The Role of Rab GTPases in Chlamydia-Infected Cells

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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.