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

The resistance of <i>Clostridioides difficile</i> to the β-lactam antibiotics cephalosporins, which target the peptidoglycan (PG) assembly, is a leading contributor to the development of <i>C. difficile</i> infections. <i>C. difficile</i> has an original PG structure with a predominance of 3→3 cross-links generated by l,d-transpeptidases (LDTs). <i>C. difficile</i> forms spores and we show that the spore cortex PG contains exclusively 3→3 cross-links. PG and spore cortex of <i>C. difficile</i> 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 <i>C. difficile</i>.