Structure and Function of IpaC from Shigella flexneri

描述（由申请人提供）：福氏志贺氏菌是一种革兰氏阴性肠道病原体，可引起细菌性痢疾（志贺氏菌病），这仍然是一个重要的全球公共卫生问题。志贺氏菌病发病机制中的一个重要步骤是细菌侵入结肠上皮细胞。侵袭质粒抗原C（IpaC）是破坏正常上皮细胞信号传导以促进志贺氏菌摄取的效应蛋白。我们的实验室在理解IpaC的结构-功能关系方面取得了一些进展;然而，迄今为止，很少关注上皮细胞侵袭过程中IpaC介导的肌动蛋白聚合的生物化学。这部分是由于纯化和处理这种蛋白质的困难。我们实验室的长期目标是确定IpaC的精确功能和结构组织，并阐明IpaC与宿主细胞骨架相互作用和破坏的分子机制和结构基础。本项目的具体目标是：1）确定IpaC与宿主GTdc 42结合的结果; 2）确定负责宿主细胞肌动蛋白直接成核的IpaC特征，其可能有助于志贺氏菌有效进入上皮细胞;和3）鉴定位于IpaC C-末端，负责其效应子活性和其促进空泡逃逸的能力。许多革兰氏阴性菌致病的共同主题是将效应蛋白特异性递送至真核细胞以破坏正常的靶细胞信号传导机制。对于鼠伤寒沙门氏这种串扰的结果是宿主细胞骨架重排和随后的细菌进入。IpaC通过相对于在其他革兰氏阴性细菌病原体中鉴定的机制非常独特的机制进行这种种间细胞通信。这项工作的完成将揭示细菌触发上皮细胞为病原体的利益而成为“吞噬细胞”的机制的重要新原理。这将有助于确定志贺氏菌和相关人类病原体感染的化疗控制目标。此外，能够调节真核生物途径的新型细菌蛋白通常是细胞生物学未来工作的创新和有价值的工具。IpaC信号传导机制的新颖性质也为它提供了一种新的有用的细胞生物学工具。

DESCRIPTION (provided by applicant): Shigella flexneri is a gram-negative enteric pathogen that causes bacillary dysentery (shigellosis), which continues to be an important worldwide public health problem. An essential step in the pathogenesis of shigellosis is bacterial invasion of the epithelial cells of the colon. Invasion plasmid antigen C (IpaC) is the effector protein that subverts normal epithelial cell signaling to promote Shigella uptake. Our lab has contributed a number of advances in understanding the structure-function relationship of IpaC; however, there has been little focus thus far on determining the biochemistry of IpaC-mediated actin polymerization during epithelial cell invasion. This is partly due to difficulties in purifying and handling this protein. The long-range goal of our laboratory is to determine the precise functional and structural organization of IpaC and to elucidate the molecular mechanism of and the structural basis for IpaC interaction with and subversion of the host cell cytoskeleton. The specific aims of this project are: 1) to determine the outcome of IpaC's association with the host GTPase Cdc42; 2) to determine the IpaC features responsible for direct nucleation of host cell actin which may contribute to efficient Shigella entry into epithelial cells; and 3) to identify the sequences and structures located at the IpaC C-terminus that are responsible for its effector activity and its ability to promote vacuolar escape. A common theme in the pathogenesis of many gram-negative bacteria is the specific delivery of effector proteins to eukaryotic cells to subvert the normal target cell signaling mechanisms. For S. flexneri, the outcome of this cross talk is host cytoskeletal rearrangement and subsequent bacterial entry. IpaC carries out this interspecies cellular communication by a mechanism that is quite unique relative to those identified in other gram-negative bacterial pathogens. Completion of this work will reveal important new principles of the mechanism by which bacteria trigger epithelial cells to become "phagocytic" for the benefit of the pathogen. This will help in identifying targets for chemotherapeutic control of infection by Shigella and related human pathogens. Moreover, novel bacterial proteins capable of modulating eukaryotic pathways are often innovative and valuable tools for future work in cell biology. The novel nature of IpaC's signaling mechanisms provide promise that it, too, represents a new and useful cell biology tool.