ER-Chlamydia Inclusion Membrane Contact Sites

描述（由申请人提供）：沙眼衣原体是一种引起巨大公共卫生关注的革兰氏阴性细菌病原体。眼血清型可导致沙眼，而生殖器血清型是发达国家细菌性传播疾病的主要原因。尽管实施了沙眼原体筛查方案，单剂量阿奇霉素治疗沙眼和无并发症的性传播衣原体感染也有效，但病例率并未下降，再感染率却在上升。如果说我们对沙眼衣原体靶向的细胞过程的了解在过去10年里有了很大的提高，那么我们才刚刚开始确定细菌发育所需的宿主和细菌因子。这种知识的缺乏是由于衣原体是专性细胞内病原体，可用的遗传工具有限。我们通过使用RNAi方法识别衣原体感染所需的宿主因子，为该领域做出了贡献。此外，根据最近描述的沙眼衣原体转化方法，我们开发了一种多功能的沙眼衣原体基因表达克隆载体。我们的遗传研究鉴定出CERT，一种参与神经酰胺非囊性运输的蛋白质，是沙眼衣原体生长所需的一个因子。我们进一步发现，CERT对包涵体的招募与靠近包涵膜的ER小管的招募相关。此外，我们发现沙眼衣原体包涵膜蛋白IncD是CERT的特异性结合伙伴。总之，这些结果使我们提出了沙眼衣原体与内质网建立直接膜接触位点并利用非囊泡运输机制的概念。这项新的R01应用的目标是进一步表征内质网包涵MCSs在沙眼衣原体感染过程中的结构和功能。我们的假设是，特定的沙眼衣原体和宿主因子定位于er包涵体MCSs，并创造一个专门的微环境，介导细菌获取必需的营养物质，如脂质。为了验证我们的假设，我们提出确定鞘磷脂合成是否发生在ER-包涵MCSs (Aim1)，表征ER-包涵MCSs （Aim2）的沙眼衣原体成分，以及表征ER-包涵MCSs （Aim3）的细胞成分。在目的1、2和3的结论中，我们将获得关于ER-Inclusion MCSs形成和功能的大量信息。我们认为，这些ER-Inclusion MCSs通过有效获取脂质等营养物质，在沙眼衣原体发育周期中发挥重要作用。我们的方法将识别定位于这些MCSs的细菌和宿主因子，从而帮助我们更好地了解ER-Inclusion MCSs在分子水平上的功能。这将进一步加深我们对感染过程中涉及的分子机制的理解，并可能揭示药物靶点，以促进预防、治疗和控制衣原体感染工具的转化研究开发。

DESCRIPTION (provided by applicant): Chlamydia trachomatis is a gram-negative bacterial pathogen of tremendous public health concern. Ocular serovars lead to trachoma and genital serovars are the leading cause of bacterial sexually transmitted disease in developed countries. Despite the implementation of C. trachomatis screening programs and the effectiveness of a single-dose of azithromycin to treat trachoma and uncomplicated sexually transmitted chlamydial infection, case rates are not declining and reinfection rates are increasing. If our knowledge of the cellular processes targeted by C. trachomatis has greatly increased over the past 10 years, we have only begun to identify the host and bacterial factors required for bacterial development. This paucity of knowledge is due to the fact that Chlamydia are obligate intracellular pathogens with limited genetic tools available. We have contributed to the field through the identification of host factors required for Chlamydia infection using the RNAi methodology. Moreover, in the light of the recently described C. trachomatis transformation method, we have developed a versatile cloning vector for gene expression in C. trachomatis. Our genetic investigations led to the identification of CERT, a protein involved in the non-vesicular trafficking of ceramide, as a factor required for C. trachomatis growth. We further showed that CERT recruitment to the inclusion correlated with the recruitment of ER tubules in close proximity of the inclusion membrane. Moreover, we identified the C. trachomatis inclusion membrane protein IncD as a specific binding partner for CERT. Altogether, these results led us to propose the notion that C. trachomatis establishes direct membrane contact sites with the ER and exploits non-vesicular transport machinery The goal of this new R01 application is to further characterize the structure and function of ER-Inclusion MCSs during C. trachomatis infection. Our hypothesis is that specific C. trachomatis and host factors localize to ER-Inclusion MCSs and create a specialized microenvironment that mediate the bacterial acquisition of essential nutrients, such as lipids. To test our hypothesis, we propose to determine whether sphingomyelin synthesis occurs at ER- Inclusion MCSs (Aim1), to characterize the C. trachomatis components of ER-Inclusion MCSs (Aim2) and to characterize the cellular components of ER-Inclusion MCSs (Aim3). At the conclusion of Aim1, 2 and 3, we will have gained a considerable amount of information on the formation and function of ER-Inclusion MCSs. We believe that these ER-Inclusion MCSs play an important role during C. trachomatis developmental cycle through the efficient acquisition of nutrients such as lipids. Our approach will identify both bacterial and host factors that localize to these MCSs and therefore help us better understand the function of ER-Inclusion MCSs at the molecular level. This will further our understanding of the molecular mechanisms involved in the infection process and may reveal drug targets to facilitate the translational research development of tools to prevent, treat and control Chlamydia infection.