Brachyspira hyodysenteriae Infection Regulates Mucin Glycosylation

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Brachyspira hyodysenteriae infection regulates mucin glycosylation synthesis inducing an increased expression of core 2 O-glycans in porcine colon Vignesh Venkatakrishnan, Macarena Paz Quintana-Hayashi, Maxime Mahu, Freddy Haesebrouck, Frank Pasmans, and Sara K. Lindén J. Proteome Res., Just Accepted Manuscript • DOI: 10.1021/acs.jproteome.7b00002 • Publication Date (Web): 16 Mar 2017 Downloaded from http://pubs.acs.org on March 20, 2017

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Journal of Proteome Research

Brachyspira hyodysenteriae infection regulates mucin glycosylation synthesis inducing an increased expression of core 2 O-glycans in porcine colon Vignesh Venkatakrishnan1, Macarena P.Quintana-Hayashi1, Maxime Mahu2, Freddy Haesebrouck2, Frank Pasmans2 and Sara K. Lindén1* 1

Department of Medical Biochemistry and Cell biology, Institute of Biomedicine, Sahlgrenska Academy, University of Gothenburg, Gothenburg, Sweden, 2Department of Pathology, Bacteriology and Poultry Diseases, Faculty of Veterinary Medicine, Ghent University, Belgium. *Corresponding author Dr. Sara K. Lindén Department of Medical Chemistry and Cell Biology University of Gothenburg Gothenburg SE-405 30, Sweden Email: [email protected] Tel: +46 31 786 3057 Fax: +46 31 786 2150

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ACS Paragon Plus Environment


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Running title: Swine dysentery induced mucin O-glycosylation changes

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Keywords Swine dysentery, Pig, Infection, Brachyspira hyodysenteriae, Colonic mucins, O-glycosylation, Core 2 Oglycans, NeuGc, Sialylation, Host-pathogen Abbreviations SD- Swine dysentery PGC- Porous graphitized carbon EIC- Extracted ion chromatogram Gal- Galactose GlcNAc- N-acetyl glucosamine GalNAc- N-acetyl galactosamine dHex- Deoxy hexose/ Fucose NeuAc- N-acetyl neuraminic acid NeuGc- N-glycolyl neuraminic acid Sul- Sulfate C1GalT- Core 1 β1-3 galactosyltransferase C2/4GnT- Core 2/4 β1-6 N-acetyl glucosaminyltransferase C3GnT- Core 3 β1-3 N-acetyl glucosaminyltransferase

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Abstract Brachyspira hyodysenteriae causes swine dysentery (SD), leading to global financial losses to the pig industry. Infection with this pathogen results in an increase of B. hyodysenteriae binding sites on mucins, along with increased colonic mucin secretion. We here predict that B. hyodysenteriae modifies the glycosylation pattern of the porcine intestinal mucus layer to optimize its host niche. We characterized the swine colonic mucin O-glycome and identified the differences in glycosylation between B. hyodysenteriae infected and non-infected pigs. O-glycans were chemically released from soluble and insoluble mucins isolated from five infected and five healthy colon tissues and analyzed using porous graphitized carbon liquid chromatography tandem mass spectrometry. In total, 94 O-glycans were identified, with healthy pigs having higher inter-individual variation although a larger array of glycan structures were present in infected pigs. This implied that infection induced loss of individual variation, and that specific infection related glycans were induced. The dominating structures shifted from core 4 type O-glycans in noninfected pigs towards core 2 type O-glycans in infected animals, which correlated with increased levels of the C2GnT glycosyl transferase. Overall, glycan chains from infected pigs were shorter and had a higher abundance of structures that were neutral and/or predominantly contained NeuGc instead of NeuAc, whereas they had a lower abundance of structures that were fucosylated, acidic, and/or sulfated than those from non-infected pigs. Therefore, we conclude that B. hyodysenteriae plays a major role in regulating colonic mucin glycosylation in pigs during SD. The changes in mucin O-glycosylation thus resulted in a glycan fingerprint in porcine colonic mucus that may provide increased exposure of epitopes important for host-pathogen interactions. The results from this study provide potential therapeutic targets and a platform for investigations of B. hyodysenteriae interactions with the host via mucin glycans.

