TRIPARTITE HEMOLYSIN BL FROM BACILLUS-CEREUS - HEMOLYTIC ANALYSIS OF COMPONENT INTERACTIONS AND A MODEL FOR ITS CHARACTERISTIC PARADOXICAL ZONE PHENOMENON

Hemolysin BL (HBL) is a unique membrane-lytic toxin from Bacillus cere us composed of three distinct proteins, designated B, L(1), and L(2). HBL produces a paradoxical zone phenomenon in gel diffusion assays in sheep blood agar, Lysis does not begin immediately adjacent to the sou rce of diffusion; rather, it begins several millimeters away. Cells ne ar the source and at intersections of lysis zones remain intact longer . Here, we developed a spectrophotometric hemolysis assay system that measures the activities of the individual HBL components and used it t o analyze the mechanisms of hemolysis and the paradoxical zone phenome non. The B component was rate-limiting, and erythrocytes were slowly p rimed by B at an optimal concentration of about 1.3 nM to rapid lytic action by the combination of the L components (L(1+2)). All of the ind ividual components bound to cells independently, and membrane-associat ed HBL components were neutralized by specific antibodies, suggesting that lysis was caused by formation of a membrane attack complex on the cell surface, Osmotic protection experiments indicate a colloid osmot ic lysis mechanism. Concentrations of the B component above 1.3 nM cau sed inhibition of L(1)-mediated lysis, and L(1) inhibited the priming reaction of B over a similar concentration range. From analyses of spe ctrophotometric and diffusion assays we constructed a basic model for the interactions between HBL components and for the paradoxical zone p henomenon in blood agar. In the latter, areas of slow lysis near diffu sion sources are caused primarily by the accumulation of inhibitory le vels of L(1) reached before cells are primed by B.