Deciphering the assembly pathway of type IV pili in Myxococcus xanthus

Type IV pili (T4P) are hairlike surface structures, present on a variety of different bacteria. They are polymers involved in diverse functions such as motility, adherence, protein secretion, DNA uptake and in many pathogens they are found to be the primary colonization factor. Especially their role in virulence makes T4P particularly relevant for studying pilus function and assembly. The T4P machinery consists of 12 conserved proteins building an envelope-spanning macromolecular machinery, which localizes polarly in Myxococcus xanthus. Although most of the proteins have been known and studied for a long time, the precise mechanism of how and in which order the individual components are assembled to generate a macromolecular machinery remain largely unknown. Here we uncovered a sequential, outside-in assembly pathway starting with the outer membrane (OM) PilQ secretin, and proceeding inwards over the periplasm and inner membrane (IM) to the cytoplasm. Specifically, by taking advantage of the cell biology tools for studying T4P in M. xanthus, we carried out one of the largest screens comprising 11 of the 12 proteins of the T4P machinery by systematically profiling the stability and localization of T4P proteins in the absence of each individual other T4P protein in combination with mapping direct protein-protein interactions. Using these approaches, we show that assembly of the T4P machinery initiates with the formation of the PilQ secretin ring, assisted by its pilotin Tgl, in the OM. Oligomeric PilQ serves as an assembly platform for further T4P components. PilQ recruits TsaP, a peptidoglycan binding protein, as well as PilP by direct interactions with PilP. PilP, in turn, recruits the IM proteins PilN and PilO. PilP/PilO/PilN likely make up a complex aligning IM and OM components of the T4P machinery. The PilP/PilO/PilN complex recruits cytoplasmic PilM by direct interaction between PilN and PilM and recruits PilC, presumably by direct interaction between PilC and PilO. Finally, the ATPases PilB and PilT that power extension and retraction of T4P, localize independently of other T4P machinery proteins. In this study, we elucidate the assembly process and functional interactions between T4P proteins. This work lays the basis for further understanding of these functionally highly versatile surface structures. Interestingly, the assembly of the type II and III secretion systems also initiates from the OM secretin and proceeds inwards. Thus, an outside-in assembly pathway is emerging as a conserved feature in secretin-containing trans-envelope export machines