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Introduction Host adapted bacterial pathogens result in a high disease burden in both humans and animals. As the result of host–pathogen co-evolution, adaptation of pathogens to a specific host generally results in a complex interaction that promotes long term pathogen sustenance in the host population. Host, pathogen and environmental determinants contribute to shaping the optimal niche for the pathogen with a variable cost on host fitness ranging from subclinical infections to severe disease and mortality. Controlling these infections using antimicrobial compounds is becoming increasingly problematic due to the global increase of acquired antimicrobial resistance, which has made the use of antimicrobial drugs and the search for alternatives a growing societal concern. Interfering with host–pathogen interactions that provide an optimal pathogen environment is a promising approach but requires in depth knowledge of host and pathogen determinants that shape the pathogen’s niche. Swine dysentery (SD) is a severe mucohemorrhagic enteric disease of pigs, causing losses by mortality and suboptimal growth performance in pig production across the world 1. The main causative agent for SD is Brachyspira hyodysenteriae, an anaerobic gram-negative spirochete, which colonizes the large intestine of pigs 2. B. hyodysenteriae infection is a re-emerging problem in the pig industry due to recent increases in antibiotic resistant strains 3. The presence of antibiotic resistant strains in pigs may lead to transfer of these resistance genes to bacteria of more zoonotic importance that colonize both pigs and humans, such as Brachyspira pilosicoli, and that cause disease in humans. The mucosal surface is covered by a mucus layer that acts as a first line of defense against invading pathogens 4. Mucus lubricates and protects the epithelial surface and trap viruses and bacteria 5. Heavily glycosylated mucin glycoproteins are the main components of the mucus layer. Invading pathogens often utilize lectin like adhesins to bind carbohydrates present on the epithelial cell surface. For example, Helicobacter pylori carries the blood group binding adhesin 6 and the sialic acid binding adhesin 7 that bind to Leb and sialylated glycans respectively in the human stomach, and Pseudomonas aeruginosa has PA IL and PA IIL 8 that bind to galactose and fucose in the human airway. 5



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Most bacteria are kept at a distance from the epithelial cell surface by the inner mucus layer, whereas the majority resides in the outer mucus layer 9. The glycans on mucins provide a vast array of structures that bacteria can adhere to, and mucins can act as a decoy for the more intimate adherence that occurs to the epithelial cell surface 10. During natural clearance of a murine colonic pathogen, Citrobacter rodentium, the inner MUC2 based mucus layer expands in an ordered fashion with striations parallel to the epithelial surface in the murine colon, indicating multiple layers of these net like structures expanding and transporting away trapped pathogens 11. Although B. hyodysenteriae infection also induces an increase in mucus thickness in the pig colon, this increase is associated with loss of the striated mucus organization, with the mucins flowing at a 45 degree angle from the epithelial surface 12. B. hyodysenteriae infection also induces de novo MUC5AC expression 12, 13, leading to the mucus being composed of both MUC2 and MUC5AC

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. This increase in the amount of mucins in addition to an increase in B. hyodysenteriae

