Probing localiztion, interactions, and effector properties of Shigella lpaD

志贺氏菌 lpaD 的定位、相互作用和效应子特性的探测

• 批准号: 7916511
• 负责人: Nicholas E Dickenson
• 金额: $ 4.76万
• 依托单位: OKLAHOMA STATE UNIVERSITY STILLWATER
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-07-22 至 2011-07-21
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7916511
• 关键词: Address; Affinity Chromatography; Anti-Infective Agents; Antibiotics; Antigens; Automobile Driving; Bacillary Dysentery; Binding; Biochemical; Cell membrane; Cells; Cellular Structures; Complex; Connexins; Cultured Cells; Cytoplasm; DNA Sequence Rearrangement; Data; Epithelial Cells; Event; Fluorescence; Fluorescence Microscopy; Fluorescence Resonance Energy Transfer; Gap Junctions; Gastroenteritis; Hela Cells; Homologous Gene; Human; Infection; Infection prevention; Injection of therapeutic agent; Intestines; Invaded; Location; Lung; Maps; Membrane; Molecular; Monitor; Mutate; Mutation; Needles; Organism; Phenotype; Plague; Plasmids; Play; Point Mutation; Process; Property; Proteins; Pseudomonas aeruginosa; Recruitment Activity; Research; Role; Salmonella; Series; Shigella; Shigella flexneri; Site; Structure; Surface; System; Techniques; Testing; Type III Secretion System Pathway; Work; Yersinia pestis; base; design; disorder prevention; innovation; monolayer; pathogen; public health relevance; research study

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.

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。弗氏沙门氏菌以及许多其他重要病原体，如鼠疫耶尔森氏菌（鼠疫）、沙门氏菌（胃肠炎）和铜绿假单胞菌（肺部感染），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的接触，并帮助招募下游效应蛋白注射到宿主细胞的膜和细胞质中，以促进细菌入侵。我们最近发现的证据表明，IpaD 可能不仅驻留在福氏链霉表面，而且还被注射到宿主细胞的细胞质中，并可能定位于间隙连接处。由于 IpaD 可能在入侵后事件（即直接细胞间传播）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。为了更好地理解 IpaD 及其来自其他系统的同源物的作用，设计了一系列实验来： a) 确定 IpaD 在培养的人上皮细胞中细胞内定位（可能通过间隙连接）的分子基础； b) 确定 IpaD 的结构特征，这些特征决定了 IpaD 能够被募集到宿主细胞内的特定位点； c) 检验 IpaD 和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。具体而言，将利用荧光共定位、FRET 和亲和层析实验来绘制 IpaD 的细胞内分布图并鉴定与其相互作用的细胞内宿主蛋白。一旦鉴定出这些蛋白质，将引入 IpaD 中的小缺失和点突变的突变分析，以绘制天然细胞内定位和与细胞蛋白质特异性相互作用所需的区域。最后，将研究天然形式和突变形式的 IpaD 在表达各种间隙连接蛋白（例如连接蛋白）的 HeLa 细胞单层中形成斑块的能力，以便更准确地了解 IpaD 作为效应器的可能作用。公共健康相关性：拟议的研究探讨了 IpaD 在细菌病原体（例如志贺氏菌）侵入和扩散到上皮细胞单层的能力中的作用。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。