UC Davis

Microbe-host interactions are mediated by protein-carbohydrate binding processes. The microbial adherence to cellular targets is a crucial step in pathogenesis. N-Glycans are oligosaccharides attached to the polypeptide of proteins and can be found on the surface of mammalian cells. Within the respiratory and intestinal tracts, highly glycosylated epithelial cells represent the primary boundary separating embedded host tissues from pathogens. Currently, there are limited methods to comprehensively explore the roles of glycans involved in these infections, therefore both virus and bacterium were employed in this study to investigate this topic. SARS‑CoV‑2, the causative agent for the COVID-19 pandemic, reaches into the respiratory tract, and Salmonella. Typhi can infect the intestinal tract and cause typhoid fever.The cell surface glycome was manipulated via metabolic engineering. N-Glycans on the cell underwent cell membrane extraction, enzymatic release, enrichment and was eventually analyzed with an Agilent 6520 Accurate Mass Q-TOF LC/MS equipped with a PGC micro-fluidic chip. Peptides and glycopeptides from host cells were obtained using cell membrane extraction and trypsin digestion. Peptides were desalted by solid-phase extraction with C18 cartridges, whereas glycopeptides were enriched with HILIC. They were then both analyzed using an Orbitrap Fusion Lumos LC-MS/MS system. Viral infection assay and confocal microscopy were employed to explore the effect of glycome on binding with SARS-Cov-2. Bacterial adherence and invasion assays were used to assess bacterial infection capacity with different types of N-glycans dominating the host cell surfaces. We established a cell-based model that enabled us to perform reliable structure-phenotype correlative experiments and compare the effect of individual N-glycan types in microbial infection. Using specific inhibitors, we created host cell surfaces that were primarily fucosylated, sialylated, undecorated, or contained mainly oligomannose structures. All the glycomic cell profiles here were confirmed via Q-TOF LC/MS. Confocal microscopy helped to reinforce the notion that HMOs, such as sialylated structures, can function as decoys to prevent SARS-Cov2 infection. After modifying the host cell glycosylation, binding assays showed the spike protein had a strong affinity towards sialylated N-Glycans. Combined with molecular dynamics simulations, our data further demonstrated that the spike protein, which recognized with host receptors to initiate viral entry, preferentially bound to sialic acids in α2-3 linkage. Bacterial adherence assay illustrated that fucosylated N-glycans on HCT116 cell surface increased significantly in the number of adhered Salmonella. In HCT116 cells, fucosylation was tunable, providing variable amounts of exogenous fucose. We then found that adherence of Salmonella is associated with the abundance of host fucosylated N-glycans. Furthermore, adherence of Salmonella to cells could be blocked by co-incubation with fucose or pretreatment of cells with fucosidase. The results proved that fucose residues on host cells bind with Salmonella. We performed qualitative and quantitative analyses of membrane proteins from host cells. The proteomic analysis showed that the metabolic engineering did not change the abundance of membrane proteins, which indicated that host protein expression did not contribute to the altered adherence. Meanwhile, glycoproteomic analysis yielded site-specific glycopeptide information. The glycopeptides were identified and quantified using a standard glycoproteomic workflow. We found that the attached N-glycans rather than glycosylation sites were manipulated in host cells. These results highlighted the importance of glycans in host-microbe interactions. This study also developed a lectin proximity oxidative labeling (Lectin PROXL) method to identify lectin-binding glycoproteins. The lectin was modified with a probe to produce hydroxide radicals. Thus, the lectin-recognized glycoproteins are oxidized and identified using a conventional proteomic process. All the lectin probes oxidized around 70% of glycoproteins. The specificity and sensitivity of each lectin were assessed utilizing glycomic and glycoproteomic data. Furthermore, the sialic acid- and fucose-binding lectins have higher specificity and sensitivity than other lectins. This approach provides an unprecedented perspective of lectin- glycoprotein interactions and protein networks mediated by distinct glycan types on cell membranes.