Structure and Function of IpaC from Shigella flexneri

描述(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世界性公共卫生问题。志贺氏菌病发病的一个重要步骤是细菌侵袭结肠上皮细胞。侵袭质粒抗原C(IPAC)是一种效应蛋白，能破坏正常的上皮细胞信号转导，促进志贺氏菌的摄取。我们的实验室在了解IPAC的结构-功能关系方面取得了一些进展；然而，到目前为止，还没有人关注IPAC介导的肌动蛋白聚合在上皮细胞侵袭过程中的生化作用。这在一定程度上是由于提纯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与宿主细胞细胞骨架相互作用和颠覆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GTP酶CDC42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，这可能有助于志贺氏菌高效进入上皮细胞；以及3)鉴定位于IPAC C末端的序列和结构，这些序列和结构负责其效应器活性和促进空泡逃逸的能力。在许多革兰氏阴性菌的发病机制中，一个共同的主题是将效应蛋白特异性地递送到真核细胞，以颠覆正常的靶细胞信号机制。对于福氏志贺氏菌来说，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通过一种与其他革兰氏阴性细菌病原体相比非常独特的机制来实现这种物种间的细胞交流。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变成“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往往是未来细胞生物学工作的创新和有价值的工具。IPAC信号机制的新颖性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.