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

Franziska Tausch,Nadja Jessberger,Richard Dietrich,Robert Janowski,Kristina Schauer,Dierk Niessing,Erwin Märtlbauer

doi:10.3390/toxins9090288

Franziska Tausch, Nadja Jessberger + Show 5 more

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https://doi.org/10.3390/toxins9090288

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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.

Highlights

The Gram-positive, facultative anaerobe and rod-shaped bacterium Bacillus cereus has become increasingly important as a cause of food poisoning outbreaks
Recombinant Hbl toxin, an approach based on the Strep-tag® purification system was used. rHbl L2 and B were expressed with an N-terminal, rHbl L1 with a C-terminal strep-tag
Each of the single components was detected by a strep-specific antibody as well as by the Hbl-specific monoclonal antibodies (mAbs). rHbl L2 and L1 were detected at their predicted molecular weight of 48.7 and 42.4 kDa, while rHbl B appeared slightly bigger in Western blot analyses than its predicted 40.9 kDa

Summary

Introduction

The Gram-positive, facultative anaerobe and rod-shaped bacterium Bacillus cereus has become increasingly important as a cause of food poisoning outbreaks. It is ubiquitous in soil, sediments, dust and plants [1]. Three types of enterotoxins are responsible for the diarrheal form. These are produced foremost in the intestine from viable bacteria that, most likely as spores, survived the stomach passage [9]. These enterotoxins are the three component complexes

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