binding sites on the mucins per se, leads to a vastly increased B. hyodysenteria binding ability in the mucus niche. However, due to the loss of mucus organization, it is not certain that the mucins can function as functional decoys. Possibly the vastly increased mucus layer may instead provide an expanded niche for the pathogen to proliferate in. Alterations in mucin glycosylation have been shown in the murine colon after a change to high fat diet, 14 and colonic biopsies from humans with ulcerative colitis had an aberrant glycosylation profile with increased levels of shorter glycan chains 15. Understanding how B. hyodysenteriae shapes its host niche opens perspectives for therapeutic strategies without use of antibiotics. Knowledge on the structural changes in glycosylation that occur during infection identifies the possible glyco-determinants that could be used for bacterial binding as well as in intervention therapies such as artificial adhesion decoys. With the advancement in the field of mass spectrometry (MS) and tandem MS over the past decade, structural elucidation of mucin glycans has become more feasible and accurate. The objectives of the present study were to characterize the mucin O-glycosylation profiles of mucins isolated from pig colons with and without B. hyodysenteriae infection, and identify the changes in glycan 6


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structures that occur during infection. Previous studies have focused on changes of particular glycan epitopes including sulfation and sialylation in diseased pigs and other animal models. To our knowledge, this is the first comprehensive glycan structural analysis determining the overall glycoepitope changes in porcine colon as a result of infection. This comprehensive mucin glycan characterization provides a reliable database of structures that are of putative importance for bacterial interactions with the host.

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Materials and Methods Ethics statement The animal experiments were approved by the ethical committee of the Faculty of Veterinary Medicine, Ghent University (EC2012/01 and EC2013/147) and complied with all ethical and husbandry regulations. Experimental inoculation and sample collection All pigs were negative for Salmonella sp. (analysed by culture) and all weakly hemolytic Brachyspira spp (analysed by culture and PCR) on arrival. Brachyspira innocens was detected in some animals of each group, and since shedding may be intermittent, was possibly present in all animals. The experimental inoculation procedure and sample collection was performed as previously described by Quintana-Hayashi et al.

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. Briefly, a total of five pigs developed swine dysentery after oral inoculation with brain heart

infusion broth (BHI) containing 108 CFU of B. hyodysenteriae strain 8dII, while control pigs received sterile BHI. Infection was confirmed by clinical signs of mucohemorrhagic diarrhea, and B. hyodysenteriae excretion in feces detected by qPCR. The scale used for fecal scores (scored twice per week) was: 0; normal, 1; softer but formed, 2; unformed semi-wet, 3; runny, 4; runny with mucus and blood. 0.5 was added to all scores if mucus or blood was present. The pigs were sacrificed at day 40 postinoculation by anesthesia with a combination of xylazine at 4.4 mg/kg (Xyl-M 2%®, VMD, Arendonk, Belgium) and zolazepam/tiletamine at 2.2 mg/kg (Zoletil® 100, Virbac, Carros, France), and final euthanasia by intracardial injection of a formulation comprising embutramide, mebezonium iodide and tetracaine (T61®, Intervet, Brussels, Belgium) at 0.3 ml/kg. Midsection samples of the spiral colon of the five infected pigs and healthy controls were collected for mucin isolation. Fecal material was removed, and the tissues were rinsed with phosphate buffered saline (PBS) containing protease inhibitor cocktail (Roche Diagnostics, Mannheim, Germany) followed snap freezing and storage at -80°C. Colon tissue samples were also collected in RNAlater® (Life Technologies, Carlsbad, CA, USA) and kept at 4°C overnight, then stored at -80°C for RNA extraction. For macroscopic imaging, sample of the apex of the colon was rinsed in cold PBS. Samples were fixed in 10% buffered formalin for histology, and to visualize the mucus layer by immunofluorescence, samples (not rinsed) were fixed in Carnoy´s fixative. 8


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Histology Fixed samples were paraffin embedded, sectioned at 4 µm and stained with hematoxylin and eosin or with Periodic Acid Schiff reagent (PAS). Images in which the crypts were perpendicular to the mucosal surface, were photographed using a Leica DM LB2 Digital (Leica Microsystems Belgium BVBA, Diegem, Belgium).

