Bacillus cereus cytotoxins Hbl, Nhe and CytK are secreted via the Sec translocation pathway

Background: Bacillus cereus and the closely related Bacillus thuringiensis are Gram positive opportunistic pathogens that may cause food poisoning, and the three secreted pore-forming cytotoxins Hbl, Nhe and CytK have been implicated as the causative agents of diarrhoeal disease. It has been proposed that the Hbl toxin is secreted using the flagellar export apparatus (FEA) despite the presence of Sec-type signal peptides. As protein secretion is of key importance in virulence of a microorganism, the mechanisms by which these toxins are secreted were further investigated. Results: Sec-type signal peptides were identified in all toxin components, and secretion of Hbl component B was shown to be dependent on an intact Sec-type signal peptide sequence. Further indication that secretion of Hbl, Nhe and CytK is dependent on the Sec translocation pathway, the main pathway on which bacterial secretion relies, was suggested by the observed intracellular accumulation and reduced secretion of the toxins in cultures supplemented with the SecA inhibitor sodium azide. Although a FEA deficient strain (a flhA mutant) showed reduced toxin expression and reduced cytotoxicity, it readily secreted overexpressed Hbl B, showing that the FEA is not required for Hbl secretion. Thus, the concurrent lack of flagella and reduced toxin secretion in the FEA deficient strain may point towards the presence of a regulatory link between motility and virulence genes, rather than FEA-dependent toxin secretion. Conclusions: The Hbl, Nhe and CytK toxins appear to be secreted using the Sec pathway, and the reduced Hbl expression of a FEA deficient strain was shown not to be due to a secretion defect.