Chlamydia trachomatis Inclusion Membrane Proteins
沙眼衣原体包涵膜蛋白
基本信息
- 批准号:6522161
- 负责人:
- 金额:$ 30.28万
- 依托单位:
- 依托单位国家:美国
- 项目类别:
- 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:
- 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
项目摘要
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):衣原体属专性细胞内细菌,是性传播疾病的最常见原因,也是全世界可预防性失明的主要原因。衣原体在非酸化液泡中复制,称为包涵体,衣原体主动修饰该液泡以防止溶酶体融合并促进细胞内存活。介导衣原体发病机制的分子决定因素在很大程度上尚未确定,这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利用致病机制来促进和维持其细胞内存活。由于衣原体仍然隔离在液泡内,因此衣原体与其宿主之间的所有相互作用都必须通过包涵膜介导。我们已经鉴定出位于包涵膜的沙眼衣原体特异性蛋白 (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 的细胞靶标来鉴定 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,作为第一个和包涵膜蛋白 IncG 的唯一细胞靶标。 14-3-3 蛋白是二聚体磷酸丝氨酸结合蛋白,可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,并确定衣原体是否靶向 14-3-3 蛋白以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 显性失活突变体和显微注射抗 IncG 抗体来破坏 14-3-3 IncG 相互作用,以检查 14-3-3 的招募是否在利用细胞信号转导和包涵体功能中发挥作用。囊泡介导的途径。其次,我们将结合使用荧光显微镜、酵母三杂交测定和免疫共沉淀实验来确定 14-3-3 蛋白是否会招募额外的信号蛋白到内含物中。第三,我们将采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/配体相互作用来改变 14-3-3 依赖性信号传导途径。具体目标 2 利用酵母双杂交测定来鉴定 IncD/E/F 的细胞靶标。识别 Incs 的细胞靶标以及这些相互作用如何促进衣原体发病机制将有助于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。
项目成果
期刊论文数量(0)
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{{ truncateString('MARCI A SCIDMORE', 18)}}的其他基金
The Role of Rab GTPases in Chlamydia-Infected Cells
Rab GTP 酶在衣原体感染细胞中的作用
- 批准号:
7727368 - 财政年份:2007
- 资助金额:
$ 30.28万 - 项目类别:
The Role of Rab GTPases in Chlamydia-Infected Cells
Rab GTP 酶在衣原体感染细胞中的作用
- 批准号:
7531052 - 财政年份:2007
- 资助金额:
$ 30.28万 - 项目类别:
The Role of Rab GTPases in Chlamydia-Infected Cells
Rab GTP 酶在衣原体感染细胞中的作用
- 批准号:
7993527 - 财政年份:2007
- 资助金额:
$ 30.28万 - 项目类别:
The Role of Rab GTPases in Chlamydia-Infected Cells
Rab GTP 酶在衣原体感染细胞中的作用
- 批准号:
7372331 - 财政年份:2007
- 资助金额:
$ 30.28万 - 项目类别:
The Role of Rab GTPases in Chlamydia-Infected Cells
Rab GTP 酶在衣原体感染细胞中的作用
- 批准号:
8197877 - 财政年份:2007
- 资助金额:
$ 30.28万 - 项目类别:
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