Clostridium difficile Has an Original Peptidoglycan Structure with a High Level of N-Acetylglucosamine Deacetylation and Mainly 3-3 Cross-links

Abstract The structure of the vegetative cell wall peptidoglycan of Clostridium difficile was determined by analysis of its constituent muropeptides with a combination of reverse-phase high pressure liquid chromatography separation of muropeptides, amino acid analysis, mass spectrometry and tandem mass spectrometry. The structures assigned to 36 muropeptides evidenced several original features in C. difficile vegetative cell peptidoglycan. First, it is characterized by a strikingly high level of N-acetylglucosamine deacetylation. In addition, the majority of dimers (around 75%) contains A2pm3 → A2pm3 (A2pm, 2,6-diaminopimelic acid) cross-links and only a minority of the more classical Ala4 → A2pm3 cross-links. Moreover, a significant amount of muropeptides contains a modified tetrapeptide stem ending in Gly instead of d-Ala4. Two l,d-transpeptidases homologues encoding genes present in the genome of C. difficile 630 and named ldtcd1 and ldtcd2, were inactivated. The inactivation of either ldtcd1 or ldtcd2 significantly decreased the abundance of 3-3 cross-links, leading to a marked decrease of peptidoglycan reticulation and demonstrating that both ldtcd1-and ldtcd2-encoded proteins have a redundant l,d-transpeptidase activity. The contribution of 3-3 cross-links to peptidoglycan synthesis increased in the presence of ampicillin, indicating that this drug does not inhibit the l,d-transpeptidation pathway in C. difficile.