Mucus-derived glycans are inhibitory signals for Salmonella Typhimurium SPI-1-mediated invasion

Mucus forms a critical barrier against enteric pathogens like Salmonella enterica serovar Typhimurium. While in vivo studies indicate that secreted, gel-forming mucins and specifically Core 3 glycosylation are protective against S. Typhimurium, the molecular mechanisms involved remain unclear. Here, we measure gene expression changes in Salmonella enterica serovar Typhimurium LT2 following growth in SPI-1 inducing medium (LB + 0.3M NaCl) with or without purified MUC2 (0.1%, w/v), MUC2 glycans (0.1%, w/v), a pool of monosaccharide components comprised of D-galactose, D-GalNAc, D-GlcNAc, D-fuc, and Neu5Ac (0.1%, w/v), or specific individual mucin sugars, namely N-acetyl galactosamine (GalNAc) (0.2%, w/v) and N-acetyl glucosamine (GlcNAc) (0.2%, w/v). Notably, we find MUC2, MUC2 glycans, and to a lesser extent,D-GalNAc and D-GlcNAc downregulate SPI-1 gene expression. S. Typhimurium LT2 overnight cultures were diluted 1:100 in 100 µL of SPI-1-inducing LB.3 medium (with or without 0.1% MUC2, 0.1% MUC2 glycans, 0.2% GalNAc, 0.2% GlcNAc, 0.1% monosaccharide pool containing equal volumes of D-galactose, D-GalNAc, D-GlcNAc, L-fucose, and Neu5AC) for 3 h at 37 °C, under static conditions. RNA was extracted with the MasterPure RNA Purification Kit (Biosearch Technologies), and DNA was eliminated using the Turbo DNA-Free Kit (Ambion). The Agilent 2100 Bioanalyzer (Agilent Technologies) was used to assess RNA integrity. rRNA was depleted with the NEBNext rRNA Depletion Kit for bacteria (NEB). The Illumina HiSeq platform was used for the sequencing mucin-, glycan-treated, and monosaccharide pool-treated samples with a single-end protocol and read lengths of 40 nucleotides. The Illumina NovaSeq platform was used for sequencing HexNAc-treated samples with a paired-end protocol and read lengths of 50 nucleotides. Reads were analyzed on the Galaxy platform with the Burrows-Wheeler algorithm, HTseq-count, and DESeq2. For all analyses of the sequencing data, we considered FDR-adjusted P values < 0.05 as significant.