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J M Reyrat

Publications and source records attributed to J M Reyrat.

At least 19 recordsLinked to original sources
PubMed2000

A structural overview of the Helicobacter cytotoxin.

VacA, the major exotoxin produced by Helicobacter pylori, is composed of identical 87-kDa monomers that assemble into flower-shaped oligomers. The monomers can be proteolytically cleaved into two moieties of 37 and 58 kDa, or P37 and P58. The most studied property of VacA is the alteration of intracellular vesicular trafficking in eukaryotic cells leading to the formation of large vacuoles containing markers of late endosomes and lysosomes. However, VacA also causes a reduction in trans-epithelial electrical resistance in polarized monolayers and forms ion channels in lipid bilayers. The ability to induce vacuoles is localized mostly but not entirely in P37, while P58 is involved in cell targeting. Here, we review the structural aspects of VacA biology.

Bacterial Proteins↗
PubMed1999

3D imaging of the 58 kDa cell binding subunit of the Helicobacter pylori cytotoxin.

Pathogenic strains of Helicobacter pylori produce a potent exotoxin, VacA, which intoxicates gastric epithelial cells and leads to peptic ulcer. The toxin is released from the bacteria as a high molecular mass homo-oligomer of a 95 kDa polypeptide which undergoes specific proteolytic cleavage to 37 kDa and 58 kDa subunits. We have engineered a strain of H. pylori to delete the gene sequence coding for the 37 kDa subunit. The remaining 58 kDa subunit is expressed efficiently and exported as a soluble dimer that is non-toxic but binds target cells in a manner similar to the holotoxin. A 3D reconstruction of the molecule from electron micrographs of quick-freeze, deep-etched preparations reveals the contribution of each building block to the structure and permits the reconstruction of the oligomeric holotoxin starting from individual subunits. In this model P58 subunits are assembled in a ring structure with P37 subunits laying on the top. The data indicate that the 58 kDa subunit is capable of folding autonomously into a discrete structure recognizable within the holotoxin and containing the cell binding domain.

Bacterial Proteins↗
PubMed1999

Helicobacter pylori vacuolating toxin forms anion-selective channels in planar lipid bilayers: possible implications for the mechanism of cellular vacuolation.

The Helicobacter pylori VacA toxin plays a major role in the gastric pathologies associated with this bacterium. When added to cultured cells, VacA induces vacuolation, an effect potentiated by preexposure of the toxin to low pH. Its mechanism of action is unknown. We report here that VacA forms anion-selective, voltage-dependent pores in artificial membranes. Channel formation was greatly potentiated by acidic conditions or by pretreatment of VacA at low pH. No requirement for particular lipid(s) was identified. Selectivity studies showed that anion selectivity was maintained over the pH range 4.8-12, with the following permeability sequence: Cl- approximately HCO3- > pyruvate > gluconate > K+ approximately Li+ approximately Ba2+ > NH4+. Membrane permeabilization was due to the incorporation of channels with a voltage-dependent conductance in the 10-30 pS range (2 M KCl), displaying a voltage-independent high open probability. Deletion of the NH2 terminus domain (p37) or chemical modification of VacA by diethylpyrocarbonate inhibited both channel activity and vacuolation of HeLa cells without affecting toxin internalization by the cells. Collectively, these observations strongly suggest that VacA channel formation is needed to induce cellular vacuolation, possibly by inducing an osmotic imbalance of intracellular acidic compartments.

Bacterial Proteins↗
PubMed1999

Towards deciphering the Helicobacter pylori cytotoxin.

