A unique combination of glycoside hydrolases in Streptococcus suis specifically and sequentially acts on host-derived Gal-epitope glycans

Infections by many bacterial pathogens rely on their ability to degrade host glycans by producing glycoside hydrolases (GHs). Here, we discovered a conserved multifunctional GH, SsGalNagA, containing a unique combination of two family 32 carbohydrate-binding modules (CBM), a GH16 domain and a GH20 domain, in the zoonotic pathogenStreptococcus suis05ZYH33. Enzymatic assays revealed that the SsCBM-GH16 domain displaysendo-(beta 1,4)-galactosidase activity specifically toward the host-derived alpha Gal epitope Gal(alpha 1,3)Gal(beta 1,4)Glc(NAc)-R, whereas the SsGH20 domain has a wide spectrum ofexo-beta-N-acetylhexosaminidase activities, includingexo-(beta 1,3)-N-acetylglucosaminidase activity, and employs this activity to act in tandem with SsCBM-GH16 on the alpha Gal-epitope glycan. Further, we found that the CBM32 domain adjacent to the SsGH16 domain is indispensable for SsGH16 catalytic activity. Surface plasmon resonance experiments uncovered that both CBM32 domains specifically bind to alpha Gal-epitope glycan, and together they had aK(D)of 3.5 mmtoward a pentasaccharide alpha Gal-epitope glycan. Cell-binding and alpha Gal epitope removal assays revealed that SsGalNagA efficiently binds to both swine erythrocytes and tracheal epithelial cells and removes the alpha Gal epitope from these cells, suggesting that SsGalNagA functions in nutrient acquisition or alters host signaling inS. suis. Both binding and removal activities were blocked by an alpha Gal-epitope glycan. SsGalNagA is the first enzyme reported to sequentially act on a glycan containing the alpha Gal epitope. These findings shed detailed light on the evolution of GHs and an important host-pathogen interaction.