Figure 4.

Flow cytometric analysis of the deposition of human C3b and C4b on fixed bacterial cells. Anti-ACWY capsule antiserum (diluted 1:50) or anti-LPS mAb EI9.2 (4 µg/mL) was used together with 5% IgG depleted human serum to investigate the effect of the mutations on antibody mediated C3b and C4b deposition. Typical results are shown for one of the reference isolates and for selected mutant isolates. The fluorescence intensity of fixed bacterial cells was determined (counts). Grey filled area: cells only incubated with secondary antibody; black thin line: negative control with 4 µg/mL of an unrelated IgG1 mAb; grey thick line: anti-ACWY capsule antiserum; black thick line: anti-LPS mAb. Reference isolate 2602: C3 and C4 deposition is caused both by anti-capsule and anti-LPS antibodies; isolate 1976 lacking capsule due to a non-synonymous SNP in the ctrE gene: no C3 and C4 deposition with anti-capsule antibodies; isolate 2008 expressing more polysaccharide than the reference isolates as a result of a non-synonymous mutation in the csaB gene: C3 and C4 deposition by anti-LPS mAb is strongly reduced; isolate 1666 expressing non-O-acetylated capsular polysaccharide: no C3 and C4 deposition caused by polyclonal anti-ACWY capsule antiserum.