Clostridioides difficile canonical L,D-transpeptidases catalyze a novel type of peptidoglycan cross-links and are not required for beta-lactam resistance

Clostridioides dif fi cile is the leading cause of antibioticassociated diarrhea worldwide with signi fi cant morbidity and mortality. This organism is naturally resistant to several betalactam antibiotics that inhibit the polymerization of peptidoglycan, an essential component of the bacteria cell envelope. Previous work has revealed that C. dif fi cile peptidoglycan has an unusual composition. It mostly contains 3-3 cross -links, catalyzed 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 three canonical Ldts encoded by C. dif fi cile (Ldt Cd1 , Ldt Cd2 , and Ldt Cd3 ) in vitro and explored their contribution to growth and antibiotic resistance. We show that two of these enzymes catalyze the formation of novel types of peptidoglycan cross -links using meso -diaminopimelic acid both as a donor and an acceptor, also observed in peptidoglycan sacculi. We demonstrate that the simultaneous deletion of these three genes only has a minor impact on both peptidoglycan structure and resistance to beta-lactams. This unexpected result therefore implies that the formation of 3-3 peptidoglycan cross -links in C. dif fi cile is catalyzed by as yet unidenti fi ed noncanonical Ldt enzymes.