Monoclonal Antibodies Neutralize Bacillus cereus Nhe Enterotoxin by Inhibiting Ordered Binding of Its Three Exoprotein Components

Abstract

The Nhe enterotoxin from Bacillus cereus is known to induce cytotoxicity on Vero and CaCo-2 cells by ordered binding of its single components NheA, NheB, and NheC. This study aimed to elucidate functional sites on NheB by identifying the epitopes of the neutralizing monoclonal antibodies 1E11 and 2B11. The binding regions of both antibodies were determined by using recombinant NheB fragments and synthetic peptides. The antigenic site of antibody 1E11 was located within the amino acids 321 to 341 of NheB, whereas reactivity of antibody 2B11 was dependent on the presence of amino acids 122 to 150 and on conformation. Both antibodies were able to bind simultaneously to NheB and did not interfere with target cell binding as shown by immunofluorescence microscopy. A set of neutralization assays revealed that antibody 2B11 most likely interfered with the interaction between NheB and NheC both on the epithelium cell surface and in solution. In contrast, antibody 1E11 inhibited association between NheA and cell-bound NheB in a competitive manner, and effectively neutralized Nhe cytotoxicity on a variety of human cell lines. This distinct mechanism further supports that NheA is the key component during the Nhe mode of action and the C-terminal epitope recognized by antibody 1E11 points to an important functional region of NheB. Bacillus cereus is a major food-borne pathogen known to produce a range of cytotoxins (for reviews, see references 27 and 28). There are four major toxins involved in food poisoning cases, namely, the emetic toxin (cereulide), a dodecadepsipeptide (1), and the three-component diarrheal toxins hemolysin BL (Hbl) (4, 5) and nonhemolytic enterotoxin (Nhe) (23). In addition, a single-component protein toxin (cytotoxin K) causing severe necrotic enteritis was identified in a rare B. cereus strain (22), for which the name "Bacillus cytotoxis" has been proposed (19). Studies on the prevalence of the nhe and hbl genes (10,15,16,26,31) in B. cereus indicate that all strains of B. cereus possess the genes of at least one of the diarrheal enterotoxins, and Nhe is the most prevalent enterotoxin harbored by B. cereus. In addition, the overall B. cereus-associated cytotoxic activity is correlated with the Nhe expression level (24).Nhe was first identified in strain NVH 0075/95, which was isolated following a large food-poisoning outbreak in Norway (23). It is a three-component toxin and consists of the exoproteins NheA (41.0 kDa), NheB (39.8 kDa), and NheC (36.5 kDa) (14). Studies using cell-based tests to assay Nhe-specific cytotoxicity demonstrated toxic effects in Vero, GH4, and CaCo-2 cells (17,20,21). The susceptibility of other cell lines has not yet been tested. It is known that Nhe has intrinsic pore-forming capacity (11) and that maximum toxicity will be reached when the ratio of the individual components is 10:10:1 for NheA, NheB, and NheC, respectively (20). NheB and NheC are mostly ␣-helical molecules with a predicted ␤-tongue, showing structural similarities to ClyA (11). The region of the predicte...

Complex Formation between NheB and NheC Is Necessary to Induce Cytotoxic Activity by the Three-Component Bacillus cereus Nhe Enterotoxin

“…Two of the MAbs (2G8 and 1E12) showed cross-reactivity with NheB, but MAbs 2F10 and 3D6 were highly specific for NheC and particularly suited for the intended studies. Recently we showed that inhibition of ordered binding of the individual Nhe components, particularly by preventing binding of NheA to NheB on the cell surface by MAb 1E11 against NheB, was an efficient way to neutralize Nhe toxicity [17]. However, none of the MAbs against NheC developed in this study, reduced Nhe toxicity, not even when NheC was pre-incubated with MAbs prior to addition of the other components.…”

“…Neutralization capacity of the monoclonal antibodies was tested on Vero cells under different experimental conditions as described earlier [17]. Neither in the simultaneous nor the consecutive assay setup neutralization of Nhe-related cytotoxic activity was observed.…”

“…The nhe genes could be found in most B. cereus isolates [24]–[27], and Nhe seems to be the most prevalent enterotoxin produced by B. cereus . Recent studies on the mode of action of Nhe [14], [17] indicated that NheC is able to bind to NheB in solution and that both components can bind to cell membranes. Additionally, it was shown that the Nhe components require a specific binding order and NheC was mandatory in the priming step to achieve maximum cytotoxicity.…”

“…Wild-type NheB was purified from 5- to 6-h culture supernatants of MHI 1672 by immunoaffinity chromatography (IAC), and purity was documented by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) [15]. The histidine-tagged recombinant NheA (rNheA) [17] was expressed in E. coli , grown in Luria-Bertani (LB) medium supplemented with the required antibiotic.…”

“…In order to analyze the neutralization capacity of the MAbs against NheC, this Vero cell assay was modified as described for neutralization of NheB-related cytotoxicity [17]. In a simultaneous assay, serial dilutions of B. cereus strain NVH 0075/95 alone or serial dilutions of purified rNheC together with a constant amount of MHI 1672 were incubated with 10 µg of the MAbs (1 mg ml −1 PBS).…”