Substrate Inhibition of VanA by d-Alanine Reduces Vancomycin Resistance in a VanX-Dependent Manner

Antimicrob Agents Chemother. 2016 Jul 22;60(8):4930-9. doi: 10.1128/AAC.00276-16. Print 2016 Aug.

Abstract

The increasing resistance of clinical pathogens against the glycopeptide antibiotic vancomycin, a last-resort drug against infections with Gram-positive pathogens, is a major problem in the nosocomial environment. Vancomycin inhibits peptidoglycan synthesis by binding to the d-Ala-d-Ala terminal dipeptide moiety of the cell wall precursor lipid II. Plasmid-transferable resistance is conferred by modification of the terminal dipeptide into the vancomycin-insensitive variant d-Ala-d-Lac, which is produced by VanA. Here we show that exogenous d-Ala competes with d-Lac as a substrate for VanA, increasing the ratio of wild-type to mutant dipeptide, an effect that was augmented by several orders of magnitude in the absence of the d-Ala-d-Ala peptidase VanX. Liquid chromatography-mass spectrometry (LC-MS) analysis showed that high concentrations of d-Ala led to the production of a significant amount of wild-type cell wall precursors, while vanX-null mutants produced primarily wild-type precursors. This enhanced the efficacy of vancomycin in the vancomycin-resistant model organism Streptomyces coelicolor, and the susceptibility of vancomycin-resistant clinical isolates of Enterococcus faecium (VRE) increased by up to 100-fold. The enhanced vancomycin sensitivity of S. coelicolor cells correlated directly to increased binding of the antibiotic to the cell wall. Our work offers new perspectives for the treatment of diseases associated with vancomycin-resistant pathogens and for the development of drugs that target vancomycin resistance.

MeSH terms

  • Alanine / metabolism*
  • Anti-Bacterial Agents / pharmacology
  • Bacterial Proteins / metabolism*
  • Carbon-Oxygen Ligases / metabolism*
  • Cell Wall / drug effects
  • Cell Wall / metabolism
  • Drug Resistance, Microbial / drug effects*
  • Enterococcus faecium / drug effects
  • Enterococcus faecium / metabolism
  • Glycopeptides / metabolism
  • Ligases / metabolism
  • Peptidoglycan / metabolism
  • Serine-Type D-Ala-D-Ala Carboxypeptidase / metabolism*
  • Streptomyces coelicolor / drug effects
  • Streptomyces coelicolor / metabolism
  • Vancomycin / pharmacology
  • Vancomycin Resistance / drug effects*

Substances

  • Anti-Bacterial Agents
  • Bacterial Proteins
  • Glycopeptides
  • Peptidoglycan
  • VanA ligase, Bacteria
  • Vancomycin
  • Serine-Type D-Ala-D-Ala Carboxypeptidase
  • VanX dipeptidase
  • Ligases
  • Carbon-Oxygen Ligases
  • lactylalanine synthetase
  • Alanine

Grant support

This research received no specific grant from any funding agency in the public, commercial, or not-for-profit sectors.