Fig. 2: S. pneumoniae actively modifies cells following primary infection.
A Supernatant, wash, and A549 cells were collected at time α (1 h) post 1° and 2° infection with MOI 35 for CFU counts, n = 5 biological replicates, lines are the mean ± SEM and statistical significance was calculated by Mann-Whitney test, ns = not significant, two-tailed **p = 0.0079. B A549 cells were collected at time α (3 h) after 1° and 2° (MOI 20) for CFU counts, n = 5 biological replicates, lines are the mean ± SEM and statistical significance was calculated by two-tailed Mann-Whitney test, ns = not significant, *p = 0,0159 (1°/2°), *p = 0,0238 (2°/2°post inactived Spn). C A549 cell metabolic activity measured by alamarBlue assay. Cells treated at time α (3 h post infection, MOI 20), 1 measure of fluorescence/hour for 12 hours. Results are expressed as resorufin fluorescence intensity. Plot shows mean ± SEM from n = 4 biological replicates. Statistical significance was determined by two-way ANOVA with FDR Benjamini-Hochberg correction for multiple comparisons Q = 0.05 for 1° (green round) versus 2° infection (round purple) ns = not significant, *p = 0.0145, ***p = 0.0004, ****p = 0.0001 and 1° (green round) versus 2°§ infection (§Spn inactived at 1° infection then Spn live at 2°, triangle pink) ns = not significant, *p = 0.0311 and 0.0170, **p = 0.0021. D Quantification of LAMP1 and colocalization with lysotracker® at time α (3 h, round) and time β (24 h, triangle) for UI, 1°, PI and 2° conditions in the A549 cells. Plots show quantifications from 60 to 75 cells by conditions in the same experiment, mean fluorescence intensity of Regions of Interest (ROI) from n = between 6000 and 22,000 lysosomes total (Fig. S2.B) ± SEM, statistical significance was determined by two-tailed Mann–Whitney test, ns = not significant, *p = 0.0357. On the right, representative immunofluorescence images by confocal microscopy of A549 cells stained with LAMP1 (GFP; green), LysoTracker® (Deep Red; red), and DAPI (gray) at time α (3 h) and β (24 h) after 1° and 2° infection. Scale bar = 20 μm. Source data are provided as a Source Data file for A–D.