VacA, the major exotoxin produced by Helicobacter pylori, is composed of identical 87 kDa monomers that assemble into flower-shaped oligomers. The monomers can be proteolytically cleaved into two moieties, one of 37 and the other of 58 kDa, named P37 and P58 respectively. The most studied property of VacA is the alteration of intracellular vesicular trafficking in eukaryotic cells leading to the formation of large vacuoles containing markers of late endosomes and lysosomes. However, VacA also causes a reduction in transepithelial electrical resistance in polarized monolayers and forms ion channels in lipid bilayers. The ability to induce vacuoles is localized mostly but not entirely in P37, whereas P58 is mostly involved in cell targeting. Until recently, H. pylori isolates were classified as tox+ or tox-, depending on whether they induced vacuoles in HeLa cells or not. Today, we know that almost all strains are cytotoxic. The major difference between tox+ and tox- resides in the cell binding domain, which exists in two allelic forms, only one of which is toxic for HeLa cells. The two forms, named m1 and m2, are found predominantly in Western and Chinese isolates respectively.

Bacterial Proteins↗
PubMed1998

The m2 form of the Helicobacter pylori cytotoxin has cell type-specific vacuolating activity.

The Helicobacter pylori toxin VacA causes vacuolar degeneration in mammalian cell lines in vitro and plays a key role in peptic ulcer disease. Two alleles, m1 and m2, of the mid-region of the vacA gene have been described, and the m2 cytotoxin always has been described as inactive in the in vitro HeLa cell assay. However, the m2 allele is associated with peptic ulcer and is prevalent in populations in which peptic ulcer and gastric cancer have high incidence. In this paper, we show that, despite the absence of toxicity on HeLa cells, the m2 cytotoxin is able to induce vacuolization in primary gastric cells and in other cell lines such as RK-13. The absence of Hela cell activity is due to an inability to interact with the cell surface, suggesting a receptor-mediated interaction. This result is consistent with the observation that the m2 allele is found in a population that has a high prevalence of peptic ulcer disease and gastric cancer. VacA is the first bacterial toxin described for which the same active subunit can be delivered by different receptor binding domains.

Alleles↗
PubMed1998

Characterisation of a monoclonal antibody and its use to purify the cytotoxin of Helicobacter pylori.

The vacuolating cytotoxin (VacA) is a major virulence factor of Helicobacter pylori which is not yet well characterised and is difficult to obtain in large quantities. Here we describe the production of a monoclonal antibody that recognises the native but not the denatured form of VacA. The antibody can be efficiently used in affinity chromatography for one-step purification of large quantities of VacA from culture supernatants. Elution at acidic pH dissociates the oligomeric molecule into monomers that reanneal in a time-dependent fashion. The purified cytotoxin is able to bind, and to intoxicate HeLa cells.

Animals↗
PubMed1998

Genetic advances for studying Mycobacterium tuberculosis pathogenicity.

Tuberculosis remains the greatest cause of death worldwide because of a single pathogen. Despite its importance, the genetic basis of the pathogenicity of Mycobacterium tuberculosis remains poorly understood, mainly because the most productive investigative approach, molecular genetic analysis, has been severely hampered by a lack of efficient tools. However, significant advances, including the development of methods for inactivating genes and studying their expression with reporter genes, have been recently made. This progress may lead to opportunities for developing new vaccines and antituberculous drugs. The aim of this review is to examine the present state of the art in mycobacterial molecular genetics and pinpoint some expected or promising areas for future research.

Animals↗
PubMed1998

Deletion of the major proteolytic site of the Helicobacter pylori cytotoxin does not influence toxin activity but favors assembly of the toxin into hexameric structures.

The Helicobacter pylori cytotoxin is proteolytically cleaved at a flexible hydrophilic loop into two subunits. Deletion of the loop sequences had no effect on biological activity of the toxin in the HeLa cell vacuolation assay but favored the organization of the protein into hexameric rather than heptameric structures.

Bacterial Proteins↗
PubMed1997

Efficient allelic exchange and transposon mutagenesis in Mycobacterium tuberculosis.

