Abstract
Helicobacter pylori vacuolating toxin (VacA) is a secreted toxin that is reported to produce multiple effects on mammalian cells. In this study, we explored the relationship between VacA-induced cellular vacuolation and VacA-induced cytochrome c release from mitochondria. Within intoxicated cells, vacuolation precedes cytochrome c release and occurs at lower VacA concentrations, indicating that cellular vacuolation is not a downstream consequence of cytochrome c release. Conversely, bafilomycin A1 blocks VacA-induced vacuolation but not VacA-induced cytochrome c release, which indicates that cytochrome c release is not a downstream consequence of cellular vacuolation. Acid activation of purified VacA is required for entry of VacA into cells, and correspondingly, acid activation of the toxin is required for both vacuolation and cytochrome c release, which suggests that VacA must enter cells to produce these two effects. Single amino acid substitutions (P9A and G14A) that ablate vacuolating activity and membrane channel-forming activity render VacA unable to induce cytochrome c release. Channel blockers known to inhibit cellular vacuolation and VacA membrane channel activity also inhibit cytochrome c release. These data indicate that cellular vacuolation and mitochondrial cytochrome c release are two independent outcomes of VacA intoxication and that both effects are dependent on the formation of anion-selective membrane channels.
Highlights
Helicobacter pylori vacuolating toxin (VacA) is a secreted toxin that is reported to produce multiple effects on mammalian cells
Acid activation of purified VacA is required for entry of VacA into cells, and correspondingly, acid activation of the toxin is required for both vacuolation and cytochrome c release, which suggests that VacA must enter cells to produce these two effects
To test whether VacA induces cytochrome c release within intact cells, monolayers of CcGFP-HeLa cells were incubated with purified acid-activated VacA (750 nM, calculated using 88-kDa VacA molecular mass [42]) for 18 h
Summary
VacA, H. pylori vacuolating cytotoxin; ATP, adenosine triphosphate; GFP, Aequorea victoria green fluorescence protein; CcGFP-HeLa, HeLa cells stably expressing cytochrome c-GFP; DIDS, 4,4Ј-diisothiocyanostilbene-2,2Ј-disulfonic acid; NPPB, epithelial cells both in vitro and in vivo, presumably by causing a defect in vesicular trafficking (10 –12). Multiple H. pylori factors have been linked to the initiation of apoptosis (29 –32), VacA was recently demonstrated to be sufficient to induce apoptosis in gastric epithelial cells [33]. Intracellular expression of VacA in transiently transfected HeLa cells or the application of purified toxin to HeLa monolayers has been reported to induce the release of cytochrome c from the intermembrane space of mitochondria, which suggests that VacA-induced apoptosis may occur via a mitochondria-dependent pathway [16]. To explore the relationship between VacA-mediated cellular vacuolation and cytochrome c release, we conducted single cell analyses using a stably transfected HeLa cell line expressing a genetically derived fusion protein comprising cytochrome c and green fluorescence protein (CcGFP-HeLa), which is localized to the mitochondria [34]. Our data indicate that cellular vacuolation and cytochrome c release are two independent cellular effects induced by VacA and that both are dependent on VacAassociated intracellular channel activity
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