MUC2 and MUC5AC immunofluorescence Tissue sections were deparaffinized and antigen retrieval was performed in 10 mM sodium citrate, pH 6.0 at 99°C for 30 min. Slides were cooled to room temperature and washed in PBS. Non-specific background was blocked with serum-free protein block (DAKO, Carpinteria, CA, USA) for 20 min. Primary antibodies anti-MUC2C3 (kindly provided by G. Hansson, University of Gothenburg, Sweden) and antiMUC5AC (45M1, Sigma-Aldrich, St. Louis, MO, USA) were diluted 1/1000 and incubated at 4°C overnight. Sections were washed with PBS and incubated with secondary antibodies conjugated with Alexa Fluor 488 (Life Technologies, Eugene, OR, USA) for MUC2, and Alexa Fluor 594 for MUC5AC, diluted 1/500 for 1 h. After washing in PBS, specimens were mounted with ProLong® antifade containing DAPI (Life Technologies, Eugene, OR, USA). Pig and human gastric specimens were used as positive controls for MUC5AC staining. We have previously demonstrated that these antibodies recognize pig MUC5AC and MUC2, even though the antibodies were raised against human mucins 12. The antibodies both recognize isolated mucins and display a similar tissue distribution in pig and human stomach and colon 12. Fluorescence in situ hybridization of formalin-fixed tissue sections The protocol was performed as previously described by Boye et al 16, with slight modifications. Briefly, tissue sections were deparaffinized and dehydrated in sequential washes of 50%, 80% and 95% ethanol for 5 min each. A hybridization buffer (0.1% sodium dodecyl sulfate, 100mM Tris pH 7.4 and 0.9 M NaCl), and 2.5 ng/µl of a Brachyspira specific probe, fluorescently labeled with Alexa fluor 488

16

, and an

eubacterial probe (EUB338) labeled with Cy3.5 17, were added on the tissue sections and incubated in a

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humidified chamber overnight at 45°C. The slides were washed in hybridization buffer at 45˚C for 15 min followed by a second wash in 100 mM Tris pH 7.4 and 0.9 M NaCl at 45˚C for 15 min. Slides were briefly washed in distilled H2O and air dried prior to mounting with ProLong® antifade containing DAPI (Life Technologies, Eugene, OR, USA). Mucin isolation and purification Mucin isolation of colon tissue samples was performed by isopycnic density gradient centrifugation, providing guanidinium chloride (GuHCl) soluble and insoluble mucins

12

. Briefly, frozen tissues were

drenched with 10 mM sodium phosphate buffer, pH 6.5, containing 0.1 mM phenylmethylsulfonyl fluoride (AppliChem, Darmstadt, Germany). Once thawed, the mucosal surfaces were scraped with a microscope slide, dispersed with a Dounce homogenizer, and stirred slowly overnight at 4°C in ice-cold extraction buffer

consisting of 6

M GuHCl (AppliChem,

Darmstadt,

Germany),

5

mM

ethylenediaminetetraacetic acid (EDTA) (Sigma-Aldrich, St. Louis, MO, USA), 5 mM N-ethylmaleimide (Alfa Aesar, Karlsruhe, Germany) and 10 mM sodium di-hydrogen phosphate at pH 6.5. GuHCl soluble mucins were obtained after centrifugation at 23000 × g for 50 min at 4°C and the remaining material was re-extracted twice by stirring overnight at 4°C in extraction buffer. The remaining pellets contained the “insoluble” mucins, which were solubilized with 10 mM dithiothreitol (DTT) in reduction buffer (6 M GuHCl, 5 mM EDTA, 0.1 M Tris/HCl, pH 8) for 5 h at 37°C. Finally, residues were alkylated overnight with 25 mM iodoacetamide (IAA, Alfa Aesar, Karlsruhe, Germany). Both the GuHCl soluble and insoluble material was dialyzed in ten volumes of extraction buffer at 4°C, changing the dialysis solution three times in 24 h. An isopycnic density-gradient centrifugation in cesium chloride (CsCl)/4 M GuHCl with a starting density of 1.39 g/ml was performed at 40000 rpm for 90 h. The mucin containing fractions (determined based on peak glycan values at a density of approximately 1.5 g/ml, see below method for the glycan and density assays) were pooled and further purified from DNA by a second gradient in CsCl/0.5 M GuHCl. Approximately 25 fractions were recovered per sample using a fraction collector equipped with a drop counter. Fractions were stored at 4°C until further analysis.

