title: Contribution of VanY D,D-carboxypeptidase to glycopeptide resistance in Enterococcus faecalis by hydrolysis of peptidoglycan precursors.
abstract: The vanR, vanS, vanH, vanA, and vanX genes of enterococcal transposon Tn1546 were introduced into the chromosome of Enterococcus faecalis JH2-2. Complementation of this portion of the van gene cluster by a plasmid encoding VanY D,D-carboxypeptidase led to a fourfold increase in the vancomycin MIC (from 16 to 64 micrograms/ml). Multicopy plasmids pAT80 (vanR vanS vanH vanA vanX) and pAT382 (vanR vanS vanH vanA vanX vanY) conferred similar levels of vancomycin resistance to JH2-2. The addition of D-alanine (100 mM) to the culture medium restored the vancomycin susceptibility of E. faecalis JH2-2/pAT80. The pentapeptide UDP-MurNAc-L-Ala-gamma-D-Glu-L-Lys-D-Ala-D-Ala partially replaced pentadepsipeptide UDP-MurNAc-L-Ala-gamma-D-Glu-L-Lys-D-Ala-D-Lac when the strain was grown in the presence of D-alanine. In contrast, resistance mediated by pAT382 was almost unaffected by the addition of the amino acid. Expression of the vanY gene of pAT382 resulted in the formation of the tetrapeptide UDP-MurNAc-L-Ala-gamma-D-Glu-L-Lys-D-Ala, indicating that a portion of the cytoplasmic precursors had been hydrolyzed. These results show that VanY contributes to glycopeptide resistance in conditions in which pentapeptide is present in the cytoplasm above a threshold concentration. However, the contribution of the enzyme to high-level resistance mediated by Tn1546 appears to be moderate, probably because hydrolysis of D-alanyl-D-alanine by VanX efficiently prevents synthesis of the pentapeptide.
authors: Unité des Agents Antibactériens, Centre National de la Recherche Scientifique, Institut Pasteur, Paris, France.
authors: M
authors: M
authors: Arthur
authors: F
authors: F
authors: Depardieu
authors: H A
authors: HA
authors: Snaith
authors: P E
authors: PE
authors: Reynolds
authors: P
authors: P
authors: Courvalin
mesh_terms: D000409:Alanine / Q000494:pharmacology; D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism*; D019729:Carbon-Oxygen Ligases; D002268:Carboxypeptidases / Q000235:genetics / Q000378:metabolism*; D002473:Cell Wall / Q000378:metabolism; D004352:Drug Resistance, Microbial; D013293:Enterococcus faecalis / Q000187:drug effects* / Q000201:enzymology*; D006020:Glycopeptides / Q000494:pharmacology*; D006868:Hydrolysis; D008025:Ligases / Q000378:metabolism; D008969:Molecular Sequence Data; D010457:Peptidoglycan / Q000378:metabolism*; D010641:Phenotype; D014640:Vancomycin / Q000494:pharmacology
chemical_list: D001426:Bacterial Proteins; D006020:Glycopeptides; D010457:Peptidoglycan; C071437:VanA ligase, Bacteria; D014640:Vancomycin; D002268:Carboxypeptidases; D008025:Ligases; D019729:Carbon-Oxygen Ligases; D000409:Alanine