Abstract
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.
Date of publication
Fall 2025
Document Type
Thesis
Language
english
Persistent identifier
http://hdl.handle.net/10950/4906
Committee members
Dr. Ali Azghani, Dr. Brent Bill, Dr. Matthew Greenwold, Dr. Andrey Komissarov
Degree
Master of Science in Biology
Recommended Citation
Obe, Feranmi Emmanuel, "Disruption of Pulmonary Epithelial Barrier Function by Pneumolysin from Streptococcus pneumoniae" (2025). Biology Theses. Paper 92.
http://hdl.handle.net/10950/4906