EFFLUX-MEDIATED RESISTANCE TO TETRACYCLINES

描述(改编自申请人的摘要)：外流已成为 越来越多的人认识到抗生素和其他药物的耐药机制 自研究人员发现四环素外排蛋白Tet以来的药物 大约20年前。今天，一些Tet外流蛋白(与初级相关) 序列)已经被描述，其具有两半，每半六个α-螺旋 跨膜结构域，由细胞质环分隔并指定交换 一个质子的四环素/阳离子络合物。主动外流是以下两种之一 四环素耐药的主要机制限制了临床应用 这一重要的抗生素家族的有效性。校长中的研究 研究人员的实验室重点放在生物化学和分子基础上 由Tn10指定的43kD B类Tet蛋白外流。这种蛋白质已经被 在洗涤剂中提纯为多组氨酸融合。最近，它一直在 成功地结晶用于2D分析。这项提案旨在确定 通过结晶方法获得Teta蛋白的三级结构， 结合2D和3D的努力，通过半胱氨酸交联的定点 半胱氨酸突变残基，并通过基因分析抑制突变。在一个 第二个目标是，他们应该寻求牵涉到域间循环的发现 利用环和半胱氨酸的突变研究Tet蛋白的功能 交叉链接研究以检查环路如何与其他域相互作用 Tet蛋白质。第三个目标将继续他们在无线电标记方面的工作 光亲和四环素类似物鉴定细菌的底物结合区 Tet蛋白质。这一方法将得到使用铁作为 四环素外排阳离子与Fenton反应生成肽 蛋白质中底物结合部位的键断裂。这些研究将继续 进一步探讨了药物外排蛋白Tet抗休克的分子基础。 对Tet蛋白的了解的进展将增强我们对Tet蛋白的了解 主要促进蛋白家族中的其他类似蛋白 它应该在传染性疾病的药物治疗领域产生广泛的影响 疾病和癌症。

DESCRIPTION (Adapted from the Applicant's Abstract): Efflux has become increasingly recognized as a mechanism of resistance to antibiotics and other drugs since the investigators' discovery of the tetracycline efflux protein Tet some 20 years ago. Today, a number of Tet efflux proteins (related by primary sequence) have been described which have two halves, each six alpha-helical transmembrane domains, separated by a cytoplasmic loop and specify an exchange of a tetracycline/cation complex for a proton. Active efflux is one of two major mechanisms of tetracycline resistance which have curtailed the clinical effectiveness of this important family of antibiotics. Studies in the Principal Investigator's laboratory focus on the biochemical and molecular basis of efflux by the 43kD class B Tet protein specified by Tn10. The protein has been purified in detergent as a polyhistidine fusion. Recently, it has been successfully crystallized for 2D analysis. This proposal seeks to determine the tertiary structure of the TetA protein through crystallization methods, combining 2D and 3D efforts, by cysteine cross-linking of site-directed cysteine mutant residues, and by genetic analysis of suppressor mutations. In a second aim, they shall pursue findings that implicate the interdomain loop in the function of the Tet protein, using mutagenesis of the loop and cysteine cross-linking studies to examine how the loop interacts with other domains of the Tet protein. A third aim will continue their work on radiolabeled photoaffinity tetracycline analogs to identify the substrate-binding region in the Tet protein. This approach will be complemented by studies that use iron as the cation for tetracycline efflux and the Fenton reaction to produce peptide bond cleavage at substrate binding sites in the protein. These studies pursue further the molecular basis for antiport by the drug efflux protein Tet. Advances in understanding of the Tet protein will enhance our knowledge of other, similar proteins in the major facilitator family of transport proteins which should have broad impact in the area of drug treatment of infectious disease and cancer.