NheABC is a pH-dependent cytotoxin that targets mitochondria and contributes to the virulence of Bacillus cereus in wound infections

Members of the Bacillus cereus group are Gram-positive, spore-forming, facultative anaerobic bacteria widely distributed in the environment. Pathogenic B. cereus group strains have been linked to a range of illnesses and infections, including severe non-gastrointestinal infections, such as wound infections. In the present study, we show that, among the pore-forming toxin (PFT)-producing bacterial strains tested, B. cereus group strain 75/95 induced pronounced membrane permeabilization in epithelial cells, prompting us to investigate the contribution of its tripartite NheABC toxin to host cell damage. NheABC exhibited potent cell membrane permeability in 2D epithelial cell cultures as well as in 3D spheroids and intestinal organoids, with prominent effects on the cell membrane and mitochondria. Functional analysis using liposome leakage assays demonstrated that cardiolipin (CL) and phosphatidylethanolamine (PE) promote NheABC-mediated membrane permeabilization, suggesting that membrane lipid composition contributes to NheABC activity and mitochondrial damage. Moreover, using erythrocytes and epithelial cells as model systems, we further demonstrated that the cytolytic activity of NheABC is pH-dependent and is markedly reduced under acidic conditions (pH 5.5). This pH dependence was independently confirmed using liposome leakage assays. Importantly, NheABC enhanced bacterial colonization and the local inflammatory response in a murine model of wound infection. Overall, our study highlights the critical role of mitochondria and pH in regulating NheABC-mediated cytolytic activity in mammalian cells, which may lead to the development of novel therapeutic strategies for managing B. cereus group-related gastrointestinal and non-gastrointestinal infections.