Opc protein contributes to ASM activation and ceramide cell surface release.

HBMEC were infected with N. meningitidis wildtype strain MC58, isogenic unencapsulated strain MC58 siaD and two isogenic strains lacking Opc expression (MC58 opc and MC58 siaD, opc) for 2 h. (A) ASM activity was measured in whole cell lysates of HBMEC infected with N. meningitidis wildtype strain MC58 or isogenic mutants. (B) ASM translocation to the outer leaflet was determined by flow cytometry. (C) In parallel, surface ceramide levels on HBMEC were determined in infected cells. All data are mean ± S.D. from three independent experiments performed in triplicate. Values are expressed as the percentage of ceramide expression, ASM translocation and activity in cells infected with Opc-deficient strains compared to parental strains. * P<0.05. (D) Expression of recombinant Opc in E. coli BL21. Expression of Opc was analyzed either in bacterial lysates by Western Blotting or by flow cytometry analysis using a monoclonal anti-Opc antibody. As a negative control E. coli not transformed with recombinant proteins were used. Representative histograms demonstrating surface expressions of Opc on E. coli BL21 (filled line) compared to parental strains (open line). Adhesion and invasion to HBMEC of an E. coli strain recombinantly expressing Opc (grey bars) or the parent strain (black bars) were determined by gentamicin protection assays. Results represent mean ± S.D. of three independent experiments done in triplicate and show the percentage of recovered bacteria compared to not transformed E. coli. ** P<0.01. (E and F) Cells were infected with Opc-expressing E. coli and parental E. coli strain and ceramide accumulation and ASM activity was determined as described above.