosynthese de lipopolysaccharides modifies
US6830908B2 (en) 1998-02-04 2004-12-14 Kyowa Hakko Kogyo Co., Ltd. Glycosyltransferase and DNA encoding the same

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CN110997714B (zh) * 2017-02-17 2024-05-03 赛诺菲 对肌营养不良蛋白聚糖和层粘连蛋白-2具有特异性的多特异性结合分子

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WO1996040893A1 (fr) * 1995-06-07 1996-12-19 Astra Aktiebolag Sequences d'acide nucleique et d'acides amines concernant helicobacter pylori, utilisees a des fins diagnostiques et therapeutiques
JPH11221079A (ja) * 1998-02-04 1999-08-17 Kyowa Hakko Kogyo Co Ltd 糖転移酵素および該酵素をコードするｄｎａ

Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6830908B2 (en) 1998-02-04 2004-12-14 Kyowa Hakko Kogyo Co., Ltd. Glycosyltransferase and DNA encoding the same
WO2002000888A1 (fr) * 2000-06-28 2002-01-03 National Research Council Of Canada Polypeptides de l'heptosyltransferase d'helicobacter pylori
WO2002000851A3 (fr) * 2000-06-28 2002-03-28 Ca Nat Research Council Helicobacter dd-heptosyltransferase
WO2002007763A3 (fr) * 2000-07-12 2002-06-20 Chiron Spa Helicobacter pylori avec biosynthese de lipopolysaccharides modifies

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