Probing localiztion, interactions, and effector properties of Shigella lpaD

描述(申请人提供)：建议的研究集中于阐明侵袭质粒抗原D(IPad)在细菌性痢疾的病原体福氏志贺氏菌的细胞入侵和细胞间传播中的可能作用。福氏杆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)等许多重要病原体一起利用III型分泌系统(TTSS)作为颠覆人类细胞正常功能的一种手段。对于志贺氏菌来说，iPad位于TTSS的暴露极，在那里它感觉到宿主细胞的接触，并帮助招募下游效应蛋白注入宿主细胞膜和细胞质，以促进细菌入侵。我们最近发现的证据表明，iPad可能不仅存在于福氏志贺氏菌的表面，而且还被注射到宿主细胞的细胞质中，在那里它可能定位于缝隙连接。由于iPad可能在入侵后事件中发挥作用(即直接细胞间传播)，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。为了更好地理解iPad及其来自其他系统的同系物的这一作用，人们设计了一系列实验来：a)确定iPad在培养的人上皮细胞中定位(可能具有缝隙连接)的分子基础；b)确定iPad的结构特征，这些结构特征是其能够被招募到宿主细胞内特定位置的原因；以及c)测试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。具体地说，将利用荧光共定位、FRET和亲和层析实验来绘制iPad的细胞内分布图，并识别与其相互作用的细胞内宿主蛋白。一旦鉴定出这些蛋白质，将进行突变分析，其中将引入iPad中的小缺失和点突变，以允许绘制天然细胞内定位和与细胞蛋白质的特定相互作用所需的区域。最后，为了更准确地理解iPad作为效应器的可能作用，将研究自然形式和突变形式的iPad在HeLa细胞单层中形成表达各种缝隙连接蛋白(例如连接蛋白)的斑块的能力。与公共卫生相关：这项拟议的研究探索了iPad在细菌病原体(如志贺氏菌)入侵和传播整个上皮细胞单层的能力中所起的作用。通过确定负责这些过程的特定机制和相互作用，将有可能量身定做特定的抗感染治疗，以防止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.