Chlamydia Effector Proteins

描述（由申请人提供）：专性细胞内细菌病原体沙眼衣原体感染眼部和生殖上皮，引起具有重要临床意义的疾病。沙眼衣原体通过在宿主和包涵膜上转运“效应”蛋白来调节宿主细胞功能。缺乏对衣原体进行经典分子遗传分析的工具，阻碍了鉴定和表征效应蛋白的进展。然而，考虑到其基因组的小尺寸，基于表达的功能方法可以被带到基因组规模，并显著加快衣原体发病机制的研究。在此，我们建议开发功能基因组工具来鉴定和表征衣原体感染期间效应蛋白的输出动态。在我们的第一个目标中，我们建议建立全面的酵母和哺乳动物表达文库，系统地调查衣原体蛋白与它们的致病性相关的功能，并应用基于免疫学的筛选来鉴定新的分泌衣原体蛋白。这些发现将与衣原体初级体的蛋白质组学数据相结合，以确定和表征介导新生致病液泡进入和生物发生的细菌因子。在我们的第二个目标中，我们扩展了功能工具的应用，以表征衣原体III型分泌（TTS）系统，这是递送效应蛋白的主要门户。通过双杂交技术和生物化学方法，我们提出构建该祖先TTS装置的蛋白质-蛋白质相互作用图谱，以鉴定核心易位、针复合体和推定的调节亚基的非保守成分。特别是，我们将重点研究与TSS系统基础结构相互作用的衣原体特异性蛋白，包括我们已经鉴定的一种新的TTS伴侣及其同源货物。

DESCRIPTION (provided by applicant): The obligate intracellular bacterial pathogen Chlamydia trachomatis infects the ocular and genital epithelium to cause diseases of significant clinical importance. C. trachomatis modulates host cellular functions by translocating "effector" proteins across host and inclusion membranes. The lack of tools to perform classical molecular genetic analysis in Chlamydia, has hampered progress in identifying and characterizing effector proteins. However, given the small size of the its genome, expression-based functional approaches can be brought to genomic scale and significantly accelerate research in Chlamydia pathogenesis. Here, we propose to develop functional genomic tools to identify and characterize effector proteins and the dynamics of effector protein export during chlamydial infection. In our first aim, we propose to generate comprehensive yeast and mammalian expression libraries to systematically survey chlamydial proteins for functions related to their pathogenicity and apply immunological-based screens to identify novel secreted chlamydial proteins. These findings will be merged with proteomic data of Chlamydia elementary bodies to identify and characterize bacterial factors mediating entry and biogenesis of the nascent pathogenic vacuole. In our second aim, we extend the application of functional tools to characterize the chlamydial Type III secretion (TTS) system, the main portal for the delivery of effector proteins. Through two-hybrid technologies and biochemical approaches we propose to construct a protein-protein interaction map of this ancestral TTS apparatus to identify non-conserved components of the core translocon, needle complex and putative regulatory subunits. In particular, we will focus our studies on Chlamydia-specific proteins that interact with the basal structure of the TSS system, including a novel TTS chaperone we have identified and its cognate cargo. PUBLIC HEALTH RELEVANCE: The extent to which the widely disseminated pathogen bacteria Chlamydia trachomatis subverts immune responses is determined by virulence factors they inject into host cells ("effector" proteins). The proposed study will develop genomic tools to identify and characterize these effector proteins. In this manner we will significantly further our understanding of chlamydial pathogenesis and generate new targets for vaccine design and therapeutic intervention.