BACILLUS SUBTILIS MULTIDRUG TRANSPORTER

枯草芽孢杆菌多药转运蛋白

• 批准号: 2187362
• 负责人: ALEX A NEYFAKH
• 金额: $ 20.38万
• 依托单位: UNIVERSITY OF ILLINOIS AT CHICAGO
• 依托单位国家: 美国
• 财政年份: 1993
• 资助国家: 美国
• 起止时间: 1993-08-01 至 1997-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2187362
• 关键词: Bacillus subtilis; bacterial genetics; bacterial proteins; binding proteins; drug metabolism; enzyme substrate; gene expression; high performance liquid chromatography; membrane transport proteins; microorganism growth; microorganism metabolism; molecular cloning; multidrug resistance; northern blottings; nucleic acid sequence; operon; oxidoreductase; polymerase chain reaction; protein structure; site directed mutagenesis; stoichiometry

The project is directed toward the understanding of the mechanism of action and physiological role of the Bacillus subtilis multidrug efflux transporter Bmr. This membrane protein is able to transport a number of structurally unrelated bacteriostatic compounds and antibiotics out of bacteria. The homologs of this protein participate in the drug resistance phenomena in clinically important bacteria. In particular, the closest known structural and functional homolog of Bmr, the Staphylococcus protein NorA, is involved in resistance of S. aureus to norfloxacin and other fluoroquinolones. The mammalian membrane pump P-glycoprotein, although being structurally different from Bmr, effluxes a similar to Bmr spectrum of drugs. This protein is involved in resistance of tumor cells to chemotherapeutic agents. The mechanism of an unusually broad substrate specificity of P-glycoprotein and Bmr remains unknown. The bacterial origin of Bmr provides an opportunity to investigate this mechanism with a large variety of molecular genetic and biochemical methods. The proposed plan of experiments includes localization of the substrate-recognition site in the Bmr molecule and the study of the fine structure of this site by using site-directed mutagenesis. These experiments will address the question, whether all the substrates of Bmr bind to the same amino acid residues of the transporter molecule, or different drugs interact with different "pockets" in the binding site. The mutagenesis studies will be comple- mented with the biochemical analysis of the drug transport process mediated by Bmr and its mutants. Other experiments will reveal the physiological role of Bmr and the nature of its cellular substrates. The bmr-containing operon will be sequenced and the homologs of the other genes of the operon will be identified in the sequence databases. Overexpression of Bmr leads to impairment of bacterial growth. The changes in metabolism and gene expression in the Bmr-overexpressing bacteria will be analyzed. Cellular genes reversing the Bmr-induced growth inhibition will be cloned and sequenced. Finally, a chromatographic analysis of substances effluxed by Bmr-overexpressing bacteria will be performed. The results of this study are expected to contribute to the understanding of both the theoretical and clinical aspects of the multidrug resistance phenomena in bacteria and tumor cells.

该项目是针对理解机制的 枯草芽孢杆菌多药外排的作用和生理作用 转运蛋白BMR。 该膜蛋白能够运输许多 从结构无关的抑菌化合物和抗生素中 细菌。 该蛋白的同源物参与该药物 临床上重要的细菌中的抗性现象。 尤其， BMR的最接近的已知结构和功能同源物， 葡萄球菌蛋白NORA与金黄色葡萄球菌的抗性有关 诺福沙星和其他氟喹诺酮类药物。 哺乳动物膜泵 P-糖蛋白，尽管结构上与BMR不同，但排出 类似于药物的BMR谱。 该蛋白参与 肿瘤细胞对化学治疗剂的抗性。 机制 P-糖蛋白和BMR的异常广泛的底物特异性 仍然未知。 BMR的细菌起源为 用大量的分子遗传和 生化方法。 拟议的实验计划包括 BMR分子中底物识别位点的定位和 通过使用站点定向的研究该站点的精细结构的研究 诱变。 这些实验将解决这个问题，是否全部 BMR的底物与相同的氨基酸残基结合 转运蛋白分子或不同的药物与不同的药物相互作用 绑定位点中的“口袋”。 诱变研究将是 对药物运输过程的生化分析进行了评估 由BMR及其突变体介导。 其他实验将揭示 BMR的生理作用及其细胞底物的性质。 这 将对含BMR的操纵子进行测序，另一个 操纵子的基因将在序列数据库中识别。 BMR的过表达导致细菌生长受损。 这 BMR过表达的代谢和基因表达的变化 细菌将进行分析。 逆转BMR诱导的细胞基因 生长抑制作用将被克隆和测序。 最后，一个 通过过表达BMR的物质的色谱分析 将进行细菌。 这项研究的结果有望 有助于理解理论和临床 细菌和肿瘤中多药耐药现象的各个方面 细胞。