Non-O1 strains of Vibrio cholerae implicated in gastroenteritis and diarrhea generally lack virulence determinants such as cholera toxin that are characteristic of epidemic strains; the factors that contribute to their virulence are not understood. Here we report that at least one-third of diarrhea-associated nonepidemic V. cholerae strains from Mexico cause vacuolation of cultured Vero cells. Detailed analyses indicated that this vacuolation was related to that caused by aerolysin, a pore-forming toxin of Aeromonas; it involved primarily the endoplasmic reticulum at early times (ϳ1 to 4 h after exposure), and resulted in formation of large, acidic, endosome-like multivesicular vacuoles (probably autophagosomes) only at late times (ϳ16 h). In contrast to vacuolation caused by Helicobacter pylori VacA protein, that induced by V. cholerae was exacerbated by agents that block vacuolar proton pumping but not by endosome-targeted weak bases. It caused centripetal redistribution of endosomes, reflecting cytoplasmic alkalinization. The gene for V. cholerae vacuolating activity was cloned and was found to correspond to hlyA, the structural gene for hemolysin. HlyA protein is a pore-forming toxin that causes ion leakage and, ultimately, eukaryotic cell lysis. Thus, a distinct form of cell vacuolation precedes cytolysis at low doses of hemolysin. We propose that this vacuolation, in itself, contributes to the virulence of V. cholerae strains, perhaps by perturbing intracellular membrane trafficking or ion exchange in target cells and thereby affecting local intestinal inflammatory or other defense responses.Many different strains of Vibrio cholerae cause diarrheal disease (6, 7) that, although not as devastating as full-blown cholera caused by epidemic strains (serogroups O1 and O139), imposes a major burden on human health, especially in developing countries (31, 41). A hallmark of epidemic V. cholerae strains is production of cholera toxin (CT), a protein that provokes a massive outpouring of body fluids directly into the intestine. The genes for CT biosynthesis are absent from most nonepidemic V. cholerae strains.Although epidemic cholera had been absent from the Americas for more than a century, it suddenly reappeared in Peru in 1991 and then quickly spread to neighboring countries (40). Soon thereafter epidemic cholera caused by the new O139 serogroup appeared in South Asia. This had particularly devastating consequences, since the new O139 strains afflicted adults with partial immunity to the previously dominant O1 strains, as well as young and immunologicaly naive children who depended on them. Given these new threats to public health, the National Institute for Diagnoses and Reference in Mexico (INDRE) began a surveillance program for O139 strains in 1993, in the event that they might also arrive in Mexico (17). Although antibody-based tests for serogroup O1 strains were well established, no such tests for O139 strains were available in Mexico at that time. Our group in Mexico therefore elected to screen cultures ...
