Conserved C-terminal tails link PilC1 and PilC2 to the Kingella kingae type IV pilus through β-strand augmentation with the core minor pilin complex

is a gram-negative bacterium that is a leading cause of septic arthritis, osteomyelitis, and a variety of other invasive diseases in young children. Infection begins with colonization of the oropharynx, which depends on type IV pili (T4P), surface appendages that promote adherence, twitching motility, and natural competence. These T4P functions are mediated by two pilus-associated proteins called PilC1 and PilC2, which are members of the PilC/PilY family of proteins. In this study, we used AlphaFold Multimer modeling to identify a conserved C-terminal tail in both PilC1 and PilC2 that is predicted to insert into a hydrophobic pocket in the FimT-PilV-PilW-PilX-PilE core minor pilin complex via β-strand augmentation. Truncation of the final 10 residues of PilC1 or PilC2 significantly impaired pilus biogenesis, adherence to epithelial cells, twitching motility, and natural transformation without altering PilC1 or PilC2 protein expression. Point mutations targeting conserved residues within the PilC2 C-terminal tail had little impact on pilus biogenesis but resulted in a marked reduction in adherence and natural transformation, as well as the elimination of twitching motility. AlphaFold modeling predicted that these conserved residues interact with a β-sheet in the globular domain of the minor pilin PilX. Consistent with this model, site-directed mutagenesis of the PilX β-strand residues predicted to contact PilC2 largely phenocopied the PilC2 C-terminal tail point mutations. Together, these findings establish that the C-terminal tail in PilC/PilY adhesins is a conserved adapter that links these adhesins to T4P through the core minor pilin complex, identifying a potential target for anti-adhesion therapeutic strategies. IMPORTANCE: produces PilC/PilY family proteins called PilC1 and PilC2, and expression of at least one of these proteins is necessary for the production of functional T4P. Using molecular modeling and mutagenesis approaches, we established that a short peptide at the C-terminal end of these proteins, which is conserved among PilC/PilY family members, is critical for PilC1 and PilC2 functions and mediates these functions via β-strand augmentation with a specific T4P-associated protein complex.