Induction of vancomycin resistance in vancomycin-resistant enterococci (VRE) involves replacement of the d-Ala-d-Ala terminus of peptidoglycan (PG) stems with d-Ala-d-Lac, dramatically reducing the binding affinity of vancomycin for lipid II. Effects from vancomycin resistance induction in Enterococcus faecalis (ATCC 51299) were characterized using a combined solid-state nuclear magnetic resonance (NMR) and liquid chromatography-mass spectrometry (LC-MS) analysis. Solid-state NMR directly measured the total amounts of d-Lac and l,d-Ala metabolized from [2-13C]pyruvate, accumulated Park's nucleotide, and changes to the PG bridge-linking density during the early exponential growth phase (OD660 = 0.4) in intact whole cells of VRE. A high level of accumulation of depsipeptide-substituted Park's nucleotide consistent with the inhibition of the transglycosylation step of PG biosynthesis during the initial phase of vancomycin resistance was observed, while no changes to the PG bridge-linking density following the induction of vancomycin resistance were detected. This indicated that the attachment of the PG bridge to lipid II by the peptidyl transferases was not inhibited by the d-Ala-d-Lac-substituted PG stem structure in VRE. Compositions of mutanolysin-digested isolated cell walls of VRE grown with and without vancomycin resistance induction were determined by LC-MS. Muropeptides with PG stems terminating in d-Ala-d-Lac were found only in VRE grown in the presence of vancomycin. Percentages of muropeptides with a pentapeptide stem terminating in d-Ala-d-Lac for VRE grown in the presence of vancomycin were 26% for the midexponential phase (OD660 = 0.6) and 57% for the stationary growth phase (OD660 = 1.0). These high percentages indicate that d-Ala-d-Lac-substituted lipid II was efficiently utilized for PG biosynthesis in VRE.

中文翻译：

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结合固态核磁共振和质谱分析法定量耐万古霉素的粪肠球菌中d-Ala-d-Lac端接的肽聚糖结构。

在耐万古霉素肠球菌（VRE）中诱导对万古霉素的耐药性涉及用d-Ala-d-Lac取代肽聚糖（PG）茎的d-Ala-d-Ala末端，从而大大降低了万古霉素对脂质II的结合亲和力。使用固态核磁共振（NMR）和液相色谱-质谱（LC-MS）分析，对粪肠球菌（ATCC 51299）中万古霉素耐药性诱导的作用进行了表征。固态NMR直接测量从[2-13C]丙酮酸代谢的d-Lac和l，d-Ala的总量，积累的Park核苷酸以及在指数增长早期阶段PG桥键密度的变化（OD660 = 0.4）完整的VRE完整细胞。十肽取代的Park'的高水平积累 观察到与在万古霉素抗性的初始阶段期间PG生物合成的转糖基化步骤的抑制一致的核苷酸，而未检测到在诱导万古霉素抗性后PG桥连接密度的变化。这表明通过肽基转移酶将PG桥连接至脂质II不受VRE中d-Ala-d-Lac取代的PG茎结构的抑制。通过LC-MS确定在有和没有万古霉素抗性诱导的情况下生长的经变溶酶消化的VRE分离的细胞壁的组成。仅在万古霉素存在的VRE中发现带有PG茎终止于d-Ala-d-Lac中的肽。在万古霉素存在下生长的VRE中，带有五肽茎终止于d-Ala-d-Lac的多肽百分比在中指数期（OD660 = 0.6）为26％，在固定生长期（OD660 = 1.0）为57％。这些高百分比表明，d-Ala-d-Lac取代的脂质II被有效地用于VRE中的PG生物合成。