Probing localiztion, interactions, and effector properties of Shigella lpaD

描述（由申请人提供）：拟议的研究重点是阐明入侵质粒抗原D （IpaD）在细菌病原体福氏志贺氏菌（细菌性痢疾的病原体）的细胞入侵和细胞间传播中的假定作用。弗氏沙门氏菌与鼠疫耶尔森氏菌（鼠疫）、沙门氏菌（胃肠炎）和铜绿假单胞菌（肺部感染）等许多其他重要病原体一起利用III型分泌系统（TTSS）作为破坏人体细胞正常功能的手段。对于志贺氏菌，IpaD位于TTSS的暴露极，在那里它感知宿主细胞接触，并帮助募集下游效应蛋白注射到宿主细胞的膜和细胞质中，促进细菌入侵。我们最近发现的证据表明，IpaD可能不仅存在于flexneri的表面，而且被注射到宿主细胞质中，并可能定位于间隙连接。由于IpaD可能在侵袭后事件（即直接的细胞间传播）中发挥作用，因此它代表了志贺氏菌TTSS功能的一个尚未描述的方面，可能是疾病预防的目标。为了更好地了解IpaD及其来自其他系统的同源物的作用，我们设计了一系列实验：a)确定IpaD在培养的人上皮细胞中细胞内定位（可能带有间隙连接）的分子基础；b)确定IpaD能够被招募到宿主细胞内特定位点的结构特征；c)验证IpaD与宿主细胞蛋白之间的相互作用影响志贺氏菌细胞间传播效率的假设。具体来说，将利用荧光共定位、FRET和亲和层析实验来绘制IpaD的细胞内分布并识别与其相互作用的细胞内宿主蛋白。一旦这些蛋白被鉴定出来，突变分析中，IpaD中的小缺失和点突变将被引入，以允许绘制天然细胞内定位和与细胞蛋白特异性相互作用所需的区域。最后，将研究原生和突变形式的IpaD在HeLa细胞单层中形成表达各种间隙连接蛋白（如连接蛋白）的斑块的能力，以便更准确地了解IpaD作为效应物的可能作用。公共卫生相关性：拟议的研究探讨了IpaD在细菌病原体（如志贺氏菌）入侵和扩散上皮细胞单层能力中的作用。通过确定导致这些过程的具体机制和相互作用，将有可能定制特定的抗感染治疗来预防这些感染，作为标准抗生素使用的替代方案。

DESCRIPTION (provided by applicant): The proposed research focuses on elucidating the putative roles of invasion plasmid antigen D (IpaD) in cellular invasion and intercellular spread of the bacterial pathogen Shigella flexneri, the causative agent of bacillary dysentery. S. flexneri along with numerous other important pathogens such as Yersinia pestis (plague), Salmonella (gastroenteritis), and Pseudomonas aeruginosa (lung infection) utilize the type III secretion system (TTSS) as a means of subverting the normal functions of human cells. For Shigella, IpaD is located at the exposed pole of the TTSS where it senses host cell contact and helps to recruit downstream effector proteins for injection into the membrane and cytoplasm of the host cell to promote bacterial invasion. We recently found evidence that IpaD may not only reside on the surface of S. flexneri, but it is injected into the host cell cytoplasm where it may be localized to gap junctions. Because IpaD may have a role in post-invasion events (i.e., direct cell-to-cell spread), it represents an as yet undescribed aspect of Shigella TTSS function that might be a target for disease prevention. In order to better understand this role of IpaD and its homologues from other systems, a series of experiments have been designed to: a) determine the molecular basis for IpaD's intracellular localization (possibly with gap junctions) in cultured human epithelial cells; b) determine the structural features of IpaD that are responsible for its ability to be recruited to specific sites within host cells; and c) test the hypothesis that interactions between IpaD and host cell proteins influences the efficiency of Shigella intercellular spread. Specifically, fluorescence co-localization, FRET, and affinity chromatography experiments will be utilized in order to both map the intracellular distribution of IpaD and identify intracellular host proteins with which it interacts. Once these proteins are identified, mutation analyses in which small deletions and point mutations in IpaD will be introduced to allow for the mapping of regions necessary for native intracellular localization and specific interaction with cellular proteins. Finally, the ability of both native and mutated forms of IpaD to form plaques in HeLa cell monolayers expressing various gap junction proteins (e.g. connexins) will be done to allow for a more precise understanding of IpaD's possible role as an effector. PUBLIC HEALTH RELEVANCE: The proposed research explores the role of IpaD in the ability of bacterial pathogens, such as Shigella, to invade and spread throughout epithelial cell monolayers. By determining the specific mechanisms and interactions that are responsible for these processes, it will be possible to tailor specific anti-infective treatments to prevent these infections as an alternative to standard antibiotic use.