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Toxins 2017, 9(9), 288; doi:10.3390/toxins9090288

Evidence for Complex Formation of the Bacillus cereus Haemolysin BL Components in Solution

1
Department of Veterinary Sciences, Faculty of Veterinary Medicine, Ludwig-Maximilians-Universität München, Schönleutnerstr 8, 85764 Oberschleißheim, Germany
2
Institute of Structural Biology, Helmholtz Zentrum München-German Research Center for Environmental Health, Ingolstädter Landstr. 1, 85764 Neuherberg, Germany
3
Biomedical Center of the Ludwig-Maximilians-Universität München, Department of Cell Biology, 82152 Planegg-Martinsried, Germany
*
Author to whom correspondence should be addressed.
Academic Editor: Alexey S. Ladokhin
Received: 16 August 2017 / Revised: 11 September 2017 / Accepted: 12 September 2017 / Published: 16 September 2017
(This article belongs to the Special Issue Cellular Entry of Binary and Pore-Forming Bacterial Toxins)
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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 L2, L1 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, L1 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 and L2 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. View Full-Text
Keywords: Bacillus cereus; complex formation; enterotoxins; haemolysin BL; monoclonal antibodies Bacillus cereus; complex formation; enterotoxins; haemolysin BL; monoclonal antibodies
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Tausch, F.; Dietrich, R.; Schauer, K.; Janowski, R.; Niessing, D.; Märtlbauer, E.; Jessberger, N. Evidence for Complex Formation of the Bacillus cereus Haemolysin BL Components in Solution. Toxins 2017, 9, 288.

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