A targeted cell lysis mechanism facilitates toxin release in Clostridioides difficile

Clostridioides difficile infection depends on the production of two large toxins, TcdA and TcdB, encoded within a pathogenicity locus alongside the phage-like holin TcdE. The mechanism of toxin secretion remains actively debated, with current models proposing either TcdE-dependent non-lytic secretion or TcdE-independent lytic release. Here, we provide evidence for a unifying model where TcdE drives lysis in a phenotypically distinct subpopulation of cells. We show that TcdE, TcdA, and TcdB expression is restricted to a small fraction of cells exhibiting markers of active lysis and establish this subpopulation as the driver of severe pathogenic outcomes in a mouse model of CDI. Overexpression of TcdR, the sigma factor regulating the pathogenicity locus, triggers TcdE-dependent lysis, even in strains previously reported to employ TcdE-independent secretion mechanisms. Correlative light and electron microscopy combined with cryo-ET reveal a distinctive ultrastructure in lytic cells. Membrane vesicles accumulate between a disrupted inner membrane and intact peptidoglycan, alongside electron-dense material containing TcdA. These observations reveal a population-level strategy in which a minority of bacteria sacrifice themselves through TcdE-mediated lysis to release toxins as a 'bet-hedging strategy'.