ACTIVATION OF VANS IN VITRO

这项建议的长期目标是研究 机会性肠球菌诱导的万古霉素耐药 并了解暴露于细胞后导致的事件顺序 到万古霉素对货车的激活，对环境感官的影响 万古霉素。在工作的最初阶段，货车和VanR的基因， 转录调控元件，在反应中被磷酸化 为了激活VANS，将在枯草芽孢杆菌和大肠杆菌中表达 Coli获得膜结合和超量表达的游离蛋白 生化研究。在这些制剂中，抗体将被检测 它们在体外激活面包车的能力，没有背景信号或 当使用抗生素作为诱导剂时得到的虚假结果 体内的抗药性。这项提议的具体目标是发展 研究VanRs激活的分子机制的体外系统 系统。具体地说，我们建议：(1)开发一个完整的 VANS嵌入到具有VANS-自动磷酸化的细胞膜中， VanR-Kinase和磷酸VanR-磷酸酶活性；以及(2)发展 将全长面包车重组为脂质体的系统。这些系统 将被用来测试货车的激活机制。 具有活性全长VAN的体外系统将使我们能够解决 三个方面的问题：(1)诱导能否在体外重组 一种酶和磷酸酶活性不同的系统 对诱导抗生素的反应？诱导者必须具备哪些属性？ 它必须是一种有效的抗生素吗？有没有免费的诱导者？ (2)万古霉素与面包车是否直接相互作用？这种可能性是 由报告建议，非活性糖肽可以在模型中诱导 核聚变报告系统。(3)细胞壁肽聚糖的中间体 合成是货车的诱导剂吗？这种可能性是由 观察非糖肽类抗生素诱导耐药。测试 这种中间体只有在无细胞系统中才是可行的，在那里 有可能确保效应器可以访问其 建议的目标。

The long-term objective of this proposal is to study the process of induced VanA-type vancomycin resistance in the opportunist Enterococcus faecium and to learn the sequence of events leading from exposure of cells to vancomycin to the activation of VanS, the environmental sensory of vancomycin. In the initial stages of work, the genes for VanS and VanR, the transcriptional regulatory element which is phosphorylated in response to VanS activation, will be expressed in Bacillus subtilis and Escherichia coli to obtain membrane-bound and over-expressed free protein for biochemical studies. In these preparations, antibodies will be tested for their ability to activate VanS, in vitro, free of background signals or spurious results which are obtained when antibiotics are used as inducers of resistance in vivo. The Specific Aims of this proposal are to develop in vitro systems to study molecular mechanisms of activation of the VanRS system. Specifically, we propose: (1) to develop a system with full-length VanS embedded in the cell membrane that has VanS-autophosphorylation, VanR-kinase, and phosphoVanR-phosphatase activities; and (2) to develop system with full-length VanS reconstituted into liposomes. These systems will be used to test mechanisms of VanS activation. The in vitro system with active full-length Van will allow us to address questions in three areas: (1) Can induction be reconstituted in vitro in a system in which the kinase and phosphatase activities are varied in response to inducing antibiotics? What attributes must an inducer possess? Does it have to be active as an antibiotic? Are there gratuitous inducers? (2) Does vancomycin interact directly with VanS? This possibility is suggested by reports that inactive glycopeptides can induce in a model fusion reporter system. (3) Can intermediates of cell wall peptidoglycan synthesis serve as inducers of VanS? This possibility is suggested by the observation that non-glycopeptide antibiotics induce resistance. Testing such intermediates will only be feasible in a cell-free system, where it is possible to have assurance that the effectors have access to their proposed targets.