Clostridioides difficile's virulence requires efficient holin-mediated toxin secretion

The human pathogen Clostridioides difficile causes pseudomembranous colitis through release of the potent TcdA and TcdB toxins. These toxins lack canonical N-terminal secretion signal sequences for translocation across the bacterial membrane, suggesting alternate mechanisms of release including targeted secretion mediated by the holin-like protein TcdE and passive release from cell lysis. Here, we show how the lack of the TcdE holin profoundly reduces toxin secretion in the high toxin-producing strains UK1 and VPI10463, independently of cell lysis. Remarkably, tcdE deletion in either strain rescued highly susceptible gnotobiotic mice from lethal infection. ΔtcdE-infected mice demonstrated undetectable levels of toxin acutely and long-term survival, despite active toxin gene expression and cytoplasmic accumulation in mutant strains. These findings demonstrate the dominant role of the TcdE holin in toxin secretion in vivo and host disease and confirm the use of a conserved and non-lytic holin-mediated toxin secretion mechanism among toxigenic species of Clostridia.