© 2002 Blackwell Science Ltd Cell vacuolization induced by Helicobacter pylori VacA cytotoxin does not depend on late endosomal SNAREs † cellular medium as a 95 kDa mature protein. VacA spon- taneously oligomerizes into inactive holigomers (Cover et al., 1994; 1997; Phadnis et al., 1994; Schmitt and Haas, 1994; Telford et al., 1994; Lupetti et al., 1996), which are activated by a short exposure to acid or alkaline pH (de Bernard et al., 1995; Yahiro et al., 1999). The acid- activated toxin becomes resistant to pepsin degradation (de Bernard et al., 1995). Cytotoxin VacA has a major effect on cells in culture, which develop intracellular vacuoles in the presence of membrane-permeant amines (Leunk et al., 1988; Cover et al., 1992; Ricci et al., 1997). Such cell alteration follows the accumulation of membrane-permeable weak bases, including dyes such as neutral red (Cover et al., 1992), because the vacuolar lumen is acidic. This provides a simple and quantitative assay of the internal volume of these compartments. These vacuoles contain membrane protein markers of late endosomes and lysosomes (Papini et al., 1994; Molinari et al., 1997) and are capable of incorporating fluid phase markers of extracellular medium (Catrenich and Chestnut, 1992; Cover et al., 1992; Papini et al., 1994). The vacuolar membrane contains an active vacu- olar ATPase proton pump (V-ATPase) (Papini et al., 1996), whose inhibition with bafilomycins prevents and reverts vacuole formation (Cover et al., 1993; Papini et al., 1993). These results indicate that vacuole forma- tion and maintenance depends on the proton pump activ- ity and suggest that intraendosomal accumulation of osmotically active molecules plays a crucial role in cell vacuolization by favouring water uptake and swelling. This hypothesis is strongly supported by the finding that VacA forms voltage-dependent, anion-selective channels in planar lipid membranes and in cells, essential to induce the transformation of late endosomal compart- ments into vacuoles (Szabò et al., 1999; Tombola et al., 1999). The formation of large vacuoles (2–5 μm) from small compartments such as late endosomes and lysosomes has to involve membrane fusion events to account for such a large increase in size. Accordingly, vacuole for- mation was found to be strictly dependent on the small GTP-binding protein Rab7 (Papini et al., 1997), which is involved in membrane trafficking at the level of late endo- somes and lysosomes (Feng et al., 1995; Meresse et al., Cellular Microbiology (2002) 4(1), 11–18 M. de Bernard, 1 * M. Moschioni, 1 A. Habermann, 2 G. Griffiths 2 and C. Montecucco 1 1 Centro CNR Biomembrane and Dipartimento di Scienze Biomediche Sperimentali, Università di Padova, Via G. Colombo 3, 35121, Padova, Italy. 2 EMBL, Meyerhofstrasse 1, Postfach 10.2209, 69012 Heidelberg, Germany. Summary Cellular vacuoles induced by the Helicobacter pylori vacuolating cytotoxin VacA originate from late endo- somal compartments. Their biogenesis requires the activity of both rab7 GTPase and the ATPase proton pump. The toxin has been suggested to cause an increased luminal osmotic pressure via its anion- specific channel activity localized on late endosomal compartments after endocytosis. Here, we show that the extensive membrane fusion that takes place in the transition from the small late endosomal com- partments to the large vacuoles does not depend on soluble N-ethylmaleimide-sensitive factor (NSF) attachment protein receptor (SNARE) proteins. The process of vacuolization leads to disappearance of the large array of internal membranes of late endo- somes. We suggest that most of the vacuole-limiting membrane derives from internal membranes. Introduction The vacuolating cytotoxin VacA is a major virulence factor of Helicobacter pylori (Salama et al., 2001), a bacterium that colonizes the stomach mucosa of the majority of the human population and induces gastroduodenal ulcers and stomach cancers in a sizeable proportion of infected patients (Warren and Marshall, 1983; Marshall et al., 1985; Parsonnet, 1998; Montecucco and Rappuoli, 2001). This toxin is synthesized as a 140 kDa precursor protein, whose C-terminal domain is removed at the bacterial surface, and the VacA toxin is released into the extra- Received 19 June, 2001; revised 17 September, 2001; accepted 28 September, 2001. *For correspondence. E-mail marina@ civ.bio.unipd.it; Tel. (+39) 049 827 6077; Fax (+39) 049 827 6049. † This article is dedicated to the memory of Professor Franco Tato.