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Analysis of mucin fractions First and second CsCl gradient mucin fractions were analyzed as previously described

12

. The mucin

density was determined by weighing a known volume using a Carlsberg pipette as a pycnometer; results were expressed as g/ml. A microtiter-based assay detecting carbohydrates as periodate-oxidizable structures

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was performed in order to determine the glycan content in the GuHCl soluble and insoluble

mucin samples. Briefly, Nunc® 96-well plates (Thermo Scientific, Waltham, MA, USA) were coated overnight at 4°C with mucin fractions diluted in 4 M and 0.5 M GuHCl. Plates were incubated with a 25 mM sodium metaperiodate solution diluted in sodium acetate (NaAc) for 20 min, and blocked with 50 mM Tris-HCl, 0.15 M NaCl, 90 µM CaCl2, 4 µM EDTA, 0.01% sodium azide (NaN3) and 2 % bovine serum albumin, at pH 8 for 1 h. The wells were then incubated for 1 h with a biotin hydrazid solution diluted 1/50 in NaAc, followed by europium labeled streptavidin diluted 1/1000 in DELFIA® Assay buffer (PerkinElmer, Waltham, MA, USA). Finally, plates were incubated with DELFIA® enhancement solution for 5 min on a shaker. Between each step the plates were washed three times with a solution containing 5 mM Tris-HCl, 0.15 M sodium chloride, 0.005 % Tween 20, and 0.01 % NaN3, at pH 7.75, except for the final step where plates were washed six times. Signal was measured in a Wallac 1420 VICTOR2 microplate reader (PerkinElmer, Waltham, MA, USA) by time-resolved fluorometry. Mucin sample preparation and concentration estimation Mucin sample preparation and concentration estimation were performed as previously described

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.

Briefly, gradient fractions containing mucins were pooled to obtain one sample for each gradient. Mucin concentration in pooled samples were determined by serial dilutions as well as a standard curve of a fusion protein of the mucin MUC1, 16TR and IgG2a Fc, starting at a concentration of 20 mg/ml and using seven 1/2 serial dilutions using the glycan detection assay described above. The mucin concentration was calculated from the standard curve. Setting the mucin concentration based on the carbohydrate content appears most appropriate as the focus of the current study is the glycans, and ensuring the same amount of glycan was used for all comparisons. Although this is not an absolute measure of glycoprotein

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concentration it can be used to ensure that the mucins are at the same concentration for comparative assays. Release of O-glycans from pig colon mucins O-glycans were released from soluble and insoluble mucins isolated from five healthy and five B. hyodysenteriae infected pigs, and analyzed using mass spectrometry as shown in 19. Approximately 100 µg colonic mucins per sample was dot blotted to a PVDF membrane (Immobilon P membranes, Millipore, Billerica, MA). Immobilized protein spots were stained and visualized with Alcian blue (Sigma-Aldrich). Stained spots were cut and subjected to reductive β-elimination with 0.5 M sodium borohydride and 50mM sodium hydroxide for 16 h at 50°C to release the glycans. The reduction reaction was quenched by the addition of glacial acetic acid to the mixtures and desalted as the non-retained fraction of strong cation exchange resin packed as a micro-column on top of hydrophobic C18 material. The solid-phase extraction removed cations and any protein or peptide component remaining. Excess borate was extracted as methyl esters by repeated evaporation. PGC-LC-MS/MS characterization of O-glycans Released O-glycans were analyzed by liquid-chromatography-tandem mass spectrometry (LC-MS/MS) using a 10 cm X 250 µm i.d. column (in-house), containing 5 µm porous graphitized carbon (PGC) particles (Thermo Scientific, Waltham, MA, USA) connected to an LTQ mass spectrometer (Thermo Scientific). O-glycans were eluted using a linear gradient from 0 to 40% acetronitrile in 10 mM ammonium bicarbonate over 40 min at a flow rate of 250 nl/min. Electrospray ionization-mass spectrometry (ESI-MS) was performed in negative ion polarity with an electrospray voltage of 3.5 kV, capillary voltage of -33.0 V, and capillary temperature of 300°C. The following scan events were used: MS full scan (m/z 380-2000) and data-dependent tandem MS (MS/MS) scans after collision-induced dissociation (CID) on precursor ions at a normalized collisional energy of 35% with a minimum signal of 300 counts, isolated width of 2.0 m/z, and activation time of 30 ms. The data were viewed and manually analyzed using Xcalibur software (version 2.2, Thermo Scientific).

