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，目前尚无疫苗可预防。 我们知识中的一个重要空白是这种专有的细胞内空泡细菌如何建立一个特权 壁龛--一个膜结合区，称为包涵体。衣原体编码一个独特的分泌性 效应器，INCS(包涵膜蛋白)，从细菌通过III型转移而来 分泌系统，并插入包涵膜。我们发现，一个早期表达的公司， 因为，它与两种不同的宿主细胞蛋白复合体结合，这两种复合体参与运输，排序连接蛋白(SNX)5和 6以及两个密切相关的Q-SNARs，Synaxin(STX)7和STX12，它们参与了晚期和早期 分别是内生体贩运。我们的结果支持一个模型，在该模型中，INCE直接与SNX5/6结合，其中 将ESCPE-1复合体(SNX5/6和SNX1/2)重定向到包涵体膜上，可能会破坏 ESCPE-1介导的人口贩运。在初步实验中，我们已经定义了Ince的区域 与STX7/12结合所必需的，并发现INCE编码另外一个不同的短基序 来自模拟R-SNARE VAMP3家族的零层基序(ZLM)的SNX5/6货物结合基序 蛋白质，已知的STX7/12结合伙伴。这一令人兴奋的观察表明，INCE可能调节 STX7/12囊泡与包涵体融合，要么刺激它，抑制它，要么起到系绳的作用。vbl.使用 RNAi，我们发现STX7和STX12在沙眼衣原体感染中起着不同的非重叠作用。我们有 创建了一个Ince零突变体，并用信息丰富的Ince变体补充了它。CT的这一英勇应用 遗传学将允许我们将INCE与SNX5/6的结合与其与STX7/12的结合分开，并测试 Single Inc.建立CT生殖道感染的动物模型。在这笔拨款中，我们建议综合采用 CT遗传学、细胞生物学、高级显微镜和生物化学：目的1.确定INCE在 沙眼衣原体感染的发病机制。我们将对Ince框架内缺失突变株的表型进行鉴定 以及使用(A)基于细胞的分析和(B)UP的小鼠模型的信息性等基因互补衍生物 生殖道感染。目的2.了解INCE的分子机制和后果：STX7/12 互动。使用信息丰富的INCE突变菌株和蛋白质，我们将(A)确定INCE：STX7/12 相互作用调节融合，使用使用新型荧光脂质探针的活细胞显微镜(B)确定 如果使用体外脂质体融合，INCE，特别是它的ZLM足以调节STX7囊泡融合 检测；(C)使用纯化的蛋白检验Ince ZLM直接与STX7和/或STX12结合的假设； AD(D)测试INCE是否可以同时与STX7/12和SNX5/6绑定；目标3.解码INCE的角色 STX7/12在调节Ct细胞内生命周期中的作用我们将调查(A)STX12如何有助于 同型包涵体融合；(B)STX7如何促进包涵体的形成和感染性后代的产生。 我们的研究将增加我们对宿主细胞生物学的知识，并揭示微生物如何颠覆这些过程。

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.