SNAREs and the biogenesis of the chlamydial inclusion membrane

 描述（由申请人提供）：50-70%的沙眼衣原体感染是细菌性性传播感染的最常见原因，无症状。这增加了广泛传播和未经治疗的感染的风险，导致盆腔炎或妇女不孕。此外，CDC估计，15至19岁的女性中有10%的衣原体检测呈阳性。因此，非常需要确定减少/预防传播、将感染限制在主要接种部位或中断/控制衣原体生长和发育的策略。在宿主细胞内，原体（EB）分化为病原体特异性寄生细胞器中的网状体（RB），称为衣原体包涵体。为了保持其自主性，衣原体包涵体与非常特异的宿主细胞途径相互作用，这最终影响包涵体的脂质和蛋白质含量。衣原体在宿主体内存活的最重要因素是生物体获得和利用宿主细胞来源的脂质的能力。这些脂质有助于衣原体包涵体的膜，以及衣原体细胞膜。众所周知，衣原体将模仿其宿主细胞的脂质组成，但生物体不会将所有可用的宿主衍生脂质并入其细胞膜中。在发育过程中，RB与衣原体包涵体的内小叶密切相关，在那里它获得脂质。这与衣原体包涵体膜与宿主细胞囊泡相互作用的特异性不仅对维持病原体特异性寄生细胞器很重要，而且最终对决定病原体的脂质含量很重要的观点一致。本实验室主要研究真核细胞SNAREs在衣原体包涵体中的功能。SNARE蛋白用于减少宿主囊泡与靶膜融合所需的能量。在这种情况下，目标膜是衣原体包涵体。我们已经证明，syntaxins 6和10和VAMP 3和4本地化衣原体包含。此外，我们已经证明，syntaxin 6和VAMP 4（2出4所需的蛋白质形成融合陷阱复合物）在衣原体包涵体相互作用。我们假设衣原体包涵体特异性地拦截多个融合SNARE复合物，以在包涵体膜中产生并维持确定的脂质组成。为了验证这一假设，首先，我们将确定真核和衣原体的结合伙伴的陷阱蛋白已知本地化的包容性，蛋白质-蛋白质相互作用往往决定的目的。其次，我们将敲除这些蛋白质，以了解这些蛋白质在决定衣原体脂质含量和随后的外膜组织中的作用。第三，我们将确定这些蛋白质的敲除如何影响衣原体的生长和发育。通过检查蛋白质-蛋白质相互作用发生在衣原体包含，我们将确定如何陷阱蛋白有助于衣原体脂质含量，并最终，衣原体发病机制。这些研究将为通过阻断脂质进入包涵体膜来改变衣原体感染性的策略奠定基础，这将最终影响衣原体的感染和传播率。

 DESCRIPTION (provided by applicant): 50-70% of Chlamydia trachomatis infections, the most common cause of bacterial sexually transmitted infections, are asymptomatic. This increases the risk of widespread transmission and untreated infections, resulting in pelvic inflammatory disease or infertility in women. Further, the CDC estimates that 10% of women between the ages of 15 to 19 test positive for Chlamydia. Hence, there is a great need to identify strategies to reduce/prevent transmission, limit infections to the primary site of inoculation or interrupt/control chlamydial growth and development. Within the host cell, elementary bodies (EBs) differentiate into reticulate bodies (RBs) in a pathogen-specified parasitic organelle termed the chlamydial inclusion. To maintain its autonomy, the chlamydial inclusion interacts with very specific host cell pathways, which ultimately influences the lipid an protein content of the inclusion. Paramount to chlamydial survival within the host is the organism's ability to obtain and utilize host cell-derived lipids. These lipids contribute to the membrane of the chlamydial inclusion, as well as, the chlamydial cell membranes. It is well established that Chlamydia will mimic the lipid composition of their host cells, but the organisms will not incorporate all available host-derived lipids into their cell membranes. During development, the RB has a close association with the inner leaflet of the chlamydial inclusion, where it acquires lipids. This is consistent with the notion that the specificity of interaction ofthe chlamydial inclusion membrane with host cell vesicles is not only important for the maintenance of the pathogen-specific parasitic organelle, but ultimately, is important for dictating the lipid content of the pathogens. Our laboratory focuses on the function of eukaryotic SNAREs at the chlamydial inclusion. SNARE proteins serve to decrease the energy required to fuse a host vesicle with a target membrane. In this case, the target membrane is the chlamydial inclusion. We have demonstrated that syntaxins 6 and 10 and VAMPs 3 and 4 localize to chlamydial inclusion. Further, we have demonstrated that syntaxin 6 and VAMP4 (2 out of 4 required proteins to form a fusogenic SNARE complex) interact at the chlamydial inclusion. We hypothesize that the chlamydial inclusion specifically intercepts multiple fusogenic SNARE complexes to create and maintain a defined lipid composition in the inclusion membrane. To test this hypothesis, first, we will identify the eukaryotic and chlamydial binding partners for SNARE proteins known to localize to the inclusion, as protein-protein interactions often dictate purpose. Second, we will knockdown these proteins to understand the role of these proteins in determining chlamydial lipid content and subsequent outer membrane organization. Third, we will determine how knockdown of these proteins affects chlamydial growth and development. By examining protein-protein interactions that occur at the chlamydial inclusion, we will determine how SNARE proteins contribute to chlamydial lipid content, and ultimately, to chlamydial pathogenesis. These studies will underlay the bases for strategies to alter chlamydial infectivity via interruption of lipid acquisition to the inclusion membrane, which will ultimately impact the infection and transmission rates of Chlamydia.