Disruption of Pulmonary Epithelial Barrier Function by Pneumolysin from Streptococcus pneumoniae

Pneumolysin (Ply), a cholesterol-dependent pore-forming toxin produced by Streptococcus pneumoniae, compromises epithelial barrier integrity and facilitates dissemination of infection. we hypothesize that Ply disrupts tight junction integrity in Calu-3 monolayers by modulating occludin expression and its interactions with other junctional components, including ZO-1 and actin filaments. To test this hypothesis, we utilized confluent bronchial epithelial monolayers of Cancer Lung cell line, clone 3 (Calu-3) cultured on a porous membrane insert that separates the apical and basal domains of a 3-dimensional (3D) cell culture model. The monolayers were exposed apically to 5 µg/mL of in-house purified recombinant pneumolysin (rPly). Transepithelial electrical resistance (TEER) was used to monitor Calu-3 monolayers’ barrier integrity over time, and tight junction proteins localization and actin remodeling were examined with a fluorescence microscope. Sodium Dodecyl Sulfate–Polyacrylamide Gel Electrophoresis (SDS-PAGE) and Western blot analysis were used to quantify protein expression levels. We found that rPly (5 µg/mL) induced a significant drop in TEER within 0.5 hour, sustained up to 1 hour, with transient recovery at 5 hours and subsequent decline at 10 hours. Removal of rPly from the monolayers at 5 hours and subsequent incubation in fresh serum-free medium led to progressive TEER restoration over 24 hours. Immunofluorescence analysis at recovery time points revealed redistribution of occludin and Zonula Occluden (ZO-1) from the cell junction into the cytoplasm, stress fiber formation at 5-10 hours, and partial restoration of occludin and ZO-1 to the cell junction, alongside cortical actin reorganization at 24 hours. Western blot analysis confirmed decreased occludin expression after 1 and 5 hours of rPly exposure, with partial restoration after 24 hours post-toxin removal. These findings suggest that rPly-induced epithelial barrier disruption is reversible through junctional reorganization and cytoskeletal remodeling, highlighting potential therapeutic avenues to prevent exacerbated pneumococcal lung injury.