Abstract
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.
Highlights
Streptococcus pneumoniae (S.pn.) is the most common bacterial pathogen causing community acquired pneumonia
Exceeding the mitochondrial capacity can result into loss of those as well as opening of the Ca2+-sensitive mitochondrial permeability transition pore, organelle swelling, and release of pro-apoptotic factors such as cytochrome C or apoptosis inducing factor (AIF)[11,12]
The resulting cellular events after [Ca2+]c overload are not uniform on the mitochondrion and depend on the biological stimulus. It remains to be elucidated if PLY induced [Ca2+]c influx has the potential to alter [Ca2+]m subsequently influencing ΔΨm, ATP generation as well as mitochondrial permeability transition pore (mPTP) associated release of mitochondrial DNA (mtDNA). Clarification of these mechanisms is of pivotal interest for the understanding of S.pn. induced immune activation since increasing evidence suggests that either cytosolically or micro-environmentally released mtDNA may be recognized by pattern recognition receptors (PRRs) as a DAMP15
Summary
Streptococcus pneumoniae (S.pn.) is the most common bacterial pathogen causing community acquired pneumonia. These roles render mitochondria to central organelles for the control of innate immune responses as well as cellular fate in the alveolar compartment and we investigated whether and how PLY affects mitochondrial function in human lung tissue and cultured pulmonary epithelial cells. The presence of PLY showed massive [Ca2+]m influx, opening of the mPTP and, by support of a novel live-cell based assay, mtDNA release independent of mitochondrial caspase activation.
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