Mucin glycans attenuate the virulence of Pseudomonas aeruginosa in infection

Abstract

A slimy, hydrated mucus gel lines all wet epithelia in the human body, including the eyes, lungs, and gastrointestinal and urogenital tracts. Mucus forms the first line of defence while housing trillions of microorganisms that constitute the microbiota¹. Rarely do these microorganisms cause infections in healthy mucus¹, suggesting that mechanisms exist in the mucus layer that regulate virulence. Using the bacterium Pseudomonas aeruginosa and a three-dimensional (3D) laboratory model of native mucus, we determined that exposure to mucus triggers downregulation of virulence genes that are involved in quorum sensing, siderophore biosynthesis and toxin secretion, and rapidly disintegrates biofilms-a hallmark of mucosal infections. This phenotypic switch is triggered by mucins, which are polymers that are densely grafted with O-linked glycans that form the 3D scaffold inside mucus. Here, we show that isolated mucins act at various scales, suppressing distinct virulence pathways, promoting a planktonic lifestyle, reducing cytotoxicity to human epithelia in vitro and attenuating infection in a porcine burn model. Other viscous polymer solutions lack the same effect, indicating that the regulatory function of mucin does not result from its polymeric structure alone. We identify that interactions with P. aeruginosa are mediated by mucin-associated glycans (mucin glycans). By isolating glycans from the mucin backbone, we assessed the collective activity of hundreds of complex structures in solution. Similar to their grafted counterparts, free mucin glycans potently regulate bacterial phenotypes even at relatively low concentrations. This regulatory function is likely dependent on glycan complexity, as monosaccharides do not attenuate virulence. Thus, mucin glycans are potent host signals that 'tame' microorganisms, rendering them less harmful to the host.

Fig. 1.. Native whole mucus suppresses virulence traits in the opportunistic pathogen P. aeruginosa.

(a) The natural mucus barrier hosts a diversity of microbes, while limiting infections at the mucosa. Mucins are the major structural component of mucus and are densely grafted with complex glycans. (b) Defects in mucus production are associated with disease and biofilm formation. (c) Representative images of GFP-expressing P. aeruginosa biofilms after 3-h treatment with buffer or native mucus reveal that native intestinal mucus reduces biofilm biomass in the wild-type (WT) strain, but not in the flagellar mutant (ΔfliD). Similar results were observed in different fields of view across three independent replicates. (d) Native intestinal mucus solutions disperse biofilm biomass into the planktonic state. Percent dispersal is based on the ratio of planktonic cells to total biomass (planktonic cells + remaining biofilm cells). Data and are from biologically independent replicates: Buffer (n = 12), intestinal mucus (n = 6). Measure of center is mean ± standard error. Significance tested with two-sided student’s t test, ****p<0.0001. (e) Native mucus solutions suppress key virulence traits relative to mucus solubilization buffer. (f) Depletion of intestinal mucus components ≥100 kDa prevents biofilm dispersal. Supplementation of mucus filtrates with exogenous purified MUC2 partially restores biofilm dispersal. Measure of center is mean ± standard error, and is calculated from 6 biologically independent replicates. Significance tested with two-sided student’s t test, ****p<0.0001. (g) Depletion of intestinal mucus components ≥100 kDa results in increased expression of virulence genes. Supplementation of mucus filtrates with exogenous purified MUC2 partially restores downregulation of virulence genes. (h) Depletion of gastric mucus components ≥100 kDa increases the expression of virulence genes. Supplementation of mucus filtrates with exogenous purified MUC5AC restores downregulation of virulence genes. (e, g, h) Data are log2-transformed qPCR measurements of relative gene expression (fold change, FC). Measure of center is mean ± standard error, and are calculated from biologically independent replicates: gastric mucus (n = 6), intestinal mucus (n = 3), saliva (n = 3). (g, h) Significance assessed with two-sided student’s t test, followed by multiple comparison correction using the Holm-Sidak method, *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001

Fig. 2.. Mucins are sufficient to attenuate P. aeruginosa virulence in vitro and in vivo.

