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Alterations in peptidoglycan precursors and vancomycin susceptibility in Tn917 insertion mutants of Enterococcus
1Rockefeller University, New York, New York 10021.
Abstract:
Derivatives of the highly vancomycin-resistant Enterococcus faecalis strain 221 (MIC, 1,024 micrograms/ml) harboring Tn917 insertions in vanR, vanH, and vanA were compared with the parent strain and the susceptible plasmid-free strain JH2-2 (MIC, 2 micrograms/ml). Cytoplasmic pools of UDP-N-acetyl-muramyl-peptide precursors of strain 221 contained the depsipeptide-terminating precursor as well as elevated levels of both the tripeptide and tetrapeptide precursors. Insertional inactivation of vanR resulted in the loss of carboxypeptidase activity, full susceptibility to vancomycin, and precursor pools similar to those of JH2-2. For the vanA insertional mutant the MBC of vancomycin was fourfold higher than that for JH2-2, and the mutant had increased levels of tripeptide and tetrapeptide precursors compared with those for JH2-2. The vanH insertional mutant showed elevated levels of these precursors, as well as a small amount of depsipeptide, and both the MIC and the MBC of vancomycin were increased compared with those for JH2-2. These findings suggest that DD-carboxypeptidase activity, under the control of vanR, results in increased pools of both tripeptide and tetrapeptide precursors, which may contribute to survival in the presence of vancomycin.
Insights
Investigating vancomycin resistance in Enterococcus faecalis, this study reveals how specific gene mutations (vanR, vanH, vanA) alter precursor pools, impacting bacterial survival against vancomycin antibiotics.
Area of Science:
- Microbiology
- Molecular Biology
- Antibiotic Resistance
Background:
- Enterococcus faecalis exhibits high-level vancomycin resistance (VRE) due to complex genetic mechanisms.
- Understanding the biochemical pathways underlying VRE is crucial for developing new therapeutic strategies.
Purpose of the Study:
- To investigate the role of vanR, vanH, and vanA genes in vancomycin resistance in E. faecalis.
- To elucidate the impact of these genes on the cytoplasmic pools of peptidoglycan precursors.
Main Methods:
- Comparative analysis of vancomycin-resistant E. faecalis strain 221 derivatives with Tn917 insertions in vanR, vanH, and vanA against the parent strain and a susceptible strain (JH2-2).
- Assessment of cytoplasmic UDP-N-acetyl-muramyl-peptide precursor pools.
- Determination of minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) of vancomycin.
Main Results:
- Strain 221 precursors included depsipeptide-terminating precursor and elevated tripeptide/tetrapeptide levels.
- vanR inactivation led to loss of carboxypeptidase activity and full vancomycin susceptibility.
- vanA and vanH mutants showed increased precursor levels and higher vancomycin MIC/MBC values, with vanH mutants also producing depsipeptide.
Conclusions:
- DD-carboxypeptidase activity, regulated by vanR, increases tripeptide and tetrapeptide precursor pools.
- These altered precursor pools likely contribute to vancomycin resistance and survival in E. faecalis.