The l,d-transpeptidation pathway is inhibited by antibiotics of the β-lactam class in Clostridioides difficile.

The resistance of Clostridioides difficile to the β-lactam antibiotics cephalosporins, which target the peptidoglycan (PG) assembly, is a leading contributor to the development of C. difficile infections. C. difficile has an original PG structure with a predominance of 3→3 cross-links generated by l,d-transpeptidases (LDTs). C. difficile forms spores and we show that the spore cortex PG contains exclusively 3→3 cross-links. PG and spore cortex of C. difficile cells were largely unaffected by the deletion of the three predicted LDTs, revealing the implication of a new family of LDTs. The d,d-carboxypeptidases producing the essential LDT substrate were inactivated by cephalosporins, resulting in the inhibition of the l,d-transpeptidation pathway. In contrast, the participation of penicillin-binding proteins (PBPs) to PG cross-linking increased in the presence of the antibiotics. Our findings highlight that cephalosporin resistance is not primarily mediated by LDTs and illustrate the plasticity of the PG biosynthesis machinery in C. difficile.