The Role of Rab GTPases in Chlamydia-Infected Cells

Rab GTP 酶在衣原体感染细胞中的作用

基本信息

批准号：
7372331
负责人：
MARCI A SCIDMORE
金额：
$ 37.76万
依托单位：
CORNELL UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2007
资助国家：
美国
起止时间：
2007-12-01 至 2012-11-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/7372331
关键词：
Affinity Chromatography Applications Grants Bacteria Binding Biological Assay Biological Process Blindness Cell physiology Cells Chlamydia Chlamydia Infections Complex Cytosol Docking Ensure Epithelial Cells Fluorescence Fluorescence Recovery After Photobleaching Fractionation GTP Binding Gene Silencing Genetic Transformation Genome Glutathione S-Transferase Goals Guanine Nucleotides Host Defense Mechanism Integration Host Factors Life Style Localized Mediating Mediator of activation protein Membrane Methods Molecular Nature Organelles Organism Pathogenesis Pathway interactions Property Proteins Public Health Recruitment Activity Research Role Severities Sexually Transmitted Diseases Specificity System Testing Transport Vesicles Two-Hybrid System Techniques Vacuole Vesicle Yeasts base design genetic regulatory protein mutant novel vaccines pathogen prevent protein function rab GTP-Binding Proteins research study therapeutic target yeast two hybrid system

项目摘要

DESCRIPTION (provided by applicant): Chlamydia species are obligate intracellular bacteria that are the most frequent cause of sexually transmitted disease as well as the leading cause of preventable blindness worldwide. Chlamydiae replicate in a non- acidified vacuole, an inclusion, which is actively modified by chlamydiae to prevent lysosomal fusion and promote intracellular survival. The molecular determinants that mediate chlamydial pathogenesis are largely undefined primarily due to the inability to manipulate the chlamydial genome. Interactions between chlamydiae and the host epithelial cell are critical not only in establishing an intracellular niche hidden from host defense mechanisms, but are also critical in determining the severity of chlamydial disease. The overall goal of this research is to identify the pathogenic mechanisms utilized by chlamydiae to promote and maintain their intracellular survival. We have demonstrated the recruitment of host Rab GTPases, which are important mediators of host vesicular-mediated pathways, to chlamydial inclusions. Due to their capacity to cycle between inactive GDP-bound and active GTP-bound states and interact with downstream effectors, Rab GTPases regulate the formation, transport and docking of transport vesicles. We propose that chlamydiae recruit Rab GTPases to establish an intracellular niche that promotes chlamydial replication and protects chlamydiae from host defense mechanisms. To achieve our goals, we propose the following: Specific Aim 1: Define the mechanisms of Rab GTPase recruitment to chlamydial inclusions. We will use fluorescence based assays including FRAP together with fractionation studies and GST pull-down experiments to determine how chlamydiae regulate and recruit Rab GTPases to the inclusion. Specific Aim 2: Identify the chlamydial proteins that recruit Rab GTPases to the inclusion. We will pursue yeast two-hybrid assays and affinity chromatography approaches to identify the chlamydial proteins that function to recruit Rab GTPases to the inclusion. Specific Aim 3: Identify biological functions of Rab GTPases in chlamydia-infected cells. We will first establish methods, including gene silencing and expression of guanine nucleotide-binding mutants, to functionally deplete Rab GTPases from infected cells, and then we will determine whether Rab GTPases are important for establishing the properties of the inclusion that promote intracellular survival and protection from host defense mechanisms. RELEVANCE: This research is relevant to public health as the identification and characterization of the complex host- pathogen interactions that facilitate the intracellular survival of chlamydiae will aid in the identification of novel vaccine candidates and therapeutic targets that can be used to help prevent and treat chlamydial disease.

描述（由申请人提供）：衣原体物种是强制性细胞内细菌，是性传播疾病的最常见原因，也是全球可预防失明的主要原因。衣原体在非酸化的液泡中复制，一种包含，被衣原体积极修饰，以防止溶酶体融合并促进细胞内存活。介导衣原体发病机理的分子决定因素主要是由于无法操纵衣原体基因组而在很大程度上不确定。衣原体与宿主上皮细胞之间的相互作用不仅在建立宿主防御机制中隐藏的细胞内生态位至关重要，而且对于确定衣原体疾病的严重程度也至关重要。这项研究的总体目标是确定衣原体用来促进和维持其细胞内生存的病原机制。我们已经证明了宿主RAB GTPases的招募，这些宿主是宿主囊泡介导的途径的重要介体，招募到衣原体夹杂物中。由于它们有能力在不活跃的GDP结合和活跃的GTP结合状态并与下游效应子相互作用之间，RAB GTPases调节了运输囊泡的形成，运输和对接。我们建议衣原体招募RAB GTPASES建立一个细胞内的小境，以促进衣原体复制并保护衣原体免受宿主防御机制的侵害。为了实现我们的目标，我们提出以下内容：特定目的1：定义RAB GTPase募集到衣原体包含物的机制。我们将使用基于荧光的测定法，包括FRAP以及分级研究和GST下拉实验，以确定衣原体如何调节RAB GTPases并募集RAB GTPases。特定目的2：确定将Rab GTPases募集到包含的衣原体蛋白。我们将采用酵母双杂交测定和亲和力色谱方法，以识别功能募集Rab GTPases的衣原体蛋白。特定目的3：确定在感染衣原体的细胞中RAB GTPases的生物学功能。我们将首先建立方法，包括鸟嘌呤核苷酸结合突变体的基因沉默和表达，以在功能上从受感染的细胞中耗尽RAB GTPase，然后我们将确定RAB GTPase是否对于建立促进细胞内存活和保护宿主防御机制的包含的特性是否很重要。相关性：这项研究与公共卫生有关，因为促进衣原体的细胞内生存的复杂宿主病原体相互作用的识别和表征将有助于鉴定新型候选疫苗候选者和可用于帮助预防和治疗衣原体疾病的治疗靶标。