Analysis of type III secreted proteins of C. trachomatis

描述（由申请人提供）：由性传播病原体沙眼衣原体引起的眼生殖器疾病表现为一系列急性和慢性后遗症，是人类发病率的重要来源。衣原体是专性细胞内病原体，在称为包涵体的膜结合液泡内发育。虽然包涵体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III型分泌系统，在其他革兰氏阴性病原体中，这是一种与发病密切相关的分泌机制。我已经确定了两种III型衣原体底物，并建议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。利用酵母双杂交试验，我建议鉴定衣原体效应物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得到证实，使用一系列交联和共沉淀试验结合质谱。这些相互作用的后果将在体外通过效应蛋白的异位表达进行研究，然后在感染细胞中对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，但尚未探索的机制，以实现宿主细胞活性的调节。通过明确阐明哪些宿主途径是目标，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.