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侵袭质粒抗原D（iPad）在细菌病原体志贺氏病原体的细胞浸润和细胞间扩散中的推定作用，这是细菌性疾病的病原体。 S. flexneri以及许多其他重要的病原体，例如耶尔森氏菌（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）作为颠覆人类细胞正常功能的一种手段。对于Shigella，iPad位于TTSS的暴露极，它可以感受宿主细胞的接触，并有助于募集下游效应蛋白，以注射到宿主细胞的膜和细胞质中，以促进细菌侵袭。我们最近发现证据表明iPad不仅可以驻留在柔性链球菌的表面上，而且还将其注入到宿主细胞细胞质中，在那里它可以定位于间隙连接。由于iPad可能在侵入后事件（即直接细胞间扩散）中起作用，因此它代表了志贺氏菌TTSS功能的尚未描述的方面，这可能是预防疾病的目标。为了更好地了解iPad及其其他系统的同源物的这种作用，已经设计了一系列实验：a）确定iPad在培养的人上皮细胞中iPad细胞内定位（可能带有间隙连接）的分子基础； b）确定iPad的结构特征，这些特征负责将其招募到宿主细胞内的特定位点的能力； c）检验以下假设：iPad和宿主细胞蛋白之间的相互作用会影响志贺氏菌间细胞扩散的效率。具体而言，将利用荧光共定位，FRET和亲和色谱实验来绘制iPad的细胞内分布并识别与其相互作用的细胞内宿主蛋白。一旦确定了这些蛋白质，将引入iPad中的小缺失和点突变的突变分析，以允许映射天然细胞内定位所必需的区域以及与细胞蛋白的特异性相互作用。最后，将完成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天然和突变形式的能力，以便对iPad作为效应子的可能作用有更精确的了解。公共卫生相关性：拟议的研究探讨了iPad在细菌病原体（例如志贺氏菌）侵入和扩散整个上皮细胞单层中的作用。通过确定负责这些过程的特定机制和相互作用，可以量身定制特定的抗感染治疗方法，以防止这些感染作为标准抗生素使用的替代方法。