Probing the role of glycocalyx in host-microbe interactions with systematic modification of the glycomic surface

Glycosylation is a conserved post-translational modification by which oligosaccharides are attached to a macromolecule, typically proteins and lipids found on the cell surface. In particular, protein glycosylation play an important role in host-microbe interactions which can further help in describing microbial pathogenicity, recognition by the host immune system and host susceptibility to infections. Glycan-mediated host-microbe interactions were empirically investigated using glycan arrays; however, characterizing the cellular glycocalyx via in situ binding with cells has not been explored. In this research, we established a metabolic-engineered host cell model that generates controlled glycan structure phenotypes on the cell surface. Specifically, we produced host cell surfaces that were primarily fucosylated, sialylated, undecorated complex-type structures, and high mannose-type structures. The manipulated host cell N-glycomes were characterized and confirmed by liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis. To validate glycans involved during infection, we employed adherence assays which showed fucosylated N-glycans on the colon cell line HCT116 cell surface significantly increased the number of adhered Salmonella enterica serovar Typhimurium (S. Typhi) compared to the other glycan subtypes. Furthermore, we showed that adherence of S. Typhi to HCT116 cells could be blocked by co-incubation with free fucose monosaccharides. The results proved that fucose residues on host cells bind with S. Typhi during bacterial infection. Proteomic analysis showed that the glycoengineering did not change the abundances of membrane proteins, indicating that host protein expression did not contribute to the increased adherence of S. Typhi. Meanwhile, glycoproteomic analysis yielded site-specific N-glycosylation information. Glycopeptides were identified and quantified using a standard glycoproteomic workflow. Collectively, our results suggested the importance of glycans in host-microbe interactions and provided a novel insight into the significance of host glycome during pathogenesis.