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 L2
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 L2
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 L1
and B, as well as L1
in solution could be shown for the first time. Surface plasmon resonance experiments with the rHbl components confirmed these results with KD
values of 4.7 × 10−7
M and 1.5 × 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.
This is an open access article distributed under the Creative Commons Attribution License
which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.