A better understanding of Mycobacterium tuberculosis virulence mechanisms is highly dependent on the design of efficient mutagenesis systems. A system enabling the positive selection of insertional mutants having lost the delivery vector was developed. It uses ts-sacB vectors, which combine the counterselective properties of the sacB gene and a mycobacterial thermosensitive origin of replication and can therefore be efficiently counterselected on sucrose at 39 degrees C. This methodology allowed the construction of M. tuberculosis transposition mutant libraries. Greater than 10(6) mutants were obtained, far exceeding the number theoretically required to obtain at least one insertion in every nonessential gene. This system is also efficient for gene exchange mutagenesis as demonstrated with the purC gene: 100% of the selected clones were allelic exchange mutants. Therefore, a single, simple methodology has enabled us to develop powerful mutagenesis systems, the lack of which was a major obstacle to the genetic characterization of M. tuberculosis.

Alleles↗
PubMed1997

Contribution of beta-lactamase production to the resistance of mycobacteria to beta-lactam antibiotics.

Mycobacterium fallax (M. fallax) is naturally sensitive to many beta-lactam antibiotics (MIC < 2 microg/ml) and devoid of beta-lactamase activity. In this paper, we show that the production of the beta-lactamase of Mycobacterium fortuitum by M. fallax significantly increased the MIC values for good substrates of the enzyme, whereas the potency of poor substrates or transient inactivators was not modified. The rates of diffusion of beta-lactams through the mycolic acid layer were low, but for all studied compounds the half-equilibration times were such that they would only marginally affect the MIC values in the absence of beta-lactamase production. These results emphasize the importance of enzymatic degradation as a major factor in the resistance of mycobacteria to penicillins.

Anti-Bacterial Agents↗
PubMed1996

Positive selection of allelic exchange mutants in Mycobacterium bovis BCG.

sacB expression is lethal to mycobacteria in the presence of sucrose. It can therefore serve as 1 counter-selectable marker for positive selection of gene replacement events as demonstrated in the fast-growing Mycobacterium smegmatis. With this methodology, a sucrose counter-selectable vector was used to deliver, into the Mycobacterium bovis BCG genome, an inactivated copy (ureC::Km) of the ureC gene encoding the mycobacterial urease. A two-step selection procedure on 2% sucrose allowed the positive selection of gene exchange mutants. This technique should thus be extremely useful for the genetic analysis of pathogenic mycobacteria.

Alleles↗
PubMed1996

Generation of unmarked directed mutations in mycobacteria, using sucrose counter-selectable suicide vectors.

The expression of sacB, the Bacillus subtilis gene encoding levansucrase, is lethal to mycobacteria in the presence of 10% sucrose. In this study, we describe the use of sacB as a marker for positive selection of gene-replacement events into Mycobacterium smegmatis. A sucrose counter-selectable suicide plasmid was used to deliver an inactivated copy of the pyrF gene (pyrF::K(m)) into the M. smegmatis genome. Only uracil auxotroph clones, resulting from replacement of the endogenous pyrF allele, survived in a one-step selection on plates containing kanamycin and 10% sucrose. This demonstrated that selection on sucrose against the maintenance of the vector bearing the sacB gene is 100% efficient, enabling the positive selection of allelic-exchange mutants. Two-step selection is also feasible; it was used to construct unmarked pyrF mutants in which the gene was inactivated by a frameshift mutation. This method of generating unmarked, directed mutations is rapid and simple, making it a powerful tool for the genetic characterization of mycobacteria.

Alleles↗
PubMed1996

Urease activity does not contribute dramatically to persistence of Mycobacterium bovis bacillus Calmette-Guérin.

Multiplication of BCGure-, an isogenic urease-negative mutant of Mycobacterium bovis BCG constructed by allelic exchange (J. M. Reyrat, F. X. Berthet, and B. Gicquel, Proc. Natl. Acad. Sci. USA 92:8768-8772, 1995), was examined in human macrophages and mice. Although ureolytic activity was not essential to BCGure-growth, a slight decrease in the multiplication and persistence of the mutated strain compared with wild-type BCG was observed in lungs of infected mice.

Animals↗