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Tausch, Franziska; Dietrich, Richard; Schauer, Kristina; Janowski, Robert; Niessing, Dierk; Märtlbauer, Erwin; Jessberger, Nadja (2017): Evidence for Complex Formation of the Bacillus cereus Haemolysin BL Components in Solution. In: Toxins, Vol. 9, No. 9, 288
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Abstract

Haemolysin BL is an important virulence factor regarding the diarrheal type of food poisoning caused by Bacillus cereus. However, the pathogenic importance of this three-component enterotoxin is difficult to access, as nearly all natural B. cereus culture supernatants additionally contain the highly cytotoxic Nhe, the second three-component toxin involved in the aetiology of B. cereus-induced food-borne diseases. To better address the toxic properties of the Hbl complex, a system for overexpression and purification of functional, cytotoxic, recombinant (r)Hbl components L-2, L-1 and B from E. coli was established and an nheABC deletion mutant was constructed from B. cereus reference strain F837/76. Furthermore, 35 hybridoma cell lines producing monoclonal antibodies (mAbs) against Hbl L-2, L-1 and B were generated. While mAbs 1H9 and 1D8 neutralized Hbl toxicity and thus, represent important tools for future investigations of the mode-of-action of Hbl on the target cell surface, mAb 1D7, in contrast, even enhanced Hbl toxicity by supporting the binding of Hbl B to the cell surface. By using the specific mAbs in Dot blots, indirect and hybrid sandwich enzyme immuno assays (EIAs), complex formation between Hbl L-1 and B, as well as L-1 and L-2 in solution could be shown for the first time. Surface plasmon resonance experiments with the rHbl components confirmed these results with K-D values of 4.7 x 10(-7) M and 1.5 x 10(-7) M, respectively. These findings together with the newly created tools lay the foundation for the detailed elucidation of the molecular mode-of-action of the highly complex three-component Hbl toxin.