Listeriolysin O and Pneumolysin: Effects on intracellular calcium homeostasis and epithelial barrier integrity
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Listeriolysin O (LLO) and pneumolysin (PLY) are two bacterial protein toxins from the family of cholesterol dependent cytolysins (CDC) that are produced by Listeria monocytogenes and Streptococcus pneumoniae, respectively. The toxins are found in all clinically relevant bacterial isolates, highlighting their importance for the virulence of these bacterial pathogens. Both LLO and PLY generate pores in the plasma membrane of host cells. High toxin concentrations directly lyse affected cells, while lower (sublytic) concentrations trigger an influx of extracellular calcium ions (Ca2+) into the cytoplasm. Ca2+ is an important intracellular second messenger. An increase in its cytoplasmic concentration ([Ca2+]i) results in the activation of signalling pathways. Both toxins were tested for their ability to create an imbalance in the Ca2+- homeostasis of epithelial cells at sublytic concentrations. It was shown that the toxins triggered a concentration-dependent increase in [Ca2+]i and caused the cells to shrink. This loss in cell surface area was dependent on the influx of extracellular Ca2+ , as it could be inhibited with LaCl3, an unspecific ion channel blocker. LLO variants harbouring single amino acid exchanges were compared to wild-type toxin to asses their functionality and gain further insights in the mechanism of pore formation. In infection experiments, it was determined that L. monocytogenes ability to invade closed epithelial monolayers was reduced by LaCl3 in a concentration-dependent manner. This implies that the LLO-induced increase in [Ca2+]i and the accompanying loss of intercellular connections are important for the bacteria to overcome epithelial barriers. Using PLY-treated epithelial cells, it was demonstrated that the toxin is able to empty intracellular Ca2+ stores independently of endogenous channels. No influx of extracellular Ca2+ was involved in this reaction, as these experiments were performed in Ca2+-free medium. Finally, the effects of PLY were compared to those of hyperosmotic conditions. It was shown that increased osmolarity and toxin administration had similar effects on the calcium homeostasis, intracellular Ca2+ stores and cell volume, which were accompanied by the translocation of the cell volume transcriptional regulator NFAT5. PLY-induced cell shrinking did not occur when PKC-alpha and MLC-phosphorylation were blocked. These inhibitors did not prevent the influx of extracellular Ca2+, suggesting that cellular signalling events are responsible for the loss in cell surface volume upon toxin treatment.Verknüpfung zu Publikationen oder weiteren Datensätzen
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Giessen: VVB Laufersweiler