Genomic and expression analysis of the vanG-like gene cluster of Clostridium difficile

Abstract

Primary antibiotic treatment of Clostridium difficile intestinal diseases requires metronidazole or vancomycin therapy. A cluster of genes homologous to enterococcal glycopeptides resistance vanG genes was found in the genome of C. difficile 630, although this strain remains sensitive to vancomycin. This vanG-like gene cluster was found to consist of five ORFs: the regulatory region consisting of vanR and vanS and the effector region consisting of vanG, vanXY and vanT. We found that 57 out of 83 C. difficile strains, representative of the main lineages of the species, harbour this vanG-like cluster. The cluster is expressed as an operon and, when present, is found at the same genomic location in all strains. The vanG, vanXY and vanT homologues in C. difficile 630 are co-transcribed and expressed to a low level throughout the growth phases in the absence of vancomycin. Conversely, the expression of these genes is strongly induced in the presence of subinhibitory concentrations of vancomycin, indicating that the vanG-like operon is functional at the transcriptional level in C. difficile. Hydrophilic interaction liquid chromatography (HILIC-HPLC) and MS analysis of cytoplasmic peptidoglycan precursors of C. difficile 630 grown without vancomycin revealed the exclusive presence of a UDP-MurNAc-pentapeptide with an alanine at the C terminus. UDP-MurNAc-pentapeptide [D-Ala] was also the only peptidoglycan precursor detected in C. difficile grown in the presence of vancomycin, corroborating the lack of vancomycin resistance. Peptidoglycan structures of a vanG-like mutant strain and of a strain lacking the vanG-like cluster did not differ from the C. difficile 630 strain, indicating that the vanGlike cluster also has no impact on cell-wall composition.

Clostridium difficile infection

“…C. difficile also demonstrates extensive acquired antimicrobial resistance 9,251 . Interestingly, C. difficile vegetative cells are sensitive to teicoplanin and vancomycin despite harbouring a genomic region that resembles a vanG glycopeptide resistance cluster 9,252 . This cluster can confer vancomycin resistance to a heterologous host, but why it is not functional in C. difficile is unclear 253 .…”

Characteristics of the Clostridium difficile cell envelope and its importance in therapeutics

“…For example, Enterococcus faecalis achieves low‐level resistance through synthesis of d ‐Ala‐ d ‐Ser precursors by enzymes encoded in the vanG operon (Depardieu et al ., ). Genes homologous to those in the vanG operon have been identified in the majority of C. difficile strains tested (Ammam et al ., ; Peltier et al ., ) and are able to synthesize d ‐Ala‐ d ‐ser precursors in response to sublethal concentrations of vancomycin (Ammam et al ., ) although this is debated (Peltier et al ., ). Introduction of the vanG cd operon into a vancomycin‐sensitive strain of E. coli conferred low‐level resistance to vancomycin and a C. difficile mutant lacking this cluster displays a slightly lower vancomycin MIC relative to wild‐type (Ammam et al ., ).…”

AsnB is responsible for peptidoglycan precursor amidation in Clostridium difficile in the presence of vancomycin

“…Vancomycin, metronidazole and fidaxomycin are the recommended antibiotics used for the treatment of C. difficile infections [10]. An orthologue gene cluster of vanG, encoding vancomycin resistance in enterococci, was reported in most C. difficile clinical isolates [11,12]. This vanG Cd cluster is composed of two operons, one made of vanR Cd and vanS Cd is involved in the regulation of the vanG Cd operon, and the second composed of vanG Cd , vanXY Cd and vanT Cd involved in the modification of PG precursors' structure.…”