Recombinant expression of type IV pilins supports new model of pilus assembly

Abstract

Type IV pili are essential virulence factors for many pathogenic bacteria including Neisseria gonorrhoeae. Their functions include: twitching motility, biofilm formation, DNA uptake, and host cell adhesion. Several proteins at the tip of the fiber – namely PilI, PilJ, PilK, and PilC – form an initiation complex that is essential to efficient pilus formation. The exact mechanism and configuration by which these proteins assemble into the fiber has previously been unknown. We have developed a new method for recombinant expression of type IV pilin proteins. By engineering a signal peptidase I sequence into the ?-helical spine of the pilins, we are able to purify them from the periplasm without the use of detergents that could disrupt protein-protein interactions. Using this method we were able to identify an obligate heterodimer formed by PilI and PilJ. Additionally, we were able to detect PilK and the C terminal half of PilC forming a complex. These results combined with alphafold modeling and cell based assays support a model where the extreme C terminus of PilC binds to PilK as the first step of pilus assembly. This PilC-PilK dimer is then able to bind to the PilI-PilJ dimer, forming the capping complex which primes pilus extension.