The sialate O-acetylesterase EstA from gut Bacteroidetes species enables sialidase-mediated cross-species foraging of 9- O-acetylated sialoglycans

Figure 4. EstA acts on 9-O- but not 7-O-acetyl esters of sialic acid. GBS capsular polysaccharide mimics host glycans and can generate high levels of sialic acid O-acetyl esters that begin at the 7-carbon position and can be migrated to the 9-carbon position. We used the intact capsule on the surface of live GBS as a model system to define the specificity of EstA. GBS sialic acids that were maintained at the 7-carbon position or induced to migrate to the 9-carbon position as described under “Experimental procedures” were used as a substrate for purified EstA. After incubation of EstA with GBS bearing 7-O- (A) or 9-O-acetylated (B) sialic acids in PBS for 30, 90, and 300 min at 37C, bacteria were washed, and their sialic acids were released with A. ureafaciens sialidase and analyzed by DMB-HPLC. The HPLC traces of mock and EstA-treated samples are from the final, 300-min time point. This experiment was performed twice with one biological replicate and three technical replicates each.