Kingella kingae KK03 Type IV Pili

Type IV Pili (T4P) are dynamic surface appendages that mediate adherence, motility, and DNA uptake in Kingella kingae, an important pediatric pathogen that causes osteoarticular infections, bacteremia, and endocarditis. While the major pilin subunit in K. kingae T4P is well characterized, the contribution of minor pilins to T4P structure and function remains unknown. Here, we used proteomics, molecular genetics, biochemical analyses, and structural modeling to identify and characterize all eight minor pilins in K. kingae. We identified a conserved operon of core minor pilin genes encoding FimT, PilV, PilW, PilX, and PilE that promotes surface piliation, adherence to epithelial cells, twitching motility, and natural transformation. Deletion of the fimTpilVWXE locus phenocopied loss of the PilC1 and PilC2 adhesins, and AlphaFold modeling combined with bacterial two-hybrid analysis suggested that FimT, PilV, PilW, and PilX form a complex at the pilus tip. The PilA2, ComP, and KK03_01180 minor pilins were dispensable for adherence and motility but promoted natural transformation and formed protein-protein interactions with the major pilin, suggesting that these proteins are incorporated throughout the pilus shaft. These findings support a new model for the architecture of the K. kingae type IV pilus, with distinct minor pilins localizing to different sites on the pilus fiber and mediating specialized functions essential for virulence.