Persistence of Helicobacter pylori VacA toxin and vacuolating potential in cultured gastric epithelial cells.
ABSTRACT The vacuolating toxin A (VacA) is one of the most important virulence factors in Helicobacter pylori-induced damage to human gastric epithelium. Using human gastric epithelial cells in culture and broth culture filtrate from a VacA-producing H. pylori strain, we studied 1) the delivery of VacA to cells, 2) the localization and fate of internalized toxin, and 3) the persistence of toxin inside the cell. The investigative techniques used were neutral red dye uptake, ultrastructural immunocytochemistry, quantitative immunofluorescence, and immunoblotting. We found that VacA 1) is delivered to cells in both free and membrane-bound form (i.e., as vesicles formed by the bacterial outer membrane), 2) localizes inside the endosomal-lysosomal compartment, in both free and membrane-bound form, 3) persists within the cell for at least 72 h, without loss of vacuolating power, which, however, becomes evident only when NH4Cl is added, and 4) generally does not degrade into fragments smaller than approximately 90 kDa. Our findings suggest that, while accumulating inside the endosomal-lysosomal compartment, a large amount of VacA avoids the main lysosomal degradative processes and retains its apparent molecular integrity.
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ABSTRACT: Extracellular secretion of products is the major mechanism by which Gram-negative pathogens communicate with and intoxicate host cells. Vesicles released from the envelope of growing bacteria serve as secretory vehicles for proteins and lipids of Gram-negative bacteria. Vesicle production occurs in infected tissues and is influenced by environmental factors. Vesicles play roles in establishing a colonization niche, carrying and transmitting virulence factors into host cells, and modulating host defense and response. Vesicle-mediated toxin delivery is a potent virulence mechanism exhibited by diverse Gram-negative pathogens. The biochemical and functional properties of pathogen-derived vesicles reveal their potential to critically impact disease.Genes & Development 12/2005; 19(22):2645-55. DOI:10.1101/gad.1299905 · 12.64 Impact Factor
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ABSTRACT: Intracellular VacA localises to the vacuolar (late endosome/lysosome) membrane, but little is known about the trafficking of the toxin beyond this region. We show that the Golgi-disturbing agent brefeldin A (BFA) enhances VacA-induced vacuolation of epithelial cells by Helicobacter pylori co-culture and, importantly, BFA treatment induces vacuolation by less toxic forms of VacA. The effect is BFA dose-dependent and occurs within 2.5 h. These data suggest that VacA may be routed deeper within the cell than the vacuole, and that vacuolation is minimised when this occurs efficiently. This may explain why some forms of VacA do not cause vacuolation and why vacuolation is minimal at the low bacteria:cell ratios observed in vivo.FEMS Microbiology Letters 09/2004; 237(1):163-70. DOI:10.1016/j.femsle.2004.06.031 · 2.72 Impact Factor
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ABSTRACT: The Helicobacter pylori vacuolating cytotoxin (VacA) intoxicates mammalian cells resulting in reduction of mitochondrial transmembrane potential (Delta Psi m reduction) and cytochrome c release, two events consistent with the modulation of mitochondrial membrane permeability. We now demonstrate that the entry of VacA into cells and the capacity of VacA to form anion-selective channels are both essential for Delta Psi m reduction and cytochrome c release. Subsequent to cell entry, a substantial fraction of VacA localizes to the mitochondria. Neither Delta Psi m reduction nor cytochrome c release within VacA-intoxicated cells requires cellular caspase activity. Moreover, VacA cellular activity is not sensitive to cyclosporin A, suggesting that VacA does not induce the mitochondrial permeability transition as a mechanism for Delta Psi m reduction and cytochrome c release. Time-course and dose-response studies indicate that Delta Psi m reduction occurs substantially before and at lower concentrations of VacA than cytochrome c release. Collectively, these results support a model that VacA enters mammalian cells, localizes to the mitochondria, and modulates mitochondrial membrane permeability by a mechanism dependent on toxin channel activity ultimately resulting in cytochrome c release. This model represents a novel mechanism for regulation of a mitochondrial-dependent apoptosis pathway by a bacterial toxin.Cellular Microbiology 03/2004; 6(2):143-54. DOI:10.1046/j.1462-5822.2003.00347.x · 4.82 Impact Factor