Mucin Glycans Attenuate the Virulence of Pseudomonas Aeruginosa in Infection

Letters https://doi.org/10.1038/s41564-019-0581-8

Mucin glycans attenuate the virulence of Pseudomonas aeruginosa in infection

Kelsey M. Wheeler1,2, Gerardo Cárcamo-Oyarce3, Bradley S. Turner3, Sheri Dellos-Nolan4, Julia Y. Co1,2, Sylvain Lehoux5, Richard D. Cummings5, Daniel J. Wozniak4 and Katharina Ribbeck 1*

A slimy, hydrated mucus gel lines all wet epithelia in the may arise from phenotypic regulation by mucus. Mucus-mediated human body, including the eyes, lungs, and gastrointestinal biofilm dispersal is dependent on an intact flagellum (Fig. 1c), indi- and urogenital tracts. Mucus forms the first line of defence cating that mucin triggers an active motility-driven escape, rather while housing trillions of microorganisms that constitute the than mechanical disruption. To test whether mucus affects only microbiota1. Rarely do these microorganisms cause infec- biofilms or whether it more broadly regulates virulence traits, we tions in healthy mucus1, suggesting that mechanisms exist in measured the expression of genes that are important for establish- the mucus layer that regulate virulence. Using the bacterium ing infection2. This revealed that intestinal mucus transcriptionally Pseudomonas aeruginosa and a three-dimensional (3D) labo- suppresses quorum sensing (lasR), siderophore biosynthesis (pvdA) ratory model of native mucus, we determined that exposure and type-3 secretion (pcrV; Fig. 1e). Native gastric and salivary to mucus triggers downregulation of virulence genes that are mucus similarly suppressed major infection-related genes (Fig. 1e), involved in quorum sensing, siderophore biosynthesis and suggesting that virulence suppression is conserved across various toxin secretion, and rapidly disintegrates biofilms—a hall- mucosal surfaces. Together, these findings demonstrate that mucus mark of mucosal infections. This phenotypic switch is trig- contains factors that modulate bacterial behaviours at the levels of gered by mucins, which are polymers that are densely grafted gene expression and phenotype. with O-linked glycans that form the 3D scaffold inside mucus. To identify specific regulatory factors within complex mucus, Here, we show that isolated mucins act at various scales, sup- we tested mucus fractions that were separated according to pressing distinct virulence pathways, promoting a planktonic molecular weight. The primary bioactive component of whole mucus lifestyle, reducing cytotoxicity to human epithelia in vitro and was larger than 100 kDa (Fig. 1f), indicating mucin polymers attenuating infection in a porcine burn model. Other viscous as possible candidates. Mucins are a major high-molecular-mass polymer solutions lack the same effect, indicating that the component of mucus that disperse cells in isolation4. Adding regulatory function of mucin does not result from its poly- purified mucins back into depleted mucus was sufficient to meric structure alone. We identify that interactions with restore biofilm dispersal and suppression of virulence genes P. aeruginosa are mediated by mucin-associated glycans (Fig. 1f–h), providing strong evidence that mucin glycopolymers (mucin glycans). By isolating glycans from the mucin back- constitute key regulatory cues inside mucus. Here the use of natively bone, we assessed the collective activity of hundreds of purified mucins was critical because commercially available complex structures in solution. Similar to their grafted coun- mucins harbour reduced chemical complexity owing to the harsh terparts, free mucin glycans potently regulate bacterial purification process5. phenotypes even at relatively low concentrations. This regu- To directly test whether mucins trigger a global transcriptional latory function is likely dependent on glycan complexity, as response, we performed RNA sequencing (RNA-seq) on P. aeruginosa monosaccharides do not attenuate virulence. Thus, mucin that were grown with or without (0.5% w/v) MUC5AC or MUC5B, glycans are potent host signals that ‘tame’ microorganisms, which are the most abundant gel-forming mucins secreted in rendering them less harmful to the host. niches colonized by P. aeruginosa, including the nasal and oral cavi- To identify mechanisms in native mucus that control pathogens, ties, respiratory tract, eyes and middle ear (Fig. 2a,b). Both mucins we tested whether exposure to native intestinal mucus alters biofilm triggered a genome-wide response (Fig. 2c, Supplementary Table integrity. Our model organism was the opportunistic pathogen 1) and suppressed many virulence pathways, including type-1, P. aeruginosa PAO1, which is not pathogenic in healthy individuals -2, -3 and -6 secretion systems, siderophore biosynthesis (pyover- (Fig. 1a), but can cause severe morbidity or death in people with dine and pyochelin) and quorum sensing (Fig. 2d, Supplementary compromised immunity, aberrant mucus production (Fig. 1b) or Table 2). Among the upregulated genes, we detected enrichment burn wounds2,3. Mature P. aeruginosa biofilms were exposed to buf- in the denitrification pathway (Supplementary Table 3), consis- fer or native intestinal mucus. Although buffer alone did not affect tent with inverse regulation by quorum sensing6. Several metabolic biofilm integrity (Fig. 1c), biofilms exposed to mucus dissociated genes were also differentially regulated, including those associated from the surface and 70% of cells shifted into the planktonic phase with fumarate metabolism and amino acid and C5-carboxylate (Fig. 1d). As the growth rate was not altered in mucus relative to transport (Supplementary Table 1). These results are consistent buffer alone (Supplementary Fig. 1), the shift to planktonic growth with metabolic changes, which often correlate with changes in

1Department of Biological Engineering, Massachusetts Institute of Technology, Cambridge, MA, USA. 2Microbiology Graduate Program, Massachusetts Institute of Technology, Cambridge, MA, USA. 3Research Laboratory of Electronics, Massachusetts Institute of Technology, Cambridge, MA, USA. 4Departments of Microbial Infection and Immunity, Microbiology, The Ohio State University, Columbus, OH, USA. 5Department of Surgery, Beth Israel Deaconess Medical Center, Harvard Medical School, National Center for Functional Glycomics, Boston, MA, USA. *e-mail: [email protected]

2146 Nature Microbiology | VOL 4 | December 2019 | 2146–2154 | www.nature.com/naturemicrobiology Nature Microbiology Letters

a Natural Gel-forming b Altered Mucin glycans mucus barrier mucins mucus barrier

c d e Buffer Mucus Intestinal mucus 100 Saliva **** 6 4 Gastric mucus 80

WT 2 0 60 –2 40 –4 –6 20 –8 Percentage dispersal

[FC] in gene expression –10 2 Δ ﬂiD 0 –12 log Buffer Intestinal –14 mucus lasR pvdA pcrV

f ghIntestinal mucus (baseline) Gastric mucus (baseline) MUC2 depleted MUC5AC depleted **** MUC2 restored MUC5AC restored 80 15 * 10 **** **** ** 60 10 5

** 40 5 * 0

20 0 –5 [FC] in gene expression [FC] in gene expression 2 2 Percentage dispersal relative to whole mucus relative to whole mucus lo g 0 lo g –5 –10 Depleted MUC2 lasR pvdA pcrV lasR pvdA pcrV restored Virulence gene Virulence gene

Fig. 1 | Native whole mucus suppresses virulence traits in the opportunistic pathogen P. aeruginosa. a, The natural mucus barrier (left) hosts a diverse range of microorganisms while limiting infections at the mucosa. Mucins (middle) are the major structural component of mucus and are densely grafted with complex glycans (right). b, Defects in mucus production are associated with disease and biofilm formation. c, Representative images of green fluorescent protein (GFP)-expressing P. aeruginosa biofilms after 3 h treatment with either buffer or native mucus reveal that native intestinal mucus reduces the biomass of biofilm 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. Scale bar, 20 μm. d, Native intestinal mucus solutions disperse biofilm biomass into the planktonic state. Percentage dispersal is based on the ratio of planktonic cells to total biomass (planktonic cells + remaining biofilm cells). Data are from n = 12 (buffer) and n = 6 (intestinal mucus) biologically independent replicates. Data are mean ± s.e.m.; significance was assessed using two-sided Student’s t-tests; ****P < 0.0001. e, Native mucus solutions suppress key virulence traits relative to mucus solubilization buffer. f, The depletion of intestinal mucus components of ≥100 kDa prevents biofilm dispersal. Supplementation of mucus filtrates with exogenous purified MUC2 partially restores biofilm dispersal. Data are mean ± s.e.m. and were calculated from six biologically independent replicates. g, The depletion of intestinal mucus components of ≥100 kDa results in increased expression of virulence genes. Supplementation of mucus filtrates with exogenous purified MUC2 partially restores the downregulation of virulence genes. *P < 0.05, **P < 0.01. h, The depletion of gastric mucus components of ≥100 kDa increases the expression of virulence genes. Supplementation of mucus filtrates with exogenous purified MUC5AC restores the downregulation of virulence genes. For e,g,h, data are log2-transformed qPCR measurements of relative gene expression (fold change (FC)); data are mean ± s.e.m. and were calculated from n = 6 (gastric mucus), n = 3 (intestinal mucus) and n = 3 (saliva) biologically independent replicates. For g,h, significance was assessed using two-sided Student’s t-tests followed by multiple comparison correction using the Holm–Sidak method. virulence7. Importantly, we found no enrichment in virulence suppresses the expression of virulence genes across a range of exper- pathways among upregulated genes (Supplementary Table 3). There imental conditions. were differences in the exact genes that were differentially regu- We validated the regulation of virulence using quantitative PCR lated, as well as differences in the magnitudes of those changes after (qPCR; Supplementary Fig. 4). To confirm the phenotypic relevance exposure to MUC5AC or MUC5B (Fig. 2c, Supplementary Fig. 2, of these changes, we combined functional biochemical assays and Supplementary Table 4), suggesting differences in mucin biochem- infection models (Fig. 2e,i). Protease activity and siderophore pro- istry and specific regulatory function. We replicated this effect with duction were lower in supernatants from P. aeruginosa exposed to various media, time points and bacterial strains (Supplementary mucin than in supernatants from bacteria grown in medium alone Fig. 3, Supplementary Tables 5–7), demonstrating that mucin (Supplementary Fig. 5). Consistent with the downregulation of

