SNAREs and the biogenesis of the chlamydial inclusion membrane

 描述(申请人提供)：50-70%的沙眼衣原体感染是细菌性传播感染的最常见原因，是无症状的。这增加了广泛传播和未经治疗的感染的风险，导致女性盆腔炎或不孕不育。此外，疾控中心估计，在15至19岁的女性中，有10%的人衣原体检测呈阳性。因此，迫切需要确定减少/防止传播、将感染限制在接种的主要部位或中断/控制衣原体生长和发育的策略。在宿主细胞内，基本体(EBS)在病原体特定的寄生细胞器中分化为网状小体(RBS)，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定的宿主细胞途径相互作用，最终影响包涵体的脂质和蛋白质含量。衣原体在宿主体内的生存最重要的是有机体获得和利用宿主细胞衍生的脂类的能力。这些脂类有助于衣原体包涵体的膜以及衣原体细胞膜的形成。众所周知，衣原体会模仿宿主细胞的脂类成分，但生物体不会将所有可用的宿主来源的脂类结合到细胞膜中。在发育过程中，RB与衣原体包涵体的内叶有密切的联系，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的寄生细胞器很重要，而且最终对决定病原体的脂质含量的观点是一致的。我们的实验室主要研究真核细胞陷阱在衣原体包涵体中的作用。SNARE蛋白用于降低将宿主囊泡与靶膜融合所需的能量。在这种情况下，靶膜是衣原体包裹体。我们已经证明了句法蛋白6和10以及VAMP 3和4定位于衣原体包涵体。此外，我们已经证明了Synaxin 6和VAMP4(形成融合SNARE复合体所需的4种蛋白质中的2种)在衣原体包涵体上相互作用。我们假设，衣原体包裹体特异性地拦截多个融合性圈套复合体，以在包涵体膜中创建和维持确定的脂成分。为了验证这一假设，首先，我们将确定已知定位于包涵体的SNARE蛋白的真核和衣原体结合伙伴，因为蛋白质-蛋白质相互作用通常决定目的。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类脂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。通过研究发生在衣原体包涵体的蛋白质-蛋白质相互作用，我们将确定SNARE蛋白如何促进衣原体的脂质含量，并最终导致衣原体的发病。这些研究将为通过阻断包涵体膜的脂质获取来改变衣原体传染性的策略奠定基础，这最终将影响衣原体的感染率和传播率。

 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.