Salmonella Degrades the Host Glycocalyx Leading to Altered Infection and Glycan Remodeling

Abstract

Complex glycans cover the gut epithelial surface to protect the cell from the environment. Invasive pathogens must breach the glycan layer before initiating infection. While glycan degradation is crucial for infection, this process is inadequately understood. Salmonella contains 47 glycosyl hydrolases (GHs) that may degrade the glycan. We hypothesized that keystone genes from the entire GH complement of Salmonella are required to degrade glycans to change infection. This study determined that GHs recognize the terminal monosaccharides (N-acetylneuraminic acid (Neu5Ac), galactose, mannose, and fucose) and significantly (p < 0.05) alter infection. During infection, Salmonella used its two GHs sialidase nanH and amylase malS for internalization by targeting different glycan structures. The host glycans were altered during Salmonella association via the induction of N-glycan biosynthesis pathways leading to modification of host glycans by increasing fucosylation and mannose content, while decreasing sialylation. Gene expression analysis indicated that the host cell responded by regulating more than 50 genes resulting in remodeled glycans in response to Salmonella treatment. This study established the glycan structures on colonic epithelial cells, determined that Salmonella required two keystone GHs for internalization, and left remodeled host glycans as a result of infection. These data indicate that microbial GHs are undiscovered virulence factors.

Figure 1. Modulation of carbohydrate-degrading enzymes alter access during invasion of Salmonella WT.

(A) Neu5Ac digestion. Methyl-β-cyclodextrin (control) disrupts lipid rafts. Sialidase treatment led to reduction in Salmonella association. Least Squares Means Differences (LSD) was used for statistical analysis. Levels not connected with the same letter are significantly different, p < 0.05. (B) Salmonella WT knockout strains characterized for the alteration in adhesion and invasion (A/I) using differentiated Caco-2 cells 60 minutes post-infection. White bars represent the CFU of Salmonella WT that adhered per Caco-2 cell. The gray bars represent the CFU of Salmonella WT that invaded per Caco-2 cell. This was done in combination with transcriptional profiling (bottom panel) of Salmonella WT during infection of Caco-2 cells to gain insights about differentially expressed carbohydrate-degrading genes. Salmonella WT genes displaying changes in gene expression levels during infection of Caco-2 cells. Colors indicate the expression of each gene induced (red) and repressed (green). LSD was used for statistical analysis. Error bars indicate SEM between 3 biological replications, *p < 0.05, **p < 0.001, ***p < 0.0001, Not Significant (NS). The statistics shown at the top indicates the statistical relevance in invasion levels of ΔinvA compared to the mutant strains.

Figure 2. Host glycome is substantially altered during infection with Salmonella WT within 60 minutes.

The bars represent the relative abundance levels of each significant glycan during infection with Salmonella. (A) Decrease in complex-fucosylated glycans; (B) Decrease in high-mannose and hybrid glycans; (C) Decrease in sialylated glycans; (D) Disappearance of sialylated glycans; (E) Accumulation of high-mannose and complex glycans; (F) New complex-fucosylated and sialylated glycans. Error bars indicate SEM between 3 biological replications, *p < 0.05, **p < 0.001, ***p < 0.0001.

Figure 3. Host responds to microbial glycan degradation by modifying its own glycan.

(A) Analysis of host pathways involved during glycan degradation of Caco-2 cells following microbial association. Canonical pathways whose biological functions were influenced based on gene expression changes are shown (Fishers exact test). Upregulated molecules in each pathway are represented as a percentage of the total canonical pathway membership. (B–E) Networks display interactions between genes involved in mannose, fucose and Neu5Ac ([D] sialidases and [E] sialyltransferases) metabolism, respectively, in Caco-2 cells treated for 60 minutes with Salmonella LT2. ST3GAL family sialyltransferases catalyzed the addition of Neu5Ac to a terminal galactose of glycoconjugates in an α-2,3 linkage. The sialyltransferases in ST6GAL family transferred alpha-2,6 linking Neu5Ac to galactose residues of N-glycans. The ST6GALNAc family sialyltransferases added Neu5Ac to terminal N-acetylgalactosamine residues of glycoproteins and glycolipids, in an α-2,6 linkage. Lastly, the ST8Sia family catalyzes the transfer of Neu5Ac in an alpha-2,8 linkage to other Neu5Ac residue present in N- or O-glycans of Neural cells. Caco-2 up-regulation of enzymes in both gene networks is indicative of microbial induced changes in host glycan biosynthesis. Gene induction is represented as a log ratio (q < 0.05) and displayed in shades of red.

Figure 4. Signature of glycan profiles and how each glycan-degrading enzyme modulates the glycans during infection.

(A) Signature of glycan profiles during infection. There is an increase in abundances of high-mannose and hybrid glycans. Also the glycans are switched from being highly sialylated to highly fucosylated (the degree of fucosylation increases and the degree of sialylation decreases); (B–E) Glycans that are highly regulated by Salmonella glycan-degrading enzymes during infection. The comparisons between CHPNeu and nanH (Sialidases) and malS and glgX (Amylases) are shown. LSD was used for statistical analysis. Error bars indicate SEM between 3 biological replications. Levels not connected with the same letter are significantly different. Statistical analysis was done within each structure.