EFFLUX MEDIATED RESISTANCE TO TETRACYCLINES

四环素类抗生素通常用于治疗多种 传染病病原体。然而，它们的功效受到了严重的影响。 由于许多不同生物体中出现耐药性而减少。这 四环素分子分析研究计划申请拨款 由主动外排介导的抗性，这是抗性中的一种常见机制， 细菌这些研究将主要致力于B类（Tn 10样） 编码的泰特蛋白作为原型外排载体超过7 相关但不同的四环素抗性决定簇类别。 这种转运蛋白有两个互补的遗传结构域， 作为多聚体。 四环素与泰特结合的活性位点将使用 保守的带电氨基酸残基的定点诱变 使用突变泰特蛋白的选择， 导致对四环素类似物的非典型耐药性，并使用定位 紫外光诱导的光亲和标记法测定了四环素的共价结合。的 两个互补的α和β结构域的作用和相互作用 泰特（包括多肽的近端和远端两半 分别）将研究使用遗传互补和显性 突变型和野生型泰特蛋白的测定。非活性回复突变体 类间杂合蛋白（其含有来自一个泰特的α结构域 蛋白质和来自另一个的β结构域）将被表征;这样的 回复突变体可能已经修复了一个不正确的α-β结构域 互动预测泰特多聚化的生化研究 单体将使用交联剂和尺寸色谱法 洗涤剂提取物。四环素转运是否伴随 质子反向转运和镁在这一过程中的作用将是 研究了泰特的功能和物理地形， 将使用泰特的多克隆抗体进一步表征膜 和泰特肽。最后，将努力净化活性泰特 并将其重组为脂质体。 外排作为耐药性的机制越来越多 认可.拟议的研究不仅对理解 对广泛使用的抗生素四环素的耐药性， 了解其他外排系统和膜转运的一般。

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.