Exploring the Arctic charr intestinal glycome: evidence for increased N

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Exploring the Arctic charr intestinal glycome: evidence for increased N-glycolylneuraminic acid levels and changed host-pathogen interactions in response to inflammation Vignesh Venkatakrishnan, János T Padra, Henrik Sundh, Kristina Sundell, Chunsheng Jin, Markus Langeland, Hanna Carlberg, Aleksandar Vidakovic, Torbjörn Lundh, Niclas G. Karlsson, and Sara K. Lindén J. Proteome Res., Just Accepted Manuscript • DOI: 10.1021/acs.jproteome.8b00973 • Publication Date (Web): 08 Mar 2019 Downloaded from http://pubs.acs.org on March 16, 2019

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Journal of Proteome Research

Exploring the Arctic charr intestinal glycome: evidence for increased N-glycolylneuraminic acid levels and changed host-pathogen interactions in response to inflammation

Vignesh Venkatakrishnan1, János T. Padra1#, Henrik Sundh2#, Kristina Sundell2, Chunsheng Jin1, Markus Langeland3, Hanna Carlberg4, Aleksander Vidakovic3, Torbjörn Lundh3, Niclas G. Karlsson1 & Sara K. Lindén1* 1Department

of Medical Chemistry and Cell Biology, Institute of Biomedicine, Sahlgrenska Academy, University of Gothenburg, Gothenburg, Sweden 2Department

of Biological and Environmental Sciences, University of Gothenburg, Gothenburg, Sweden

3Department

of Animal Nutrition and Management, Swedish University of Agricultural Science, Uppsala,

Sweden Department of Wildlife, Fish, and Environmental Studies, Swedish University of Agricultural Science, Umeå, Sweden 4

#These authors contributed equally *Corresponding author Prof. 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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Abbreviations 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 FFSB- Full fat grounded soy bean GuHCl- Guanidinium Hydrochloride PMSF- Phenylmethylsulfonyl fluoride

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Abstract Disease outbreaks are a limiting factor for sustainable development of the aquaculture industry. The intestinal tract is covered by a mucus layer mainly comprised by highly glycosylated proteins called mucins. Mucins regulate pathogen adhesion, growth and virulence and the glycans are vital for these functions. We analyzed intestinal mucin O-glycans on mucins from control and full-fat extruded soy bean (known to cause enteritis) fed Arctic charr using liquid chromatography-tandem mass spectrometry. In total, 56 glycans were identified on Arctic charr intestinal mucins, with a high prevalence of core 5 type and sialylated O-glycans. Disialic acid epitope containing structures including NeuAcɑ2,8NeuAc, NeuAc(Gc)ɑ2,8NeuGc(Ac) and NeuGcɑ2,8NeuGc were the hallmark of Arctic charr intestinal mucin glycosylation. Arctic charr fed with soy bean meal diet had lower i) number of structures detected, ii) inter-individual variation and iii) Nglycolylneuraminic acid containing glycans compared to control Arctic charr. Furthermore, Aeromonas salmonicida grew less in response to mucins from inflamed Arctic charr than from the control group. The Arctic charr glycan repertoire differed from that of Atlantic salmon. In conclusion, loss of Nglycolylneuraminic acid may be a biomarker for inflammation in Arctic char, and inflammation induced glycosylation changes affect host-pathogen interactions.

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Keywords Arctic charr, Atlantic salmon, Core 5 O-glycans, Disialic acids, Distal intestine, Liquid chromatographytandem Mass spectrometry, Mucin O-glycans, NeuGc, Sialic acid, Soybean diet

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Introduction Aquaculture is one of the fastest growing food sectors in the world, and accounts for more than 50% of the fish consumed globally (FAO, 2018). Arctic charr (Salvelinus alpinus) is interesting for the aquaculture in cold climates since it is the most cold-adapted species within the salmonid family 1. The Arctic charr has therefore become an economically important aquaculture species in the temperate countries of the Northern Hemisphere. The gastrointestinal tract of fish is important for regulation of ion and water balance, nutrient uptake and as a barrier towards the environment. The GI-tract is also a major route for several pathogenic infections 2-3. Its function as a barrier is likely to be affected by changes in diet, which underlines the importance of appropriate composition and nutrition in feed for cultured fish. Soybean meal (SBM) in replacement for fish meal, is a common protein source in aqua feeds due to its high protein content and favorable amino acid profile. Atlantic salmon, (Salmo salar L.) fed full-fat or solvent extracted SBM diet develop enteritis in the distal part of the intestine characterized by shorter mucosal folds, a widening of the central stroma within the mucosal folds, increased amounts of connective tissue, increased infiltration of inflammatory cells in the lamina propria and increased amounts of goblet cells 4-6. These histological symptoms appear concurrent with impaired intestinal barrier function towards ions and small uncharged molecules and a state of diarrhea 6.

