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1.
Toxins (Basel) ; 11(5)2019 05 20.
Article in English | MEDLINE | ID: mdl-31137585

ABSTRACT

A major virulence factor involved in Bacillus cereus food poisoning is the three-component enterotoxin hemolysin BL. It consists of the binding component B and the two lytic components L1 and L2. Studying its mode of action has been challenging, as natural culture supernatants additionally contain Nhe, the second three-component enterotoxin, and purification of recombinant (r) Hbl components has been difficult. In this study, we report on pore-forming, cytotoxic, cell binding and hemolytic activity of recently generated rHbl components expressed in E. coli. It is known that all three Hbl components are necessary for cytotoxicity and pore formation. Here we show that an excess of rHbl B enhances, while an excess of rHbl L1 hinders, the velocity of pore formation. Most rapid pore formation was observed with ratios L2:L1:B = 1:1:10 and 10:1:10. It was further verified that Hbl activity is due to sequential binding of the components B - L1 - L2. Accordingly, all bioassays proved that binding of Hbl B to the cell surface is the crucial step for pore formation and cytotoxic activity. Binding of Hbl B took place within minutes, while apposition of the following L1 and L2 occurred immediately. Further on, applying toxin components simultaneously, it seemed that Hbl L1 enhanced binding of B to the target cell surface. Overall, these data contribute significantly to the elucidation of the mode of action of Hbl, and suggest that its mechanism of pore formation differs substantially from that of Nhe, although both enterotoxin complexes are sequentially highly related.


Subject(s)
Bacterial Adhesion , Bacterial Proteins/toxicity , Hemolysin Proteins/toxicity , Animals , Bacillus cereus , Cell Survival/drug effects , Chlorocebus aethiops , Erythrocytes/drug effects , Hemolysis/drug effects , Sheep , Vero Cells
2.
Toxins (Basel) ; 9(9)2017 09 16.
Article in English | MEDLINE | ID: mdl-28926954

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.


Subject(s)
Bacterial Proteins , Hemolysin Proteins , Recombinant Proteins , Animals , Antibodies, Monoclonal/pharmacology , Antibodies, Neutralizing/pharmacology , Bacillus cereus/genetics , Bacillus cereus/metabolism , Bacterial Proteins/genetics , Bacterial Proteins/immunology , Bacterial Proteins/metabolism , Bacterial Proteins/toxicity , Cell Line , Cell Survival/drug effects , Chlorocebus aethiops , Escherichia coli/genetics , Female , Hemolysin Proteins/genetics , Hemolysin Proteins/immunology , Hemolysin Proteins/metabolism , Hemolysin Proteins/toxicity , Mice, Inbred BALB C , Recombinant Proteins/genetics , Recombinant Proteins/immunology , Recombinant Proteins/metabolism , Recombinant Proteins/toxicity , Solutions , Vero Cells
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