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

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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.