The quorum sensing com system regulates pneumococcal colonisation and invasive disease in a pseudo-stratified airway tissue model

Abstract

AbstractStreptococcus pneumoniae (Spn) colonises respiratory epithelia but can also invade lung cells causing pneumonia. We developed an ex vivo model with human airway epithelial (HAE) cells harvested from lung biopsies to study Spn colonisation and translocation. Flow-cytometry, confocal imaging and electron microscopy studies identified the epithelial lineage with signs of differentiation (beating cilia, mucus, and tight junctions). HAE cells were challenged with Spn wild-type TIGR4 (wtSpn) or its isogenic ΔcomC quorum sensing-deficient mutant. ΔcomC mutant colonised significantly less than wtSpn at 6 h post-inoculation but at significantly higher levels at 19 h and 30 h. Translocation correlated inversely with colonisation density. Transepithelial electric resistance (TEER) decreased after pneumococcal infection and correlated with increased translocation for both strains.Confocal imaging illustrated colocalisation of intracellular Spn with both cilia and zonulin-1 and prominent microcolony formation with wtSpn but disintegration of microcolony structures over time with ΔcomC mutant. ΔcomC caused a more pronounced release of both zonulin-1 and lactate dehydrogenase into the supernatant at later time points than wtSpn, suggesting that cytotoxicity is likely not the mechanism leading to translocation. There was a density- and time-dependent increase of inflammatory cytokines from human HAE cells infected with ΔcomC compared with wtSpn, including increased levels of the NLRP3 inflammasome-related IL-18.In conclusion, our experiments indicate that ComC system allows a higher organisational level of population structure resulting in microcolony formation, increased early colonisation and subsequent translocation. We propose that ComC inactivation unleashes a very different and possibly more virulent phenotype that merits further investigation.