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，具有巨大的公共卫生问题。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C.随着沙眼筛查计划的实施以及阿奇霉素单剂量治疗沙眼和单纯性传播衣原体感染的有效性的提高，病例率没有下降，而再感染率却在上升。如果我们对C.虽然沙眼的感染率在过去10年中大大增加，但我们才刚刚开始确定细菌发展所需的宿主和细菌因素。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C.沙眼衣原体转化方法，我们已经开发了一种通用的克隆载体，用于在沙眼衣原体中表达基因。沙眼我们的遗传学研究导致了CERT的鉴定，CERT是一种参与神经酰胺非囊泡运输的蛋白质，是C。沙眼生长。我们进一步表明，CERT募集到包涵体与ER小管的募集紧密接近包涵体膜。此外，我们还鉴定了C.沙眼衣原体包涵体膜蛋白IncD作为CERT的特异性结合伴侣。总之，这些结果使我们提出了C。沙眼衣原体建立与ER的直接膜接触位点，并利用非囊泡转运机制。这种新的R 01应用的目的是进一步表征沙眼衣原体过程中ER-包涵体MCS的结构和功能。沙眼感染我们的假设是，特定的C。沙眼衣原体和宿主因子定位于ER-包涵体MCS，并产生介导细菌获得必需营养素（如脂质）的专门微环境。为了验证我们的假设，我们建议确定鞘磷脂合成是否发生在ER-包涵体MCSs（Aim 1），以表征C。本发明的目的是为了鉴定ER-包涵体MCS的沙眼组分（Aim 2）并表征ER-包涵体MCS的细胞组分（Aim 3）。在目标1、2和3的结论中，我们将获得关于ER-包涵体MCS的形成和功能的大量信息。我们认为这些ER-包含MCSs在C.沙眼衣原体通过有效地获取营养物质如脂质来完成发育周期。我们的方法将识别定位于这些MCS的细菌和宿主因子，从而帮助我们更好地理解ER-包涵体MCS在分子水平上的功能。这将进一步加深我们对感染过程中所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转化研究开发。