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The monosaccharide compositions and each O-glycan structures were manually analyzed using monoisotopic mass, MS/MS CID fragmentation profiles and PGC-LC retention time. The relative abundance of each glycan was calculated based on the integrated peak areas obtained from LC-MS chromatograms and was represented as a percentage of the total peak area to be used for relative quantitation. Each identified glycan was characterized based on its retention on LC column and the fragmentation pattern on MS/MS

20, 21

. Common MS/MS diagnostic ions to distinguish isomer variations

and structural determination were used based on previously available knowledge-base and in-silico fragmentation pattern from Glyco Workbench 22-24. In order to identify the overall length of the glycans in control and infected pigs, the number of monosaccharides in each identified glycan was added and the relative abundances were calculated. qPCR analysis for core enzyme expression Isolation of RNA from pig colon tissue samples was performed using Trizol (Life Technologies, Carlsbad, CA, USA) according to the manufacturer's instructions. RNA yield and purity was assessed through UV spectroscopy (NanoDrop, Thermo Scientific, MA, USA). Total RNA (5 µg) was DNase treated at 37°C for 45 min, followed by the addition of 5 mM EDTA and heat inactivation of DNase at 75°C for 10 min prior to cDNA synthesis. Magnesium chloride was added to a 5 mM final concentration, and this RNA was used for cDNA synthesis with oligo-dT and Superscript III (Life Technologies, Carlsbad, CA, USA) at 50°C for 2 h. The cDNA was used in a real-time PCR reaction using EvaGreen® supermix (Bio-Rad Laboratories Inc., Hercules, CA, USA) and primers for pig core 1 synthase glycoprotein-Nacetylgalactosamine,

3-beta-galactosyltransferase1

acetylglucosaminyltransferase

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genes

designed

and using

UDP-GlcNAc:betaGal the

Primer3

program

beta-1,3-N(available

at

http://frodo.wi.mit.edu/primer3/). qPCR data were normalized using the expression levels of ACTB and RPL4 reference genes

25

. Samples were amplified in triplicate, and a negative control without reverse

transcriptase was included to verify the absence of contaminating genomic DNA. Data acquisition and analysis was performed using the CFX manager 3.1 software (Bio-Rad Laboratories Inc., Hercules, CA, USA). 13



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Statistical analysis Statistical analysis was performed using GraphPad Prism version 6 software (La Jolla, CA, USA). Comparisons of qualitative frequency data were performed using Fisher’s exact test (two tailed) and quantitative data are expressed as the median ± interquartile range and analyzed with the Mann-Whitney U-test. The Pearson product-moment correlation coefficient was calculated on means for the groups for each glycan to investigate how similar data sets from different groups were. P values ≤ 0.05 were considered as statistically significant.

