Molecular determinants of Kingella kingae translocation across polarized respiratory epithelium

Kingella kingae is the leading cause of osteoarticular infections in children under 36 months of age. The pathogenesis of K. kingae disease begins with colonization of the oropharynx followed by translocation across the respiratory epithelium, entry into the bloodstream, and dissemination to distant sites. The bacterial factors that facilitate translocation across the respiratory barrier are poorly understood. In this study, we used polarized Calu-3 cells differentiated at an air-liquid interface (ALI) as a model of the respiratory epithelium combined with molecular genetics, barrier integrity measurements, cytotoxicity assays, and RNA sequencing to interrogate the molecular mechanism by which K. kingae breaches the respiratory epithelium. We found that the pore-forming RtxA toxin drives disruption of the epithelial barrier and is a major contributor to translocation. We observed that type IV pili (T4P) also contribute to translocation and mediate a toxin-independent translocation mechanism. Strains lacking both RtxA and T4P were unable to translocate, confirming that at least one or the other of these factors is required for translocation. Transcriptomic profiling of K. kingae identified 512 differentially expressed bacterial genes during infection and suggested a new role for HprK and a novel esterase in the process of translocation. This study establishes that multiple bacterial factors influence K. kingae translocation across the respiratory epithelial barrier and provides insight into important gene expression changes during K. kingae interaction with host cells.