A Clostridioides difficile endolysin modulates toxin secretion without cell lysis

The Clostridia produce and secrete Large Clostridial Glucosylating Toxins (LCGTs) responsible for disease symptoms, but the secretion mechanism is largely unknown. Recently, a holin-like protein was shown to be essential for toxin secretion. Holins, typically bacteriophage-specific proteins, are part of the holin-endo(lysin) system that releases phage progeny. To determine if the clostridia also use a lysin, we investigated two conserved putative lysins, M7404_01910 and M7404_02200, in the release of the LCGTs TcdA and TcdB from a Clostridioides difficile ribotype 027 strain, M7404. Sequence analysis and structural modelling indicates that both proteins are related to N-acetylmuramoyl-l-alanine amidases, similar to CD27L, a lysin from the C. difficile phage ΦCD27. Disruption of these genes reveal that only M7404_02200 contributes to toxin secretion and does so in a non-lytic fashion. Peptidoglycan hydrolysis assays show that recombinant M7404_02200 is an active peptidoglycan amidase, confirming its role in TcdA and TcdB secretion in C. difficile M7404.