EFFLUX MEDIATED RESISTANCE TO TETRACYCLINES

四环素类抗生素通常用于治疗广泛的 传染病病原体。然而，它们的效果是严重的。 在许多不同的生物体中出现抗药性而受到抑制。这 一项提案寻求资助四环素的分子分析 主动外排介导的抗性--抗性的一种常见机制 细菌。这些研究将主要针对B类(类似Tn10) 编码Tet蛋白作为原型外排载体已超过7年 相关但不同的四环素耐药决定因素。 这种转运蛋白有两个互补的遗传域， 充当复制者。 将使用以下方法寻找四环素与Tet结合的活性部位 保守的带电氨基酸残基的定点突变 利用突变的Tet蛋白的选择，预测在膜内 导致对四环素类似物的非典型耐药性，并使用定位 紫外光诱导光亲和标记法与四环素共价结合。这个 的两个互补的α和β结构域的作用和相互作用 Tet(包括多肽的近端和远端半部分 将使用遗传互补和显性来研究 突变型和野生型Tet蛋白的检测。非活性的活性反转体 类间杂交蛋白(包含来自一个TET的α结构域 蛋白质和来自另一个的β结构域)将被表征； 回复者可能修复了一个错误的α-β结构域 互动。Tet预测多聚体的生物化学研究 单体将使用交联剂和尺寸层析进行 洗涤剂萃取物。四环素转运是否伴随着 质子对流和镁在这一过程中的作用 调查过了。TET的功能和物理地貌 膜将使用Tet多克隆抗体进行进一步鉴定 和Tet多肽。最后，将努力净化活性TET 并将其重组为脂质体。 外排作为一种耐药机制正变得越来越 被认可了。拟议的研究不仅对理解 对广泛使用的抗生素四环素产生抗药性，但对 了解其他外排系统和膜转运的一般情况。

The tetracyclines are antibiotics commonly used to treat a wide range of infectious disease agents. However, their efficacy has been severely curtailed by the emergence of resistance in many different organisms. This proposal seeks funding for the molecular analysis of tetracycline resistance mediated by an active efflux, a common mechanism in resistant bacteria. The studies will be devoted primarily to the Class B (Tn10-like) encoded Tet protein as a prototype efflux carrier for more than seven related, but different, classes of tetracycline resistance determinant. This transport protein has two complementing genetic domains and appears to act as a multimer. The active site for binding of tetracycline to Tet will be sought using site-directed mutagenesis of conserved, charged amino acid residues predicted to be within the membrane, using selection of mutant Tet proteins leading to atypical resistance to tetracycline analogs, and using location of tetracycline bound covalently by UVinduced photoaffinity labeling. The role and interactions of the two complementary alpha and beta domains of Tet (comprising the proximal and distal halves of the polypeptide respectively) will be studied using genetic complementation and dominance assays of mutant and wild type Tet proteins. Active revertants of inactive interclass hybrid proteins (which contain an alpha domain from one Tet protein and a beta domain from another) will be characterized; such revertants presumably have remedied an incorrect alpha - beta domain interaction. Biochemical studies on the predicted multimerization of Tet monomers will be done using cross-linking agents and size chromatography of detergent extracts. Whether tetracycline transport is accompanied by proton antiport and the role of magnesium in this process will be investigated. The functional and physical topography of Tet within the membrane will be further characterized using polyclonal antibodies to Tet and Tet peptides. Finally, efforts will be directed at purifying active Tet and reconstituting it into liposomes. Efflux as a mechanism for drug resistance is becoming increasingly more recognized. The proposed studies are important for understanding not only resistance to the widely used antibiotic tetracycline, but also for understanding other efflux systems and membrane transport in general.