Neisseria meningitidis (Nm) can cause life-threatening meningitis after crossing the blood-brain barrier (BBB). The small lyso-phospholipid sphingosine 1-phosphate (S1P) is an important regulator of BBB integrity which is formed by sphingosine kinases (SphKs). The barrier function of the BBB is mainly exerted by brain endothelial cells (BECs). BECs express 3 types of S1P receptors (S1PR1-3), which, upon binding to S1P, can positively or negatively influence BBB permeability. We hypothesize that Nm manipulates S1P-S1PR signaling to favor uptake into BECs as a first step to cross the BBB.

We used LC-MS/MS to generate a time-resolved picture of sphingolipid metabolism and secretion after infecting BEC cell line hCMEC/D3 with Nm serogroup B strain MC58. qPCR in combination with phospho-specific western blots of BEC subcellular fractions was applied to determine transcriptional and (post-)translational regulation of S1P-metabolizing enzymes and receptors. SphK enzymatic activity assays were used to determine bacterial virulence factors as well as cellular interactors leading to an increase of S1P after Nm infection. Antibiotic killing assays and differential fluorescent staining detected with confocal microscopy were conducted to identify the role of S1P and S1P signaling during bacterial adherence and invasion. ELISA and qPCR experiments shed light on the role of S1P signaling during BEC inflammation.

We found that Nm infection of BECs induced continuous production and secretion of S1P. S1P production was facilitated by increased SphK enzymatic activity, which could be ascribed to neisserial type IV pilus' (Tfp) interaction with cellular surface receptor CD147. Activation appeared due to transcriptional upregulation and phospho-activation of isozyme SphK1. Phospho-activation of EGFR, a pre-requisite for Nm invasion, was dependent S1P on secretion. Moreover, we found that inhibiting S1P production or signaling over S1PR2 prevented uptake of Nm into BECs and reduced inflammatory cytokine expression and release.

Taken together, our results highlight the SphK-S1P-S1PR signaling axis as a potential target for adjuvant therapy during Nm meningitis.