Abstract
Meningococcal meningitis is a severe central nervous system infection that occurs when Neisseria meningitidis (Nm) penetrates brain endothelial cells (BECs) of the meningeal blood-cerebrospinal fluid barrier. As a human-specific pathogen, in vivo models are greatly limited and pose a significant challenge. In vitro cell models have been developed, however, most lack critical BEC phenotypes limiting their usefulness. Human BECs generated from induced pluripotent stem cells (iPSCs) retain BEC properties and offer the prospect of modeling the human-specific Nm interaction with BECs. Here, we exploit iPSC-BECs as a novel cellular model to study Nm host-pathogen interactions, and provide an overview of host responses to Nm infection. Using iPSC-BECs, we first confirmed that multiple Nm strains and mutants follow similar phenotypes to previously described models. The recruitment of the recently published pilus adhesin receptor CD147 underneath meningococcal microcolonies could be verified in iPSC-BECs. Nm was also observed to significantly increase the expression of pro-inflammatory and neutrophil-specific chemokines IL6, CXCL1, CXCL2, CXCL8, and CCL20, and the secretion of IFN-γ and RANTES. For the first time, we directly observe that Nm disrupts the three tight junction proteins ZO-1, Occludin, and Claudin-5, which become frayed and/or discontinuous in BECs upon Nm challenge. In accordance with tight junction loss, a sharp loss in trans-endothelial electrical resistance, and an increase in sodium fluorescein permeability and in bacterial transmigration, was observed. Finally, we established RNA-Seq of sorted, infected iPSC-BECs, providing expression data of Nm-responsive host genes. Altogether, this model provides novel insights into Nm pathogenesis, including an impact of Nm on barrier properties and tight junction complexes, and suggests that the paracellular route may contribute to Nm traversal of BECs.
Highlights
Neisseria meningitidis or meningococcus (Nm) is a Gramnegative human-exclusive pathogen that asymptomatically colonizes the upper respiratory tract of 10–40% of the world population (Rouphael and Stephens, 2012)
No significant difference in invasion could be observed in a pilus-deficient pilE mutant (Supplementary Figure 2B), we found that the pilusoverexpressing pilT mutant is hyper invasive in induced pluripotent stem cells (iPSCs)-brain endothelial cells (BECs) compared to invasion rates of the WT strain, suggesting a role for the pilus during infection (Figure 1D)
These results demonstrate that various strains and mutants of Nm can interact with iPSC-BECs following similar patterns and phenotypes to previously described models, and that iPSC-BECs possess an important receptor for virulence
Summary
Neisseria meningitidis or meningococcus (Nm) is a Gramnegative human-exclusive pathogen that asymptomatically colonizes the upper respiratory tract of 10–40% of the world population (Rouphael and Stephens, 2012). Meningitis is a disease that is still associated with high mortality despite administration of modern antibiotic therapy (Rouphael and Stephens, 2012). Binding of type IV pili to host receptor CD147 triggers the formation of highly ordered CD147/β2-adrenergic receptor clusters assembled by the scaffolding protein α-actinin-4 (Maïssa et al, 2017). The activation of this receptor promotes the recruitment of the polarity complex to the bacterial adhesion site together with adherens and tight junction proteins, leading to the opening of a paracellular route that may facilitate bacterial passage (Coureuil et al, 2009; Bernard et al, 2014). The primary transit route of BECs by Nm has yet to be determined, highlighting the need for further investigation
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