Streptococcus pneumoniae (the pneumococcus) causes systemic infections in humans. Pneumococci produce several pathogenicity factors such as the cytotoxic pore-forming toxin pneumolysin, which mediates vascular injury during pneumococcal infection.
Here, we present a novel cell culture infection technique, which we used to determine the impact of pneumococci and of purified pneumolysin on endothelial cell migration, cell morphology and bacterial attachment during infection. For the so- called chamber separation cell migration assay (CSMA), we used specific inserts that ensured a defined cell-free area in the monolayer of primary endothelial cells (HUVEC). The CSMA enabled time courses of live cell infection studies with pneumococci. Additionally, differential immunofluorescence staining followed by confocal microscopy visualizes the effects of bacterial cell attachment. Incubation of HUVEC with wild-type pneumococci significantly reduced the velocity of cell migration and significantly inhibited endothelial gap closure. Similar results were determined by employing purified pneumolysin protein, which identifies this cytotoxin as a potent inhibitor of endothelial cell migration. With the aim to analyse the effect of pneumococcal infection on endothelial wound healing under physiological flow conditions, we combined the CSMA with a microfluidic pump system, which enables the application of defined shear stress values, thereby mimicking the vascular blood flow. Equally as in infection under static conditions, circulating pneumococci prevented endothelial gap closure at a defined shear stress of 10 dyn/cm2.
Concluding, the novel combined in vitro infection technique provides valuable insight into the effect of pneumococci on endothelial cell migration and on barrier integrity.