Resistance to glycopeptide antibiotics in the teicoplanin producer is mediated by van gene homologue expression directing the synthesis of a modified cell wall peptidoglycan

Abstract

Glycopeptide resistance has been studied in detail in enterococci and staphylococci. In these microorganisms, high-level resistance is achieved by replacing the C-terminal D-alanyl-D-alanine of the nascent peptidoglycan with D-alanyl-D-lactate or D-alanyl-D-serine, thus reducing the affinities of glycopeptides for cell wall targets. Reorganization of the cell wall is directed by the expression of the van gene clusters. The identification of van gene homologs in the genomes of several glycopeptide-producing actinomycetes suggests the involvement of a similar self-resistance mechanism to avoid suicide. This report describes a comprehensive study of self-resistance in Actinoplanes teichomyceticus ATCC 31121, the producer of the clinically relevant glycopeptide teicoplanin. A. teichomyceticus ATCC 31121 showed a MIC of teicoplanin of 25 microg/ml and a MIC of vancomycin of 90 microg/ml during vegetative growth. The vanH, vanA, and vanX genes of A. teichomyceticus were found to be organized in an operon whose transcription was constitutive. Analysis of the UDP-linked peptidoglycan precursors revealed the presence of UDP-glycomuramyl pentadepsipeptide terminating in D-alanyl-D-lactate. No trace of precursors ending in d-alanyl-d-alanine was detected. Thus, the van gene complex was transcribed and expressed in the genetic background of A. teichomyceticus and conferred resistance to vancomycin and teicoplanin through the modification of cell wall biosynthesis. During teicoplanin production (maximum productivity, 70 to 80 microg/ml), the MIC of teicoplanin remained in the range of 25 to 35 microg/ml. Teicoplanin-producing cells were found to be tolerant to high concentrations of exogenously added glycopeptides, which were not bactericidal even at 5,000 microg/ml.

Figures

FIG. 1.

LC-MS analysis of the cell wall cytoplasmatic precursor pool in ramoplanin-treated mycelium (B) or untreated mycelium (A) of A. teichomyceticus. Bacterial cultures were grown, harvested, and extracted as reported in Materials and Methods. Detection was by ESI-MS. A major peak, detectable only in ramoplanin-treated cells at a retention time of 17.8 min (indicated by the arrow), corresponded to the cell wall precursor UDP-N-glycolylmuramyl-Gly-d-Glu-mDap-d-Ala-d-Lac (as identified by ESI-MS). No trace of precursors terminating in d-Ala-d-Ala was detected. The other peaks present on the HPLC plot showed MS profiles not related to soluble peptidoglycan precursors.

FIG. 2.

(A) Full-scan mass spectrum (negative ion current) of the peak eluted at 17.8 min. The molecular ion [M-H]− at m/z 1195.2 corresponded to the UDP-N-glycolylmuramyl-Gly-d-Glu-mDap-d-Ala-d-Lac and double-charged [M-H]2− at m/z 597.1. UDP-N-glycolylmuramyl-Gly-d-Glu-m-3-hydroxy-Dap-d-Ala-d-Lac is identifiable by the molecular ion [M-H]− at 1211.0 and double-charged [M-2H]2− at m/z 605.0. The ion at m/z 402.9 corresponded to UDP formed directly in the MS source. (B) MS-MS analysis of the quasimolecular ion [M-H]− at m/z 1195.2. (C) Chemical structure of the UDP-N-glycolylmuramyl-Gly-d-Glu-mDap-d-Ala-d-Lac (R = H) or of the UDP-N-glycolylmuramyl-Gly-d-Glu-m-3-hydroxy-Dap-d-Ala-d-Lac (R = OH) and structural assignments for the main fragment ions. Note that the masses of the observed fragment ions (numbers above arrows) are 1 Da smaller than the masses of the neutral structures due to the loss of H+.

FIG. 3.

Teicoplanin MICs (thick bars) in A. teichomyceticus mycelium prepared from fermentation flasks during teicoplanin production. The levels of production of teicoplanin (reported as mg/liter ⧫) and growth (reported as g/liter [dry weight] of mycelium ▴) at the different fermentation intervals are indicated. The results are the averages of three independent experiments. Bars represent ± 1 standard deviation.