Nature Microbiology | VOL 4 | December 2019 | 2146–2154 | www.nature.com/naturemicrobiology 2147 Letters Nature Microbiology

a bc d Liquid culture 80 MUC5AC Upregulated Downregulated virulence pathways MUC5B Eye Middle ear 60 MUC5AC MUC5B + MUC5AC MUC5B Apr type-1 secretion system MUC5AC 1,233 302 264 Xcp type-2 secretion system Oral cavity Lung 40 Type-3 secretion MUC5B [FDR] MUC5B + 10 P < 10–44 HIS-I type-6 secretion system MUC5AC Pyoverdine synthesis –log 20 Downregulated Pyochelin synthesis Stomach Intestinal Phenazine biosynthesis MUC5AC Quorum sensing tract 0 Native 3D mucin 532 379 218 Biofilm formation networks MUC2 –10 –5 0510 –10 –8 –6 –4 –2 0 P < 10–177 log10[FDR] log2[FC] in gene expression ef g h Cell culture HT-29 P. aeruginosa Cell death Merge 25 50 IC50 = 0.044% 20 40 15 30 alone

** Medium 10 *** 20 of total CFU) 5 *** cell (percentage

Cytotoxicity (%) 10

Attachment to host **** 0 0

0.0010.010.1 1 0.1% Mucin + 0.01 0.10 0.50

0.05 MUC5AC epithelial cells MUC5AC (%) MUC5AC (%) No MUC5AC

i jk Live animal 1 d after infection 7 d after infection 1 × 1012 1010 1010 10 9 1 × 10 10 109 8 108 108 1 × 10 –1 7 7 tissu e tissu e 10 10 1 × 106 –1 –1 6 6 10 10 4 ABTGC CFU ml 1 × 10 105 105 CFU g CFU g 2 MUC5AC Mucin + 4 4 10 10 1 × 10 host cells + 3 3 * immune system 10 10 1 0510 15 20 25 Time (h)

No treatment No treatment 0.5% MUC5AC 0.5% MUC5AC 0.05% MUC5AC 0.05% MUC5AC

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. The 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-change values and false discovery rate (FDR)-adjusted P values is provided in Supplementary Table 1. Fold-change data are average measurements. FDR-adjusted P values were determined using the Benjamini–Hochberg P-value adjustment method. Data are from n = 6 (no mucin treatment), n = 3 (MUC5AC-treated) and n = 3 (MUC5B-treated) biologically independent replicates. Correspondence plots of the fold-change values are provided in Supplementary Fig. 2. Principal component analysis of expression data is provided in Supplementary Fig. 13. The Venn diagrams contain the total number of genes that are differentially expressed (FDR-adjusted P < 0.05) after exposure to 0.5% w/v MUC5AC (purple) or MUC5B (orange). The significance of overlap was assessed using a hypergeometric test. Functional enrichment analysis of the non-overlapping regions of the Venn diagrams is provided Supplementary Fig. 2. d, Functional enrichment analyses identify key virulence pathways among downregulated genes. Significance of enrichment was assessed using one-sided Mann–

Whitney U–tests, for which ranking was calculated on the basis of mean log2-transformed fold changes from n = 6 (no mucin treatment), n = 3 (MUC5AC- treated) and n = 3 (MUC5B-treated) biologically independent replicates. Bars, FDR-adjusted P values. The red dashed line indicates FDR-adjusted P = 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 inhibited P. aeruginosa attachment to HT-29 cells. The centre line indicates the median, the box limits indicate the upper and lower quartiles and the whiskers indicate 1.5× the interquartile range. Data are from n = 7 (no MUC5AC), n = 4 (0.01% MUC5AC), n = 4 (0.05% MUC5AC), n = 4 (0.1% MUC5AC) and n = 7 (0.5% MUC5AC) biologically independent replicates. Significance was assessed in relation to the medium-alone control using ordinary one-way analysis of variance (ANOVA), followed by Dunnett’s multiple comparisons test; ***P < 0.001. 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 ± s.e.m.; n = 4 biologically independent replicates. IC50, half- maximum inhibitory concentration. 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, medium alone) or 6 h (bottom, medium with MUC5AC) as indicated. Similar results were observed in different fields of view across three independent replicates. Scale bar, 20 μm. 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 d. Symbols represent P. aeruginosa PAO1 burden on six individual biopsies collected from two pigs following no treatment (circle), treatment with 0.05% MUC5AC (square) or treatment with 0.5% MUC5AC (triangle). The centre bars indicate the mean bacterial burden. Significance was assessed using Kruskal–Wallis tests followed by Dunn’s multiple comparisons test. k, MUC5AC in isolation does not alter P. aeruginosa viability relative to medium alone. Data are mean CFU ± s.e.m., n = 3 biologically independent replicates. biofilm formation, MUC5AC suppressed P. aeruginosa PAO1 asso- epithelial cells in a concentration-dependent manner (Fig. 2f). ciation to surfaces made of glass (Supplementary Fig. 6) and plastic Mucins promoted a larger vertical distribution of P. aeruginosa cells (Supplementary Fig. 7) and reduced attachment to live HT-29 human than those grown in medium alone, shifting them to a suspended

2148 Nature Microbiology | VOL 4 | December 2019 | 2146–2154 | www.nature.com/naturemicrobiology Nature Microbiology Letters a

Baseline Unknown phenotype phenotype

Non-virulent Mucin phenotype network WT ΔmotABCD Δpslpel b WT c Regulatory ΔmotABCD Quorum sensing Siderophorec-di-GMP cAMP/vfrGac/Rsm 5 cascade Δpslpel 0 Regulatory SiaA, SadC, RoeA, LasR, RhlR, MvfRFur, PvdS, PchR CyaA/B, Vfr, PilA GacS, RsmA components WspR –5 [FC] in gene 2 –10 Phenazine Bioﬁlm formation log

expression in the Downstream Type-1 secretion Pyoverdine Type-3 secretion Biofilm formation presence of mucin Biofilm dispersal regulon Type-2 secretion Pyochelin Motility Type-6 secretion Siderophores –15 Biofilm formation lasR pvdA pcrV Virulence gene d Quorum sensing Pyoverdine Type-3 secretion Biofilm Type-6 secretion 4 Log

lasR siaD PA0070 2 4 PA2254 [FC] in gene expression mvfR PA1694 pslA PA0076 phnB PA2256 pslD 2 PA0080 2 PA1303 PA1700 pslG phzE2 pvdQ pslJ PA0086 rhlI pcrV pvdL pelG PA1844 0 0 phzC1 secA exsA pelD PA3484 pvdF PA4911 pelA PA1719 hsiB3 aprX wspE –2 PA2424 clpV3 –2 PA0852 PA1725 wspA clpV2 [FC] in gene expression lasB pvdA bifA 2 in the presence of CM C –4 MUC5AC MUC5B MUC5AC MUC5B MUC5ACMUC5B MUC5ACMUC5B MUC5ACMUC5B –4 lo g lasR pvdA pcrV Virulence gene The virulence regulon of mucin

