Figure 1
From: Pneumolysin induced mitochondrial dysfunction leads to release of mitochondrial DNA
Pneumolysin induces mitochondrial fragmentation and disrupts mitochondrial motility in epithelial cells and human lung tissue. (A) A549 cells were labelled with MitoTrackerOrange and subsequently infected with S.pn. D39Δcps or S.pn. D39ΔcpsΔply for 5 hours, stimulated with 1.0 µg/ml PLY for 15 min or left untreated. Cells were fixed, stained with DAPI and mitochondrial morphology was analysed by structured illumination microscopy. Mitochondria were pseudocoloured using YellowHot LUT and a reconstructed widefield image of the nucleus is shown in blue. Scale bar represents 5 µm. (B,C) Quantification of mitochondrial morphology in control and PLY treated cells exemplified in (A). The mitochondrial network was examined and quantified in stochastically selected mitochondria-rich parts of the cells. Integrative network/shape analysis (number of branches per mitochondrion (B) and branch length per mitochondrion (C)) was performed (mean ± SD from n = 3 independent experiments, *P < 0.05, one-tailed Mann-Whitney test). (D) Quantification of mitochondrial mean velocity in A549 cells immediately before and five minutes after stimulation with 1 µg/ml PLY (mean ± SD from n = 4 independent experiments, *P < 0.05, one-tailed Mann-Whitney test). (E) Human lung tissue was labelled with MitoTrackerOrange (cyan) and caspase-3/7 sensor (magenta) and was left untreated (ctrl, left), stimulated with 1.0 µg/ml PLY for 1 h (PLY, middle) or infected with S.pn. D39-GFP for 12 h (D39-GFP, right). The tissue was analysed by spectral confocal microscopy. Autofluorescence of collagen fibres is shown in grey. In addition GFP-pneumococci are shown in red (arrowheads) and PLY in green (open arrowheads). The asterisk indicates accumulation of caspase-3/7 sensor in the nucleus. Scale bars, 5 µm.
