Cell Vacuolation, a Manifestation of the El Tor Hemolysin of Vibrio cholerae

Abstract

Culture supernatants of nontoxigenic nonepidemic clinical strains of Vibrio cholerae belonging to diverse serogroups were found to induce vacuolation of nonconfluent HeLa cells. The vacuoles became prominent 18 h after introduction of culture supernatant, and vacuolated cells survived for 48 h and then died. Only a fraction of the vacuolated cells took up neutral red dye, implying that there were differences in the vacuolar microenvironment. Further tests showed that the factor responsible for vacuolation was heat labile and proteinaceous. Vacuolating activity was completely neutralized by antibody to hemolysin of V. cholerae but not by antibody to vacuolating cytotoxin of Helicobacter pylori. Partial purification of the vacuolating factor led to elution of fractions, which showed both hemolytic and vacuolating activity. PCR amplification and cloning of the hemolysin structural gene (hlyA) into Escherichia coli DH5␣ led to isolation of clones producing cell vacuolating factor in a cell-associated form. Further, a null insertion mutation in the hlyA gene of a high-vacuolatingfactor-producing strain led to complete abolition of both cell vacuolating and hemolytic activities. These analyses establish vacuolation as a potentially important but previously unrecognized property of V. cholerae El Tor hemolysin.Much of the virulence of bacterial pathogens is initiated by secreted factors that induce specific biochemical changes in host tissue, culminating in pathology and disease. Cholera toxin (CT), the potent secretory toxin produced by toxigenic strains of Vibrio cholerae, is critically involved in key features of the disease cholera. Other putative factors, including zonula occludens toxin (Zot), accessory cholera enterotoxin (Ace), El Tor hemolysin, Shiga-like toxin, and heat-stable toxin, are reportedly produced by virulent strains of V. cholerae and are thought to contribute to the disease process (12). Despite attenuation of several virulence genes in recombinant candidate vaccine strains of V. cholerae, a safe and efficacious vaccine still eludes us. The residual diarrhea caused by genetically attenuated live oral vaccine strains of V. cholerae O1 prompted us to look for new factors secreted by V. cholerae.One means of assessing the toxicity of the secreted products of bacterial pathogens is to study the effect of culture supernatants on eukaryotic cell lines (12). In this context, we have previously reported that cell-free culture supernatants of V. cholerae strains induced morphological changes, including elongation and rounding in eukaryotic cells (18,21,23). We initiated the present study to examine the mechanism as to how nontoxigenic nonepidemic V. cholerae strains are able to cause a disease that resembles cholera in absence of already known virulence determinants, especially CT, found in their toxigenic epidemic causing counterparts. While attempting this, we observed that some clinical strains of V. cholerae induce vacuolation on HeLa cells, which seemed reminiscent of that induced by VacA cytotoxin of...

Genetic Diversity of Vibrio cholerae O1 in Argentina and Emergence of a New Variant

“…As deduced from the results of the cytotoxicity inhibition assay on Vero cells, El Tor hemolysin was responsible for the cytotoxic activity. Furthermore, the diluted culture supernatants of the Tucumán strains caused a vacuolization effect similar to that described recently in association with El Tor hemolysin (7,21). This effect was also abolished by incubation with the corresponding antiserum.…”

“…They also demonstrated that these strains exhibited cytotoxic activity, caused by El Tor hemolysin of V. cholerae, which evoked a unique vacuolating effect on Vero cells (7). This effect of El Tor hemolysin was also observed in a group of isolates of V. cholerae non-O1 recovered in India (21), suggesting that this virulence factor may play an important role in the development of diarrhea, especially in strains lacking other well-defined toxins, e.g., CT.…”

“…Like other hemolysins, El Tor hemolysin has been historically described as a pore-forming toxin (14,37), and its effect has been demonstrated on erythrocytes and epithelial cells (38,40). Although its role in diarrhea has been repeatedly suggested (7,21,36,40), there is only one report demonstrating cytolethal activity of El Tor hemolysin on human intestinal cells up to now (39). Here, by means of the experiments conducted in the Ussing chamber, we went on to prove that the deleterious effects introduced in the cells actually induce an inhibition of water absorption concomitant with an increase in Isc in the human small intestine.…”

Detection of RTX Toxin Gene in Vibrio cholerae by PCR

“…Evidence of the inflammatory response due to a V. cholerae O1 El Tor strain from which all known toxin genes excluding the rtx gene cluster have been deleted has also been reported (16). The vacuolating activity of the V. cholerae El Tor hemolysin in nucleated mammalian cells may be associated with gastrointestinal symptoms caused by nontoxigenic V. cholerae (11). A recent investigation (4) demonstrated that the RTX toxins of V. cholerae caused actin depolymerization and cross-linking in HEp-2 cells.…”

Structural Requirements of Cholesterol for Binding to Vibrio cholerae Hemolysin

“…Some studies have shown that VCH induces vacuolation in tissue culture cells (6,17,18). Therefore, it seems likely that the vacuolating activity is provoked by VCH-mediated pore formation.…”