Role of the PilC1 and PilC2 pilus-associated proteins in Kingella kingae pathogenicity - Kingella kingae is an invasive gram-negative pathogen that is a leading cause of bone and joint infections in young children, accounting for up to 88% of osteoarticular cases in children <4 years old. In addition, K. kingae is an important cause of invasive bloodstream infections in young children. Complications of osteoarticular infections in children include abnormal bone growth, decreased joint mobility, unstable joint articulation, and chronic joint dislocation, with residual skeletal dysfunction in 10-25% of cases. Complications of invasive bloodstream infections include multi-organ injury and mortality. Roughly 25% of K. kingae isolates possess β-lactamase activity, and many of these isolates are resistant to other antibiotics as well, raising concern about approaches to treatment in the future and underscoring the need for novel therapeutics. The pathogenesis of K. kingae disease begins with colonization of the oropharynx, followed by invasion of the bloodstream and spread to bones, joints, and other sites. We have established that K. kingae produces type IV pili (T4P), which play a critical role in adherence to epithelial cells and extracellular matrix proteins and augment the RtxA toxin in promoting efficient translocation across polarized epithelial monolayers, important steps in colonization and invasion of the bloodstream. We have discovered that K. kingae T4P pili contain pilus-associated adhesins called PilC1 and PilC2, which promote pilus biogenesis and mediate adherence and twitching motility. PilC1 and PilC2 share some structural homology but have little amino acid homology and possess different binding and twitching motility properties. Based on mass spectrometry analysis of purified T4P, homology analysis, and structural modeling, we have found that K. kingae T4P also contain multiple minor pilins, including the FimT, PilV, PilW, PilX, and PilE minor pilins encoded by the fimTpilVWXE locus. Bacterial 2-hybrid results and AlphaFold3 predictions suggest that PilC1 and PilC2 form a heterodimer and interact with a minor pilin complex. In this proposal, deploying high- resolution electron and fluorescent microscopy, biochemical techniques, and molecular and cell biological methods, we will elucidate the physical relationship of PilC1 and PilC2 with each other, characterize the physical relationship of PilC1-PilC2 and with the pilus fiber, and define the roles of PilC1 and PilC2 in K. kingae trafficking across polarized respiratory epithelial cells. The proposed studies will provide insights into the design of novel therapeutics against K. kingae and potentially other pathogens that produce T4P and may also shed light on other closely related systems such as the type II secretion system in gram-negative bacteria, the competence system for DNA uptake in gram-positive bacteria, and flagella in archaea.
