Purification and characterization of an extracellular secretogenic non-membrane-damaging cytotoxin produced by clinical strains of Vibrio cholerae non-O1

Abstract

Some clinical strains of Vibrio cholerae non-O1 produce an extracellular factor that evokes a rapid and dramatic cytotoxic response which manifests as cell rounding of Chinese hamster ovary (CHO) and HeLa cells without accompanying membrane damage. This study was performed to establish the identity of the non-membrane-damaging cytotoxin (NMDCY), which was not inhibited by antitoxins against cholera toxin, heat-labile toxin of enterotoxigenic Escherichia coli, El Tor hemolysin, Shiga-like toxin I, and Shiga-like toxin II, indicating that NMDCY did not bear an apparent immunological relationship with the above toxins and hemolysin. Brain heart infusion broth and AKI medium supported the maximal production of NMDCY; culture supernatant of AKI medium was found to be free of hemolysin activity, whereas in brain heart infusion broth hemolysin was coproduced with NMDCY. Maximal production of NMDCY in AKI medium was observed at 37 degrees C under shaking conditions with the pH of the medium adjusted to 8.5. NMDCY was purified to homogeneity by a three-step purification procedure which increased the specific activity of the cytotoxin by 1.7 X 10(5)-fold. The denatured molecular weight of the purified toxin was 35,000, and the cytotoxin was heat labile and sensitive to trypsin. Purification of the cytotoxin revealed an enterotoxic activity as reflected by its ability to accumulate fluid in the rabbit ileal loop. Both the cytotoxic and enterotoxic activities of NMDCY could be inhibited or neutralized by antiserum raised against purified cytotoxin but not by preimmune serum. Immunodiffusion test between purified NMDCY and antiserum gave a single well-defined precipitin band which showed reactions of complete identity, while, in an immunoblot assay, a well-defined single band was observed in the 35-kDa region. Our results indicate that the cytotoxic and enterotoxic activities expressed by NMDCY appear to contribute to the pathogenesis of the disease associated with V. cholerae non-O1 strains which produce this cytotoxin.