Characteristics of the Protein Complexes and Pores Formed by Bacillus cereus Hemolysin BL.

Bacillus cereus Hemolysin BL is a tripartite toxin responsible for a diarrheal type of food poisoning. Open questions remain regarding its mode of action, including the extent to which complex formation prior to cell binding contributes to pore-forming activity, how these complexes are composed, and the properties of the pores formed in the target cell membrane. Distinct complexes of up to 600 kDa were found on native gels, whose structure and size were primarily defined by Hbl B. Hbl L1 and L2 were also identified in these complexes using Western blotting and an LC-MS approach. LC-MS also revealed that many other proteins secreted by B. cereus exist in complexes. Further, a decrease of toxic activity at temperatures ≥60 °C was shown, which was unexpectedly restored at higher temperatures. This could be attributed to a release of Hbl B monomers from tight complexation, resulting in enhanced cell binding. In contrast, Hbl L1 was rather susceptible to heat, while heat treatment of Hbl L2 seemed not to be crucial. Furthermore, Hbl-induced pores had a rather small single-channel conductance of around 200 pS and a probable channel diameter of at least 1 nm on planar lipid bilayers. These were highly instable and had a limited lifetime, and were also slightly cation-selective. Altogether, this study provides astonishing new insights into the complex mechanism of Hbl pore formation, as well as the properties of the pores.

Binding to The Target Cell Surface Is The Crucial Step in Pore Formation of Hemolysin BL from Bacillus cereus.

A major virulence factor involved in Bacillus cereus food poisoning is the three-component enterotoxin hemolysin BL. It consists of the binding component B and the two lytic components L1 and L2. Studying its mode of action has been challenging, as natural culture supernatants additionally contain Nhe, the second three-component enterotoxin, and purification of recombinant (r) Hbl components has been difficult. In this study, we report on pore-forming, cytotoxic, cell binding and hemolytic activity of recently generated rHbl components expressed in E. coli. It is known that all three Hbl components are necessary for cytotoxicity and pore formation. Here we show that an excess of rHbl B enhances, while an excess of rHbl L1 hinders, the velocity of pore formation. Most rapid pore formation was observed with ratios L2:L1:B = 1:1:10 and 10:1:10. It was further verified that Hbl activity is due to sequential binding of the components B - L1 - L2. Accordingly, all bioassays proved that binding of Hbl B to the cell surface is the crucial step for pore formation and cytotoxic activity. Binding of Hbl B took place within minutes, while apposition of the following L1 and L2 occurred immediately. Further on, applying toxin components simultaneously, it seemed that Hbl L1 enhanced binding of B to the target cell surface. Overall, these data contribute significantly to the elucidation of the mode of action of Hbl, and suggest that its mechanism of pore formation differs substantially from that of Nhe, although both enterotoxin complexes are sequentially highly related.