Tripartite haemolysin BL: isolation and characterization of two distinct homologous sets of components from a single Bacillus cereus isolate

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Abstract
Haemolysin BL (HBL), a three-component enterotoxic/necrotizing/vascular permeability toxin, is a likely virulence factor of Bacillus cereus diarrhoeal food poisoning and necrotic infections. This paper describes the isolation of two distinct homologous sets of all three HBL components from a single B. cereus isolate, MGBC 145. The proteins of one set (designated HBL, consisting of B, L1 and L2), were about 87–100% identical in N-terminal amino acid sequences to their respective prototype components from strain F837/76, and the proteins of the homologous set (HBLa, consisting of Ba, L1a and L2a) were all about 62–65% identical. Only the latter homologues differed immunochemically and physicochemically from the prototypes. HBL and HBLa exhibited similar haemolytic and vascular permeability potencies, and the homologues could be interchanged freely. There were no notable differences in activity between the L component homologues. However, components B and Ba were significantly different. Both were secreted as monomers, but unlike B, Ba was isolated as a relatively inactive complex that could be reactivated with urea. When Ba was substituted for B in gel-diffusion assays the distinct discontinuous haemolysis pattern typical of the presence of B did not occur. In suspension assays, excess B inhibited the haemolysis of B-primed cells by L1 (as previously described), but not that of Ba-primed cells. Excess Ba had the opposite effect and enhanced lysis of Ba-primed cells, but not that of B-primed cells. These differences reveal details about how the toxin components interact on target cell membranes. The authors’ observations indicate that HBL represents a new family of multicomponent toxins that was generated by a process of gene and operon duplication that occurred either intracellularly or by horizontal transfer, and raise the possibility of the existence of other related toxins in the genetically diverse B. cereus taxonomic group.