Export & Import of Lethal Agents Mediated by TolC

描述(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，如大肠杆菌，转运发生在两个膜上，外膜是第一个渗透屏障。外膜蛋白介导的转运需要将营养物质带进体内，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剂，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，它介导了一系列不同分子的扩散，包括α-溶血素的输出和抗生素的外流。尽管TolC的三维结构已经被解析，但单个残基的作用以及它完成一组不同分子的运输的机制仍然知之甚少。此外，TolC如何与内膜的其他蛋白质相互作用形成促进双向转运活动的复合体尚不清楚。本研究旨在研究TolC在出口(抗生素)和进口(结肠素E1)中的作用，以及它与运输复合体的其他蛋白质的相互作用。鉴定TolC的功能关键残基和完成不同转运活动所必须发生的各种蛋白质-蛋白质相互作用将通过结合遗传、结构和生化方法来实现；关于该项目各个方面的现有初步数据强烈反映了它们的可行性。TolC及其同系物在其他革兰氏阴性细菌中的存在表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，它还能介导病原菌分泌多种毒力因子，使其成为医学上重要的研究对象。

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.