Clostridioides difficile canonical L,D-transpeptidases catalyse a novel type of peptidoglycan crosslinks

Clostridioides difficile is the leading cause of antibiotic-associated diarrhoea worldwide with significant morbidity and mortality. This organism is naturally resistant to several beta-lactam antibiotics that inhibit the polymerisation of peptidolgycan, the essential component of the bacteria cell envelope. C. difficile can cause infections following a dysbiosis induced by a treatment with these drugs. Previous work has revealed that C. difficile peptidoglycan has an unusual composition. It mostly contains 3-3 crosslinks, catalysed by enzymes called L,D-transpeptidases (Ldts) that are poorly inhibited by beta-lactams. It was therefore hypothesized that peptidoglycan polymerization by these enzymes could underpin antibiotic resistance. Here, we investigated the catalytic activity of the 3 canonical Ldts encoded by C. difficile (LdtCd1, LdtCd2 and LdtCd3) in vitro and explored their contribution to growth and antibiotic resistance. We showed that two of these enzymes catalyse the formation of novel types of peptidoglycan crosslinks and demonstrated that the simultaneous deletion of these 3 genes only has a minor impact on both peptidoglycan structure and resistance to beta-lactams. This unexpected result therefore revealed that the formation of 3-3 peptidoglycan crosslinks in C. difficile is mediated by yet unidentified non-canonical Ldt enzymes.