Utilizing CMP-Sialic Acid Analogs to Unravel Neisseria gonorrhoeae Lipooligosaccharide-Mediated Complement Resistance and Design Novel Therapeutics
Fig 5
IgG, IgM, and complement components C3, C4 and FB deposition on N. gonorrhoeae (Ng) F62 that have incorporated each indicated sialic acid analog into lipooligosaccharide (LOS).
Ng F62 ΔlgtD was grown in media alone (open bars), or media supplemented with 20 μg/ml (~30 μM) of each of the indicated CMP-Sias (NulOs). Bacteria were incubated with either 3.3% or 10% normal human serum (NHS) at 37°C for 10 min and IgG and IgM binding, and C3, C4 and factor B (FB) deposited on bacteria were measured by whole cell ELISA. Data with 3.3% and 10% NHS is shown using hatched and solid bars, respectively. mAb 2-8C-4-1 [62] that detects the Neisserial lipoprotein H.8 was similar across all groups and confirmed similar capture of bacteria in all wells (bottom right graph). Measurement of gonococcal H.8 lipoprotein antigen was performed to assess similarity of bacterial capture across microtiter wells. The mean (SD) of three observations is shown. Significance of differences in Ig or complement component binding/deposition using each analog vs, Neu5Ac is indicated for results that used 10% NHS only (for simplicity): *, P<0.05; **, P<0.01; ***, P<0.001; ****, P<0.0001 (ANOVA). Controls with bacteria alone yielded OD405nm readings <0.1 (negative).