Impact of peptidoglycan-remodeling enzymes on antibiotic and stress resistance in Clostridioides difficile – REMODIFF

Clostridioides difficile infection (CDI) is the primary cause of nosocomial antibiotic-associated diarrhoea and imposes a significant burden on healthcare. ß-lactam antibiotics, particularly cephalosporins, are recognized as major causative agents of CDI but the mechanism of intrinsic resistance of C. difficile to cephalosporins remains unclear. ß-lactams are broad-spectrum antibiotics targeting cell wall peptidoglycan (PG) assembly. PG is composed of glycan chains cross-linked by short peptides and provides protection against environmental stresses. In most bacteria, peptide linkages are primarily created by D,D-transpeptidases (DDT) belonging to the family of penicillin-binding proteins (PBPs) and primary targets of most ß-lactam antibiotics. However, most of the PG cross-links are synthesized by non-classical and essential transpeptidases in C. difficile, namely the L,D-transpeptidases (LDTs), that are insensitive to ß-lactams with the exception of carbapenems. C. difficile is the first and so far, the only bacterium in which LDTs are essential for viability. This reveals an original mode of PG expansion and implies that inhibition of the LDT activity could be deleterious to C. difficile. Whereas DDTs use native PG precursors containing a pentapeptide as acyl donors to generate cross-links, LDTs requires a tetrapeptide stem in the acyl donor substrate and their activity can therefore be limited by their substrate availability. Tetrapeptide stems are produced by PG-remodeling enzymes including D,D-carboxypeptidases that cleave off the terminal residue of pentapeptide stems and specific endopeptidases and they can also be degraded into tripeptide stems by L,D-carboxypeptidases. The objective of this project is to characterize the role of the major PG-remodeling enzymes in C. difficile in controlling LDT activity, and define their implication in PG homeostasis, ß-lactam resistance and adaptation to environmental stresses. This will greatly enhance our current understanding of PG dynamics in link with ß-lactam resistance in C. difficile. These enzymes could be leveraged as targets of vulnerability to inform approaches to combat C. difficile infections.