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（由申请人提供）：沙眼衣原体是一种革兰氏阴性细菌病原体，对公共卫生的关注很大。眼部血清导致沙眼瘤，生殖器血清射手是发达国家细菌性传播疾病的主要原因。尽管实施了气管梭状芽胞庭筛查程序以及单剂量的阿奇霉素治疗沙眼瘤和简单的性传播衣原体感染的有效性，但病例率并没有下降，再感染率正在增加。如果我们对过去10年中沙眼齿状囊肿的细胞过程的了解大大增加，那么我们才开始确定细菌发育所需的宿主和细菌因素。知识的匮乏是由于衣原体具有较少的细胞内病原体，具有有限的遗传工具。我们通过使用RNAi方法来鉴定衣原体感染所需的宿主因素为领域做出了贡献。此外，鉴于最近描述的沙眼转化方法，我们开发了一种用于气管梭菌中基因表达的多功能克隆载体。我们的遗传研究导致了证书的鉴定，CERT是一种与神经酰胺的非腔贩运有关的蛋白质，是沙眼梭状芽孢杆菌生长所需的因素。我们进一步表明，募集的纳入与纳入膜的募集相关。此外，我们确定了沙眼包含膜蛋白蛋白INCD是CERT的特定结合伙伴。总的来说，这些结果使我们提出了这样一种观念：沙眼梭状芽孢杆菌与ER建立直接的膜接触位点并利用非敏感运输机械的目标是该新R01应用的目标是进一步表征甲状腺梭菌感染期间ER灌注MCSS的结构和功能。我们的假设是，特定的沙眼梭菌和宿主因素本地化为ER包含MCS，并创建了一种专门的微环境，可介导细菌的必需营养素（例如脂质）。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纳入MCSS（AIM1），以表征ER插入MCSS的沙眼成分（AIM2）（AIM2）并表征ER-灌注MCSS的细胞成分（AIM3）（AIM3）。在AIM1，2和3的结论结束时，我们将获得有关ER包含MCS的形成和功能的大量信息。我们认为，通过有效地获得脂质（例如脂质），这些ER含量MCS在沙眼发育周期中起着重要作用。我们的方法将确定将其定位于这些MCS的细菌和宿主因素，因此有助于我们更好地理解ER包含MCS在分子水平上的功能。这将进一步理解感染过程中涉及的分子机制，并可能揭示药物靶标，以促进工具的转化研究开发，以预防，治疗和控制衣原体感染。