Type IV pili (Tfp) are one of the most widespread virulence factors in prokaryotes. Their inherent capacity to mediate an astonishing array of functions differentiates them from other pili and contributes to the pathogenesis of many important human pathogens. Previous intensive efforts by our group in Neisseria meningitidis identified 23 proteins dedicated to Tfp biology, 15 of which are essential for pilus biogenesis. Though these proteins are widely accepted to exert their functions within a large multiprotein complex, the mechanisms governing the biogenesis and functionality of these organelles remained poorly defined. Consequently, the first objective of my project was to perform a large-scale analysis to identify fundamental interactions between 11 Pil proteins from N. meningitidis. To achieve this, we employed the bacterial adenylate cyclase two-hybrid system, which uncovered 20 different binary interactions, many of which are novel and represents the most complex interaction network between Pil proteins reported to date. Significantly, this study revealed that PilE, PilM, PilN and PilO involved in pilus assembly, indeed interact and provided us with a strong foundation to proceed to our main objective, which was to perform a detailed functional analysis of this poorly characterized subcomplex. Using a battery of assays we determined the membrane topology of PilN and PilO, mapped the interaction domains between PilE, PilM, PilN and PilO, and showed that a widely conserved N-terminal motif in PilN is essential for both PilM-PilN interactions and pilus assembly. Furthermore, we established by stability and co-immunoprecipitation studies that PilP (another protein involved in pilus assembly) forms a complex with PilM, PilN and PilO. Finally, we attempted to reconstitute a minimal Tfp assembly machinery in E. coli, however these efforts necessitate further improvements. Taken together, this study has shed light on the molecular mechanisms of Tfp biology and provides a useful blueprint for future studies.