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

The human pathogen <i>Clostridioides difficile</i> 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, <i>tcdE</i> deletion in either strain rescued highly susceptible gnotobiotic mice from lethal infection. Δ<i>tcdE</i>-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 <i>in vivo</i> and host disease and confirm the use of a conserved and non-lytic holin-mediated toxin secretion mechanism among toxigenic species of <i>Clostridia</i>.