Role of EPEC secreted protein EspF in pathogenesis

描述（由申请人提供）： 肠致病性大肠杆菌（EPEC）是发展中国家常见的肠道病原体，也是婴儿死亡的重要原因。EPEC附着于肠上皮细胞，破坏其功能，并产生特征性的“附着和消退（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。EspF是一种206个氨基酸的蛋白质，具有独特的N-末端序列，随后是三个富含脯氨酸的47-氨基重复序列。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。转位的EspF导致跨上皮阻力的损失，增加单层通透性，并重新分配TJ蛋白，occludin。此外，EspF似乎还激活促炎转录因子NF-κ B。我们的初步研究表明，EspF与各种宿主蛋白质相互作用，包括细胞角蛋白18（CK 18）。此外，宿主CK 18被重新分配，并且其与14-3-3调节蛋白的相互作用在EPEC感染后改变。14-3-3是一个丰富的、普遍表达的蛋白质家族，其调节CK 18溶解度和分布、细胞周期检查点、信号传导途径和凋亡。该提案的长期目标是确定EspF介导宿主效应的机制。直接目标是建立相互作用所需的EspF区域或域，以及相互作用的相应功能意义。将通过在酵母双杂交测定和GST下拉测定中使用espF的缺失克隆来评价与CK 18和可能的其他宿主蛋白相互作用所需的EspF结构域。改变这种相互作用的功能后果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。将探索CK 18和14-3-3之间相互作用的基础，并评估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是否与14-3-3直接相互作用，以及14-3-3对EPEC诱导的屏障功能和炎症反应改变的影响。

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.