ER-Chlamydia Inclusion Membrane Contact Sites

ER-衣原体包涵体膜接触位点

• 批准号: 8629920
• 负责人: ISABELLE DERRE
• 金额: $ 41.63万
• 依托单位: YALE UNIVERSITY
• 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/8629920
• 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; 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; public health relevance; 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(申请人提供)：沙眼衣原体是一种极具公共卫生意义的革兰氏阴性细菌。在发达国家，眼部血清型导致沙眼，生殖器血清型是细菌性传播疾病的主要原因。尽管实施了沙眼衣原体筛查计划，单剂阿奇霉素治疗沙眼和无并发症的性传播衣原体感染有效，但病例比率并未下降，再感染率正在上升。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10年里有了很大的增长，那么我们才刚刚开始识别细菌发育所需的宿主和细菌因素。这种知识的匮乏是由于衣原体是细胞内的专性病原体，可用的遗传工具有限。我们通过使用RNAi方法确定衣原体感染所需的宿主因素，为这一领域做出了贡献。此外，根据最近描述的沙眼衣原体转化方法，我们开发了一个通用的沙眼衣原体基因表达克隆载体。我们的遗传学研究发现CERT是沙眼衣原体生长所必需的一种蛋白质，参与神经酰胺的非囊泡运输。我们进一步表明，CERT对包涵体的募集与内质网小管在包涵膜附近的募集相关。此外，我们还确定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的膜接触部位并利用非囊泡运输机制的概念。这一新的R01应用的目标是进一步表征沙眼衣原体感染过程中内质网包涵体MCSs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内质网包涵体MCSs，并创建一个专门的微环境，介导细菌获得必要的营养，如脂质。为了验证我们的假设，我们建议确定内质网包含的MCSs(Aim1)是否发生鞘磷脂合成，表征ER包含型MCSs的沙眼衣原体成分(AIM2)和表征ER包含型MCSs的细胞成分(Aim3)。在Aim1、2和3的结论中，我们将获得关于内质网包涵体MCSs的形成和功能的大量信息。我们认为，这些内质网包涵体MCS通过有效地获取脂质等营养物质，在沙眼衣原体发育周期中发挥着重要作用。我们的方法将识别定位于这些MCSs的细菌和宿主因素，从而帮助我们在分子水平上更好地了解内质网包涵体MCSs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的工具的翻译研究开发。