MenC isolates are less efficient at activating ASM and membrane ceramide release on HBMEC.
(A) Adherent and (B) intracellular bacteria were evaluated in HBMEC infected with N. meningitidis wildtype strain MC58, four MenC strains (WUE2121, DE7017, FAM18, DE6904) and two further MenB strains (DE7901 and α4) for 4 hrs by gentamicin protection assays. The graphs represent mean value ± S.D. of four independent experiments done in duplicate and show the percentage of recovered bacteria compared to MC58. * P<0.05. (C) Surface ceramide levels on infected HBMEC were determined by flow cytometry. Representative histograms demonstrating surface expressions of ceramide on HBMEC infected with DE7901, α4, WUE2121, DE7017, FAM18, DE6904 (green lines) compared to N. meningitidis strain MC58 (orange lines), uninfected cells served as control (filled grey). (D) Ceramides were detected on HBMEC left uninfected (control) or infected with GFP-expressing strain WUE2121 for 2 hrs by immunofluorescence analysis. (E) Whole cell lysates of cells infected with the four MenC strains and strain MC58 were analyzed for ASM activity. Data (mean ± S.D.) are compiled from three independent experiments performed in triplicate. * P<0.05. SgC = serogroup C meningococci.