Abstract
Diarrheal disease caused by Bacillus cereus is driven by pore-forming enterotoxins, with hemolysin BL (Hbl) and non-hemolytic enterotoxin (Nhe) as major virulence factors. Toxin structure and mode of action are well described, while especially human intestinal epithelial responses remain incompletely characterized. With the co-culture system of Caco-2 and mucus-producing HT29-MTX cells (9:1), this study describes a suitable model for investigating enterotoxin effects. Cell line-, dose- and enterotoxin-dependent changes in cell viability were detected. Undifferentiated Caco-2 generally showed higher susceptibility than HT29-MTX cells. Twenty-four-hour co-cultures were more susceptible than 28-day co-cultures but displayed partial recovery after toxin removal. Enterotoxin exposure caused a time-dependent decline in transepithelial electrical resistance, consistent with barrier dysfunction. In Ussing chamber experiments, enterotoxin-containing supernatants, especially Nhe+Hbl, increased short-circuit currents, indicating altered electrogenic ion transport consistent with pro-secretory phenotypes relevant to diarrhea. Transcriptome analyses demonstrated a pronounced time- and dose-dependent response toward Nhe+Hbl. Two-hour sublethal enterotoxin exposure elicited few transcriptional changes, whereas lethal enterotoxin exposure rapidly suppressed proliferation- and cell-cycle-associated programs. Pathway analyses consistently highlighted TNFα/NF-κB-driven inflammation, MAPK-associated stress signaling, and p53/apoptosis pathways; metabolic remodeling and senescence-/autophagy-related processes became more apparent at longer exposure (8 h) and/or higher toxin levels. Dose-dependent caspase-3/7 activation, LDH release, and minimal caspase-1 activation were also consistent with early inflammatory response, apoptotic cell death and membrane damage. Collectively, this study integrates barrier function, ion transport, cell viability, and transcriptomics to define host intestine epithelial response programs triggered by B. cereus enterotoxins and to connect toxin exposure to barrier dysfunction and diarrheal pathophysiology.