Figure 5.

Exposure of Neisseria gonorrhoeae to VLY-liberated HeLa contents increases sialylation-dependent recruitment of human FH. A, Schematic of chimeric FH protein S2534 (Fc3/FH*). S2534 consists of human IgG3 Fc receptor (Fc3) fused to the 3 C-terminal domains 18–20 of human FH. A point mutation in FH domain 19 (D to G at position 1119), which was introduced to abrogate binding to host cells, does not interfere with binding of the C-terminus of FH to sialylated N. gonorrhoeae. B, Binding of Fc3/FH* to N. gonorrhoeae F62 WT and Δlst following incubation with either 0 μM or 1 μM CMP-Neu5Ac. Data are presented as the percentage of gated events positive for FITC. C and D, Fc3/FH* binding after N. gonorrhoeae F62 WT and Δlst were incubated with the supernatants of HeLa cells that had been exposed to either 1 μg/mL VLY or a parallel dilution of the pET28a vector-only control. C, Representative dot-plot of Fc3/FH* binding to N. gonorrhoeae WT and Δlst after exposure to VLY-extracted HeLa contents. D, Fc3/FH* binding presented as the percentage of gated events positive for FITC. B and D, Data are a combination of 2 independent experiments, with 2–3 (B) or 4–5 (D) biological replicates per experiment. Error bars represent standard deviation. Significance determined by 1-way ANOVA. ****P < .0001. Abbreviations: CMP-Neu5Ac, cytidine-5′-monophospho-N-acetyl neuraminic acid; FH, Factor H; FITC, fluorescein isothiocyanate; lst, lipooligosaccharide-sialyltransferase; ns, not significant; SSC, side scatter; VLY, vaginolysin; WT, wild type.