Multi-functional minor pilins coordinate type IV pilus assembly, adherence, motility, and DNA uptake in the pediatric pathogen <i>Kingella kingae</i>

Type IV pili (T4P) are dynamic surface appendages that mediate adherence, motility, and DNA uptake in <i>Kingella kingae</i>, an important pediatric pathogen that causes osteoarticular infections, bacteremia, and endocarditis. While the major pilin subunit in <i>K. kingae</i> 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 <i>K. kingae</i>. 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 <i>fimTpilVWXE</i> 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 <i>K. kingae</i> type IV pilus, with distinct minor pilins localizing to different sites on the pilus fiber and mediating specialized functions essential for virulence.IMPORTANCE<i>Kingella kingae</i> is an emerging pediatric pathogen and a leading cause of osteoarticular infections in children 6 months to 4 years of age. To establish infection, <i>K. kingae</i> relies on T4P, dynamic surface structures that mediate host cell adherence, motility, and DNA uptake. T4P are expressed by a wide range of bacterial pathogens beyond <i>K. kingae</i>, including <i>Pseudomonas aeruginosa</i>, <i>Neisseria gonorrhoeae</i>, <i>Neisseria meningitidis</i>, and <i>Legionella pneumophila</i>, among others. The type IV pilus is composed of pilin subunits, including a major pilin that displays significant antigenic diversity and low-abundance minor pilins that are highly conserved. This study demonstrates the importance of eight minor pilins in <i>K. kingae</i> virulence properties. Given the conservation of minor pilins across diverse bacterial species, targeting minor pilin complexes may provide a foundation for a new class of broad-spectrum antivirulence therapies that prevent bacterial colonization and disease.