Figure 1

Pneumolysin induces cell DNA damage and cell lysis.

(A) Lysate of pneumococcal protoplast induces DNA damage in alveolar epithelial cells. S. pneumoniae serotypes 19F, 3 and 4, grown in bacteria media, are re-suspended in cell wall digestion buffer to isolate the protoplasts, which are then physically lysed by Bead beater in F12-K culture media. The pneumococcal lysates are then incubated with alveolar epithelial cells for 7 h and analyzed for γH2AX. Representative images of three independent experiments showing γH2AX (red) and nuclei (blue). (B,C) Pneumolysin induces double strand breaks (DSBs) in alveolar epithelial cells. (B) Recombinant pneumolysin was incubated with alveolar epithelial cells at 0.1 μg/ml and 1 μg/ml concentration for 12 h and analyzed for γH2AX and 53BP1. Representative images showing DSBs in alveolar epithelial cells with nuclei (blue), γH2AX (red), 53BP1 (green) and colocalized foci (yellow). (C) Alveolar epithelial cells exposed to pneumolysin were analyzed for γH2AX and 53BP1 at 4, 8, 12, 24 and 48 h of incubation. γH2AX and 53BP1 positive cells (≥5 foci per nucleus) were quantified and expressed as percentage positive cells. Cytotoxicity was quantified by using LDH assay and expressed as percent of total LDH release during lysis by 1% Triton X-100. (D) Representative images showing DSBs in alveolar epithelial cells with nuclei (blue), γH2AX (red), MDC1 (green) and colocalized foci (yellow). ‘Media’ indicates negative control without any pneumolysin. Results show mean ± SEM for three independent experiments.