(a) Gene expression was evaluated in liquid culture with or without the native mucin network. (b) The predominant gel-forming mucins secreted into mucosal niches throughout the body. Sources of mucin used in this study are highlighted. (c) MUC5AC and MUC5B elicit global transcriptional responses in P. aeruginosa PAO1. A complete list of fold-changes (FC) and false discovery rates (FDR) appear in Table S1. Fold-change data are average measurements. FDR was determined using the Benjamini Hochberg p-value adjustment method. Data are biologically independent replicates: no mucin treatment (n = 6), MUC5AC-treated (n = 3), MUC5B-treated (n = 3). Correspondence plots of the FC values are presented in Figure S2. Principal component analysis of expression data appears in Figure S13. Venn diagrams contain the total number of genes differentially expressed (FDR < 0.05) after exposure to 0.5% w/v MUC5AC (purple) or MUC5B (orange). Significance of overlap was tested using the hypergeometric test. Functional enrichment analysis of the non-overlapping regions of the Venn diagrams appears in Figure S2. (d) Functional enrichment analyses identify key virulence pathways among downregulated genes. Significance of enrichment was assessed using the one-sided Mann-Whitney U test, where ranking was based on mean log2-transformed fold changes from biologically independent replicates: no mucin treatment (n = 6), MUC5AC-treated (n = 3), MUC5B-treated (n = 3). Bars, FDR. Red line, FDR = 0.05. (e) P. aeruginosa pathogenicity was evaluated in cell culture (containing a single human epithelial cell type, HT-29). (f) Exposure to increasing MUC5AC concentrations inhibits P. aeruginosa attachment to HT-29 cells. CFU, colony-forming units. Center line, median; box limits, upper and lower quartiles; whiskers, 1.5x interquartile range. Data are biologically independent replicates: no mucin (n = 7), 0.01% mucin (n = 4), 0.05% mucin (n = 4), 0.1% mucin (n = 4), 0.5% mucin (n = 7). Significance was tested in relation to the medium-alone control by ordinary one-way ANOVA, followed by Dunnett’s multiple comparisons test, **p<0.01, ***p<0.001, ****p<0.0001. (g) MUC5AC protects HT-29 epithelial cells from death in a concentration-dependent manner. Dotted lines indicate the 95% confidence interval for the dose-response curve. Data are based on bulk measurements of propidium iodide fluorescence 7.5 h after infection. Data are mean ± standard error, n = 4 biologically independent replicates. (h) MUC5AC maintains the intact epithelial cell monolayer and prevents the onset of HT-29 cellular rounding, bacterial attachment, and HT-29 death. Representative confocal microscopy of HT-29 epithelial cells (bright field), GFP-expressing P. aeruginosa PAO1 cells (green), and propidium iodide staining (red) after exposing HT-29 cells to P. aeruginosa for 5 h (top) to 6 h (bottom) with MUC5AC as indicated. Similar results were observed in different fields of view across three independent replicates. (i) Bacterial viability was monitored in a live dermal wound model (containing living tissue, immune cells, and secreted factors). (j) Bacterial burden on porcine burn wounds decreases after treatment with 0.5% MUC5AC for 7 days. Symbols represent P. aeruginosa PAO1 burden on 6 individual biopsies collected from two pigs following no treatment (circle), treatment with 0.05% MUC5AC (square), or treatment with 0.5% MUC5AC (triangle). Center bar indicates the mean bacterial burden. Significance tested with the Kruskal-Wallis test, followed by Dunn’s multiple comparisons test, *p<0.05. (k) MUC5AC in isolation does not alter P. aeruginosa viability relative to medium alone. Data are mean CFU, colony-forming units, ± standard error, n = 3 biologically independent replicates.

Fig. 3.. The virulence systems suppressed by mucin are downstream of multiple, distinct regulatory cascades, and regulation of these systems is independent of bacterial motility and aggregation.

(a) Mucin promotes a motile, non-aggregated phenotype and suppresses virulence in P. aeruginosa PAO1. To determine whether changes to virulence are caused by a shift in motility or aggregation, we monitored the virulence phenotype of non-motile (ΔmotABCD) and non-aggregative (Δpslpel) mutants after mucin exposure. (b) Downregulation of virulence gene expression by mucin does not require a shift in motility or aggregation, indicating that it is a parallel effect of mucin. Data are log2-transformed qPCR measurements of relative gene expression ± standard error, n = 3 biologically independent replicates. Significance was assessed by multiple two-tailed t test, followed by Holm-Sidak correction for multiple comparisons, no significant difference in log2(fold change) between the WT and mutants. (c) Mucin’s virulence regulon is downstream of multiple, interconnected regulatory cascades. (d) Carboxymethylcellulose (CMC) does not differentially regulate virulence genes. Data are log2-transformed qPCR measurements of relative gene expression ± standard error, n = 3 biologically independent replicates. Significance was assessed by one sample two-tailed t test, followed by Bonferroni correction for multiple comparisons, no significant difference in log2(fold change) between CMC and medium alone.

