Although chlamydial infections are treatable with antibiotics, current prevention strategies fail to reduce new infections and subsequent sequelae. Hence, there is a great need to further understand chlamydial survival mechanisms within the host to identify efficacious targets to prevent infection, increase the ease of detection or interrupt/control chlamydial growth. Paramount to chlamydial survival within the host is the organism's ability to obtain and utilize host cell-derived lipids. Within the host cell, EBs differentiate into RBs in a membrane-bound vacuole termed the chlamydial inclusion. The inclusion intercepts a subset of Golgi-derived exocytic vesicles containing sphingomyelin and cholesterol. Utilizing a polarized epithelial cell model to study directional trafficking of lipidsand proteins to the inclusion, we demonstrated that the chlamydial inclusion preferentially intercepts basolaterally targeted exocytic vesicles, suggesting that C. trachomatis specifically interact with a subset of basolateral trafficking machinery. Subsequent studies demonstrated that trans-Golgi SNARE protein syntaxin 6 colocalizes with the chlamydial inclusion in a manner that is conserved across chlamydial species, requires chlamydial protein synthesis and utilizes a eukaryotic signal sequence, YGRL. The YGRL signal sequence returns syntaxin 6 to the trans-Golgi from the plasma membrane;the requirement of this signal sequence for syntaxin 6 localization to the inclusion suggests that syntaxin 6 cycles on and off the inclusion membrane. Recently, we demonstrated that a second trans-Golgi SNARE protein, syntaxin 10 colocalizes to the chlamydial inclusion in a manner distinct from the localization of syntaxin 6. Syntaxin 10 colocalizes to the inclusion in association with Golgi structural proteins, indicating that insteadof docking vesicles to the inclusion, it may dock Golgi-structures. Based on the localization of both syntaxin 6 and syntaxin 10, we propose that these proteins may be interacting with distinct pools of proteins thereby affecting their function at the chlamydial inclusion. The driving hypothesis of this proposal is that Chlamydia recruit syntaxin 6 and 10 to traffic lipids to or fro the inclusion and the organisms themselves;as the precise composition of host lipids in chlamydial organisms is linked to the ability of Chlamydia to limit host defense during an infection.
In Aim 1, we will interrogate protein-protein interactions and the trafficking dynamics f syntaxins 6 and 10 at the inclusion. For the first time, recycling events will be examined at the chlamydial inclusion membrane.
In Aim 2, we will correlate the localization of specific syntaxin proteins with distinct changes in chlamydial lipid composition, and then, link the changes in chlamydial lipid composition to host response by examining the effect of lipid-altered chlamydial organisms on the activation of specific cell signaling pathways. Studies in this proposal will examine discrete mechanisms of chlamydial acquisition, using novel imaging techniques. Significantly, we will interrogate how eukaryotic lipids that are incorporated into the chlamydial cell wall impact host response to infection.
Current strategies to prevent and limit chlamydial infection are ineffective, resulting in $700 million medical costs. A strategy that Chlamydia uses to survive in the host is the acquisition host derived lipids. Studies in this proposal seek to understand chlamydial mechanisms of lipid acquisition and the role these lipids play in chlamydial pathogenesis and host response to infection.