The Role of Rab GTPases in Chlamydia-Infected Cells

描述（由申请人提供）：衣原体是专性细胞内细菌，是性传播疾病的最常见原因，也是世界范围内可预防失明的主要原因。衣原体在非酸化液泡中复制，这是一种被衣原体积极修饰的包涵体，以防止溶酶体融合并促进细胞内存活。介导衣原体发病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。衣原体与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，而且在决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促进和维持其细胞内生存的致病机制。我们已经证明了宿主Rab gtp酶的招募，这是宿主囊泡介导途径的重要介质，衣原体包涵体。由于Rab GTPases能够在不活跃的gtp结合状态和活跃的gtp结合状态之间循环，并与下游效应物相互作用，因此可以调节运输囊泡的形成、运输和对接。我们提出，衣原体招募Rab gtpase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。为达致上述目标，我们提出以下建议：

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.