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 the 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 exposure to mucin. b, Downregulation of the expression of virulence genes 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 ± s.e.m.; n = 3 biologically independent replicates. Significance was assessed using two-tailed t-tests followed by Holm–Sidak correction for multiple comparisons; no significant difference in log2[fold change] was found between the WT and mutants. c, The virulence regulon of mucin is downstream of multiple interconnected regulatory cascades. d, CMC does not differentially regulate virulence genes. Data are log2-transformed qPCR measurements of relative gene expression ± s.e.m.; n = 3 biologically independent replicates. Significance was assessed using one-sample two-tailed t-tests followed by Bonferroni correction for multiple comparisons; no significant difference in log2[fold change] was found between CMC and medium alone. and less-aggregated planktonic-like state with cells still detectable at of microbial phenotypes, which attenuates pathogens and thereby ≥50 μm from the glass surface (Supplementary Fig. 6). Collectively, facilitates host-mediated clearance. these data highlight two facets of mucin regulatory function—one It is tempting to speculate that mucin triggers this phenotypic in which mucins induce a global transcriptional response that ‘dis- switch by maintaining cells in the planktonic non-aggregate form. arms’ P. aeruginosa by downregulating important virulence genes, To test this hypothesis, we evaluated whether mucins still suppressed and another in which mucins suppress aggregation and bacterial virulence genes in mutants lacking flagellar motility (ΔmotABCD) attachment to surfaces. Mucin-mediated changes were sufficient or the ability to aggregate (Δpslpel; Fig. 3a). Both mutants responded to neutralize antagonistic interactions between P. aeruginosa and to mucins (Fig. 3b), indicating that the virulence-attenuating func- human cells. By monitoring the survival of human epithelial cells tion of mucin is not a downstream consequence of changes to over time, we determined that MUC5AC reduced P. aeruginosa- motility or aggregation, but rather a parallel effect. Integrating our mediated epithelial cell death in a dose-dependent manner (Fig. 2g) RNA-seq results with the major genetic components of the P. aerugi- while maintaining the morphology and confluency of the epithelial nosa virulence network8,9 (Fig. 3c) revealed that the broad virulence- cell monolayer (Fig. 2h). attenuating effect of mucin probably involves multiple regulatory To better understand the relevance of the regulatory function systems. Polymer solutions such as mucin have complex structural of mucin in a complex biological system of multiple cell types and and biochemical properties that could directly or indirectly trigger an active immune system, we exposed P. aeruginosa-infected por- signalling events through many sensory systems in P. aeruginosa. cine burn wounds to a wound dressing containing MUC5AC and One possibility is that mucins trigger a general response through quantified the bacterial burden over time by counting colony- their electrostatic or hydrophobic properties or by creating geo- forming units (CFU; Fig. 2i, Supplementary Fig. 8). Exposure to metric constraints. An alternative hypothesis is that the observed MUC5AC resulted in two-log reductions in CFU in wounds one response is triggered by sensing specific biochemical moieties that week after infection, compared with no reduction in the mucin-free are presented on secreted mucins. To distinguish between these two mock treatment (Fig. 2j). The sustained clearance of P. aeruginosa possibilities, we tested whether carboxymethylcellulose (CMC)—a detected here is probably not due to direct killing by MUC5AC, as well-established mucin mimetic with charge and viscoelastic prop- viability was not inhibited by mucins in isolation (Fig. 2k). Rather, erties that are similar to those of native mucins10—could elicit mucin likely mediates bacterial clearance through the regulation changes in P. aeruginosa virulence. Importantly, exposure to CMC

Nature Microbiology | VOL 4 | December 2019 | 2146–2154 | www.nature.com/naturemicrobiology 2149 Letters Nature Microbiology

abc GalNAc GlcNAc Gal Fuc

APTS dye Glc1 Glc2 Glc3 Glc4 Glc5 Glc6 Glc7 Malt GalNAcManNAcGlcNAcManGlc Xyl Fuc Gal GalA 6 3 Glucose polymer standards 2 nmol standards 4 2 120 2 1 4×

RF U 3× 0 0 90 4 5 6 78910 11 354 678910 3× 6 3 2× MUC5AC-glycan MUC5AC-glycan 60 pool 5× 4 2 components Relative intensity (%)

RFU 30 2 1

0 0 0

456 7 8 910 11 354 678910 1,000 2,000 3,000 4,000 Migration time (min) Migration time (min) m/z d e fhg Medium alone Mucin glycans Monosaccharides 120 * 3 9 * 20 1 × 10 y = 0.12 × x – 0.007 WT * 2 P < 0.0001 [FC ] ) 2 8 × 108 15 3 90 ) ΔfliD

WT 1 –1 8 6 × 10 60 10 0 4 × 108 Aggregate volume ( µ m (CFU ml 8 5 30 –1 2 × 10 Dispersed cells

in gene expression –2 0 Attachment to host cel l 0 0 0.01% glycans lo g (percentage of total CFU) Δ ﬂi D –3 –9 –6 –3 0369

0.5% MUC5AC log2[FC] Medium alone Medium alone Medium alone in gene expression MUC5AC glycansMonosaccharides MUC5ACMonosaccharides glycans MUC5ACMonosaccharides glycans

i jlMedium alone k Downregulated virulence pathways Monosaccharide pool 1 1 d after infection 7 d after infection ) 3 Mucin glycans 9 9

Apr type-1 secretion system 600 10 10

A Monosaccharide pool

( 2 Xcp type-2 secretion system 0.1 8 8 Type-3 secretion 10 10 1

HIS-I type-6 secretion system tissue 0.01 7 7 Pyochelin synthesis –1 10 10 Pyoverdine synthesis 0 [FC] in gene 2

expressio n 6 Phenazine biosynthesis 0.001 106 10

–1 CFU g

Quorum sensing lo g

Biofilm formation Optical density 5 5 0.0001 –2 10 10 * –24 –18 –12 –6 0 02468 lasR pvdA pcrV Time (h) log10[FDR] Virulence gene

No treatment No treatment

0.1% MUC5AC0.1% Monosaccharides glycans 0.1% MUC5AC0.1% Monosaccharides glycans

Fig. 4 | Complex O-linked glycans are the major regulatory component of MUC5AC. a, Oligosaccharides released by alkaline β-elimination were resolved using capillary electrophoresis. The mucin glycan pool includes extended chains consisting of >7 residues (bottom). Top, glucose (Glc) polymer standards. b, Monosaccharide composition of mucin glycans was assessed using capillary electrophoresis (bottom). These mucin oligosaccharides are predominantly O-linked, as evidenced by the ratio of mannose (Man; N-linked) to N-acetylgalactosamine (GalNAc; O-linked). The red labels indicate the quantification standard maltose (Malt) and the migration standard galacturonic acid (GalA). Migration times for monosaccharide standards (top) were as follows: N-acetylgalactosamine, 4.5 min; N-acetylmannosamine (ManNAc) from N-acetylneuraminic acid, 4.95 min; N-acetylglucosamine (GlcNAc), 5.26 min; mannose, 6.08 min; glucose (Glc), 6.33 min; xylose (Xyl), 6.99 min; fucose (Fuc), 7.29 min; and galactose (Gal), 7.67 min. c, MALDI-TOF spectrum of O-linked glycans from MUC5AC. The complete list of O-linked glycan structures with experimental and theoretical masses is provided in Supplementary Table 8. For a–c, similar results were observed in three independent replicates. d, Representative images of GFP-expressing P. aeruginosa biofilms after a 3 h treatment with medium alone, 0.01% mucin glycans or a 0.01% pool of monosaccharides. Glycan solutions reduce biofilm biomass in the WT strain, but not the flagellar mutant (ΔfliD). Similar results were observed in different fields of view across three independent replicates. Scale bar, 20 μm. e, Mucin glycans, but not monosaccharides, disperse biofilm biomass into the planktonic state for the WT strain, but not the flagellar mutant (ΔfliD). Data are mean ± s.e.m.; n = 3 biologically independent replicates. Significance was assessed in relation to the medium-alone control using ordinary one-way ANOVA followed by Dunnett’s multiple comparisons test. f, Mucin glycans inhibit bacterial attachment to human epithelial HT-29 cells. The centre line indicates the median, the box limits indicate the upper and lower quartiles and the whiskers indicate 1.5× the interquartile range. Data are from n = 6 (medium alone), n = 3 (MUC5AC glycans) and n = 6 (monosaccharides) biologically independent replicates. Significance was assessed in relation to the medium-alone control using ordinary one-way ANOVA, followed by Dunnett’s multiple comparisons test. g, Relative size distributions of aggregates were identified using live 3D confocal microscopy and analysed using IMARIS. 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. The centre line indicates the median, the box limits indicate the upper and lower quartiles and the whiskers indicate 1.5× the interquartile range. Data are average aggregate sizes compiled from six separate z-stacks. Significance was assessed using the Kruskal–Wallis test followed by Dunn’s multiple comparisons test. 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 three biologically independent replicates. Significance was assessed using a regression slope test. i, Free glycans suppress the same virulence pathways as whole mucin. The significance of enrichment was assessed using Mann–Whitney U–tests, for which ranking was calculated on the basis of mean log2-transformed fold changes from three biologically independent replicates. j, Growth is not altered by the presence of the monosaccharide components in mucin glycans. Data are mean optical density at 600 nm (OD600) ± s.e.m.; 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 ± s.e.m., n = 3 biologically independent replicates. l, Bacterial burden on porcine burn wounds decreases after treatment with 0.1% mucin glycans for 7 d. Symbols represent the burden of P. aeruginosa PAO1 on individual biopsies collected from burn wounds following no treatment (circle, n = 6), treatment with 0.1% MUC5AC glycans (square, n = 3) or treatment with 0.1% monosaccharides (triangle, n = 3). The centre bar indicates the mean bacterial burden. Significance was assessed in relation to the no-treatment control using Kruskal–Wallis tests followed by Dunn’s multiple comparisons test.

