Export & Import of Lethal Agents Mediated by TolC

描述（由申请人提供）：分子在生物膜上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，如埃希氏螺旋菌，转运发生在两层膜上，其中外膜是第一个渗透性屏障。需要外膜蛋白介导的运输将营养物质带入体内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。本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流出。尽管已经确定了TolC的三维结构，但个体残基的作用以及它实现多种分子运输的机制仍然知之甚少。此外，目前尚不清楚TolC如何与内膜的其他蛋白质相互作用，形成促进双向运输活性的复合物。目前正在进行的研究是为了检查TolC在出口（抗生素）和进口（粘菌素E1）中的作用，以及检查它与运输复合物的其他蛋白质的相互作用。鉴定功能关键的TolC残基和完成各种运输活动必须发生的各种蛋白质-蛋白质相互作用将通过采用遗传、结构和生化方法的组合来实现；有关该项目各方面的现有初步数据有力地反映了其可行性。TolC及其同源物在其他革兰氏阴性菌中的存在证明了这种独特的多功能蛋白的广泛相关性。除了TolC在抗生素耐药中的明显作用外，其介导病原菌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.