Analysis of type III secreted proteins of C. trachomatis

描述（由申请方提供）：性传播病原体沙眼衣原体诱导的眼生殖器疾病表现为一系列急性和慢性后遗症，是人类发病的重要来源。衣原体是专性细胞内病原体，在称为包涵体的膜结合空泡内发育。虽然内含物代表了一个特殊的生态位，但隔离的衣原体具有调节宿主细胞功能的能力，以创造和维持一个允许的生长环境。衣原体表达一种III型分泌系统，在其他革兰氏阴性病原体中，该分泌系统是一种与发病机制密切相关的分泌机制。我已经确定了两个衣原体III型基板，并建议确定这些基板与宿主分子的具体相互作用，并描绘这些相互作用的后果。使用酵母双杂交检测，我建议确定衣原体效应和宿主蛋白质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试验结合质谱法进行确认。这些相互作用的后果将通过效应蛋白的异位表达进行体外研究，然后对感染细胞中检测到的效应进行生化确认。衣原体III型分泌系统代表了一个有吸引力的，但尚未开发的，机制，以实现宿主细胞活动的调制。通过具体阐明哪些宿主途径是靶向的，我将深入了解i）衣原体用于塑造其细胞内环境的分子机制，以及ii）哪些宿主细胞过程对衣原体存活很重要。

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.