Cell vacuolation, a manifestation of the El Tor hemolysin of Vibrio cholerae

Culture supernatants of nontoxigenic nonepidemic clinical strains of Vibrio cholerae belonging to diverse serogroups Here found to induce vacuolation of nonconfluent HeLa cells, The vacuoles became prominent 18 h after introd uction of culture supernatant, and vacuolated cells survived for 18 h and t hen died. Only a fraction of the vacuolated cells took up neutral red dye, implying that there Here differences in the vacuolar microenvironment. Furt her tests showed that the factor responsible for vacuolation Has heat labil e and proteinaceous. Vacuolating activity Has completely neutralized by ant ibody to hemolysin of V. cholerae but not by antibody to vacuolating cytoto xin of Helicobacter pylori. Partial purification of the vacuolating factor led to elution of fractions, which showed both hemolytic and vacuolating ac tivity, PCR amplification and cloning of the hemolysin structural gene (hly A) into Escherichia coli DH5 alpha led to isolation of clones producing cel l vacuolating factor in a cell-associated form. Further, a null insertion m utation in the hlyA gene of a high-vacuolating-factor-producing strain led to complete abolition of both cell vacuolating and hemolytic activities. Th ese analyses establish vacuolation as a potentially important but previousl y unrecognized property of V. cholerae El Tor hemolysin.