2150 Nature Microbiology | VOL 4 | December 2019 | 2146–2154 | www.nature.com/naturemicrobiology Nature Microbiology Letters did not differentially regulate signature virulence genes (Fig. 3d), or human cells (Fig. 4f), or differentially regulate quorum sensing prevent surface attachment (Supplementary Fig. 9) or protect (lasR), siderophore production (pvdA) or type-3 secretion (pcrV) epithelial cells from P. aeruginosa-induced death (Supplementary genes (Fig. 4k). Monosaccharide exposure also did not suppress Fig. 9). These results suggest that the polymeric structure of mucin the production of virulence factors, even with increasing concen- is not sufficient to mediate these virulence-attenuating effects. trations of monosaccharides (Supplementary Fig. 12), although a Rather, specific biochemistry that is present in mucins, but not in non-significant effect on bacterial burden in vivo was detected CMC, is necessary to attenuate P. aeruginosa virulence. (Fig. 4l). On the basis of these data, we conclude that the complex Native mucins display a plethora of complex glycan structures arrangement and particular stereochemistry of these sugar residues that are covalently linked to serine and threonine11–13, creating a are critical to their function as regulatory signals. wealth of biochemical information with the potential to influence The diversity of O-linked glycans on mucins exceeds even that microbial gene expression. P. aeruginosa uses many strategies to on tissue surfaces16–18, and their complexity makes them ideal for sense and respond to host signals, enabling it to coordinate the encoding biological information with a high degree of specificity. In switch between its pathogenic and host-compatible states8,9. The this way, mucins present and retain a myriad of potential regulatory sensing of signature mucin-glycan motifs may be an effective mech- cues. We propose that microorganisms probably evolved mecha- anism to limit the production of metabolically costly virulence fac- nisms to recognize, process, uptake and respond to specific moieties tors in niches in which virulence would not be advantageous. The within the complex array of mucin glycans19. The question remains potential of complex mucin glycans to regulate microbial behav- as to how mucin glycans interact with, and are sensed by, P. aerugi- iour has been largely overlooked owing to fundamental technical nosa at the molecular level. One possibility is that glycans directly limitations, including the difficulty of purifying intact mucins, non- serve as a signal through a carbohydrate binding site in a global standard methods for isolating O-linked glycans and the analytical regulatory system, such as those affecting the secondary messen- complexity of predicting glycan structures14. To determine whether ger c-di-GMP or the non-coding RNAs rsmY and rsmZ8,9. Potential glycans contribute to the virulence-neutralizing capability of mucin, glycan sensors have been identified in P. aeruginosa that are thought we assessed the degree to which glycans isolated from the MUC5AC to feed into regulatory virulence pathways and have annotated car- protein backbone recapitulate the mucin response in P. aeruginosa. bohydrate binding sites, such as the two-component sensor LadS and We isolated mucin glycans by alkaline β-elimination, which pre- the diguanyulate cyclase NicD20,21. Mucin glycans may also trigger served the unique sequences in the oligosaccharide chains and metabolic changes by serving as a nutritional substrate, or regulate yielded a pooled library of O-linked glycans (Fig. 4a,b). We then signalling pathways through interactions with specific P. aeruginosa analysed glycans using matrix-assisted laser desorption/ionization lectins or surface adhesins. We speculate that the structural diver- time-of-flight (MALDI-TOF). After processing for annotation and sity of mucins and mucin-associated glycans enables them to inter- assignment on the basis of mass and known core structures, we act with several bacterial receptors and mediate distinct functions. identified >90 glycan structures (Fig. 4c, Supplementary Table 8), Collectively, our findings reveal a previously unrecognized role not including distinct isomeric forms, which would probably for mucin glycans as potent host-derived regulators of a bacterial increase the library diversity to several hundred structures. The phenotype that has broad implications for how the body prevents diversity of our glycan library is comparable to those of published mucosal infections while maintaining a diverse microbiota. The libraries for mammalian gastric and respiratory mucins11–13,15. reason why diseased mucus no longer retains the ability to attenuate Specifically, glycans in our library were predominantly built on virulence22–26 remains an open question. Based on our findings that core-2 (Galβ1-3(GlcNAcβ1-6)GalNAcα1-) and, to a lesser extent, glycans trigger a switch in bacterial phenotype, we postulate that core-1 (Galβ1-3GalNAcα1-) structures, with a high level of fucosyl- changes to mucin glycosylation patterns in disease, such as increased ation and relatively little sialylation11–13,15, where hyphens refer to the sialylation15, will alter both the binding properties of mucin with glycosidic linkages between sugar monomers. microorganisms and its protective function. By isolating a mucin- Like native mucus and intact mucins, mucin glycans triggered glycan library, we have established the conceptual and technical the active motility-dependent dispersal of bacterial cells from framework to systematically address these open questions about the mature biofilms (Fig. 4d, Supplementary Fig. 10) into the planktonic glycan regulatory code. We posit that cracking this code will explain state (Fig. 4e). Mucin glycans also prevented bacterial attachment to the influence of glycans on the virulence of microorganisms that glass surfaces (Supplementary Fig. 11) and to human cells (Fig. 4f) interact with the host through mucosal surfaces. Identification of and yielded bacterial aggregates smaller than those formed in specific bioactive glycans will likely reveal a class of therapeutics for medium alone or with monosaccharides (Fig. 4g). To determine the treating intractable bacterial infections and may inspire treatment mucin-regulated pathways that were specifically altered in response strategies that tune host glycan signals to attenuate virulence and to mucin glycans, we exposed P. aeruginosa to a pool of potential stabilize the healthy microbiota. glycan cues and monitored changes in gene expression. RNA- seq revealed that P. aeruginosa that was exposed to relatively low Methods concentrations (0.01% w/v) of free MUC5AC glycans undergoes Strains and growth conditions. Batch cultivation of P. aeruginosa strains a transcriptional response that mirrors that associated with expo- (Supplementary Table 9) was carried out under shaking at 37 °C in Luria–Bertani broth (LB; Difco). Gentamicin (30 μg ml−1) was added to the medium for strains sure to 0.5% (w/v) whole native mucin that comprised up to 0.4% with gentamicin-resistant plasmids. (w/v) grafted glycans (Fig. 4h). Functional enrichment analyses For whole-mucus experiments, overnight cultures of PAO1 were diluted confirmed that the same virulence pathways suppressed by intact tenfold in ABTGC (ABT minimal medium, described previously27, supplemented MUC5AC were also suppressed by MUC5AC glycans (Fig. 4i), with 5 g glucose and 5 g casamino acids) and grown for 4 h. Then, 75 μl of these further indicating that an integral part of mucin, rather than cultures was exposed to 75 μl of solubilized mucus or solubilization buffer (described in the ‘Preparation of whole mucus’ section) for 1 h at 37 °C in a static another mucus-associated factor, is the primary virulence-neu- 96-well microtitre plate (Cellstar). tralizing agent in mucus. We anticipate that increasing the glycan For experiments involving liquid culture, overnight cultures of PAO1 concentration to levels present on mucin will improve the dynamic were diluted to an OD600 of 0.01 in 150 μl ABTGC with or without MUC5AC range of the transcriptional response. Importantly, the viability of (0.01%–0.5%), MUC5B (0.5%), CMC (0.5%; Sigma), MUC5AC glycans P. aeruginosa was not altered by the presence of monosaccharides (0.01%) or monosaccharide mixture (0.01%), and grown for 5 h at 37 °C in a static 96-well microtitre plate. Note that, on the basis of the data shown in that are present in mucin glycans (Fig. 4j). Exposure of P. aeruginosa Fig. 4b, the monosaccharide mixture contained equal weights of galactose, to a pool of these monosaccharides did not trigger dispersal N-acetylglucosamine, N-acetylgalactosamine, fucose and sialic acid (all of the (Fig. 4d,e), prevent attachment to glass (Supplementary Fig. 11) sugars were obtained from Sigma). The concentration of glycans was selected on

