Institute of Disease Control and Prevention, Academy of Military Medical Sciences, Beijing, China.
J Proteome Res. 2010 Apr 5;9(4):1772-85. doi: 10.1021/pr901216e.
To understand the molecular mechanisms of bacteria resistance to glycopeptides, we obtained proteomic profiles of vancomycin-resistant Enterococcus faecalis V583 (reference strain) and V309 (clinical isolate) passaged with and without the drug. The specificity and reversibility of vancomycin resistance genes induced in V583 and V309 were further studied over time. By semiquantitative RT-PCR of vancomycin-treated versus untreated samples of both strains, 28 (V583) or 20 (V309) up-regulated proteins, 8 (V583) or 6 (V309) down-regulated proteins, and 1 (V583) or 4 (V309) proteins with mobility changes in 2-DE gel analysis were identified. Some of these proteins have known vancomycin resistance functions or are related to virulent factors, stress, metabolism, translation, and conjunction, which would help Enterococcus survive under drug selection. Vancomycin induced specifically and reversibly VanA, VanX, VanB, and VanXB. Notably, 6 proteins (Pgm, Ldh, Gap-2, RpsB, EF2076, and sex pheromone cAD1 precursor lipoprotein) exhibited clear post-translational modifications. Vancomycin induced phosphorylation of Ser/Thr in Ldh, Gap-2, and sex pheromone cAD1 precursor lipoprotein (EF3256), newly identified here as enterococcal phosphoproteins. Our data suggest that phosphorylated EF3256 is normally active in E. faecelis, whereas EF3256-P together with oppA-like protein may play a key role in the regulation of pheromone and transmission of conjugation plasmids.
为了理解细菌对糖肽类抗生素耐药的分子机制，我们获得了万古霉素耐药粪肠球菌 V583（参考株）和 V309（临床分离株）经药物和未经药物传代后的蛋白质组图谱。我们还进一步研究了万古霉素耐药基因在 V583 和 V309 中诱导的特异性和可逆转性。通过对两株菌的万古霉素处理和未处理样本进行半定量 RT-PCR，在 2-DE 凝胶分析中，V583 有 28 个（或 20 个）上调蛋白、8 个（或 6 个）下调蛋白和 1 个（或 4 个）迁移率变化蛋白，V309 有 20 个（或 14 个）上调蛋白、8 个（或 6 个）下调蛋白和 1 个（或 4 个）迁移率变化蛋白。其中一些蛋白具有已知的万古霉素耐药功能，或者与毒力因子、应激、代谢、翻译和结合有关，这有助于肠球菌在药物选择下存活。万古霉素诱导了特异性和可逆性的 VanA、VanX、VanB 和 VanXB。值得注意的是，有 6 种蛋白（Pgm、Ldh、Gap-2、RpsB、EF2076 和性信息素 cAD1 前体脂蛋白）表现出明显的翻译后修饰。万古霉素诱导 Ldh、Gap-2 和性信息素 cAD1 前体脂蛋白（EF3256）中 Ser/Thr 磷酸化，这是新发现的肠球菌磷酸蛋白。我们的数据表明，磷酸化的 EF3256 在粪肠球菌中通常是活跃的，而 EF3256-P 与类似 oppA 的蛋白可能在性信息素调节和接合质粒传递中发挥关键作用。
