Role of EPEC secreted protein EspF in pathogenesis

EPEC分泌蛋白EspF在发病机制中的作用

• 批准号: 6561210
• 负责人: V K VISWANATHAN
• 金额: $ 10.72万
• 依托单位: UNIVERSITY OF ILLINOIS AT CHICAGO
• 依托单位国家: 美国
• 财政年份: 2003
• 资助国家: 美国
• 起止时间: 2003-05-01 至 2006-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6561210
• 关键词: Escherichia coli; Escherichia coli infections; bacteria infection mechanism; bacterial proteins; cell adhesion; gastrointestinal epithelium; gene deletion mutation; inflammation; membrane permeability; nuclear factor kappa beta; protein localization; protein protein interaction; protein structure function; tight junctions; yeast two hybrid system

DESCRIPTION (provided by applicant): Enteropathogenic Escherichia coli (EPEC) is a frequently isolated diarrheal pathogen in the developing world, and a significant cause of mortality in infants. EPEC attaches to intestinal epithelial cells, subverts their function, and produces the characteristic "attaching and effacing (A/E) lesion". Pathogenesis requires the type III secretion system that directly injects effector proteins into host cells. One of these, EspF, is a 206 amino acid protein with a unique N-terminal sequence followed by three proline-rich 47-amino repeat sequences. EspF is critical for mediating tight junction (TJ) alterations, although it is not required for bacterial viability or A/E lesion formation. Translocated EspF causes a loss in transepithelial resistance, increases monolayer permeability, and redistributes the TJ protein, occludin. In addition EspF also appears to activate the pro-inflammatory transcription factor, NF-kappaB. Our preliminary studies indicate that EspF interacts with various host proteins, including cytokeratin 18 (CK18). Furthermore, host CK18 is redistributed and its interaction with 14-3-3 regulatory proteins is altered following infection with EPEC. 14-3-3 is an abundant, ubiquitously expressed family of proteins that regulate CK18 solubility and distribution, cell-cycle checkpoints, signaling pathways and apoptosis. The long-term goal of this proposal is to determine the mechanism by which EspF mediates host effects. The immediate objectives are to establish the regions or domains of EspF required for interactions, and the corresponding functional significance of the interactions. The domains of EspF required for interaction with CK18, and possibly other host proteins, will be evaluated by using deletion clones of espF in yeast two-hybrid assays and GST pull-down assays. The functional consequences of altering such interactions will be evaluated by assessing epithelial barrier function and inflammation responses following infection with an EspF mutant complemented with the corresponding deletion constructs. The basis of the interaction between CK18 and 14-3-3 will be explored, and the significance of the altered interaction between these two proteins following EPEC infection will be assessed. This proposal will also evaluate the role of 14-3-3 in EPEC infection, assess if EspF directly interacts with 14-3-3, and the effect of 14-3-3 on EPEC induced alterations in barrier function and inflammatory responses.

描述（由申请人提供）： 肠病性大肠杆菌 (EPEC) 是发展中国家常见的腹泻病原体，也是婴儿死亡的重要原因。 EPEC附着在肠上皮细胞上，破坏其功能，并产生特征性的“附着和抹去（A/E）病变”。发病机制需要III型分泌系统直接将效应蛋白注入宿主细胞。其中之一，EspF，是一种 206 个氨基酸的蛋白质，具有独特的 N 末端序列，后跟三个富含脯氨酸的 47 个氨基酸重复序列。 EspF 对于介导紧密连接 (TJ) 改变至关重要，尽管它不是细菌活力或 A/E 病变形成所必需的。易位的 EspF 会导致跨上皮阻力丧失，增加单层通透性，并重新分配 TJ 蛋白 Occludin。此外，EspF 似乎还可以激活促炎转录因子 NF-kappaB。我们的初步研究表明 EspF 与多种宿主蛋白​​相互作用，包括细胞角蛋白 18 (CK18)。此外，感染 EPEC 后，宿主 CK18 会重新分布，其与 14-3-3 调节蛋白的相互作用也会发生改变。 14-3-3 是一个丰富、普遍表达的蛋白质家族，可调节 CK18 溶解度和分布、细胞周期检查点、信号传导途径和细胞凋亡。该提案的长期目标是确定 EspF 介导宿主效应的机制。近期目标是建立相互作用所需的 EspF 区域或域，以及相互作用相应的功能意义。与 CK18 以及可能的其他宿主蛋白相互作用所需的 EspF 结构域将通过在酵母双杂交测定和 GST Pull-down 测定中使用 espF 的缺失克隆进行评估。将通过评估用相应缺失构建体补充的 EspF 突变体感染后的上皮屏障功能和炎症反应来评估改变这种相互作用的功能后果。将探索 CK18 和 14-3-3 之间相互作用的基础，并将评估 EPEC 感染后这两种蛋白质之间相互作用改变的意义。该提案还将评估 14-3-3 在 EPEC 感染中的作用，评估 EspF 是否直接与 14-3-3 相互作用，以及 14-3-3 对 EPEC 诱导的屏障功能和炎症反应改变的影响。