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 A(2)pm(3) → A(2)pm(3) (A(2)pm, 2,6-diaminopimelic acid) cross-links and only a minority of the more classical Ala(4) → A(2)pm(3) cross-links. Moreover, a significant amount of muropeptides contains a modified tetrapeptide stem ending in Gly instead of D-Ala(4). Two L,D-transpeptidases homologues encoding genes present in the genome of C. difficile 630 and named ldt(cd1) and ldt(cd2), were inactivated. The inactivation of either ldt(cd1) or ldt(cd2) significantly decreased the abundance of 3-3 cross-links, leading to a marked decrease of peptidoglycan reticulation and demonstrating that both ldt(cd1)-and ldt(cd2)-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.
Highlights
Peptidoglycan (PG)2 of Gram-positive bacteria usually consists of long linear glycan strands cross-linked by short stem peptides [5, 6]
The PGs of C. difficile should have some specificities regarding the effect of antibiotics inhibiting PG biosynthesis; C. difficile, susceptible to -lactams, exhibits higher minimal inhibitory concentrations than in other Clostridium species such as Clostridium perfringens [24], and it displays a preserved susceptibility to vancomycin despite the presence of a vanG-like operon [25]
Vegetative Cell Wall Peptidoglycan Composition of C. difficile 630—Muropeptides of C. difficile 630 were obtained from PG digestion by muramidase mutanolysin, reduced with sodium borohydride, and separated by reverse-phase high performance liquid chromatography (RP-HPLC)
Summary
Bacterial Strains and Culture Conditions—The bacterial strains used in this study are listed in supplemental Table S1. PMTL007::Cdi-ldtcd1-1231s and pMTL007::Cdi-ldtcd2-480s plasmids (supplemental Table S2) retargeted the group II intron to insert into ldtcd and ldtcd genes in the sense orientation immediately after the 1231th and 480th nucleotides in the coding sequence, respectively, within the DNA sequence encoding the catalytic domain. These derivative pMTL007 plasmids were transformed into the conjugative donor E. coli HB101 (RP4) and transferred via conjugation into the C. difficile 630⌬erm. Genomic DNA was isolated from single thiamphenicol-resistant colonies and subjected to PCR using primer ldtcd1-F with the EBS universal primer (supplemental Table S2) to determine the presence of an intron insertion. Positive individual colonies were tested by PCR using ldtcd1-flanking primers (supplemental Table S2)
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