MODULATION OF PULMONARY EPITHELIAL PARACELLULAR PERMEABILITY BY PNEUMOLYSIN FROM STREPTOCOCCUS PNEUMONIAE

Pneumolysin (PLY), a key virulence factor of Streptococcus pneumoniae (S. pneumoniae), disrupts airway epithelial barriers, yet its cellular and molecular mechanisms remain poorly understood. We aim to elucidate recombinant pneumolysin (rPLY)’s effects on Calu-3 bronchial epithelial barrier integrity and underlying pathways. This study investigates the effects of non-lytic and lytic PLY concentrations (5, 15, 30 µg/mL) to test the hypothesis that PLY increases epithelial paracellular permeability by affecting the anatomy and physiology of epithelial tight junctions. To address the hypothesis that PLY increases paracellular permeability through disruption of tight junction integrity in Calu-3 cells in a time- and dose-dependent manner, genomic DNA was isolated from virulent S. pneumoniae strain D39, and specific regions were amplified using PCR. The amplified DNA was ligated into the pET28b plasmid, and Escherichia coli (E. coli) BL21 cells were transformed to support the recombinant vector and produce a viable pneumolysin product. The PLY protein was purified via FPLC-NGC and further purified via the De-Salting column. Analysis by SDS-PAGE revealed a final product of ~53 kDa, corresponding to the molecular weight of the natural pneumolysin. The non- lethal concentration of the purified recombinant protein was determined via LDH assay. Confluent monolayers of Calu-3 cell line were cultured on porous membrane inserts and used to investigate PLY's impact on epithelial paracellular permeability. Transepithelial electrical resistance (TEER) was used to evaluate the integrity of epithelial junctional complexes. The TEER data revealed a significant reduction in resistance post-PLY exposure with various concentrations of PLY (5, 15, and 30 µg/mL) for up to 50 minutes. TEER data indicated a significant decrease in epithelial resistance at 50 minutes for the highest concentration (Triplicate inserts, 4 independent experiments, t-test, p< 0.01), indicating a compromised barrier integrity. Fluorescence microscopy images indicated subtle alterations in the expression and localization of the tight junction protein occludin. In conclusion, our data revealed the potential mechanism of S. pneumoniae-induced pulmonary injury at the cellular level.