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 A 2 pm 3 3 A 2 pm 3 (A 2 pm, 2,6-diaminopimelic acid) cross-links and only a minority of the more classical Ala 4 3 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.Clostridium difficile, a Gram-positive spore-forming bacterium, is the major cause of intestinal diseases associated with antibiotic therapy such as ampicillin, clindamycin, and cephalosporins, which disrupt the barrier intestinal flora and allow C. difficile colonization (1, 2). Clinical manifestations range from asymptomatic colonization or mild diarrhea to pseudomembranous colitis (3). The main virulence factors have been identified as toxin A and B (4). Recent outbreaks have led to increasing morbidity and mortality and have been associated with a new highly virulent strain (BI/NAP1/027) of C. difficile.Antibiotic treatment of C. difficile-associated disease requires metronidazole or vancomycin therapy.Peptidoglycan ( (9), which were originally detected in Enterococcus faecium (Ldt fm ) (10) and then in other Gram-positive bacteria (11,12), in mycobacteria (13-15), and in Escherichia coli (16,17). Ldts use acyl donors containing a tetrapeptide stem (9) and were consequently expected to confer resistance to ␤-lactams (10, 18).Another possible variation of the PG structure is the occurrence of N-deacetylation or O-acetylation of glycan strands, either on GlcNAc or on MurNAc residues (19,20). N-Deacetylation in Listeria monocytogenes (21) or Streptococcus pneumoniae (22) and O-acetylation in Staphylococcus aureus have been linked to lysozyme resistance (23).The PGs of C. difficile should have some specificities regarding the effect of antibiotics inhibiting PG biosynthesis; C. difficile, although susceptible to ␤-lactams, exhibits higher minimal inhibitory concentrations than in oth...

Clostridium difficile Extracytoplasmic Function σ Factor σ V Regulates Lysozyme Resistance and Is Necessary for Pathogenesis in the Hamster Model of Infection

“…The resulting plasmids, pTHE519 (csfV 63 ), pCE356 (cd1607 117 ), and pCE358 (cd0739 48 ), were moved into C. difficile JIR8094 via the E. coli conjugation donor HB101/pRK24 (12). The csfV 63 , cd0739 48 , and cd1607 117 intron insertion mutants were generated as previously described (31,41). The insertion of introns into csfV, cd0739, and cd1607 was confirmed by PCR using primers complementary to the chromosome and/or intron.…”

“…To induce expression of genes under the control of P tet , strains bearing P tet vectors were grown in 2 g/ml of tetracycline. Peptidoglycan was purified using a modified B. subtilis peptidoglycan purification protocol (24,47,48). Briefly, cells were harvested by centrifugation, and the supernatant was discarded.…”

“…Teichoic acids were removed by resuspending the pellet in 1 ml of 49% hydrofluoric acid (Thermo Scientific) and incubated with rocking for 48 h at 4°C. The isolated peptidoglycan was digested with mutanolysin for 16 h at 37°C, and the solubilized muropeptides were reduced and separated by reverse-phase high-pressure liquid chromatography (HPLC) as previously described (47,48). Muropeptides separated using a phosphate buffer and methanol gradient were collected and further purified using HPLC and a trifluoroacetic acid-acetonitrile buffer system.…”

“…To construct the csfV, cd0739, and cd1607 mutants, we retargeted plasmid pCE240 (31) or pBL100 (39) using oligonucleotides designed by a targetron algorithm generously provided by Rob Britton (Michigan State University) or ClosTron (40). The intron was PCR amplified using Taq colE1 bla cat repA orfB oriT aad9 xylR P xyl -csfV pCE382 P tet -pdaV colE1 bla cat repA orfB oriT aad9 pCE400 P tet -prsA2 colE1 bla cat repA orfB oriT aad9 pCE383 P tet -pdaVprsA2 colE1 bla cat repA orfB oriT aad9 polymerase (NEB) from a targetron template using the EBS universal primer CDE914 in combination with the following primer sets: for csfV 63 , CDEP958, CDEP956, and CDEP957; for cd0739 48 , CDEP1497, CDEP1498, and CDEP1499; and for cd1607 117 , CDEP1494, CDEP1495, and CDEP1496. The resulting PCR products were digested with HindIII and BsrGI and cloned into pCE240 or pBL100 digested with the same restriction enzymes.…”

Nonclassical Transpeptidases of Mycobacterium tuberculosis Alter Cell Size, Morphology, the Cytosolic Matrix, Protein Localization, Virulence, and Resistance to β-Lactams

“…The ␤-lactams act by inhibiting D,D-transpeptidases (also known as penicillin-binding proteins), a class of enzymes that catalyze the formation of transpeptide linkages between the fourth amino acid of one peptide stem and the third amino acid of another stem. Recently, a complementary class of enzymes, namely, L,D-transpeptidases, that transfer peptide linkage between the L and D centers of the third and the fourth amino acids of donor peptide to the third amino acid of acceptor peptide has been identified in numerous bacteria, including Enterococcus faecium, Bacillus subtilis, Mycobacterium tuberculosis, Clostridium difficile, and E. coli (6,(13)(14)(15)(16)(17)(18). Despite the significance of D,D-transpeptidases, which serve as targets to the most widely used class of antibiotics, the study of the relevance of L,D-transpeptidases to the physiology of the cell and susceptibility to drugs has only recently begun.…”