title: Large-scale study of the interactions between proteins involved in type IV pilus biology in Neisseria meningitidis: characterization of a subcomplex involved in pilus assembly.
abstract: The functionally versatile type IV pili (Tfp) are one of the most widespread virulence factors in bacteria. However, despite generating much research interest for decades, the molecular mechanisms underpinning the various aspects of Tfp biology remain poorly understood, mainly because of the complexity of the system. In the human pathogen Neisseria meningitidis for example, 23 proteins are dedicated to Tfp biology, 15 of which are essential for pilus biogenesis. One of the important gaps in our knowledge concerns the topology of this multiprotein machinery. Here we have used a bacterial two-hybrid system to identify and quantify the interactions between 11 Pil proteins from N. meningitidis. We identified 20 different binary interactions, many of which are novel. This represents the most complex interaction network between Pil proteins reported to date and indicates, among other things, that PilE, PilM, PilN and PilO, which are involved in pilus assembly, indeed interact. We focused our efforts on this subset of proteins and used a battery of assays to determine the membrane topology of PilN and PilO, map the interaction domains between PilE, PilM, PilN and PilO, and show that a widely conserved N-terminal motif in PilN is essential for both PilM-PilN interactions and pilus assembly. Finally, we show that PilP (another protein involved in pilus assembly) forms a complex with PilM, PilN and PilO. Taken together, these findings have numerous implications for understanding Tfp biology and provide a useful blueprint for future studies.
authors.affiliation: Section of Microbiology, Imperial College London, London, UK.
authors.forename: Michaella
authors.initials: M
authors.lastname: Georgiadou
authors.forename: Marta
authors.initials: M
authors.lastname: Castagnini
authors.forename: Gouzel
authors.initials: G
authors.lastname: Karimova
authors.forename: Daniel
authors.initials: D
authors.lastname: Ladant
authors.forename: Vladimir
authors.initials: V
authors.lastname: Pelicic
mesh_terms: D058947:Bacterial Secretion Systems; D010861:Fimbriae, Bacterial / Q000378:metabolism*; D046911:Macromolecular Substances / Q000378:metabolism; D026901:Membrane Transport Proteins / Q000378:metabolism*; D009345:Neisseria meningitidis / Q000378:metabolism / Q000502:physiology*; D011485:Protein Binding; D054730:Protein Interaction Domains and Motifs; D025941:Protein Interaction Mapping; D055503:Protein Multimerization; D020798:Two-Hybrid System Techniques
chemical_list: D058947:Bacterial Secretion Systems; D046911:Macromolecular Substances; D026901:Membrane Transport Proteins