Dissecting the role of the Inclusion membrane protein IncE, a master multi-tasking scaffolding protein, in the pathogenesis of Chlamydia trachomatis infections

摘要 沙眼衣原体（Ct）感染是人类疾病的重要原因，目前尚无疫苗。 我们知识中的一个重要空白是这种专性细胞内空泡细菌如何建立一个特权的 壁龛--一种被称为内含物的膜结合区室。衣原体编码一个独特的分泌型 效应物Incs（包涵体膜蛋白），从细菌通过III型转运 分泌系统，并插入包涵体膜。我们发现，一个早期表达的公司， 因斯结合两种不同的宿主细胞蛋白复合物，其参与运输、分选连接蛋白（SNX）5和 6以及两个密切相关的Q-陷阱，Syntaxin（STX）7和STX 12，它们参与了晚期和早期 内体运输。我们的结果支持因斯直接结合SNX 5/6的模型， 将ESCPE-1复合物（SNX 5/6和SNX 1/2）重定向至包涵体膜， ESCPE-1介导的贩运。在初步实验中，我们已经定义了因斯的区域， 并发现因斯编码一个额外的不同的短基序， 来自SNX 5/6货物结合基序，其模拟R-SNARE的VAMP 3家族的零层基序（ZLM） 蛋白质，STX 7/12的已知结合伴侣。这一令人兴奋的观察表明，因斯可能调节 STX 7/12囊泡与包涵体融合，或者通过刺激它，抑制它，或者作为系链发挥作用。使用 通过RNAi，我们发现STX 7和STX 12在Ct感染中发挥不同的非重叠作用。我们有 创建了因斯无效突变体，并用信息性因斯变体对其进行补充。这种CT的英勇应用 遗传学将使我们能够将因斯与SNX 5/6的结合与STX 7/12的结合分开，并测试IncE在SNX 5/6中的作用。 在Ct生殖道感染的动物模型中的单个Inc。在这项资助计划中，我们建议采用以下方法： CT遗传学、细胞生物学、高级显微镜学和生物化学：目标1。确定因斯在 Ct感染的发病机制。我们将表征因斯框内缺失突变株的表型 使用（A）基于细胞的测定和（B）小鼠模型， 生殖道感染目标2.了解因斯：STX 7/12的分子机制和后果 交互.使用提供信息的因斯突变菌株和蛋白质，我们将（A）确定因斯：STX 7/12 相互作用调节融合，使用采用新型荧光脂质探针（B）的活细胞显微镜测定 如果因斯，特别是其ZLM，足以调节STX 7囊泡融合，使用体外脂质体融合 （C）使用纯化的蛋白质测试因斯ZLM直接结合STX 7和/或STX 12的假设; ad（D）测试因斯是否可以同时结合STX 7/12和SNX 5/6;目的3.解码角色 STX 7/12在调节Ct细胞内生命周期中的作用我们将研究（A）STX 12如何有助于 同型包涵体融合;（B）STX 7如何促成包涵体形成和感染性子代的产生。 我们的研究将增加我们对宿主细胞生物学的了解，并揭示微生物如何破坏这些过程。

Abstract Chlamydia trachomatis (Ct) infections are important causes of human disease for which no vaccine exists. An important gap in our knowledge is how this obligate intracellular vacuolar bacterium establishes a privileged niche--a membrane bound compartment termed the inclusion. Chlamydia encode a distinctive family of secreted effectors, the Incs (Inclusion membrane proteins), which are translocated from the bacteria through the type III secretion system and inserted into the inclusion membrane. We have discovered that an early expressed Inc, IncE, binds to two different host cell protein complexes that are involved in trafficking, sorting nexins (SNX)5 and 6 as well as two closely related Q-SNARES, Syntaxin(STX)7 and STX12, which are involved in late and early endosome trafficking, respectively. Our results support a model wherein IncE directly binds SNX5/6, which redirects the ESCPE-1 complex (SNX5/6 together with SNX1/2) to the inclusion membrane, potentially disrupting ESCPE-1-mediated trafficking. In preliminary experiments, we have defined the regions of IncE that are necessary for binding to STX7/12 and discovered that IncE encodes an additional distinct short motif separate from the SNX5/6 cargo binding motif that mimics the zero layer motif (ZLM) of the VAMP3 family of R-SNARE proteins, known binding partners of STX7/12. This exciting observation suggests that IncE may modulate STX7/12 vesicle fusion with the inclusion, either by stimulating it, inhibiting it, or functioning as a tether. Using RNAi, we have discovered that STX7 and STX12 serve distinct non-overlapping roles in Ct infection. We have created an IncE null mutant and complemented it with informative IncE variants. This heroic application of Ct genetics will allow us to separate IncE binding to SNX5/6 from its binding to STX7/12, and to test the role of a single Inc in an animal model of Ct genital tract infection. In this grant, we propose to employ a combination of Ct genetics, cell biology, advanced microscopy, and biochemistry to: Aim 1. Determine the role of IncE in the pathogenesis of Ct infections. We will characterize the phenotypes of the IncE in-frame deletion mutant strain and informative isogenic complemented derivatives using (A) cell-based assays and (B) a mouse model of upper genital tract infection. Aim 2. Understand the molecular mechanism and consequences of IncE:STX7/12 interactions. Using informative IncE mutant strains and proteins, we will (A) determine whether IncE:STX7/12 interactions modulate fusion, using live cell microscopy that employs novel fluorescent lipid probes (B) determine if IncE, and specifically its ZLM, is sufficient to modulate STX7 vesicle fusion, using an in vitro liposome fusion assay; (C) test the hypothesis that the IncE ZLM binds directly to STX7 and/or STX12, using purified proteins; ad (D) Test whether IncE can bind simultaneously to STX7/12 and SNX5/6; Aim 3. Decode the roles of STX7/12 in regulating the Ct intracellular lifecycle. We will investigate (A) how STX12 contributes to homotypic inclusion fusion; (B) how STX7 contributes inclusion formation and production of infectious progeny. Our studies will increase our knowledge of host cell biology and reveal how microbes subvert these processes.