Probing mechanism and outcome of Chlamydia trachomatis response to antimicrobial insults

沙眼衣原体对抗菌药物损伤反应的探讨机制和结果

• 批准号: 9808730
• 负责人: LI SHEN
• 金额: $ 22.05万
• 依托单位: LSU HEALTH SCIENCES CENTER
• 依托单位国家: 美国
• 财政年份: 2019
• 资助国家: 美国
• 起止时间: 2019-06-01 至 2021-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9808730
• 关键词: Acute; Area; Automobile Driving; Bacteria; Bacterial Infections; Biological Assay; Biology; Cell Communication; Cell Culture Techniques; Cell Separation; Cells; Cellular biology; Chlamydia; Chlamydia Infections; Chlamydia trachomatis; Chronic; Chronic Disease; Clinical; Data; Development; Ectopic Pregnancy; Ensure; Epithelial Cells; Excision; Exposure to; Female infertility; Foundations; Gene Expression Profile; Genes; Genetic; Grant; Human; Immune; Immunity; In Situ; In Vitro; Infection; Interferon Type II; Intrinsic factor; Knowledge; Life Style; Link; Measures; Metabolic; Metabolic Pathway; Metabolism; Methods; Modeling; Molecular; Molecular Biology; Outcome; Pathogenesis; Pelvic Inflammatory Disease; Pelvic Pain; Penicillins; Phenotype; Physiological; Population; Proteins; Publishing; Recovery; Regulator Genes; Reporter; Reporter Genes; Research Personnel; Role; Sexual Transmission; Signal Pathway; Stress; System; Techniques; Technology; Testing; Vacuole; Woman; Work; antimicrobial; antimicrobial drug; biological adaptation to stress; chronic infection; combat; fitness; innovation; insight; novel; novel therapeutics; promoter; protein expression; reproductive; reproductive tract; response; stress tolerance; success; tool; transcriptome sequencing; transcriptomics; treatment strategy; tubal infertility

Abstract An obligate intracellular bacterium, Chlamydia trachomatis (Ct), is the leading sexually transmitted bacterial infection worldwide. Chronic Ct infection leads to serious reproductive complications in women, including pelvic inflammatory disease, pelvic pain, tubal infertility, and ectopic pregnancy. Recent compelling molecular data support historical studies showing that Ct can establish chronic infections even with repeated antimicrobial treatments. Yet, the mechanisms underpinning Ct adaptation, survival, and persistence during this onslaught and host immune-imposed antimicrobial insults are unknown. To evaluate antimicrobial responses of intracellular bacteria, it is desirable to establish quantitative relations between the fitness of growing or persisting bacteria and the target host cells. These important relations are challenging to study during Chlamydia infection due to the current limitations in techniques. To overcome this barrier, we have created a robust endogenous promoter- fluorescent protein reporter system. Building on our recent success in probing the central aspect of a unique Ct developmental cycle in situ, we hypothesize that Ct adapts to, and modulates, host cell metabolic pathways to maintain viability during exposure to external and host immune-imposed antimicrobial insults. To test this hypothesis, we propose two Aims: 1. To develop a novel in situ method using fluorescent protein expression levels to quantify interchangeable Ct developmental cycle in live cells. This powerful tool will allow us to quantitatively probe divergent chlamydial forms in a single Ct-containing vacuole and in a culture composed of physiologically and phenotypically different cells; and 2. To characterize key signaling pathways, in Ct and its host cells, that are vital to Ct adaptation and survival, in the presence of antimicrobial insults. Our innovative studies will be achieved using advanced technologies, including gene reporter assays, florescence activated cell sorting (FACS), and dual RNA sequencing (FACS-dual-RNAseq). Greater understanding of how Ct adapts, survives, and persists in the presence of host immunity or antimicrobial agents, as well as reactivates from persistence may provide important new insights into chlamydial pathogenesis. The findings from these studies will provide the foundation to develop new treatment strategies targeting both acute and the chronic, transmissible reservoirs of Ct infection.

摘要 一种专性细胞内细菌，沙眼衣原体（Ct），是主要的性传播细菌 感染全球。慢性Ct感染可导致女性严重的生殖并发症，包括盆腔 炎症性疾病、盆腔疼痛、输卵管不孕和异位妊娠。最近引人注目的分子数据 支持历史研究表明，即使反复使用抗菌药物，Ct也可以建立慢性感染， 治疗。然而，在这场冲击中，支持Ct适应、生存和持久性的机制 和宿主免疫施加的抗微生物损伤是未知的。评价细胞内的抗菌反应， 对于细菌，需要建立生长或持续存在的细菌的适合度之间的定量关系 和靶宿主细胞。这些重要的关系是具有挑战性的研究在衣原体感染，由于 目前技术上的局限性。为了克服这个障碍，我们创造了一个强大的内源性启动子- 荧光蛋白报告系统。在我们最近成功探索独特的Ct的中心方面的基础上， 在原位发育周期中，我们假设Ct适应并调节宿主细胞代谢途径， 在暴露于外部和宿主免疫施加的抗微生物损伤期间保持活力。为了验证这一 假设，我们提出两个目标：1。建立一种新的荧光蛋白原位表达方法 水平来量化活细胞中可互换的Ct发育周期。这个强大的工具将使我们能够 在单个含Ct的液泡和由以下组成的培养物中定量探测不同的衣原体形式 生理上和表型上不同的细胞;和2.为了表征关键信号通路，在Ct及其 宿主细胞，这是至关重要的Ct适应和生存，在存在的抗微生物损伤。我们的创新 研究将使用先进的技术，包括基因报告分析，荧光激活细胞， 分选（FACS）和双重RNA测序（FACS-dual-RNAseq）。更好地理解Ct如何适应， 在宿主免疫或抗微生物剂存在下存活并持续存在， 持久性可能为衣原体发病机制提供重要的新见解。这些研究的结果 将为开发针对急性和慢性的新治疗策略提供基础， Ct感染的可传播储库。