Character and origin of vacuoles induced in mammalian cells by the cytotoxin of Helicobacter pylori

Abstract

Summary. Cytotoxic activity of culture supernates of Helicobacter pylori is manifested by vacuolation of mammalian cells in vitro. The formation and maturation of toxin-induced vacuoles in HeLa cells has been studied to examine the possibility that they are autophagosomal in nature. Observation by light microscopy revealed that vacuoles originate in a perinuclear position, increasing in number and size until cell degeneration and lysis occur after 48 h. Ultrastructural study of mature vacuoles indicated the presence of a bounding membrane with contents consisting of degenerate cytoplasmic components and acid phosphatase activity. Confocal fluorescence imaging demonstrated acridine orange accumulation in the vacuoles of toxin-treated cells, indicating an acidic intravacuolar pH. These features are characteristic of autophagosomes. In addition, the size of vacuoles in living, acridine orange-stained cells tended to be inversely proportional to fluorescence intensity. Fluid phase endocytic markers were observed only rarely within nascent vacuoles. Over the succeeding 24 h, labelling of most vacuoles with these dyes was observed. This, along with the observation of intravacuolar acid phosphatase activity, provides evidence that vacuoles communicate at some point during their development with endocytically derived compartments. These observations provide direct evidence for an autophagic origin of these structures.

Cellular vacuoles induced by Helicobacter pylori originate from late endosomal compartments.

“…There is evidence that the autophagocytic pathway converges with the endosomal pathway at a prelysosomal level (37,38). On the basis ofmorphological exmination, vacuoles have been suggested to originate from endosomes (7) or autophagosomes (39,40). If this latter proposal were correct, increased autophagocytosis would enhance the effect of vacA.…”

Effect ofHelicobacter pylori’s vacuolating cytotoxin on the autophagy pathway in gastric epithelial cells

“…9,12,17 Several of the earliest studies describing the action of VacA on cultured epithelial cell lines reported that broth culture supernatants from vacA + H. pylori strains induced the formation of intracellular vesicles that visibly stained with acridine orange, initially excluded the endocytic marker Lucifer yellow, and, appeared to contain cytoplasmic content. 18,19 Although these are classical characteristics of autophagosomes, neither the capacity of H. pylori to induce autophagy nor the relationship of these autophagic-like vesicles to VacAdependent vacuoles has been systematically evaluated. Autophagy is an evolutionarily-conserved cellular mechanism for the degradation of cellular components in the cytoplasm.…”

Helicobacter pylori toxin VacA induces vacuole formation by acting in the cell cytosol

“…Moreover, orally administered VacA induces gastric mucosa degeneration and inflammatory cell recruitment Marchetti et al, 1995). This toxin causes vacuolation of cells in culture (Leunk et al, 1988;Catrenich and Chestnut, 1992) and has therefore been named vacuolating cytotoxin (VacA) (Cover and Blaser, 1992). These vacuoles are acidic and their membrane contains the vacuolar ATPase proton pump and the small GTP-binding protein rab7; therefore, they have been suggested to arise from late compartments of the endocytic pathway (Cover et al, 1993;Papini et al, 1993;.…”