Molecular mechanism of Type 3 protein targeting and secretion

Type III secretion (T3S) is a specialized protein export and delivery system for bacterial protein toxins. These effectors are directly injected in the cytoplasm of host eukaryotic cells, through a dedicated nanosyringe, the injectisome, that spans two bacterial and one eukaryotic membrane. T3SS is one of the most complex protein secretion systems known. To become functional, up to 40 different proteins, in hundreds of copies, interact and assemble in a hierarchical manner. The role of the ATPase complex/C-ring in sorting needs to be clarified. The primary energy source for T3SS secretion is thought to derive from the proton motive force across the inner membrane [16], while ATP hydrolysis is believed to dissociate chaperone/secretory protein complexes [17-20]. Nonetheless, the exact energizing mechanism required for protein translocation remains elusive. Trying to map protein interactions and determining affinities in solution using membrane protein domains and secretory protein fragments resulted in no interactions detected or 30-fold lower affinities compared to the ones probed using membrane vesicles with functional injectisomes [11, 13]. The lack of purified and reconstituted translocase is a critical obstacle. Monitoring protein translocation in vitro, is a major step towards improving our understanding of the fundamental aspects of T3SS function and the molecular basis of the system’s regulation. The complex and dynamic nature of interactions between T3SS components requires novel approaches to dissect the order of events during secretion. The proposed research combines molecular bacteriology, biochemistry, cutting-edge structural dynamics methods, such as single molecule FRET (smFRET) and Hydrogen-deuterium Exchange Mass Spectrometry (HDX-MS), to decode the role of conformational dynamics of injectisome components, with high resolution structural analysis, in vitro and in vivo functional assays and reconstitution of the protein release mechanism