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Many bacterial toxins are important virulence factors, capable of instigating marked changes in the physiology of susceptible cells and tissues. Mechanisms of membrane attack by Vibrio toxins were examined on target cells, using cell physiological techniques, in particular, assays of haemolysis and radioisotope movement.Kanagawa haemolysin (KH; commercially available preparation of the thermostable direct haemolysin (TDH)) of V. parahaemolyticus caused lysis of human (but not horse or hagfish) erythrocytes that occurred (a) after colloid osmosis due to raised cation permeability, (b) independently of the KH:red cell ratio, and (c) with a monovalent cation selectivity series (reversed Eisenman VIII with a small K+ anomaly). The binding phase of KH was longer than the 1 - 2 minutes suggested by other workers.The KH-induced cation leak was (a) rapid in onset, (b) of a magnitude higher in the first ten minutes of treatment than subsequently, (c) of a multi-hit nature, (d) unaffected by a variety of membrane-active agents, and (e) inhibited by Zn2+, Cd2+ or mixing of toxin with dibutyl phthalate. Neuraminidase treatment of HRBC enhanced KH-induced cation influx and haemolysis, suggesting that additional receptors for TDH were unmasked by this treatment. In the presence of subhaemolytic KH, physiological levels of extracellular Ca2+ increased K+ influx by the Gardos channel, and Mg2+ (1.5 mM) decreased flux by the Na+/K+/2Cl cotransporter. There were no significant changes to sodium pump activity.TDH and El Tor haemolysin (ETH) were purified from culture supernatants of V. parahaemolyticus and non-Ol V. cholerae, respectively. Highly purified TDH was derived from KH and used to confirm that the identified features of KH action were attributable to TDH. Although ETH also caused haemolysis by colloid osmosis secondary to increased cation permeability, it differed from TDH in its lability to air/vibration, relative magnitude of induced influx with respect to time, and selectivity series of induced lesion. Concentrated supernatants, from V. cholerae strains deleted of known virulence factors, caused morphological changes of Chinese hamster ovary cells, suggesting the presence of unidentified factors capable of perturbing cell physiology.Confirming its potential as an enterotoxin, KH (albeit at a high dose) increased the efflux rate constant for 86Rb+ from rabbit jejunocytes. These findings are discussed in the context of a possible pathogenic role for TDH in the gastrointestinal tract. |
