The binding regions of both antibodies were determined by using recombinant NheB fragments and synthetic peptides. target cell binding as shown by immunofluorescence microscopy. A set of neutralization assays revealed that antibody 2B11 most likely interfered with the conversation between NheB and NheC both around the epithelium cell surface and in answer. 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 unique mechanism further supports that NheA is the important component during the Nhe mode of action and the C-terminal epitope recognized by antibody 1E11 points to an important functional region of NheB. == INTRODUCTION == Bacillus cereusis a major food-borne pathogen known to produce a range of cytotoxins (for reviews, see recommendations27and28). You will find 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 recognized in a rareB. cereusstrain (22), for which the name Bacillus cytotoxis has been proposed (19). Studies around the prevalence of thenheandhblgenes (10,15,16,26,31) inB. cereusindicate that all strains ofB. cereuspossess the genes of at least one of the diarrheal enterotoxins, and Nhe is the most prevalent enterotoxin harbored byB. cereus. In addition, the overallB. cereus-associated cytotoxic activity is usually 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 RAC1 (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 assessments to assay Nhe-specific cytotoxicity exhibited 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 usually 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 predicted -tongue in NheC is necessary for cell binding but not for conversation with NheB in answer (21). Binding between NheB and NheC could be demonstrated in answer, and both components are able to bind to cell membranes. In contrast, NheA does not bind to cellsper se, nor will it seem to interact with NheB and NheC in answer. The presence of NheA is usually, however, required in the final step of the Nhe mode of action in order to trigger toxicity, indicating a specific binding order of the individual components (21). In these fundamental studies monoclonal antibodies (MAbs) against NheB have been used to neutralize Nhe toxicity in Vero and Caco-2 cells. Therefore, we thought that identification of the binding regions of these antibodies on NheB could lead to significant functional implications. == MATERIALS AND METHODS == == B. cereusstrains, culture medium, and culture conditions. == B. cereusstrains used in the present study were as follows: NVH 0075/95 (fully cytotoxic), MHI1672 (generating NheA and NheB, low cytotoxic), and MHI1761 (generating NheB and NheC, not cytotoxic). The latter food isolates bear a preliminary quit codon in the 5 end of thenheCornheAgene, respectively, as published earlier (21). Cells were produced in CGY medium supplemented with 1% glucose for toxin production, exactly as explained previously (21). All strains lacked bothhblandcytK, as exhibited by PCR, immunoassay, and cell culture assay (31). == Cloning of recombinant full-length Nhe components and NheB deletion mutants. == The full-length NheA gene was amplified and cloned into pBAD102 Directional TOPO Expression system (Invitrogen). Recombinant NheA was then expressed inEscherichia coli(LMG-194). Expression and purification of NheC was performed as explained elsewhere (20). Concentration of recombinant protein preparations was determined CA inhibitor 1 by in-house enzyme immunoassay (EIA) using MAb 1A8 CA inhibitor 1 for NheA and polyclonal rabbit serum for NheC (8). Truncated NheB genes were PCR amplified, cloned into the pBAD102 directional TOPO CA inhibitor 1 expression system and expressed inE. coli(LMG-194) according to the manufacturer’s (Invitrogen) recommendations. Corresponding recombinant proteins showed N-terminal deletions of 30, 60, 92, 121, and 151 amino acids (for additional information, seeTable 1). The reactivity of deletion mutants was assayed with MAbs 2B11 and 1E11 by EIA and Western blotting. For further epitope mapping of MAb 1E11, three peptide fragments comprising the C-terminal sequence of NheB (amino acids 205 to 372; seeTable 1) were generated in the same way. == Table 1..
The binding regions of both antibodies were determined by using recombinant NheB fragments and synthetic peptides