Polymicrobial extracellular vesicles reduce the innate immune response of human cystic fibrosis bronchial epithelial cells

Adults with cystic fibrosis (CF) have chronic antibiotic-resistant polymicrobial lung infections, the leading cause of death in CF. We developed a polymicrobial culture model containing four genera that represents a ‘pulmotype’ detected in ~34% of lung infections in people with CF (pwCF), and accounts for 27% of the variability in lung function. This community, comprised of Pseudomonas aeruginosa, Staphylococcus aureus, Streptococcus sanguinis, and Prevotella melaninogenica, is grown in synthetic CF media (SCFM2) under anoxic conditions that mimic the environment in mucus plugs in CF. We have shown that Pseudomonas in monoculture communicates with primary human bronchial epithelial cells (pHBEC) by secreting bacterial extracellular vesicles (bEVs) that diffuse through mucus and deliver virulence factors, DNA, and RNA to pHBEC. We report herein that each bacterial genus in the polymicrobial community secretes bEVs containing proteins and RNAs predicted to promote the establishment of chronic infection by enhancing virulence and biofilm formation, and upregulating the stress response and pro-inflammatory pathways in pHBEC. This response is most pronounced in CF pHBEC. Trikafta, a highly effective drug, does not ameliorate the response or return it to WT levels. Bacterial EVs also inhibited Trikafta-stimulated CFTR Cl- currents by CF pHBEC. These studies provide insight into why Trikafta does not eliminate polymicrobial lung infections and a hyperinflammatory lung environment in pwCF.