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: 7727368
• 负责人: MARCI A SCIDMORE
• 金额: $ 37.41万
• 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/7727368
• 关键词: Affinity Chromatography; Bacteria; Binding; Biological Assay; Biological Process; Blindness; Cells; Chlamydia; Chlamydia Infections; Complex; Docking; Epithelial Cells; Fluorescence; Fractionation; GTP Binding; Gene Silencing; Genome; Goals; Guanine Nucleotides; Host Defense Mechanism; Mediating; Mediator of activation protein; Methods; Minor; Molecular; Organizational Change; Pathogenesis; Pathway interactions; Property; Proteins; Public Health; Recruitment Activity; Research; Research Proposals; Role; Severities; Sexually Transmitted Diseases; Text; Transport Vesicles; Two-Hybrid System Techniques; Vacuole; Yeasts; base; mutant; novel vaccines; pathogen; prevent; protein function; rab GTP-Binding Proteins; research study; therapeutic target; vaccine candidate

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）：衣原体物种是专性细胞内细菌，是性传播疾病的最常见原因，也是全世界可预防性失明的主要原因。衣原体在非酸化液泡（一种包涵体）中复制，衣原体主动修饰该液泡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尚未确定，这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对于建立隐藏于宿主防御机制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促进和维持其细胞内存活的致病机制。我们已经证明了宿主 Rab GTP 酶（宿主囊泡介导途径的重要介质）招募到衣原体包涵体中。由于 Rab GTP 酶能够在非活性 GDP 结合状态和活性 GTP 结合状态之间循环并与下游效应子相互作用，因此可调节运输囊泡的形成、运输和对接。我们建议衣原体招募 Rab GTPases 来建立细胞内生态位，促进衣原体复制并保护衣原体免受宿主防御机制的影响。为了实现我们的目标，我们提出以下建议： 具体目标 1：确定 Rab GTP 酶募集到衣原体包涵体的机制。我们将使用基于荧光的测定（包括 FRAP）以及分级分离研究和 GST 下拉实验来确定衣原体如何调节和招募 Rab GTPases 来包含在内。 具体目标 2：鉴定将 Rab GTPases 募集到包涵体中的衣原体蛋白。我们将采用酵母双杂交分析和亲和层析方法来鉴定能够招募 Rab GTP 酶到内含物中的衣原体蛋白。 具体目标 3：确定 Rab GTPases 在衣原体感染细胞中的生物学功能。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，以功能性地消除感染细胞中的 Rab GTP 酶，然后我们将确定 Rab GTP 酶对于建立促进细胞内存活和免受宿主防御机制影响的内含物特性是否重要。 相关性：这项研究与公共卫生相关，因为对促进衣原体细胞内存活的复杂宿主-病原体相互作用的识别和表征将有助于识别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。