Export & Import of Lethal Agents Mediated by TolC

描述（由申请人提供）：分子穿过生物膜的运输是所有活细胞共有的基本过程。在革兰氏阴性细菌中，如大肠杆菌，转运发生在两个膜上，其中外膜代表第一个渗透屏障。需要外膜蛋白介导的运输将营养物质带入内部并排出不需要的代谢物和抑制剂。具有讽刺意味的是，存在于细菌生存的恶劣环境中的有毒生物分子和感染因子经常利用这些外膜蛋白进入细胞。这项研究的重点是一种独特的外膜蛋白TolC，它介导多种分子的扩散，包括α-溶血素的输出和抗生素的外排。尽管TolC的三维结构的分辨率，单个残基的作用和它完成不同的分子组的运输的机制仍然知之甚少。此外，还不知道TolC如何与内膜的其他蛋白质相互作用以形成促进双向转运活性的复合物。目前的研究正在进行检查的作用TolC在出口（抗生素）和进口（大肠杆菌素E1），以及检查其与其他蛋白质的运输复合物的相互作用。鉴定功能关键的TolC残基和各种蛋白质-蛋白质相互作用，必须发生以完成不同的运输活动，将通过采用遗传，结构和生物化学方法的组合来实现;关于该项目所有方面的现有初步数据强烈反映了其可行性。TolC及其同系物在其他革兰氏阴性菌中的存在，阴性细菌证明了这种独特的多功能蛋白质的更广泛的相关性。除了TolC在抗生素耐药性中的明显作用外，其介导病原菌中多种毒力因子分泌的能力使其成为医学上重要的研究课题。

DESCRIPTION (provided by applicant): Transport of molecules across biological membranes is a fundamental process shared by all living cells. In Gram-negative bacteria, such as Escherichia coil, transport occurs across two membranes, of which the outer membrane represents the first permeability barrier. Outer membrane protein-mediated transport is needed to bring nutrients inside and expel unwanted metabolites and inhibitors. Ironically, toxic biomolecules and infectious agents, present in the hostile environment in which bacteria live, frequently exploit these outer membrane proteins to gain entry into the cell. The focus of this research is a unique outer membrane protein, TolC, which mediates the diffusion of a diverse group of molecules including the export of alpha-hemolysin and the efflux of antibiotics. Despite the resolution of TolC's three-dimensional structure, the role of individual residues and the mechanism by which it accomplishes the transport of a diverse group of molecules remains poorly understood. Moreover, it is unknown how TolC interacts with other proteins of the inner membrane to form the complexes that facilitate the two-way transport activity. The present study is being undertaken to examine the role of TolC in export (antibiotic) and import (colicin E1 ), as well as to examine its interactions with other proteins of the transport complexes. Identification of functionally critical TolC residues and the various protein-protein interactions that must occur to accomplish the diverse transport activities will be achieved through employing a combination of genetic, structural, and biochemical approaches; available preliminary data concerning all aspects of the project strongly reflect their feasibility.The presence of TolC and its homologues in other Gram-negative bacteria demonstrates the broader relevance of this unique multifunctional protein. Besides the obvious role of TolC in antibiotic resistance, its ability to mediate the secretion of a variety of virulence factors in pathogenic bacteria makes it a medically important subject of investigation.