Deciphering the Regulation and Functional Mechanisms of the Type Three Secretion Apparatus

许多影响农作物、牲畜和人类的细菌性病原体利用分泌系统向宿主传递强毒因子。其中最普遍的是第三型分泌系统（T3SS）。T3SS由充当转录调节因子、伴侣和效应剂的蛋白质和一种被称为三型分泌装置（T3SA）的类似注射器的纳米机器组成，它在接触宿主细胞时转移效应剂。后一种现象是许多人类肠道病原体（如沙门氏菌、肠结耶尔森菌、肠致病性大肠杆菌和志贺氏菌）潜在毒力的关键因素，这些病原体每年在全世界总共造成数亿例病例。尽管它很重要，但T3SA功能的许多基本方面在很大程度上仍然未知。事实上，T3SA组装和功能机制的详细动态视图是最迫切缺乏的，以有效地利用这种结构知识，以开发新的抗感染药物。由于全球变暖，对抗生素耐药性和传染病死灰复燃或蔓延的担忧日益增加，这凸显了开展这些研究工作的重要性。目前的研究计划旨在通过开发尖端方法来监测T3SA的活性，并以志贺氏菌为模型，揭示T3SA内部工作的动态性质，从而成为其中的一部分。我们将利用志贺氏菌T3SA的遗传和结构数据，利用各种发光、荧光和标记法实时监测T3SA活性。例如，我们将建立一种基于原位酶的生物素标记策略，以绘制在失活T3SA与活性T3SA出口装置形成的蛋白-蛋白复合物。这些数据将被用来获得T3SA机制的动态视图。为此，Förster共振能量转移（FRET）检测将被设计用于实时监测T3SA活性，并为识别导致T3SA激活和失活的分子线索铺平道路。基于我们最近在使用基于转录的荧光分泌活性报告（fluoTSAR）监测T3SA活性方面的进展，我们将鉴定与T3SA组装相关的t3ss无关蛋白，并开发一种专为筛选T3SA抑制剂的遗传和小分子文库而设计的发光试验。由于药物靶向T3SA活性代表了目前用于治疗这些细菌感染的抗生素治疗的有希望的替代品，我们的基础研究活动将出现许多应用。在此资助期内将达到的里程碑以及由此产生的高影响力出版物将为我的研究计划提供坚实的基础。受训者将获得专门为公共和私营部门各种不同职业量身定制的技术和智力技能。

Many bacterial pathogens affecting crops, livestock and humans use secretion systems to deliver potent virulence factors to their host. One of the most prevalent is the Type Three Secretion System (T3SS). T3SS are constituted of proteins acting as transcription regulators, chaperones and effectors, and of a syringe-like nanomachine known as the Type Three Secretion Apparatus (T3SA), which transfer effectors upon contacting host cells. The latter phenomenon is the key element of the virulence potential of many human enteropathogens (e.g. Salmonella spp., Yersinia enterecolitica, enteropathogenic Escherichia coli and Shigella spp.), which are collectively responsible for hundreds of millions of cases worldwide each year. Despite its importance, many basic aspects of T3SA function are still largely unknown. Indeed, a detailed dynamic view of T3SA assembly and functional mechanisms is most acutely lacking to efficiently exploit this structural knowledge in order to develop novel anti-infectious agents. Raising concerns of antibiotic resistance and infectious diseases resurgence or spreading due to global warming underline the importance of conducting these research efforts. The current research proposal is aiming to be part of these by developing cutting-edge methods to monitor T3SA activity and shedding light on the dynamic nature of T3SA inner workings, using Shigella as a model. We will monitor T3SA activity in real-time using a variety of luminescent, fluorescent and tagging assays that will take advantage of genetic and structural data about Shigella T3SA. For example, we will establish an in situ enzyme-based biotin labeling strategy to map protein-protein complexes that are formed at the export apparatus of inactive versus active T3SA. This data will be harnessed to obtain a dynamic view of T3SA mechanisms. To do this, Förster Resonance Energy Transfer (FRET) assays will be designed to monitor T3SA activity in real-time and pave the way to the identification of molecular cues leading to T3SA activation and inactivation. Building on our recent progress in monitoring T3SA activity using the fluorescent Transcription-based Secretion Activity Reporter (fluoTSAR), we will identify T3SS-unrelated proteins implicated in T3SA assembly and develop a luminescent assay tailored to screen genetic and small-molecules libraries for T3SA inhibitors. Since pharmacologically targeting T3SA activity represents a promising replacement for antibiotic therapy currently used to treat these bacterial infections, numerous applications will emerge from our fundamental research activities. Milestones that will be reached in this funding period and high impact publications stemming from it will provide a solid foundation for my research program. Trainees implicated will acquire technical and intellectual skills tailored for a wealth of different career in the public and private sectors.