Few therapeutic alternatives remain for the treatment of infections due to multiresistant Mycobacterium abscessus. Here we show that the peptidoglycans of the "rough" and "smooth" morphotypes contain predominantly 3?3 cross-links generated by l,d-transpeptidases, indicating that these enzymes are attractive targets for the development of efficient drugs.
The peptidoglycan layer is a vital component of the bacterial cell wall. The existing paradigm describes the peptidoglycan network as a static structure that is cross-linked predominantly by 4-->3 transpeptide linkages. However, the nonclassical 3-->3 linkages predominate the transpeptide networking of the peptidoglycan layer of nonreplicating Mycobacterium tuberculosis. The molecular basis of these linkages and their role in the physiology of the peptidoglycan layer, virulence and susceptibility of M. tuberculosis to drugs remain undefined. Here we identify MT2594 as an L,D-transpeptidase that generates 3-->3 linkages in M. tuberculosis. We show that the loss of this protein leads to altered colony morphology, loss of virulence and increased susceptibility to amoxicillin-clavulanate during the chronic phase of infection. This suggests that 3-->3 cross-linking is vital to the physiology of the peptidoglycan layer. Although a functional homolog exists, expression of ldtMt2 is dominant throughout the growth phases of M. tuberculosis. 4-->3 transpeptide linkages are targeted by one of the most widely used classes of antibacterial drugs in human clinical use today, beta-lactams. Recently, meropenem-clavulanate was shown to be effective against drug-resistant M. tuberculosis. Our study suggests that a combination of L,D-transpeptidase and beta-lactamase inhibitors could effectively target persisting bacilli during the chronic phase of tuberculosis.
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 beta-lactams, the most broadly used class of antibiotics. To gain insight into the mechanism of beta-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 beta-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 beta-lactam resistance in C. jeikeium.
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