Chlamydia trachomatis Inclusion Membrane Proteins

描述(申请人提供)：衣原体是专性细胞内细菌，是性传播疾病最常见的原因，也是全球可预防失明的主要原因。衣原体在未酸化的空泡中复制，称为包涵体，被衣原体主动修饰，以防止溶酶体融合，促进细胞内生存。介导衣原体致病的分子决定因素在很大程度上是未知的，主要是由于无法操纵衣原体基因组。这项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。因为衣原体仍然被隔离在液泡中，所以衣原体与宿主之间的所有相互作用都必须通过包涵体膜来调节。我们已经鉴定了定位于包涵膜的沙眼衣原体特异性蛋白(IncD/E/F/G)。它们在细胞内的定位使它们有可能通过与宿主蛋白的直接相互作用来调节宿主与病原体的相互作用。为了实现我们的总体目标，我们建议通过鉴定和鉴定INCD/E/F/G的细胞靶标来确定INCD/E/F/G的生物学功能。我们已经鉴定出哺乳动物14-3-3蛋白是第一个也是唯一一个包涵膜蛋白INcG的细胞靶标。14-3-3蛋白是二聚体磷酸丝氨酸结合蛋白，通过信号复合体的亚细胞定位调控不同的信号转导途径。具体目的1：实验旨在确定14-3-3IncG相互作用的生物学功能，并确定衣原体是否以14-3-3蛋白为靶标来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显性负性突变体和微量注射抗IncG抗体来阻断14-3-3-3IncG的相互作用，以检测14-3-3‘S在开发细胞信号转导和囊泡介导的通路中是否具有包涵体功能。其次，我们将使用荧光显微镜、酵母三杂交试验和免疫共沉淀实验相结合的方法来确定14-3-3蛋白是否招募了额外的信号蛋白到包涵体中。第三，我们将使用免疫共沉淀实验来确定衣原体是否通过改变宿主14-3-3/配体的相互作用来改变14-3-3依赖的信号通路。特异性目标2利用酵母双杂交试验来确定INCD/E/F的细胞靶点。识别INCS的细胞靶点以及这些相互作用如何在衣原体致病中起作用将有助于更好地理解促进衣原体细胞内生存的复杂的宿主-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.