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

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.

Consumed Foodstuffs Have a Crucial Impact on the Toxic Activity of Enteropathogenic Bacillus cereus

“…In this study, a set of 20 enteropathogenic and apathogenic B. cereus strains was used, which has been extensively characterized ( Kniehl et al, 2003 ; Jessberger et al, 2015 , 2017 ). For toxicity studies, a nheABC deletion strain ( Tausch et al, 2017 ) was additionally used. Overnight cultures were grown in CGY medium (casein-glucose-yeast: 2% casein hydrolysate, 0.6% yeast extract, 0.2% ammonium sulfate, 1.4% K 2 HPO4, 0.6% KH 2 PO4, 0.1% sodium citrate × 2H 2 O, 0.2% MgSO 4 × 7H2O, 1% glucose) at 32°C under continuous agitation.…”

Structural and Mechanistic Features of ClyA-Like α-Pore-Forming Toxins

“…These striking similarities mirror current models on how each subunit contributes to the mechanism of pore formation. Indeed, pairwise binding experiments in solution could show stable complexes between NheB and NheC [ 32 ], as well as Hbl-B and Hbl-L1 [ 33 ]. Moreover, experiments have suggested that NheA and Hbl-L2 recruitment represent the final step in pore assembly for Nhe and Hbl toxins, respectively [ 16 , 34 ].…”

Cellular Entry of Binary and Pore-Forming Bacterial Toxins

“…This Special Issue of Toxins , entitled “Cellular Entry of Binary and Pore-Forming Bacterial Toxins,” gives a sense of the recent advances in characterizing the functional and structural aspects of this broad scientific problem that goes beyond the classical field of toxinology and microbiology and spills into the general areas of biochemistry, biophysics, and molecular and cell biology. The contributions to this Special Issue include several experimental articles, employing sophisticated techniques to gain important insights into the mechanism of cellular entry [ 1 , 2 , 3 , 4 , 5 , 6 ]; a thought-provoking perspective comment [ 7 ]; and two conceptual reviews, one on apicomplexan pore-forming toxins [ 8 ] and one on clostridial binary toxins [ 9 ]. What have we learned about the field from this collection?…”

“…Deciphering complex pathways requires integration of various approaches . Cellular entry of bacterial toxins utilizes a complex mechanism [ 8 , 9 ] that involves multiple protein partners interacting with each other [ 3 , 9 ] and with a lipid bilayer [ 1 , 2 , 6 ]. Key players often undergo profound conformational changes, both in aqueous [ 5 ] and membranous environments [ 1 , 2 ].…”