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

Abstract

The nonhemolytic enterotoxin (Nhe) is known as a major pathogenicity factor for the diarrheal type of food poisoning caused by Bacillus cereus. The Nhe complex consists of NheA, NheB and NheC, all of them required to reach maximum cytotoxicity following a specific binding order on cell membranes. Here we show that complexes, formed between NheB and NheC under natural conditions before targeting the host cells, are essential for toxicity in Vero cells. To enable detection of NheC and its interaction with NheB, monoclonal antibodies against NheC were established and characterized. The antibodies allowed detection of recombinant NheC in a sandwich immunoassay at levels below 10 ng ml−1, but no or only minor amounts of NheC were detectable in natural culture supernatants of B. cereus strains. When NheB- and NheC-specific monoclonal antibodies were combined in a sandwich immunoassay, complexes between NheB and NheC could be demonstrated. The level of these complexes was directly correlated with the relative concentrations of NheB and NheC. Toxicity, however, showed a bell-shaped dose-response curve with a plateau at ratios of NheB and NheC between 50:1 and 5:1. Both lower and higher ratios between NheB and NheC strongly reduced cytotoxicity. When the ratio approached an equimolar ratio, complex formation reached its maximum resulting in decreased binding of NheB to Vero cells. These data indicate that a defined level of NheB-NheC complexes as well as a sufficient amount of free NheB is necessary for efficient cell binding and toxicity. Altogether, the results of this study provide evidence that the interaction of NheB and NheC is a balanced process, necessary to induce, but also able to limit the toxic action of Nhe.

The Bacillus cereus Hbl and Nhe Tripartite Enterotoxin Components Assemble Sequentially on the Surface of Target Cells and Are Not Interchangeable

“…We are uncertain about the physiological consequences of this binding. A more recent study suggested that at higher concentrations, NheB attaches unspecifically to Vero cells, which may result in an inability to further form active complexes upon binding of NheC and NheA [23]. Our findings parallel these results.…”

“…In a later study, however, the authors identified NheB binding using one assay but not another; additionally, NheC was described as a binding moiety [22]. Additionally, it was recently shown that in solution, NheB and NheC form complexes, and it was suggested that this complex, in addition to NheC alone, may bind to the cellular surface [23]. Thus, for both the Hbl and Nhe toxins, the binding order of toxin components and the mechanisms of pore formation are not clear.…”

HIV-1 gp140 epitope recognition is influenced by immunoglobulin DH gene segment sequence

“…The PEPperPRINT ® chip allows an assessment of binding to 849 possible overlapping 13 amino acid linear epitopes within the protein sequence of gp160 (Heilkenbrinker et al, 2013; Shukla et al, 2012). Based on averaged median foreground intensities, intensity maps were generated and antibody binding to the peptide map was highlighted by an intensity color code with red for high spot intensities (Fig.…”

Bacillus cereus phospholipases, enterotoxins, and other hemolysins

“…The optimal ratio between the NheA, NheB, and NheC components appear to be about 10:10:1, respectively [87]. A ratio lower than 5:1 and higher than 50:1 between NheB and NheC strongly reduces cytotoxicity [70,86]. The complex formation between NheB and NheC is directly correlated with the relative concentrations.…”

“…Under natural conditions, NheB and NheC form a complex that seem to be necessary to induce cytotoxic activity, and to reach maximum cytotoxicity, the binding of the components follows a specific order on the cell membrane [70]. Due to the complex formation with NheB in solution, free NheC have never been detected in natural culture supernatants of B. cereus strains [66,70,81,87]. The optimal ratio between the NheA, NheB, and NheC components appear to be about 10:10:1, respectively [87].…”