Introduction

Neisseria meningitidis (Nm) is a human-specific pathogen that can interact with and penetrate brain endothelial cells (BECs) of the blood-brain barrier, and cause meningitis. Nm can modulate the sphingolipid metabolism in BECs and hijack the sphingolipid balance in the host to promote cellular invasion. We found that dihydroceramide (dhCer) levels are significantly increased during infection of BECs with Nm and therefore aimed to investigate their role in the interaction of Nm with BECs.

Methods

BECs (hCMEC/D3s) were infected with Nm and transcriptional regulation of enzymes of the de novo synthesis pathway was assessed by qRT-PCR. Protein levels of dihydroceramide desaturase (DEGS) of infected hCMEC/D3s were investigated by western blotting. DEGS activity was determined by addition of deuterated dhCer during infection and measurement of deuterated dhCer and Cer with LC-MS/MS. Furthermore, subcellular localization of dhCer production after infection was examined by live-cell confocal microscopy using clickable ω-azido-dihydrosphingosine in combination with wheat germ agglutinin-AF Plus 405 for plasma membrane staining in mApple-Sec61b-C1 transfected hCMEC/D3s to detect ER localization. Biotin/streptavidin-AF 647 staining was used to localize Nm.

Results

We found that the increase in dhCer after Nm infection is not caused by transcriptional regulation of enzymes of the de novo synthesis pathway (including orosomucoid-like protein, serine palmitoyltransferase, 3-ketodihydrosphingosine reductase, ceramide synthase, ceramidase and DEGS) as well as by DEGS protein levels. DEGS enzymatic activity was, however, reduced after 8 h of Nm infection. Interestingly, we detected ER localization of clicked sphingolipid species and translocation to the plasma membrane after Nm infection.

Summary

Taken together, we showed that Nm leads to an increase in dhCer levels in BECs. This increase was not caused by the transcriptional regulation of enzymes of the de novo synthesis pathway but is due to reduced DEGS activity. Moreover, clickable ω-azido-dihydrosphingosine can be used to investigate the localization of dhCer within the infected host cells by confocal microscopy.