Nature Microbiology | VOL 4 | December 2019 | 2146–2154 | www.nature.com/naturemicrobiology 2151 Letters Nature Microbiology the basis of the mucin dose response curves shown in Fig. 2; in these experiments, Medical Center as previously described36. Mass spectrometry data were acquired we found that 0.01% was the lowest mucin concentration with measurable effects using an UltraFlex II MALDI-TOF mass spectrometer (Bruker Daltonics). The (Fig. 2f). Owing to the technical challenges of preparative-scale purification of reflective positive mode was used, and data were recorded between 500 m/z mucin glycans, we used the minimal inhibitory concentration in this research. and 6,000 m/z. The mass spectrometry O-linked glycan profile was acquired by aggregating at least 20,000 laser shots. Mass peaks were manually annotated and Collection of human saliva. Submandibular saliva was collected from human assigned to a particular O-linked glycan composition on the basis of known volunteers using a custom vacuum pump, pooled, centrifuged at 2,500g for 5 min core structures. and protease inhibitors were added as previously described28. Samples of human saliva were collected after explaining the nature and possible consequences of the RNA preparation. Total RNA was extracted using the MasterPure RNA Purification studies, obtaining informed consent and receiving approval from the institutional kit (Lucigen) and residual DNA was removed using the Turbo DNA-free kit review board and Massachusetts Institute of Technology’s Committee on the Use of (Ambion). The integrity of the total RNA was assessed using an Agilent 2100 Humans as Experimental Subjects under protocol no. 1312006096. Bioanalyzer (Agilent Technologies). 16S, 23S and 5S rRNA were removed using the Ribo-Zero Magnetic Kit (Bacteria; Epicentre). Preparation of whole mucus. Mucus was scraped from fresh pig stomachs and intestines and solubilized (1 g scrapings in 5 ml) in 0.2 M sodium chloride RNA-seq. Gene expression analysis was performed using Illumina RNA-seq. buffer with protease inhibitors (5 mM benzamidine hydrochloride, 1 mM RNA-seq was conducted for three biological replicates. The libraries were produced dibromoacetophenone, 1 mM phenylmethylsulfonylfluoride and 5 mM EDTA, using the KAPA RNA HyperPrep kit (Kapa Biosystems). The libraries were pH 7) and 0.04% sodium azide (Sigma). Cellular debris and food waste was sequenced using the Illumina HiSeq platform with a single-end protocol and read removed using low-speed centrifugation at 8,000g (7,000 r.p.m., Sorvall GS-3 rotor) lengths of 40 or 50 nucleotides. for 30 min at 4 °C. Analysis of sequencing data. Sequencing reads were mapped onto the Mucin purification. This study used native porcine gastric mucins (MUC5AC), P. aeruginosa PAO1 reference genome, which is available for download from the porcine intestinal mucins (MUC2) and human salivary mucins (MUC5B), which Pseudomonas Genome Database (http://www.pseudomonas.com) using the Galaxy differ from industrially purified mucins in their rheological properties and server37. Gene expression values were normalized on the basis of library size and bioactivity4,5,28–30. Native mucins were purified as described previously4,28–31. differentially expressed genes were identified using a negative binomial test with an In brief, mucus was scraped from fresh pig stomachs and intestines, solubilized FDR-adjusted P < 0.05. Expression changes in signature virulence genes identified in sodium chloride buffer (described above) and insoluble material was removed by RNA-seq were validated using qPCR, which revealed strong concordance by ultracentrifugation at 190,000g RCF for 1 h at 4 °C (40,000 r.p.m., Beckman between the two methods (Supplementary Fig. 4). 50.2 Ti rotor with polycarbonate bottles). Submandibular saliva was collected Functional category (pathway) assignments were downloaded from the from human volunteers using a custom vacuum pump, pooled, centrifuged and Pseudomonas Genome Database. Pathway enrichment analysis was performed in protease inhibitors were added28. Mucins were purified using size-exclusion MATLAB (R2016b) using one-sided Mann–Whitney U-tests, for which ranking chromatography on separate Sepharose CL-2B columns. Mucin fractions were then was calculated on the basis of the log2-transformed fold change. desalted, concentrated and lyophilized for storage at −80 °C. Lyophilized mucins Heat maps and scatter plots of gene expression data were constructed using were reconstituted by shaking gently at 4 °C overnight in the desired medium. GraphPad Prism. Principle component analysis was performed in R (v.3.4.0) using Mass spectrometry is routinely used to monitor the composition of purified mucin the DESeq2 workflow38. For all analyses of the sequencing data, P values were extracts30,31. This type of analysis has shown that mucin extracts purified from adjusted for multiple comparisons using Benjamini–Hochberg correction to obtain porcine stomach mucus, for example, are composed predominantly of MUC5AC, FDR-adjusted P values. with small quantities of MUC2, MUC5B and MUC6, as well as histones, actin and albumin30,31. RT–qPCR analysis. A list of the primers used in this study is provided in Supplementary Table 9. qPCR with reverse transcription (RT–qPCR) was Isolation of mucin oligosaccharide. Here we applied non-reductive alkaline performed using a two-step method. First-strand cDNA was synthesized from β-elimination ammoniolysis to dissociate non-reduced glycans from mucins32. total RNA using the ProtoScript II First Strand cDNA Synthesis kit (NEB). The Purified mucins were dissolved in ammonium hydroxide saturated with cDNA was used as a template for RT–qPCR using a SYBR PowerUp Master Mix ammonium carbonate and incubated at 60 °C for 40 h to release oligosaccharide kit (Applied Biosystems by Life Technologies) on a Roche LightCycler 480 real- glycosylamines and partially deglycosylated mucins. Volatile salts were removed time PCR system. Primers for RT–qPCR were designed on the basis of previously using repeated centrifugal evaporation and the oligosaccharide glycosylamines published literature or using the NCBI Primer-BLAST tool (https://www.ncbi.nlm. were separated from residual deglycosylated mucins by centrifugal filtration nih.gov/tools/primer-blast/). The genes rpoD and proC were used as endogenous through 3–5 kDa molecular weight cut-off membranes in accordance with the controls. The elimination of contaminating DNA was confirmed using qPCR manufacturer’s instructions (Amicon Ultracel). The resulting oligosaccharide amplification of rpoD on control samples that did not have reverse transcriptase glycosylamines were converted to reducing oligosaccharide hemiacetals by added during cDNA synthesis. Melting-curve analyses were used to verify single- treatment with boric acid. Residual boric acid was removed by repeated centrifugal product amplification. Changes in gene expression were calculated on the evaporation from methanol. Oligosaccharides were further purified using solid- basis of mean change in qPCR cycle threshold (∆Ct) using the ∆∆Ct method ΔΔC phase extraction using Hypercarb mini-columns (ThermoFisher) and residual (fold change = 2À t ). solvents were removed through centrifugal evaporation33. I Dispersal of static P. aeruginosa biofilms. Under static conditions, P. aeruginosa Capillary electrophoresis of oligosaccharides. Reducing oligosaccharides released biofilm dispersal was assayed as previously described4, with slight modifications. from mucins were labelled by reductive amination using the fluorescent tag In brief, an overnight culture of PAO1-GFP or PAO1-GFP ΔfliD was diluted 8-aminopyrene-1,3,6-trisulfonic acid, sodium cyanoborohydride and citric acid. in ABTGC medium to an initial OD600 of 0.01, added to a glass-bottom or Labelled oligosaccharides were analysed using polyvinylalcohol coated N-CHO plastic 96-well plate and incubated for 48 h at 37 °C under static conditions. The capillaries in accordance with the manufacturer’s (SciEx/Beckman) protocol supernatant containing non-adherent cells was removed from the plate and the using a PA800 (Beckman) capillary electrophoresis instrument, detected with biofilm remaining in each well was washed at least three times with 0.9% NaCl. laser-induced fluorescence and analysed using the 32 Karat software. The relative The biofilms were exposed to whole mucus, the solubilization buffer, ABTGC sizes of separated oligosaccharides were determined by comparison with the medium alone, or ABTGC medium containing either 0.01% mucin glycans or migration times of glucose polymer standards. Chromatograms were constructed monosaccharides. The biofilms were statically incubated at 37 °C for 3 h. The plates in GraphPad Prism (v.7.04). were washed three times with 0.9% NaCl and resuspended in ABTGC medium, then examined using microscopy to determine the remaining biofilm biomass. Capillary electrophoresis of monosaccharides. The monosaccharide composition Viable dispersed cells were quantified using CFU counts on LB agar plates. Image of released mucin glycans was determined using established methods34,35. In brief, acquisition was performed using a confocal laser scanning microscope (LSM 800, neutral monosaccharides were obtained by hydrolysis in trifluoracetic acid for Zeiss) equipped with a ×63/1.4 NA oil-immersion or a ×100/1.4 NA oil-immersion 1 h at 80 °C. The liberated monosaccharides were labelled with 8-aminopyrene- objective. The excitation wavelength for GFP was 488 nm. At least five stacks were 1,3,6-trisulfonic acid using reductive amination as described for oligosaccharides. recorded for each well and at least three independent wells. Biofilm quantification Monosaccharides were analysed with capillary electrophoresis on a bare silica was performed using IMARIS (v.7.7.2). column and detected using laser-induced fluorescence. Monosaccharides were identified and quantified by comparison with a maltose internal standard, and Measurement of protease activity. Bacterial cultures were pelleted by migrations times and standard curves were generated using purified standard centrifugation (13,200g for 3 min at room temperature), and the supernatants were sugars. Chromatograms were constructed in GraphPad Prism (v.7.04). filter-sterilized using 0.2 μm filters.

