PURIFICATION AND CHARACTERIZATION OF A HEMOLYSIN PRODUCED BY VIBRIO-MIMICUS

Vibrio mimicus is a causative agent of human gastroenteritis. This pat hogen secretes a pore-forming toxin, V. mimicus hemolysin (VMH), which causes hemolysis by three sequential steps: binding to an erythrocyte membrane, formation of a transmembrane pore, and disruption of the ce ll membrane, VMH with a molecular mass of 63 kDa was purified by ammon ium sulfate precipitation and column chromatography with phenyl Sephar ose HP and Superose 6 HR, The hemolytic reaction induced by VMH contin ued up to disruption of all erythrocytes in the assay system, Moreover , VMH that bound preliminarily to erythrocyte ghosts showed a sufficie nt ability to attack intact erythrocytes. These results suggest revers ible binding of the toxin molecule to the membrane, The final cell-dis rupting stage was effectively inhibited by various divalent cations, A dditionally, some cations, such as Zn2+ and Cu2+, blocked the pore-for ming stage at high concentrations, Although VMH could disrupt all kind s of mammalian erythrocytes tested, those from horses were most sensit ive to the hemolysin, Horse erythrocytes were found to have the most t oxin-binding sites and to be hemolyzed by the least amount of membrane -bound to?rin molecules, suggesting that toxin binding to and pore for mation on erythrocytes are more effective in horses than in other mamm als, Purified VMH induced fluid accumulation in a ligated rabbit ileal loop in a dose-dependent manner, In addition, the antibody against th e hemolysin obviously reduced enteropathogenicity of living V, mimicus cells, These findings clearly demonstrate that VMH is probably involv ed in the virulence of this human pathogen.