Efflux-mediated resistance to tetracyclines

四环素类药物治疗感染性疾病的用途已经被广泛应用。 由于抵抗而急剧减少。在四环素（Tc）抗性的两种主要机制中， 主动外排是最常见的，由超过20种不同的Tc决定子代表。由于我们 大约25年前发现了主动Tc外排，该实验室专注于原型外排 蛋白，由Tn10指定的B类泰特A（TetA（B））蛋白。该项目主要有三个方面 旨在更好地理解泰特蛋白外排活性的分子基础。1.成长和 分析TetA（B）蛋白的2D和3D晶体。这项工作将受益于最近的事态发展， 膜蛋白晶体结构工作和泰特蛋白纯化的改进， 使我们有能力重复获得晶体。2.加大努力，以确定基质 结合位点：a.制备并使用光亲和标记的Tc来鉴定特定的氨基酸， 包括Tc结合位点。B.使用FeTc复合物和芬顿反应切割附近的蛋白质 该结合位点因此限定了第二个这样的切割位点。C.进行定点诱变 “a”中鉴定的残基，以观察对Tc抗性和Tc转运的影响。3.继续遗传 TetA（B）蛋白的分析，集中于结构域间环。通过抑制突变，氨基酸 替代和交联的研究，我们将研究的物理邻居和功能的作用， 环的外排和底物特异性。深入了解分子结构和功能 TetA蛋白是一种主要易化因子超家族的外排蛋白原型， 了解这种常见的耐药性机制，并提出克服耐药性的方法。 反过来，这一知识可能有助于四环素家族抗生素的复兴， 长期以来，它们的耐受性和安全性都很好。这样的理解也会增强 努力扭转用于治疗传染病的其他抗生素和药物的外流，包括 细菌、真菌、寄生虫和癌症。

Use of tetracyclines to treat infectious diseases has been sharply curtailed because of resistance. Among two major mechanisms for tetracycline (Tc) resistance, active efflux is the most common, represented by more than 20 different Tc determinants. Since our discovery of active Tc efflux almost 25 years ago, this laboratory has focused on a prototype efflux protein, the class B Tet A (TetA(B)) protein specified by Tn10. The proposed project has three major aims directed at better understanding the molecular basis for Tet protein efflux activity. 1. Grow and analyze 2D and 3D crystals of the TetA(B) protein. This work will benefit from recent developments in membrane protein crystal structure work and improvements in the purification of Tet protein that have led to our ability to reproducibly obtain crystals. 2. Extend efforts aimed at identifying the substrate binding site(s): a. Prepare and use photoaffinity labeled Tcs to identify particular amino acids which comprise the Tc binding site. b. Use a FeTc complex and the Fenton reaction to cleave the protein near the binding site and so define the second such cleavage site. c. Perform site-directed mutagenesis of residues identified in "a" to see the effect on Tc resistance and Tc transport. 3. Continue the genetic analysis of the TetA(B) protein, focusing on the interdomain loop. By suppressor mutations, amino acid replacements and cross-linking studies, we shall study the physical neighborhood and functional role of the loop in terms of efflux and substrate specificity. Insights into the molecular structure and function of TetA protein, a prototype of an efflux protein of the Major Facilitator Superfamily, will help in understanding this common mechanism of drug resistance and suggests ways it could be overcome. This knowledge, in turn, could help lead to a renaissance for the tetracycline family of antibiotics which have a long history of being generally well-tolerated and safe. Such understanding will also enhance efforts to reverse efflux of other antibiotics and drugs used to treat infectious diseases, including bacteria, fungi and parasites, and cancer.