The Role of Rab GTPases in Chlamydia-Infected Cells

Rab GTP 酶在衣原体感染细胞中的作用

• 批准号: 7531052
• 负责人: MARCI A SCIDMORE
• 金额: $ 37.81万
• 依托单位: CORNELL UNIVERSITY
• 依托单位国家: 美国
• 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/7531052
• 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; Goals; Guanine Nucleotides; Host Defense Mechanism; Integration Host Factors; Life Style; 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; vaccine candidate; yeast two hybrid system

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）：衣原体属是专性细胞内细菌，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。衣原体在非酸化的空泡（一种内含物）中复制，其被衣原体主动修饰以防止溶酶体融合并促进细胞内存活。介导衣原体发病机制的分子决定因素在很大程度上是不确定的，主要是由于无法操纵衣原体基因组。衣原体和宿主上皮细胞之间的相互作用不仅在建立隐藏于宿主防御机制的细胞内生态位方面至关重要，而且在确定衣原体疾病的严重程度方面也至关重要。本研究的总体目标是确定衣原体利用的致病机制，以促进和维持其细胞内的生存。我们已经证明了招聘的主机拉布GTP酶，这是重要的调解人的主机囊泡介导的途径，衣原体夹杂物。由于它们能够在非活性GDP结合和活性GTP结合状态之间循环并与下游效应物相互作用，Rab GTP酶调节运输囊泡的形成、运输和对接。我们建议，衣原体招募Rab GTP酶，以建立一个细胞内的小生境，促进衣原体复制和保护衣原体宿主防御机制。为了实现我们的目标，我们提出以下建议： 具体目标1：确定衣原体包涵体中Rab GT抗体募集的机制。我们将使用基于荧光的测定，包括FRAP连同分馏研究和GST下拉实验，以确定衣原体如何调节和招募Rab GTP酶的纳入。 具体目标2：鉴定将Rab GTP酶募集到包涵体的衣原体蛋白。我们将继续进行酵母双杂交试验和亲和层析方法，以确定衣原体蛋白的功能，招募拉布GTP酶的列入。 具体目标3：鉴定衣原体感染细胞中Rab GTP酶的生物学功能。我们将首先建立方法，包括基因沉默和鸟嘌呤核苷酸结合突变体的表达，从感染的细胞中功能性地消耗Rab GTP酶，然后我们将确定Rab GTP酶是否对建立促进细胞内存活和保护宿主防御机制的内含物的性质很重要。 相关性：这项研究与公共卫生有关，因为鉴定和表征促进衣原体细胞内存活的复杂宿主-病原体相互作用将有助于鉴定可用于帮助预防和治疗衣原体疾病的新型候选疫苗和治疗靶点。