Pneumolysin induced mitochondrial dysfunction leads to release of mitochondrial DNA

Streptococcus pneumoniae (S.pn.) is the most common bacterial pathogen causing community acquired pneumonia. The pore-forming toxin pneumolysin (PLY) is the major virulence factor of S.pn. and supposed to affect alveolar epithelial cells thereby activating the immune system by liberation of danger-associated molecular patterns (DAMP). To test this hypothesis, we established a novel live-cell imaging based assay to analyse mitochondrial function and associated release of mitochondrial DNA (mtDNA) as DAMP in real-time. We first revealed that bacterially released PLY caused significant changes of the cellular ATP homeostasis and led to morphologic alterations of mitochondria in human alveolar epithelial cells in vitro and, by use of spectral live-tissue imaging, in human alveoli. This was accompanied by strong mitochondrial calcium influx and loss of mitochondrial membrane potential resulting in opening of the mitochondrial permeability transition pore and mtDNA release without activation of intrinsic apoptosis. Moreover, our data indicate cellular mtDNA liberation via microvesicles, which may contribute to S.pn. related pro-inflammatory immune activation in the human alveolar compartment.


IMS-mEOS plasmid construction
mEOS targeted to the mitochondrial inner membrane space (IMS) was generated by amplifying the region coding for the IMS sequence (comprising bp 1-147 of Smac-1) from the plasmid pBabe-puro-IMS-RP (Addgene plasmid #24535) using the primers Next, the MitoTracker channel was spatially processed using a Gaussian filter with a radius of 1, followed by applying a linear contrast stretch and binarization using Otsu method in Fiji/ImageJ. Using this mask, the intensity in the Syto-82 channel was measured in the mitochondrial area.
If indicated, images were deconvolved using Huygens ® Essential 15.10 (Scientific Volume Imaging, Hilversum, NL) and 3D-stacks are displayed as maximum intensity projections (MIPs) and adjusted for brightness and contrast in Zen or ImageJ/Fiji.

Quantification of mitochondrial morphology and motility
Quantification of mitochondrial morphology was done as described previously 1 . In brief, 4-5 randomly chosen field of views containing 3-5 cells were imaged resulting in 14-18 analysed cells per independent experiment. Maximum intensity projections of SIM 3D stacks in stochastically selected mitochondria-rich parts of the cells were skeletonized following thresholding using the Huang method and using the "Skeletonize 2D/3D" plugin in Fiji/ImageJ. The resulting vectorised mitochondrial skeleton was used to identify and count/measure branches, branch points and end points.
For quantification of mitochondrial motility, images were collected directly before and 5 min after stimulation with PLY in one randomly chosen field of view containing 14-29 cells per independent experiment. Images were acquired every 3.13 seconds over a total imaging time of 2.04 min at a resolution of 0.132 µm/pixel (1024 × 1024 pixels).
Tracks with a duration less than 0.5 min were excluded.

Quantification of anti-DNA staining in mitochondria
Quantification of anti-DNA staining was performed using Imaris software (v8.1.2, Bitplane AG, Zürich, Switzerland) in 3 randomly chosen areas containing approx. 20 cells per experiment. First, a mask was generated based on the DAPI signal and applied to the anti-DNA channel to exclude the nuclear DNA signal. Next, the mitochondrial volume was calculated based on the MitoTracker signal using an appropriate intensity thresholding. All volumes smaller than 0.1 µm 3 were excluded.
Finally, the fluorescence intensity within the mitochondrial volume in the masked anti-DNA channel was quantified.

Subcellular fractionation and western blotting
Cytosolic and mitochondrial fractions were generated using the Qproteome

Supplementary Note: Visualization of mtDNA in living alveolar cells by Syto82
Opening of the mitochondrial permeability transition pore (mPTP) is associated with potential release of mtDNA fragments 2 , which may promote immune activation. In order to visualize mtDNA in real-time, we established a novel live-cell imaging assay based on Syto82, a nucleic acid dye belonging to the dye Syto ® family.
It has been demonstrated that some members of the Syto ® dye family of fluorescent nucleic acid dyes can incorporate into mitochondria 3 Fig. 3D). Therefore, Syto82 staining is a sensitive and accurate new staining method for quantifying mtDNA in live-cell imaging.
The ability of Syto82 to stain mtDNA may relate to its cationic charge, which may allow preferential accumulation of the dye within mitochondria in a similar manner to ethidium bromide 6 . To further validate Syto-82 staining we used PicoGreen. Staining of mtDNA by this dye has been suggested to be unaffected by the mitochondrial membrane potential once the dye is in the mitochondria 7  MitoTrackerOrange are pseudocolored in cyan, caspase-3/7 is shown in magenta, and autofluorescence is shown in grey. In addition PLY was stained after fixation with antibodies in green. Scale bar, 5 µm.