ACTIVATION OF VANS IN VITRO

本建议的长期目标是研究 诱导机会肠球菌产生VanA型万古霉素耐药 并了解细胞暴露导致的事件顺序 万古霉素的激活，环境感官的 万古霉素在工作的最初阶段，VanS和VanR的基因， 在应答中磷酸化的转录调节元件 到VanS激活，将在枯草芽孢杆菌和大肠杆菌中表达 大肠杆菌，以获得膜结合和过量表达的游离蛋白， 生化研究。在这些制剂中，将检测抗体 它们在体外激活VanS的能力，没有背景信号，或 当使用抗生素作为诱导剂时， 在体内的抵抗力。该提案的具体目标是发展 研究VanRS激活的分子机制的体外系统 系统具体而言，我们建议：（1）开发一个系统， 包埋在细胞膜上的VanS具有VanS-自磷酸化， VanR-激酶和磷酸VanR-磷酸酶活性;和（2）开发 用全长VanS重构成脂质体的系统。这些系统 将用于测试VanS激活机制。 具有活性全长货车的体外系统将允许我们解决 在三个方面的问题：（1）诱导可以在体外重建， 一个系统，其中激酶和磷酸酶活性是不同的， 对诱导抗生素的反应？诱导者需要具备什么属性？ 它必须像抗生素一样有效吗？有没有免费的诱因？ (2)万古霉素是否与VanS直接相互作用？这种可能性 报告表明，无活性糖肽可以在模型中诱导 融合报告系统(3)细胞壁肽聚糖中间体 合成作为VanS？这种可能性是由 观察到非糖肽类抗生素诱导耐药性。测试 这样的中间体仅在无细胞系统中是可行的，其中它 可以确保效应器可以访问其 提出的目标。

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.