Tripartite Hemolysin BL from Bacillus cereus: HEMOLYTIC ANALYSIS OF COMPONENT INTERACTIONS AND A MODEL FOR ITS CHARACTERISTIC PARADOXICAL ZONE PHENOMENON

Abstract

Hemolysin BL (HBL) is a unique membrane-lytic toxin from Bacillus cereus composed of three distinct proteins, designated B, L1, and L2. HBL produces a paradoxical zone phenomenon in gel diffusion assays in sheep blood agar. Lysis does not begin immediately adjacent to the source of diffusion; rather, it begins several millimeters away. Cells near 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 to analyze the mechanisms of hemolysis and the paradoxical zone phenomenon. The B component was rate-limiting, and erythrocytes were slowly primed 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 individual components bound to cells independently, and membrane-associated 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 osmotic lysis mechanism. Concentrations of the B component above 1.3 nM caused inhibition of L1-mediated lysis, and L1 inhibited the priming reaction of B over a similar concentration range. From analyses of spectrophotometric and diffusion assays we constructed a basic model for the interactions between HBL components and for the paradoxical zone phenomenon in blood agar. In the latter, areas of slow lysis near diffusion sources are caused primarily by the accumulation of inhibitory levels of L1 reached before cells are primed by B.