CATION FLUX STUDIES OF THE LESION-INDUCED IN HUMAN ERYTHROCYTE-MEMBRANES BY THE THERMOSTABLE DIRECT HEMOLYSIN OF VIBRIO-PARAHAEMOLYTICUS

Vibrio parahaemolyticus, an important agent of seafood-borne gastroent eritis, expresses several putative virulence factors that could accoun t for the disease symptoms of infected humans, namely, diarrhea, nause a, and abdominal cramps. The pathogenicity of V. parahaemolyticus corr elates well with the Kanagawa phenomenon (the hemolytic ability of str ains grown on Wagatsuma blood agar), implicating the thermostable dire ct hemolysin (TDH) as the predominant toxin responsible for pathogenic ity. TDH-induced hemolysis could be inhibited by the addition of the o smolyte sorbitol to the extracellular solution, supporting the hypothe sis that hemolysis occurs through colloid osmosis secondary to an incr ease in the cation permeability of the membrane. The effect of TDH on cation permeability was investigated by measuring K+ (congener, Rb-86) influx into human erythrocytes in which the endogenous cation transp orters had been blocked (by use of ouabain, bumetanide, and nitrendipi ne). TDH increased K+ influx into these cells; this increase was rapid in onset and constant in magnitude, suggesting a direct action by TDH on the membrane. The kinetics of leak generation were examined; the r elationship between counts accumulated and hematocrit indicated that t he TDH-induced lesion is multihit in nature. TDH-induced K+ influx was sensitive to Zn2+. Time courses of hemolysis in isosmotic solutions o f monovalent cation chlorides were used to obtain the selectivity seri es for the TDH-induced leak: Cs+ > Li+ > K+ > Rb+ > Na+. Both the Zn2 sensitivity and this selectivity series were obtained for crude cultu re supernatants, suggesting that TDH is the predominant leak-inducing agent. Thus, we have identified several features of the TDH-induced le ak likely to be important in the diarrhetic action of V. parahaemolyti cus in the human intestine.