Stem cell-derived brain-like endothelial cells to interrogate <i>Streptococcus pneumoniae</i> interaction with brain endothelium

<i>Streptococcus pneumoniae</i> (pneumococcus) is an opportunistic pathogen that remains the leading cause of bacterial meningitis worldwide. For meningitis to occur, pneumococcus must breach the blood-brain barrier (BBB), a highly specialized network of brain endothelial cells that comprise the microvasculature of the brain. Here, we report the use of human induced pluripotent stem cell-derived brain-like endothelial cells (iBECs) to model the BBB during pneumococcal infection. iBECs were infected with the <i>S. pneumoniae</i> strain TIGR4. Adherence assays showed that pneumococcal adherence to iBECs was a saturable process. Moreover, deletion of two pneumococcal adhesins resulted in an adherence defect, supporting a receptor-mediated interaction between pneumococcus and iBECs. Next, the integrity of several tight junction components was assessed via western blot and RT-qPCR, revealing the loss of abundance and expression in iBECs during infection with pneumococcus. Simultaneously, the expression of <i>VEGFA</i> and the tight junction repressor <i>SNAI1</i> was upregulated. Semi-automated analysis of junction images also demonstrated a loss of ZO-1 and occludin continuity during pneumococcal infection. Consistent with these findings, the loss of TEER and the increase in barrier permeability were observed in pneumococcus-infected iBECs. The toxin pneumolysin (Ply) was important for this disruption, as the loss of Ply in pneumococcus partially arrested the reduction of TEER and the increase in permeability. Finally, RT-qPCR showed that pneumococcus was sufficient to upregulate a panel of inflammatory cytokines in iBECs. Taken together, these findings show that pneumococcus interacts with and disrupts iBECs during infection, supporting iBECs as an important model for studying pneumococcus-BBB interactions.