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

许多影响农作物、牲畜和人类的细菌病原体利用分泌系统向宿主传递强大的毒力因子。其中最普遍的是三型分泌系统(T3SS)。T3SS由作为转录调节因子、伴侣和效应器的蛋白质以及被称为T3SA的注射器状纳米机器组成，T3SA在与宿主细胞接触时转移效应器。后一种现象是许多人类肠道病原体(如沙门氏菌、肠结肠耶尔森氏菌、致病性大肠杆菌和志贺氏菌)潜在毒力的关键因素，这些病原体每年共同导致全球数亿例病例。尽管它很重要，但T3SA功能的许多基本方面仍然很大程度上是未知的。事实上，T3SA组装和功能机制的详细动态视图非常缺乏，无法有效地利用这种结构知识来开发新的抗感染药物。人们对抗生素耐药性和全球变暖导致的传染病死灰复燃或蔓延的担忧，突显了开展这些研究工作的重要性。目前的研究方案旨在通过开发尖端方法来监测T3SA活性，并以志贺氏菌为模型揭示T3SA内部工作的动态性质，从而成为这些研究的一部分。我们将使用各种发光、荧光和标记分析来实时监测T3SA的活性，这些分析将利用志贺氏菌T3SA的遗传和结构数据。例如，我们将建立一种基于酶的原位生物素标记策略，以绘制在非活性T3SA与活性T3SA的出口装置形成的蛋白质-蛋白质复合体的图谱。这些数据将被利用来获得T3SA机制的动态视图。为此，Förster Resonance Energy Transfer(FRET)分析将被设计为实时监控T3SA活性，并为识别导致T3SA激活和失活的分子线索铺平道路。在我们使用基于荧光转录的分泌活性报告程序(荧光TSAR)监测T3SA活性方面的最新进展的基础上，我们将识别与T3SA组装有关的T3SS无关蛋白，并开发一种专门用于筛选T3SA抑制剂的遗传和小分子文库的发光分析方法。由于针对T3SA活性的药理作用是目前用于治疗这些细菌感染的抗生素疗法的一种有前途的替代品，我们的基础研究活动将出现许多应用。在这一资助期将达到的里程碑和由此产生的高影响力的出版物将为我的研究计划提供坚实的基础。参与培训的学员将获得为公共和私营部门不同职业量身定做的技术和智力技能。

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.