ER-Chlamydia Inclusion Membrane Contact Sites

ER-衣原体包涵体膜接触位点

• 批准号: 9064073
• 负责人: ISABELLE DERRE
• 金额: $ 39.19万
• 依托单位: UNIVERSITY OF VIRGINIA
• 依托单位国家: 美国
• 财政年份: 2014
• 资助国家: 美国
• 起止时间: 2014-06-15 至 2019-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9064073
• 关键词: Azithromycin; Bacterial Sexually Transmitted Diseases; Binding; Cell membrane; Cell physiology; Cells; Ceramides; Chlamydia; Chlamydia Infections; Chlamydia trachomatis; Cloning Vectors; Developed Countries; Development; Dose; Drug Targeting; Foundations; Funding; Gene Expression; Genetic; Genital system; Goals; Golgi Apparatus; Grant; Growth; Health; Infection; Integration Host Factors; Investigation; Kinetics; Knowledge; Laboratories; Lead; Lipids; Mediating; Membrane; Membrane Proteins; Methodology; Methods; Molecular; Nutrient; Pathogenesis; Pathway interactions; Phosphorylation; Play; Process; Program Effectiveness; Proteins; Public Health; RNA Interference; Role; Route; STIM1 gene; Sexually Transmitted Diseases; Site; Sphingomyelins; Structure; Testing; Trachoma; Translational Research; Vesicle; design; insight; novel; novel therapeutics; pathogen; prevent; research and development; research study; screening; sphingomyelin synthase; tool; trafficking

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.

描述（由申请人提供）：沙眼衣原体是一种革兰氏阴性细菌病原体，引起极大的公共卫生关注。眼部血清型导致沙眼，而生殖器血清型是发达国家细菌性性传播疾病的主要原因。尽管实施了沙眼衣原体筛查计划以及单剂阿奇霉素治疗沙眼和单纯性性传播衣原体感染的有效性，但病例率并未下降，再感染率仍在增加。如果说我们对沙眼衣原体所针对的细胞过程的了解在过去十年中已经大大增加，那么我们才刚刚开始识别细菌发育所需的宿主和细菌因子。这种知识的匮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 方法鉴定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，我们开发了一种用于沙眼衣原体基因表达的多功能克隆载体。我们的基因研究发现了 CERT（一种参与神经酰胺非囊泡运输的蛋白质），它是沙眼衣原体生长所需的因子。我们进一步表明，CERT 对包涵体的招募与紧邻包涵体膜的 ER 小管的招募相关。此外，我们确定了沙眼衣原体包涵膜蛋白 IncD 作为 CERT 的特异性结合伴侣。总而言之，这些结果使我们提出这样的观点：沙眼衣原体与 ER 建立直接膜接触位点并利用非囊泡运输机制。这一新的 R01 应用的目标是进一步表征 ER-Inclusion MCS 的结构和功能沙眼衣原体感染。我们的假设是，特定的沙眼衣原体和宿主因子定位于 ER-Inclusion MCS，并创造一个特殊的微环境，介导细菌获取必需的营养物质，如脂质。为了检验我们的假设，我们建议确定鞘磷脂合成是否发生在 ER-包含 MCS (Aim1)、表征 ER-包含 MCS 的沙眼衣原体成分 (Aim2) 以及表征 ER-包含 MCS 的细胞成分 (Aim3) ）。完成目标 1、2 和 3 后，我们将获得有关 ER-Inclusion MCS 的形成和功能的大量信息。我们相信，这些 ER-Inclusion MCS 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 MCS 的细菌和宿主因子，从而帮助我们在分子水平上更好地了解 ER 包含 MCS 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。