Utilizing CMP-Sialic Acid Analogs to Unravel Neisseria gonorrhoeae Lipooligosaccharide-Mediated Complement Resistance and Design Novel Therapeutics
Fig 2
Substrate specificity of gonococcal LOS sialyltransferase (Lst).
A. N. gonorrhoeae (Ng) strain F62 ΔlgtD was grown in gonococcal media alone (‘No sialic acid’) or in media supplemented with CMP salts of the indicated sialic acid (Sia), each at a concentration of 20 μg/ml (30 μM), for 2 h at 37°C. Bacteria were washed, pellets treated with protease K, lysed in 4× LDS buffer and lysates were separated on 12% Bis-Tris gels using MES running buffer. LOS was transferred to a PVDF membrane and probed with monoclonal antibody (mAb) 3F11 that recognizes the lacto-N-neotetraose only in the non-sialylated state; the addition of a Sia residue abrogates mAb 3F11 binding (upper panel). LOS was also visualized by silver staining following electrophoresis on 16.5% tricine gels (lower panel). B. Incorporation of NulO by LNnT LOS was assessed using whole cell ELISA with mAb 3F11, *** P<0.001. C. Wild-type Ng F62 was grown with (shaded) or without (solid line) 30 μM CMP-Neu5Gc for 3 hours and analyzed by flow cytometry using a polyclonal Neu5Gc-specific chicken IgY Ab followed by a FITC conjugated donkey anti-chicken IgY secondary Ab. As a negative control, wild-type Ng F62 was grown in CMP-Neu5Gc as above and incubated with the secondary Ab only (dashed line).