Fig. 4.. Complex O-glycans are the major regulatory component of MUC5AC.

(a) Oligosaccharides released by alkaline β-elimination were resolved via capillary electrophoresis. The mucin-glycan pool includes extended chains consisting of >7 residues. (b) Monosaccharide composition of mucin-glycans were assessed via capillary electrophoresis. These mucin oligosaccharides are predominantly O-linked, as evidenced by the Man (N-linked) to GalNAc (O-linked) ratio. Red labels indicate the quantitation standard maltose (Malt) and the migration standard galacturonic acid (GalA). Abbreviations and migration times (in minutes) for monosaccharide standards: N-acetylgalactosamine (GalNAc) 4.5, N-acetylmannosamine (ManNAc) from N-acetylneuraminic acid 4.95, N-acetylglucosamine (GlcNAc) 5.26, mannose (Man) 6.08, glucose (Glc) 6.33, xylose (Xyl) 6.99, fucose (Fuc) 7.29, galactose (Gal) 7.67. (c) MALDI-TOF spectrum of O-glycans from MUC5AC. m/z, mass/charge values. The complete list of O-glycan structures with experimental and theoretical masses appears in Table S8. (a-c) Similar results have been observed in 3 independent replicates. (d) Representative images of GFP-expressing P. aeruginosa biofilms after a 3-h treatment with medium alone, with 0.01% mucin-glycans, or with a 0.01% pool of monosaccharides. Glycan solutions reduce biofilm biomass in the wild-type (WT) strain, but not the flagellar mutant (ΔfliD). Similar results were observed in different fields of view across three independent replicates. (e) Mucin-glycans, but not monosaccharides, disperse biofilm biomass into the planktonic state for the wild-type (WT) strain, but not the flagellar mutant (ΔfliD). Center line, mean ± standard error, n = 3 biologically independent replicates. Significance was tested in relation to the medium-alone control by ordinary one-way ANOVA, followed by Dunnett’s multiple comparisons test, *p<0.05 (f) Mucin-glycans inhibit bacterial attachment to human epithelial HT-29 cells. Center line, median; box limits, upper and lower quartiles; whiskers, 1.5x interquartile range. Data are biologically independent replicates: medium alone (n = 6), MUC5AC-glycans (n = 3), monosaccharides (n = 6). Significance was tested in relation to the medium-alone control by ordinary one-way ANOVA, followed by Dunnett’s multiple comparisons test, *p<0.05. (g) Relative size distributions of aggregates identified via live 3D confocal microscopy and analyzed by COMSTAT. In medium alone and medium with monosaccharides, P. aeruginosa biomass is concentrated in large surface-associated aggregates, whereas MUC5AC-glycans suppress the formation of aggregates. Center line, median; box limits, upper and lower quartiles; whiskers, 1.5x interquartile range. Data are average aggregate sizes compiled from six separate z-stacks. Significance tested with the Kruskal-Wallis test, followed by Dunn’s multiple comparisons test, *p<0.05. (h) Low concentrations of MUC5AC-glycans elicit a transcriptional response that positively correlates with transcriptional changes elicited by whole MUC5AC. Fold-change data are average measurements from 3 biologically independent replicates. Significance tested with the regression slope test. (i) Free glycans suppress the same virulence pathways as whole mucin. Significance of enrichment was assessed using the Mann-Whitney U test, where ranking was based on mean log2-transformed fold changes from 3 biologically independent replicates. (j) Growth is not altered by the presence of the monosaccharide components in mucin-glycans. Data are mean OD₆₀₀ ± standard error, n = 3 biologically independent replicates. (k) Complex mucin-glycans, but not their monosaccharide components, induce expression changes in signature virulence genes. Data are qPCR measurements of relative gene expression ± standard error, n = 3 biologically independent replicates. (l) Bacterial burden on porcine burn wounds decreases after treatment with 0.1% mucin-glycans for 7 days. Symbols represent P. aeruginosa PAO1 burden on individual biopsies collected burns following no treatment (circle, n = 6), treatment with 0.1% MUC5AC-glycans (square, n = 3), or treatment with 0.1% monosaccharides (triangle, n = 3). Center bar indicates the mean bacterial burden. Significance tested in relation to the no-treatment control with the Kruskal-Wallis test, followed by Dunn’s multiple comparisons test, *p<0.05.