MALDI-TOF and prediction of mucin-glycan structure. β-Eliminated glycans Protease IV activity. The activity of protease IV in cell-free culture supernatants was were permethylated and analysed at the Glycomics Core at Beth Israel Deaconess measured by breakdown of the chromogenic substrate Chromozym PL (Roche),

2152 Nature Microbiology | VOL 4 | December 2019 | 2146–2154 | www.nature.com/naturemicrobiology Nature Microbiology Letters which reacts specifically with protease IV in P. aeruginosa culture supernatants39. Epithelial cytotoxicity assay. The killing of HT-29 cells by P. aeruginosa was In brief, 10 µl pretreated sample and 3 µl Chromozym PL (7 mM) were combined in measured using the membrane-impermeable nuclear stain propidium iodide, reaction buffer (20 mM Tris-HCl, pH 8.0) to a total volume of 100 µl in a microwell which enables continuous quantitative measurement of cell viability over time plate. The plates were assayed on a SpectraMax M Series Multi-Mode Microplate owing to its optimal linear DNA-binding, without background from bacterial Reader (Molecular Devices) pre-equilibrated to 30 °C by measuring the rate of cell death (Supplementary Fig. 14). After co-culture for 1–10 h, propidium increase in absorbance at 405 nm at 3 min intervals for 30 min. Protease IV activity iodide fluorescence (excitation/emission 535 nm/617 nm) was measured using was calculated using the following equation: a SpectraMax M Series Multi-Mode Microplate Reader (Molecular Devices). Between measurements, plates were kept in an incubator at 37 °C, 5% CO and 95% F ´ E ´ d ´ ΔA=min 2 air. At the end of the experiment, maximal fluorescence was measured following treatment of each well with 1% Triton X-100 to permeabilize all cells and label all where F is the dilution factor, E is the extinction coefficient (which is 10.4 nuclei with propidium iodide, which corresponds to 100% cell death. Background at 405 nm), d is the path length (at 100 l volume in a microtitre plate, path µ fluorescence of propidium iodide was measured in uninfected control cells length 0.53 cm) and A/min is the maximum change in absorbance min−1. The ≈ Δ (exposed to medium alone or the appropriate concentration of MUC5AC) at the protease activity was normalized to account for variation in bacterial cell growth on beginning of the experiment. Percentage cytotoxicity was calculated as: the basis of the OD600 of the culture at 5 h. Relative changes were calculated on the basis of the protease activity in medium alone. f f = M f ´ 100 ðÞ� 0 ðÞ� 0

Alkaline protease activity. Alkaline protease activity was tested using a modified where f0 is the initial fluorescence, M is the maximum fluorescence after addition method that was described previously40. Samples containing 1 mg of Hide powder of Triton X-100 and f is the fluorescence at any given time. Mucin dose response azure (Sigma) dissolved in buffer (0.075 ml) consisting of 20 mM Tris-HCl, pH 8.0 curves were assessed at 7.5 h. and 1 mM CaCl2 were mixed with 0.025 ml of the culture supernatants. The reaction mixtures (0.1 ml) were incubated at 37 °C for 1 h. Undissolved substrate Confocal imaging. Image acquisition was performed using a confocal laser was removed by centrifugation at 4,000g for 5 min. The absorbance of the reaction scanning microscope (LSM 800; Zeiss) equipped with a ×63/1.4 NA oil-immersion mixtures was determined at 595 nm using a SpectraMax M Series Multi-Mode or a ×100/1.4 NA oil-immersion objective. Images were analysed using Zeiss ZEN Microplate Reader (Molecular Devices). Protease activity was expressed in terms v.2.1 imaging software. The excitation wavelengths for GFP and propidium iodide −1 −1 of protease U ml , where one unit is equivalent to an increase of 1.0 OD595 h were 488 nm and 535 nm, respectively. The 3D images of attached and unattached at 37 °C. Protease activity was normalized to account for variation in bacterial cells were created with IMARIS v.7.7.2. Plots were generated in GraphPad Prism. cell growth on the basis of the OD600 of the culture at 5 h. Relative changes were Quantification of bacterial aggregate volume was performed using IMARIS v.7.7.2. calculated on the basis of the protease activity in the medium alone. Examination of bacterial interactions with porcine burn wounds. Interactions Measurement of siderophore fluorescence. Pyoverdine and pyochelin levels were between P. aeruginosa and full-thickness burn wounds were assessed as previously simultaneously quantified on the basis of characteristic fluorescence spectra, as described43,44. In brief, female Yorkshire pigs (n = 4) weighing between 32 and 36 kg previously described41. In brief, fluorescence was measured on a SpectraMax M were anaesthetized and the dorsal trunk was shaved and surgically prepared. Under Series Multi-Mode Microplate Reader (Molecular Devices). Pyoverdine production aseptic conditions, six 5.08 × 5.08 cm full-thickness burn wounds were created on was quantified using an excitation wavelength of 400 nm and an emission the back of the pig using an electrically heated burn device with controlled pressure wavelength of 460 nm. Pyochelin fluorescence was quantified using an excitation delivery for 50 s. One day after the thermal injury, mid-log phase cultures of wavelength of 350 nm and an emission wavelength of 410 nm. To account for the P. aeruginosa were topically inoculated onto the wound site at a concentration background fluorescence of pyoverdine, pyochelin production was calculated using of 1 × 108 CFU in 250 μl of 20% PF-127 (Sigma) prepared in PBS with 0%, 0.05% the following equation: (0.5 mg ml−1) or 0.5% (5.0 mg ml−1) MUC5AC, or 0.01% (0.1 mg ml−1) MUC5AC glycans or monosaccharides. The inoculated and treated wound was rubbed with a z w 3E 7 ´ y2 0:0413 ´ y ¼ � � � sterile spatula for 30 s. On days 1 and 4 after inoculation, treatment was reapplied to the wound. On days 1 and 7 after inoculation, full-thickness wound-tissue where z is the actual value of pyochelin production, w is the fluorescence measured biopsies were collected for microbiological analysis using a 6 mm sterile disposable at an excitation/emission of 350 nm/410 nm and y is the pyoverdine fluorescence punch biopsy tool. Treatments for each wound site were randomized, and were not measured at an excitation/emission of 400 nm/460 nm. performed blinded. Viable bacterial counts were determined from three randomly selected 6 mm punch biopsies from each wound site. Biopsies were weighed and Quantification of bacterial growth and polystyrene-attached biomass. Growth placed in separate sterile polypropylene culture test tubes containing 1 ml of PBS. curves were measured in microtitre plates on a SpectraMax M Series Multi-Mode All of the samples were homogenized using a Pro Scientific Bio-Gen Series Pro200 Microplate Reader (Molecular Devices) by measuring the absorbance at 600 nm 42 hand-held homogenizer for 45 s. The resulting solutions were serially diluted and (OD ) and by CFU counts. Adherent biomass was quantified using crystal violet . −1 600 plated on Pseudomonas isolation agar (Difco) with rifampicin (100 μg ml ) in at least Cells attached to the wells of the plate were washed three times with 0.9% NaCl and triplicate and incubated at 37 °C overnight. CFUs were calculated per gram of tissue. stained with 0.01% crystal violet for 15 min at room temperature. The wells were then washed three times with 0.9% NaCl and ethanol was then added to each well. Calculation of sample size. Statistical experts in the OSU Center for Biostatistics After 15 min, crystal violet staining was quantified by measuring absorbance at performed sample size calculations on the basis of power analyses. To determine 595 nm (OD595) and then normalized to the density of the culture (OD600) at 5 h. the minimum sample sizes necessary to detect some of the effects under investigation, data from previous preliminary studies were used. Although power Human cell culture. Authenticated mycoplasma-free HT-29 cells (ATCC, HTB38), analysis (based on α = 0.05, two-tailed tests) indicated that six animals per group a human carcinoma cell line with epithelial morphology, were obtained directly would be required to achieve statistical significance, 1–2 animals per group showed from the American Type Culture Collection (ATCC). Cell morphology was significant effects. As the data were not normally distributed, nonparametric routinely monitored to verify cell-line authenticity, and cells were periodically tested Kruskal–Wallis one-way ANOVA was used. for contamination by PCR. The cells were cultured in DMEM with GlutaMAX All care of laboratory animals was in accordance with institutional guidelines, (Gibco) supplemented with 10% fetal bovine serum (FBS, Gibco) at 37 °C in and approved by the Ohio State University Institutional Animal Care and Use 5% CO2 and 95% air. Cells were split 1:12 and passaged as the culture reached Committee (IACUC) under protocol 2012A00000041-R1. confluence. To prepare for co-culture with bacteria, HT-29 cells were detached from the growth surface with 0.25% trypsin and 1 mM EDTA (Gibco), resuspended in Statistical analysis. Unless noted otherwise, experiments were performed with at DMEM and FBS, counted, diluted to the appropriate density in DMEM and FBS, least three biological replicates consisting of at least three technical replicates, and and seeded in 96-well plates to confluence (approximately 5 105 HT-29 cells). × results are presented as mean ± s.e.m. Statistical significance was assessed using ordinary one-way ANOVA followed by Dunnett’s multiple comparisons test, or Examination of bacterial interactions with epithelial cells. In all of the one-sample Student’s t-tests for normalized data unless otherwise noted. Adjusted experiments, control and experimental HT-29 cells were treated identically except P values of <0.05 were considered to be significant. that the co-culture medium did not contain bacteria in the uninfected controls. Bacteria were co-cultured with HT-29 cell monolayers at an initial multiplicity of Reporting Summary. Further information on research design is available in the infection of 20 (1 107 CFU) for 1–8 h. Subsequently, HT-29 cells were processed × Nature Research Reporting Summary linked to this article. and assayed in the cell-function assays described below.

