Analysis of type III secreted proteins of C. trachomatis

沙眼衣原体III型分泌蛋白分析

基本信息

批准号：
6930624
负责人：
KENNETH A FIELDS
金额：
$ 10.8万
依托单位：
UNIVERSITY OF MIAMI SCHOOL OF MEDICINE
依托单位国家：
美国
项目类别：
财政年份：
2004
资助国家：
美国
起止时间：
2004-08-01 至 2007-07-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/6930624
关键词：
Chlamydia trachomatis HeLa cells SDS polyacrylamide gel electrophoresis bacteria infection mechanism bacterial proteins host organism interaction immunoprecipitation mass spectrometry matrix assisted laser desorption ionization nucleic acid sequence protein protein interaction protein quantitation /detection sexually transmitted diseases vesicle /vacuole western blottings yeast two hybrid system

Chlamydia trachomatis HeLa cells SDS polyacrylamide gel electrophoresis bacteria infection mechanism bacterial proteins host organism interaction immunoprecipitation mass spectrometry matrix assisted laser desorption ionization nucleic acid sequence protein protein interaction protein quantitation /detection sexually transmitted diseases vesicle /vacuole western blottings yeast two hybrid system

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项目摘要

DESCRIPTION (provided by applicant): Oculogenital disease induced by the sexually transmitted pathogen Chlamydia trachomatis manifests as a range of acute and chronic sequelae that represent a significant source of morbidity in humans. Chlamydiae are obligate intracellular pathogens that develop within a membrane-bound vacuole termed an inclusion. Although inclusions represent a privileged niche, sequestered chlamydiae possess the ability to modulate host-cell functions in order to create and maintain a permissive growth environment. Chlamydiae express a type III secretion system that, in other gram-negative pathogens, is a secretion mechanism strongly associated with pathogenesis. I have identified two chlamydial type III substrates and propose to identify specific interactions of these substrates with host molecules and delineate the consequences of those interactions. Using yeast two-hybrid assays, I propose to identify specific interactions between chlamydial effectors and host proteins. These elucidated in vitro interactions will be confirmed in chlamydial infections using a series of crosslinking and coprecipitation assays in combination with mass spectroscopy. The consequences of these interactions will be investigated in vitro by ectopic expression of effector proteins followed by biochemical confirmation of detected effects in infected cells. The chlamydial type III secretion system represents an attractive, yet unexplored, mechanism to achieve modulation of host cell activities. By specifically elucidating which host pathways are targeted I will gain insight into i) molecular mechanisms employed by Chlamydia to sculpt their intracellular environment and ii) what host cellular processes are important to chlamydial survival.

描述（由申请人提供）：性传播的病原体沙丘瘤引起的眼源性疾病表现为一系列急性和慢性后遗症，代表了人类发病的重要来源。衣原体是在膜结合的液泡中发育的务实的细胞内病原体，称为包容性。尽管夹杂物代表了特权的利基市场，但隔离的衣原体具有调节宿主 - 细胞功能以创建和维持宽松的增长环境的能力。衣原体表达了III型分泌系统，在其他革兰氏阴性病原体中，它是与发病机理密切相关的分泌机制。我已经确定了两种衣原体III型底物，并提出要确定这些底物与宿主分子的特定相互作用，并描述这些相互作用的后果。我建议使用酵母两种杂交测定法，以确定衣原体效应子和宿主蛋白之间的特定相互作用。这些阐明的体外相互作用将在衣原体感染中使用一系列交联和共沉淀测定法与质谱相结合。这些相互作用的后果将在体外通过异位表达效应蛋白的异位表达，然后对感染细胞中检测到的作用进行生化确认。衣原体III型分泌系统代表了一种有吸引力但未开发的机制，可以调节宿主细胞活性。通过明确阐明哪种宿主途径的目标，我将深入了解i）衣原体用来雕刻其细胞内环境的分子机制； ii）ii）哪些宿主细胞过程对衣原体生存至关重要。