Chlamydia trachomatis Inclusion Membrane Proteins

沙眼衣原体包涵膜蛋白

• 批准号: 6637090
• 负责人: MARCI A SCIDMORE
• 金额: $ 31.32万
• 依托单位: CORNELL UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2002
• 资助国家: 美国
• 起止时间: 2002-07-01 至 2007-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6637090
• 关键词: Chlamydia trachomatis; HeLa cells; bacteria infection mechanism; biological signal transduction; fluorescence microscopy; gene mutation; host organism interaction; immunoprecipitation; mass spectrometry; matrix assisted laser desorption ionization; membrane proteins; mitogen activated protein kinase; protein binding; protein protein interaction; protein structure function; vesicle /vacuole; yeast two hybrid system

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.

描述（由申请人提供）：衣原体物种是强制性细胞内细菌，是性传播疾病的最常见原因，也是全球可预防失明的主要原因。衣原体在非酸化的液泡中复制，称为包含，该液体被衣原体积极修饰，以防止溶酶体融合并促进细胞内存活。介导衣原体发病机理的分子决定因素主要是由于无法操纵衣原体基因组而在很大程度上不确定。这项研究的总体目标是确定衣原体利用的致病机制来促进和维持其细胞内生存。由于衣原体在液泡中保持隔离，因此衣原体与其宿主之间的所有相互作用都必须通过包含膜进行介导。我们已经确定了局部在包含膜的沙眼特异性蛋白（INCD/E/F/G）。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进行了宿主 - 病原体相互作用的潜在介体。为了实现我们的整体目标，我们建议通过鉴定和表征INCD/E/F/G的细胞靶标确定INCD/E/F/G的生物学功能，我们已经确定了哺乳动物14-3-3蛋白，是包含膜蛋白的第一个也是唯一的细胞靶标。 14-3-3蛋白是二聚体磷酸塞蛋白结合蛋白，通过信号复合物的定向亚细胞定位来调节各种信号转导途径。具体目标1：实验旨在定义14-3-3 INCG相互作用的生物学功能，并确定衣原体靶向14-3-3蛋白是否旨在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显性阴性突变体的表达和抗浓度抗体的显微注射来破坏14-3-3 INCG的相互作用，以检查14-3-3的募集是否在包含在细胞信号传输中的募集功能是否功能。其次，我们将使用荧光显微镜，酵母三杂交测定法和共免疫沉淀实验的组合来确定14-3-3蛋白是否募集了其他信号蛋白来包含。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依赖的信号传导途径。特定的目标2利用酵母两杂交测定法来识别INCD/E/F的细胞靶标。鉴定INC的细胞靶标以及这些相互作用如何促进衣原体发病机理将使人们更好地理解促进衣原体细胞内存活的复杂宿主 - 病原体相互作用。