Chlamydia trachomatis Inclusion Membrane Proteins

描述（由申请人提供）：衣原体是专性细胞内细菌，是性传播疾病的最常见原因，也是世界范围内可预防失明的主要原因。衣原体在一个被称为包涵体的非酸化液泡中复制，包涵体被衣原体积极修饰，以防止溶酶体融合，促进细胞内存活。介导衣原体发病的分子决定因素在很大程度上是不确定的，主要是由于无法操纵衣原体基因组。本研究的总体目标是确定衣原体促进和维持其细胞内生存的致病机制。由于衣原体被隔离在液泡内，因此衣原体与宿主之间的所有相互作用都必须通过包裹膜介导。我们已经鉴定出沙眼衣原体特异性蛋白（IncD/E/F/G）定位在包涵膜上。它们的细胞内定位使它们通过与宿主蛋白的直接相互作用成为宿主-病原体相互作用的潜在介质。为了实现我们的总体目标，我们建议通过鉴定和表征IncD/E/F/G的细胞靶点来鉴定IncD/E/F/G的生物学功能。我们已经鉴定出哺乳动物14-3-3蛋白，作为包涵膜蛋白IncG的第一个也是唯一的细胞靶点。14-3-3蛋白是二聚体磷酸丝氨酸结合蛋白，通过信号复合物的定向亚细胞定位调节多种信号转导途径。具体目的1：实验旨在确定14-3-3 IncG相互作用的生物学功能，并确定衣原体是否靶向14-3-3蛋白利用宿主信号和囊泡介导的途径。首先，我们将通过表达14-3-3显性阴性突变体和显微注射抗IncG抗体来破坏14-3-3 -IncG相互作用，以研究14-3-3 -包涵体的募集是否在利用细胞信号转导和囊泡介导途径中起作用。其次，我们将结合荧光显微镜、酵母三杂交试验和共免疫沉淀实验来确定14-3-3蛋白是否会招募额外的信号蛋白加入包涵体。第三，我们将采用共免疫沉淀实验来确定衣原体是否通过改变宿主14-3-3/配体相互作用来改变14-3-3依赖的信号通路。Specific Aim 2利用酵母双杂交法鉴定IncD/E/F的细胞靶点。鉴定Incs的细胞靶点以及这些相互作用如何促进衣原体的发病机制将有助于更好地理解促进衣原体细胞内生存的复杂宿主-病原体相互作用。

DESCRIPTION (provided by applicant): Chlamydiae species are obligate intracellular bacteria that are the most frequent cause of sexually transmitted disease as well as the leading cause of preventable blindness worldwide. Chlamydiae replicate in a non-acidified vacuole, termed an inclusion, which is actively modified by chlamydiae to prevent lysosomal fusion and promote intracellular survival. The molecular determinants that mediate chlamydial pathogenesis are largely undefined primarily due to the inability to manipulate the chlamydial genome. The overall goal of this research is to identify pathogenic mechanisms utilized by chlamydiae to promote and maintain their intracellular survival. Because chlamydiae remain sequestered within a vacuole, all interactions between chlamydiae and their host must be mediated through the inclusion membrane. We have identified Chlamydia trachomatis-specific proteins (IncD/E/F/G) that are localized to the inclusion membrane. Their intracellular localization makes them potential mediators of host-pathogen interactions via direct interactions with host proteins. To achieve our overall goals, we propose to identify biological functions of IncD/E/F/G through identification and characterization of cellular targets of IncD/E/F/G We have identified mammalian 14-3-3 proteins, as the first and only cellular targets of an inclusion membrane protein, IncG. 14-3-3 proteins are dimeric phosphoserine binding proteins that regulate diverse signal transduction pathways through directed subcellular localization of signaling complexes. Specific Aim 1: Experiments are designed to define biological functions of 14-3-3 IncG interactions and determine whether chlamydiae target 14-3-3 proteins to exploit host signal and vesicular-mediated pathways. First, we will disrupt 14-3-3 IncG interactions through expression of 14-3-3 dominant negative mutants and microinjection of anti-IncG antibodies to examine whether 14-3-3's recruitment to the inclusion functions in exploitation of cellular signal transduction and vesicular-mediated pathways. Second, we will use a combination of fluorescence microscopy, yeast tri-hybrid assays and co-immunoprecipitation experiments to determine whether 14-3-3 proteins recruit additional signaling proteins to the inclusion. And third, we will employ co-immunoprecipitation experiments to determine whether chlamydiae alter 14-3-3-dependent signaling pathways by altering host 14-3-3/ligand interactions. Specific Aim 2 utilizes yeast two-hybrid assays to identify cellular targets of IncD/E/F. Identification of cellular targets of Incs and how these interactions contribute to chlamydial pathogenesis will lead to a better understanding of the complex host-pathogen interactions that facilitate chlamydial intracellular survival.