© 2002 Blackwell Science Ltd The Vibrio cholerae haemolysin anion channel is required for cell vacuolation and death tioning of the Cystic Fibrosis Transmembrane Regulator (CFTR) chloride channel and of other cellular proteins (Hogenauer et al., 2000). The zona occludens toxin alters the tight junctions that control the paracellular permeabil- ity of the polarized epithelial monolayer (Marinaro et al., 1999). The V. cholerae heat-stable toxin causes rapid fluid accumulation after activation of the guanylyl cyclase and the influx of Ca 2+ ions across the plasma membrane (Visweswariah et al., 1992; Hoque et al., 2001). In addition, many V. cholerae strains secrete a haemolytic toxin of 63 kDa, termed V. cholerae cytolysin (VCC) or haemolysin (Honda and Finkelstein, 1979; Ramamurthy et al., 1993; Dalsgaard et al., 1995). VCC causes lysis of vertebrate erythrocytes and cultured cells (Honda and Finkelstein, 1979; Zitzer et al., 1997a). It also increases the vascular permeability of rabbit skin and it is lethal to mice (Yamamoto et al., 1984). VCC forms transmembrane anion-selective pentameric channels with a diameter of 0.9–1.0 nm, leading to osmotic swelling of the cell and lysis (Krasilnikov et al., 1992; Zitzer et al., 1995, 1997a; 1999). The membrane- permeabilizing action of VCC has been documented in planar lipid bilayers and liposomes (Krasilnikov et al., 1992; Zitzer et al., 1993; Menzl et al., 1996). A pathogenic role for VCC in enteritis is suggested by the observation that the purified protein elicits fluid accumulation in rabbit ileal loops (Zitzer et al., 1993). Furthermore, a cytocidal action of VCC on human intestinal cells was attributed to VCC oligomerization and pore formation on cell mem- branes (Zitzer et al., 1997b; 1999). Recently, extensive cell vacuolation of HeLa and Vero cells was shown to be induced by V. cholerae filtrates and the vacuolating activ- ity was linked to VCC (Coelho et al., 2000; Mitra et al., 2000). Such cell vacuolating effect was reminiscent of that induced by the VacA cytotoxin of Helicobacter pylori (Cover and Blaser, 1992; Montecucco and Rappuoli, 2001; Morbiato et al., 2001). On the other hand, some dif- ferences among the cellular vacuoles induced by the two toxins were apparent. Vibrio cholerae cytolysin-induced vacuoles were found to uptake the weak base dye, neutral red, indicating that they are acidic like VacA-induced vacuoles. However, the specific vacuolar ATPase proton pump inhibitor bafilomycin A1, which is able to inhibit vacuole formation in VacA treated cells, did not prevent vacuole formation in VCC exposed cells (Cover et al., 1993; Papini et al., 1993; Coelho et al., 2000; Mitra et al., 2000; Figueroa-Arredondo et al., 2001). Cellular Microbiology (2002) 4(7), 397–409 Monica Moschioni, 1 Francesco Tombola, 1 Marina de Bernard, 1 Ana Coelho, 2 Alexander Zitzer, 3 Mario Zoratti 1 and Cesare Montecucco 1 * 1 Centro CNR Biomembrane and Dipartimento di Scienze Biomediche Sperimentali, Università di Padova, Via G. Colombo 3, 35121, Padova, Italy. 2 Departamento de Genetica, Universidade Federal do Rio de Janeiro RJ21944-970, Brazil. 3 Institute of Medical Microbiology and Hygiene, Johannes Gutenberg University, 55101 Mainz, Germany. Summary Several strains of Vibrio cholerae secrete a haemolytic toxin of 63 kDa, termed V. cholerae cytolysin (VCC). This toxin causes extensive vacuo- lation and death of cells in culture and forms an anion-selective channel in planar lipid bilayers and in cells. Here, we identify inhibitors of the VCC anion channel and show that the formation of the anion channel is necessary for the development of the vacuoles and for the cell death induced by this toxin. Using markers of cell organelles, we show that vacuoles derive from different intracellular com- partments and we identify the contribution of late endosomes and of the trans-Golgi network in vacuole biogenesis. Introduction The Gram-negative bacterium Vibrio cholerae is an intestinal non-invasive pathogen that adheres to the apical side of the small intestine (Salyers and Whitt, 1994) thereby inducing cholera, an acute watery diarrhoea disease of humans (Kaper et al., 1995). Vibrio cholerae produces several virulence factors that are involved in colonization and adhesion and also releases protein toxins that co-operate to induce the alteration of ion and water fluxes across the intestinal epithelium, which is the basis of the massive loss of fluids characteristic of cholera (Kaper et al., 1995). Cholera toxin enters into epithelial cells and elevates the cAMP level, which affects the func- Received 23 January, 2002; revised 9 April, 2002; accepted 9 April, 2002. *For correspondence. E-mail: cesare@civ.bio.unipd.it; Tel. (+39) 049 8276058; Fax: (+39) 049 8276049.