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

许多影响作物、牲畜和人类的细菌病原体利用分泌系统将强毒力因子传递给其宿主。其中最常见的是三型分泌系统（T3 SS）。T3 SS由充当转录调节剂、伴侣和效应物的蛋白质以及被称为三型分泌装置（T3 SA）的类螺旋桨纳米机器组成，其在接触宿主细胞时转移效应物。后一种现象是许多人类肠道病原体（例如沙门氏菌属，肠结肠耶尔森氏菌（Yersinia enterecolitica）、肠致病性大肠杆菌（Escherichia coli）和志贺氏菌（Shigella spp.），这些组织每年在全世界范围内造成数亿起病例。尽管它的重要性，T3 SA功能的许多基本方面仍然是未知的。事实上，T3 SA组装和功能机制的详细动态视图是最严重缺乏的，以有效地利用这种结构知识，以开发新的抗感染剂。由于全球变暖，人们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，这突出了开展这些研究工作的重要性。目前的研究提案旨在通过开发尖端方法来监测T3 SA活性，并以志贺氏菌为模型，揭示T3 SA内部工作的动态性质。我们将利用志贺氏菌T3 SA的遗传和结构数据，使用各种发光、荧光和标记检测方法实时监测T3 SA的活性。例如，我们将建立一个原位酶为基础的生物素标记策略，以映射蛋白质-蛋白质复合物，形成在出口装置的非活性与活性T3 SA。这些数据将被用来获得T3 SA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T3 SA活性，并为识别导致T3 SA激活和失活的分子线索铺平道路。基于我们最近在使用荧光基于转录的分泌活性报告基因（fluoTSAR）监测T3 SA活性方面的进展，我们将鉴定T3 SA组装中涉及的T3 SS无关蛋白，并开发一种荧光检测方法，用于筛选T3 SA抑制剂的遗传和小分子文库。由于靶向T3 SA活性的抗生素是目前用于治疗这些细菌感染的抗生素疗法的有希望的替代品，因此我们的基础研究活动将产生许多应用。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。参与培训的学员将获得专为公共和私营部门的各种职业而量身定制的技术和知识技能。**

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. **