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Results Experimental design Soluble and insoluble mucins were isolated and purified from colonic tissues from five experimentally B. hyodysenteriae infected pigs and five non-infected healthy control pigs. Infected pigs developed clinical signs of SD, including mucoid and/or hemorrhagic diarrhea and the pigs excreted B. hyodysenteriae in their feces (Figure 1A and B). Necrotic lesions and excessive mucus was visible to the eye (Figure 1C) and histologically the tissue displayed elongated crypts and an abundance of PAS positive material (stains mainly mucin glycans, Figure 1D). One of the five infected pigs (Pig 1) had experienced a short duration of SD with milder diarrhea and milder clinical signs at the time of necropsy, compared to the majority of the infected pigs having acute dysentery (Figure 1). Pig 1 showed lesions in the proximal and mid colon: thickened rugae with flocks of mucus were present as well as small areas of necrosis. Pig 2 showed thickened rugae and patches of necrosis in the proximal colon. In the mid colon large areas of necrosis were seen and the mucosa was covered with fibronecrotic material. Pig 3 showed lesions in the proximal and mid colon, consisting of thickened rugae, small areas of necrosis and excessive mucus. Pig 4 showed lesions in the entire colon, though more prominent in the mid colon. Thickened rugae and excessive mucus were noted. Pig 5 showed lesions mainly consisting of thickened mucosa and patches of necrosis in the proximal and mid colon. In our previous study on mucins from these pigs we identified an increase in mucin (MUC5AC and MUC2) expression and mucin binding ability to B. hyodysenteriae in inoculated pigs with clinical signs of SD compared to healthy pigs 12. In Table 1, we have summarized factors such as duration of SD (all pigs were harvested day 40 post infection, but individuals differed in time to onset of disease), level of mucin expression and B. hyodysenteriae binding for each pig, and we relate the glycosylation results in the current study to these data. From the isolated soluble and insoluble mucins from ten pigs, O-glycans were released by reductive β-elimination and analyzed on PGC-LC-ESI-negative ion-tandem mass spectrometry (MS/MS). One of the insoluble mucin samples from a healthy pig (Pig A), was excluded due to poor quality spectral data. Molecular mass, tandem mass spectra and retention on

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PGC column along with the theoretical monosaccharide composition were used to elucidate the O-glycan structures. The set of validating MS/MS spectra can be found in an annotated form in Supplementary file S1. Identified glycan compositions without good tandem mass spectra with low confident structural identification were not included for qualitative or relative quantitative analysis. These putative structures constituted less than 4% of the soluble mucin glycans and less than 3% of the insoluble mucin glycans. B. hyodysenteriae is present in the mucus, at the epithelial surface and within epithelial cells B. hyodysenteriae has been previously demonstrated to adhere to rat and pig intestinal epithelial cell lines (IEC-18 and IPEC-J2) but not to a human colorectal cell line (HRT-18)

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, indicating it can adhere to

epithelial surfaces in a host specific manner. Furthermore, B. hyodysenteriae adheres to mucins in a manner that differs between pigs

12

. Here we show that B. hyodysenteriae locates to the mucus, the

epithelial surface and sometimes even within cells (Figure 2), which makes it important to study mucin glycosylation in B. hyodysenteriae infected pigs. A total of 94 O-glycan structures were identified, whereof the infected pigs carried more structures but had less individual variation In total, 94 O-glycan structures were identified from five infected and five healthy soluble and insoluble pig mucin samples. Out of those, 78 glycans were common to both the infected and healthy pig groups (i.e. present at least in one pig in each pig group, Figure 3a), 14 structures were identified in infected pigs only whereas two glycans were identified in healthy pigs only (Figure 3a). Removing infected pig 1 (which only experienced one day of SD and below is shown to have a glycosylation pattern more similar to the healthy pigs) from the group of infected pigs only increased the number of glycans present in the healthy pig group to four. The inter-individual variation of glycan structures was higher among healthy pigs than among infected pigs (P

Brachyspira hyodysenteriae Infection Regulates Mucin Glycosylation

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