The β-lactam-sensitive d,d-carboxypeptidase activity of Pbp4 controls the l,d and d,d transpeptidation pathways in Corynebacterium jeikeium

Corynebacterium jeikeium is an emerging nosocomial pathogen responsible for vascular catheters infections, prosthetic endocarditis and septicemia. The treatment of C. jeikeium infections is complicated by the multiresistance of clinical isolates to antibiotics, in particular to β-lactams, the most broadly used class of antibiotics. To gain insight into the mechanism of β-lactam resistance, we have determined the structure of the peptidoglycan and shown that C. jeikeium has the dual capacity to catalyse formation of cross-links generated by transpeptidases of the d , d and l , d specificities. Two ampicillin-insensitive cross-linking enzymes were identified, Ldt_Cjk1, a member of the active site cysteine l , d -transpeptidase family, and Pbp2c, a low-affinity class B penicillin-binding protein (PBP). In the absence of β-lactam, the PBPs and the l , d -transpeptidase contributed to the formation of 62% and 38% of the cross-links respectively. Although Ldt_Cjk1 and Pbp2C were not inhibited by ampicillin, the participation of the l , d -transpeptidase to peptidoglycan cross-linking decreased in the presence of the drug. The specificity of Ldt_Cjk1 for acyl donors containing a tetrapeptide stem accounts for this effect of ampicillin since the essential substrate of Ldt_Cjk1 was produced by an ampicillin-sensitive d , d -carboxypeptidase (Pbp4_Cjk). Acquisition and mutational alterations of pbp2C accounted for high-level β-lactam resistance in C. jeikeium .