Bacterial anti-inflammatory lipid mediators

项目摘要 先天性免疫系统对感染的快速反应依赖于病原体相关免疫缺陷的检测。 分子模式（PAMPs），如脂多糖，激活强烈的炎症反应， 入侵生物我的研究项目集中在一个不受重视的对策部署， 减弱对PAMP的免疫应答的肠道细菌和病原体。油酸水合酶（OhyA） 是一种在大肠杆菌和重要的人类病原体葡萄球菌中表达的酶 金黄色。OhyA立体特异性地水合不饱和脂肪酸的9-顺式双键以产生10-顺式双键。 然而，表达OhyA的细菌不合成不饱和脂肪酸。 OhyA底物仅在宿主组织的界面处遇到，在那里不饱和脂肪酸是 丰富。我推测，由细菌或病原体产生的hFAs作为信号传导， 减弱对PAMP的先天免疫反应的分子。事实上，S。aureus ohyA敲除不能 在小鼠中建立感染，说明OhyA和hFAs对S.金黄色葡萄球菌毒力因此，hFA， 这是首次在肠道微生物组中发现，可能提供了一个重要的机制， 细菌，以减轻胃肠道炎症和创造宽容，共生环境。我的飞行员 实验和一些先前的报道为hFA的抗炎作用提供了初步证据， 但hFA的免疫抑制机制尚不清楚。为了弥补这一缺陷，我将 通过课程作业和在保罗博士实验室的实践培训接受正式的免疫学指导 托马斯，一个专家免疫学家，在K99阶段。我们将确定hFAs是否直接拮抗 细胞外PAMP结合免疫细胞受体，结合T细胞受体的CD 1脂质呈递分子 识别，以拮抗T细胞活化，或阻断PAMP-受体活化下游的信号传导， 刺激G蛋白偶联受体和/或PAPR抑制剂。这些实验的结果将揭示 hFA修饰免疫细胞应答的机制基础。OhyA没有哺乳动物同源物， 结合OhyA的X射线结构，验证OhyA对毒力的重要性将提供关键的 确定OhyA是否是抗微生物药物发现的候选靶标所需的信息。期间 在R 00阶段，我将检验OhyA被包装到由S. 金黄色葡萄球菌与周围的宿主组织相互作用，在感染部位周围形成hFA介质的光环。我 这些发现将为OhyA如何结合和影响膜提供原子机制的理解 结构以促进囊泡形成。K99/R 00研究计划旨在利用我的专业知识， 生物化学和结构生物学，同时接受免疫学的新培训，以促进理解 细菌的免疫调节，并开始一个富有成效和成功的职业生涯，我将领导一项研究， 计划阐明细菌脂质产物在控制宿主反应中的作用。

Project Summary The rapid response of the innate immune system to infections relies on the detection of pathogen-associated molecular patterns (PAMPs), such as lipopolysaccharides, that activate a robust inflammatory response to invading organisms. My research program focuses on an unappreciated countermeasure deployed by commensal bacteria and pathogens that attenuates the immune response to PAMPs. Oleate hydratase (OhyA) is an enzyme that is expressed in commensal bacteria and the important human pathogen Staphylococcus aureus. OhyA stereospecifically hydrates the 9-cis double bond of unsaturated fatty acids to produce 10- hydroxy-fatty acids (hFAs); however, bacteria expressing OhyA do not synthesize unsaturated fatty acids. OhyA substrates are only encountered at the interface of host tissues, where unsaturated fatty acids are abundant. I hypothesize that hFAs produced by commensal or pathogenic organisms serve as signaling molecules that blunt the innate immune response to PAMPs. Indeed, S. aureus ohyA knockouts fail to establish infections in mice, illustrating the importance of OhyA and hFAs to S. aureus virulence. Thus, hFAs, which were first discovered in the gut microbiome, may provide an important mechanism for commensal bacteria to attenuate gastrointestinal inflammation and create tolerant, symbiotic environments. My pilot experiments and some previous reports provide preliminary evidence for the anti-inflammatory action of hFAs, but the mechanism(s) of immune suppression by hFAs are unknown. To address this shortcoming, I will receive formal immunology instruction through coursework and hands-on training in the laboratory of Dr. Paul Thomas, an expert immunologist, during the K99 phase. We will determine whether hFAs directly antagonize extracellular PAMP binding to immune cell receptors, bind to CD1 lipid-presenting molecules for T-cell receptor recognition to antagonize T cell activation, or intercept signaling downstream of PAMP-receptor activation by stimulating G-protein coupled receptors and/or PAPR. The results of these experiments will uncover the mechanistic basis for hFA modification of immune cell responses. OhyA has no mammalian homolog, and validating the importance of OhyA for virulence coupled with the X-ray structures of OhyA will provide the key information required to determine whether OhyA is a candidate target for antimicrobial drug discovery. During the R00 phase, I will test the hypothesis that OhyA is packaged into extracellular vesicles released by S. aureus to interact with surrounding host tissues to form a halo of hFA mediators around the infection site. My findings will provide an atomistic mechanistic understanding of how OhyA binds to and influences membrane structure to promote vesicle formation. The K99/R00 research plan is designed to leverage my expertise in biochemistry and structural biology, while receiving new training in immunology, to advance the understanding of immune regulation by bacteria and launch a productive and successful career in which I will lead a research program to elucidate the role bacteria lipid products play in controlling host responses.