Functional analysis of the flagellar type III secretion apparatus in Salmonella enterica serovar Typhimurium

The ability of many bacteria to swim trough liquids is not only essential to access more favorable environments but is also crucial for many pathogens to establish a successful infection. Swimming motility is dependent on a macromolecular propeller, the flagellum. Flagellum synthesis requires the translocation of thousands of flagellar subunits across the bacterial cell envelope. This export process depends on a complex nanomachine located at the base of the flagellum, the flagellar Type III secretion system (fT3SS). This apparatus is similar to the virulence associated T3SS (vT3SS) used by Gramm-negative pathogens to inject toxins into the host cell. The fT3SS is composed of six cytoplasmic and seven membrane proteins previously described as essential for export. FliGMN and FliHIJ form the cytoplasmic ring and the flagellar specific ATPase complex, respectively. FlhBA and FliOPQR form the export gate surrounded by the MS-ring made of FliF. Except for FliO, the membrane components are highly conserved among species, and have clear homologs in the vT3SS. Despite intense study on the function and the synthesis of the flagellum, the molecular details of protein export via the fT3SS is poorly understood. It remains unclear which proteins are essential for flagellar substrate export and the function of several of them is unknown. In this thesis, we contributed to untangle the mechanism underlying flagellar Type III secretion. In the first part, we reconstituted a minimal T3SS apparatus, only composed of proteins required and sufficient for export. We uncoupled export of flagellar substrate from synthesis, to measure export in a plasmid- and a chromosomally-encoded minimal T3SS. We determined that FlhBA, FliF and FliPQR are essential and that FliO is only dispensable in presence of excess FliP. In the second part, we investigated the link between FliO and FliP. We demonstrated that FliO prevents Lon-mediated proteolysis of FliP and that FliO facilitates the formations of FliP complexes required for flagellar assembly. We observed that FliO is not part of the completed flagellum. We propose that FliO acts as a specific chaperone for flagellar assembly. In the last part, we examined the role of the cleavable signal peptide of FliP and the effect of its cleavage on motility and flagellar synthesis. We observed that the presence of the signal peptide enhances FliP expression but is not crucial for motility, while cleavage is required for flagellar assembly.