Analysis of bacterial attachment. After 1 h of co-culture, non-adherent bacteria in Data availability the supernatant were aspirated and quantified by serial dilution as CFUs. HT-29 cell High-throughput sequencing data presented in Figs. 1 and 4 are deposited in the monolayers were washed three times with phosphate-buffered saline (PBS, Gibco) Gene Expression Omnibus (GEO) under accession number GSE136097. All other and cells were lysed with 1% Triton X-100 (Sigma) and removed from the growth data that support the findings of this study are available from the corresponding surface. The attached bacteria were serially diluted in PBS and quantified as CFUs. author on reasonable request.

Nature Microbiology | VOL 4 | December 2019 | 2146–2154 | www.nature.com/naturemicrobiology 2153 Letters Nature Microbiology

Received: 19 September 2018; Accepted: 9 September 2019; 28. Frenkel, E. S. & Ribbeck, K. Salivary mucins protect surfaces from colonization Published online: 14 October 2019 by cariogenic bacteria. Appl. Environ. Microbiol. 81, 332–338 (2015). 29. Kavanaugh, N. L., Zhang, A. Q., Nobile, C. J., Johnson, A. D. & Ribbeck, K. Mucins suppress virulence traits of Candida albicans. mBio 5, References e01911-14 (2014). 1. Schroeder, B. O. Fight them or feed them: how the intestinal mucus layer 30. Caldara, M. et al. Mucin biopolymers prevent bacterial aggregation by manages the gut microbiota. Gastroenterol. Rep. 7, 3–12 (2019). retaining cells in the free-swimming state. Curr. Biol. 22, 2325–2330 (2012). 2. Sadikot, R. T., Blackwell, T. S., Christman, J. W. & Prince, A. S. Pathogen-host 31. Lieleg, O., Lieleg, C., Bloom, J., Buck, C. B. & Ribbeck, K. Mucin biopolymers interactions in Pseudomonas aeruginosa pneumonia. Am. J. Respir. Crit. Care as broad-spectrum antiviral agents. Biomacromolecules 13, 1724–1732 (2012). Med. 171, 1209–1223 (2005). 32. Huang, Y., Mechref, Y. & Novotny, M. V. Microscale nonreductive release of 3. Parsek, M. R. & Singh, P. K. Bacterial bioflms: an emerging link to disease O-linked glycans for subsequent analysis through MALDI mass spectrometry pathogenesis. Annu. Rev. Microbiol. 57, 677–701 (2003). and capillary electrophoresis. Anal. Chem. 73, 6063–6069 (2001). 4. Co, J. Y. et al. Mucins trigger dispersal of Pseudomonas aeruginosa bioflms. 33. Packer, N. H., Lawson, M. A., Jardine, D. R. & Redmond, J. W. A general NPJ Bioflms Microbiomes 4, 23 (2018). approach to desalting oligosaccharides released from glycoproteins. 5. Wagner, E., Wheeler, K. M. & Ribbeck, K. Mucins and their role in shaping Glycoconj. J. 15, 737–747 (1998). the functions of mucus barriers. Annu. Rev. Cell Dev. Biol. 34, 189–215 (2018). 34. Chen, F.-T. A. & Evangelista, R. A. Profling glycoprotein N-linked 6. Toyofuku, M. et al. Quorum sensing regulates denitrifcation in Pseudomonas oligosaccharide by capillary electrophoresis. Electrophoresis 19, aeruginosa PAO1. J. Bacteriol. 189, 4969–4972 (2007). 2639–2644 (1998). 7. Rohmer, L., Hocquet, D. & Miller, S. I. Are pathogenic bacteria just looking 35. Guttman, A. Analysis of monosaccharide composition by capillary for food? Metabolism and microbial pathogenesis. Trends Microbiol. 19, electrophoresis. J. Chromatogr. A 763, 271–277 (1997). 341–348 (2011). 36. Taniguchi, T. et al. N-Glycosylation afects the stability and barrier function 8. Jimenez, P. N. et al. Te multiple signaling systems regulating virulence in of the MUC16 mucin. J. Biol. Chem. 292, 11079–11090 (2017). Pseudomonas aeruginosa. Microbiol. Mol. Biol. Rev. 76, 46–65 (2012). 37. Afgan, E. et al. Te Galaxy platform for accessible, reproducible and 9. Balasubramanian, D., Schneper, L., Kumari, H. & Mathee, K. A dynamic and collaborative biomedical analyses: 2018 update. Nucleic Acids Res. 46, intricate regulatory network determines Pseudomonas aeruginosa virulence. W537–W544 (2018). Nucleic Acids Res. 41, 1–20 (2013). 38. Love, M. I., Huber, W. & Anders, S. Moderated estimation of fold change and 10. Van Der Reijden, W. A., Veerman, E. C. I. & Nieuw Amerongen, A. V. dispersion for RNA-seq data with DESeq2. Genome Biol. 15, 550 (2014). Rheological properties of commercially available polysaccharides with 39. Caballero, A., Tibodeaux, B., Marquart, M., Traidej, M. & O’Callaghan, R. potential use in saliva substitutes. Biorheology 31, 631–642 (1994). Pseudomonas keratitis: protease IV gene conservation, distribution, and 11. Jin, C. et al. Structural diversity of human gastric mucin glycans. Mol. Cell. production relative to virulence and other Pseudomonas proteases. Investig. Proteom. 16, 743–758 (2017). Ophthalmol. Vis. Sci. 45, 522–530 (2004). 12. Karlsson, N. G., Nordman, H., Karlsson, H., Carlstedt, I. & Hansson, G. C. 40. Howe, T. R. & Iglewski, B. H. Isolation and characterization of alkaline Glycosylation diferences between pig gastric mucin populations: a protease-defcient mutants of Pseudomonas aeruginosa in vitro and in a comparative study of the neutral oligosaccharides using mass spectrometry. mouse eye model. Infect. Immun. 43, 1058–1063 (1984). Biochem. J. 326, 911–917 (1997). 41. Dumas, Z., Ross-Gillespie, A. & Kümmerli, R. Switching between apparently 13. Holmen Larsson, J. M., Tomsson, K. A., Rodriguez-Pineiro, A. M., redundant iron-uptake mechanisms benefts bacteria in changeable Karlsson, H. & Hansson, G. C. Studies of mucus in mouse stomach, small environments. Proc. R. Soc. B 280, 20131055 (2013). intestine, and colon. III. Gastrointestinal Muc5ac and Muc2 mucin O-glycan 42. Junker, L. M. & Clardy, J. High-throughput screens for small-molecule patterns reveal a regiospecifc distribution. Am. J. Physiol. Gastrointest. inhibitors of Pseudomonas aeruginosa bioflm development. Antimicrob. Liver Physiol. 305, G357–G363 (2013). Agents Chemother. 51, 3582–3590 (2007). 14. Cummings, R. D. & Pierce, J. M. Te challenge and promise of glycomics. 43. Roy, S. et al. Mixed-species bioflm compromises wound healing by Chem. Biol. 21, 1–15 (2014). disrupting epidermal barrier function. J. Pathol. 233, 331–343 (2014). 15. Xia, B., Royall, J. A., Damera, G., Sachdev, G. P. & Cummings, R. D. Altered 44. Borlee, B. R. et al. Pseudomonas aeruginosa uses a cyclic-di-GMP-regulated O-glycosylation and sulfation of airway mucins associated with cystic fbrosis. adhesin to reinforce the bioflm extracellular matrix. Mol. Microbiol. 75, Glycobiology 15, 747–775 (2005). 827–842 (2010). 16. Nakano, M., Saldanha, R., Göbel, A., Kavallaris, M. & Packer, N. H. Identifcation of glycan structure alterations on cell membrane proteins in desoxyepothilone B resistant leukemia cells. Mol. Cell. Proteom. 10, Acknowledgements M111.009001 (2011). We thank S. Lory for comments and Life Science Editors for editing assistance. This 17. Lee, S. H. et al. Core2 O-glycan structure is essential for the cell surface research was supported by NIBIB/NIH grant R01 EB017755-04 (OSP 6940725), the expression of sucrase isomaltase and dipeptidyl peptidase-IV during intestinal National Science Foundation Career award PHY-1454673, funding from the Deshpande cell diferentiation. J. Biol. Chem. 285, 37683–37692 (2010). Center for Technological Innovation and the MRSEC Program of the National Science 18. Yamada, K. & Kinoshita, M. Comparative studies on the structural features of Foundation under award DMR-1419807 (to K.R.), NIH grant P41GM103694 (to R.D.C.) O-glycans between leukemia and epithelial cell lines. Proteome Res. 8, and NIEHS/NIH grant P30-ES002109. D.J.W. is supported by NIH grants R01AI34895 521–537 (2009). and R01AI097511. This material is based on research supported by the National Science 19. Varki, A. Biological roles of glycans. Glycobiology 27, 3–49 (2017). Foundation Graduate Research Fellowship under grant no. 1745302 and the MIT/ 20. Ventre, I. et al. Multiple sensors control reciprocal expression of Pseudomonas NIGMS Biotechnology Training Program grant 5T32GM008334-28 (to K.M.W.). G.C.-O. aeruginosa regulatory RNA and virulence genes. Proc. Natl Acad. Sci. USA is supported by the Early Postdoc Mobility Fellowship of the Swiss National Science 103, 171–176 (2006). Foundation (grant no. P2ZHP3_164844). 21. Basu Roy, A. & Sauer, K. Diguanylate cyclase NicD-based signalling mechanism of nutrient-induced dispersion by Pseudomonas aeruginosa. Author contributions Mol. Microbiol. 94, 771–793 (2014). K.M.W., G.C.-O., B.S.T., S.D.-N., J.Y.C., D.J.W., R.D.C. and K.R. designed the experiments. 22. Landry, R. M., An, D., Hupp, J. T., Singh, P. K. & Parsek, M. R. Mucin– K.M.W., G.C.-O., B.S.T., S.D.-N., J.Y.C. and S.L. conducted experiments. All of the authors Pseudomonas aeruginosa interactions promote bioflm formation and analysed the data and contributed to writing the manuscript. antibiotic resistance. Mol. Microbiol. 59, 142–151 (2006). 23. Secor, P. R., Michaels, L. A., Ratjen, A., Jennings, L. K. & Singh, P. K. Competing interests Entropically driven aggregation of bacteria by host polymers promotes The authors declare no competing interests. antibiotic tolerance in Pseudomonas aeruginosa. Proc. Natl Acad. Sci. USA 115, 10780–10785 (2018). 24. Cattoir, V. et al. Transcriptional response of mucoid Pseudomonas aeruginosa Additional information to human respiratory mucus. mBio 3, e00410-12 (2012). Supplementary information is available for this paper at https://doi.org/10.1038/ 25. Duan, K. & Surette, M. G. Environmental regulation of Pseudomonas aeruginosa s41564-019-0581-8. PAO1 las and Rhl quorum-sensing systems. J. Bacteriol. 189, 4827–4836 (2007). 26. Arora, S. K., Ritchings, B. W., Almira, E. C., Lory, S. & Ramphal, R. Te Correspondence and requests for materials should be addressed to K.R. Pseudomonas aeruginosa fagellar cap protein, FliD, is responsible for mucin Reprints and permissions information is available at www.nature.com/reprints. adhesion. Infect. Immun. 66, 1000–1007 (1998). Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in 27. Chua, S. L. et al. In vitro and in vivo generation and characterization of published maps and institutional affiliations. Pseudomonas aeruginosa bioflm-dispersed cells via c-di-GMP manipulation. Nat. Protoc. 10, 1165–1180 (2015). © The Author(s), under exclusive licence to Springer Nature Limited 2019

Corresponding author(s): Katharina Ribbeck

Reporting Summary Nature Research wishes to improve the reproducibility of the work that we publish. This form provides structure for consistency and transparency in reporting. For further information on Nature Research policies, see Authors & Referees and the Editorial Policy Checklist.

Statistical parameters When statistical analyses are reported, confirm that the following items are present in the relevant location (e.g. figure legend, table legend, main text, or Methods section). n/a Confirmed The exact sample size (n) for each experimental group/condition, given as a discrete number and unit of measurement An indication of whether measurements were taken from distinct samples or whether the same sample was measured repeatedly The statistical test(s) used AND whether they are one- or two-sided Only common tests should be described solely by name; describe more complex techniques in the Methods section. A description of all covariates tested A description of any assumptions or corrections, such as tests of normality and adjustment for multiple comparisons A full description of the statistics including central tendency (e.g. means) or other basic estimates (e.g. regression coefficient) AND variation (e.g. standard deviation) or associated estimates of uncertainty (e.g. confidence intervals)

For null hypothesis testing, the test statistic (e.g. F, t, r) with confidence intervals, effect sizes, degrees of freedom and P value noted Give P values as exact values whenever suitable.

For Bayesian analysis, information on the choice of priors and Markov chain Monte Carlo settings For hierarchical and complex designs, identification of the appropriate level for tests and full reporting of outcomes Estimates of effect sizes (e.g. Cohen's d, Pearson's r), indicating how they were calculated

Clearly defined error bars State explicitly what error bars represent (e.g. SD, SE, CI)

Our web collection on statistics for biologists may be useful.

Software and code Policy information about availability of computer code Data collection Zeiss ZEN 2.1 imaging software (Thornwoods, NY, USA).

Data analysis IMARIS 7.7.2 (Bitplane, Switzerland). PRISM (GraphPad Software). MATLAB R2016b. RStudio version 3.4.0 and the DESeq2 workflow (Love et al. 2014). Galaxy Servers (Afgan et al. 2016).

For manuscripts utilizing custom algorithms or software that are central to the research but not yet described in published literature, software must be made available to editors/reviewers upon request. We strongly encourage code deposition in a community repository (e.g. GitHub). See the Nature Research guidelines for submitting code & software for further information.

Data April 2018 Policy information about availability of data All manuscripts must include a data availability statement. This statement should provide the following information, where applicable: - Accession codes, unique identifiers, or web links for publicly available datasets - A list of figures that have associated raw data - A description of any restrictions on data availability

High throughput sequencing data presented in Figures 1, 4, and S1 were deposited to the Gene Expression Omnibus (GEO), accession code is included in the text and data will be available upon publication.

1 nature research | reporting summary Field-specific reporting Please select the best fit for your research. If you are not sure, read the appropriate sections before making your selection. Life sciences Behavioural & social sciences Ecological, evolutionary & environmental sciences For a reference copy of the document with all sections, see nature.com/authors/policies/ReportingSummary-flat.pdf

Life sciences study design All studies must disclose on these points even when the disclosure is negative. Sample size RNA-seq data were generated from 3 biological replicates. Supporting RNA-seq data presented in the supplement were generated using 2 biological replicates. All experiments performed in 96-well plates were performed in at least biological triplicate (exact number of replicates indicated by dots overlaying bars), and each replicate measure comprised of 3 technical replicates. In vivo experiments were performed on two pigs, 3 punch biopsies were collect from each and analyzed for bacterial load for a total of 6 replicates.

Data exclusions No data were excluded from the analysis.

Replication Experiments were performed at least in triplicate unless noted otherwise. Media, strain, and time points were varied for the RNA-seq experiments to confirm reproducibility under different experimental conditions.

Randomization Treatment application sites were randomized in the porcine burn wound infection experiments.

Blinding Blinding was not used in our study.

Reporting for specific materials, systems and methods

Materials & experimental systems Methods n/a Involved in the study n/a Involved in the study Unique biological materials ChIP-seq Antibodies Flow cytometry Eukaryotic cell lines MRI-based neuroimaging Palaeontology Animals and other organisms Human research participants

Unique biological materials Policy information about availability of materials Obtaining unique materials MUC5AC and MUC5B were purified by standard procedures described in the manuscript.

Eukaryotic cell lines Policy information about cell lines Cell line source(s) ATCC

Authentication HT-29 cells obtained from ATCC were certified authentic, authenticity was monitored based on morphology. April 2018 Mycoplasma contamination HT-29 cells obtained from ATCC were certified mycoplasma free.

Commonly misidentified lines No commonly misidentified lines were used. (See ICLAC register)

2 Animals and other organisms nature research | reporting summary Policy information about studies involving animals; ARRIVE guidelines recommended for reporting animal research Laboratory animals Yorkshire pigs

Wild animals This study did not involve wild animals.

Field-collected samples This study did not involve field-collected samples.

Human research participants Policy information about studies involving human research participants Population characteristics Whole saliva samples were collected from healthy volunteers. Volunteers were not screened for a particular age or gender. MUC5B was isolated and purified from pooled saliva samples.

Recruitment Participants were recruited by word-of-mouth, which could lead to selection bias. Specifically, participants were largely adults in their 20s-30s. This is unlikely to affect salivary mucin composition or the outcomes of this work. April 2018