Replacing a 100% fish meal (FM) diet with 50% SBM in aqua feed for rainbow trout (Oncorhynchus

mykiss), lead to enteritis-like effects with damaged villi, microvilli and an altered microbiota 7, though the enteritis symptoms are described to be milder in rainbow trout compared to Atlantic salmon 8. The intestinal epithelium is covered by secreted mucus that act as an extrinsic barrier and protects the epithelial surface cells 9. The mucus layer consists of secreted, heavily O-glycosylated proteins called mucins and is the first line of defense against incoming pathogens 9-13. The molecular weight of mucins can range in MDa, and mucins carry regions rich in serine and threonine, which get extensively O-glycosylated, leading to that glycans comprise over 70% of the mucin weight 11.

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Mucin O-glycans are central to host-pathogen interactions, affecting bacterial adhesion and proliferation 14: Helicobacter pylori binds to blood group antigens and sialylated glycans on the human gastric mucosa

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and Pseudomonas aeruginosa has affinity to bind to galactosylated and fucosylated glycan structures on human lung mucins 16. Furthermore, the fish pathogen Aeromonas salmonicida adheres differentially to skin and intestinal mucins of Atlantic salmon in a sialic acid dependent manner 17 and Atlantic salmon intestinal mucins enhance A. salmonicida growth through N-acetylhexosamine containing O-glycans 18. Alterations in mucin glycosylation can occur during infection and/or inflammation in mammals, which in turn may affect the host-pathogen interaction: H. pylori infection in primates induces time dependent glycosylation changes of mucin structures H. pylori adheres to 13 and Brachyspira hyodysenteriae infection in pigs result in increased binding sites for the pathogen and increased core-2 O-glycans in the porcine colon 19-20. In fish, changes in mucus composition have been detected after exposure to bacteria and stress 21-23, but information regarding the effect of intestinal inflammation and/or infection on mucin O-glycan repertoire is lacking. Lectin binding and histochemistry have been used to identify glycans in the fish gastrointestinal tract 24, but the only available detailed structural teleost (Atlantic salmon) mucin O-glycome was recently characterized 25.

O-glycosylation signatures of skin and intestinal mucins in Atlantic salmon were distinctly different, with

skin having shorter and less diverse structures compared to intestine. Atlantic salmon intestinal mucins carried N-acetylneuraminic acid (NeuAc) core 5 as the most dominant glycan, and N-glycolylneuraminic acid (NeuGc) structures were not identified 25. Three O-glycans with di-NeuAc (or disialic acid) motif were identified in distal intestinal mucins purified from Atlantic salmon 25. In another study of Atlantic salmon skin, two cysteine proteinase inhibitors were identified, carrying a disialic acid motif (NeuAcα2,8NeuAc) on N-glycans and sialylated core 1 O-glycans 26. An oligo-sialic acid motif was also identified in N- and Oglycans released from the eggs of zebrafish (Danio rerio) 27-28. The main aims of the present study were to characterize the Arctic charr intestinal mucin O-glycosylation in health and during inflammation and to investigate if inflammation induced O-glycosylation changes affect host-pathogen interactions. This was accomplished through the following objectives: to assess the 6


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impact of full-fat ground soy bean (FFSB) on intestinal health of Arctic charr, define the Arctic charr intestinal mucin O-glycosylation pattern, to determine how it is affected by inflammation as well as to investigate how detected differences in glycosylation affect A. salmonicida growth response and adhesion to mucins. Finally, the Arctic charr intestinal mucin O-glycosylation repertoire was compared with that of Atlantic salmon.

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Methodology Experimental set-up and intestinal sampling The Arctic charr experiment was carried out at Kälarne Research Station (Vattenbrukscentrum Norr AB, Sweden) using Arctic charr from the Swedish breeding programme designated ‘Arctic superior’ 29. In total, 120 fish (183.3g ± 5.1 (mean ± SEM)) were anesthetised in buffered MS-222 (100 mg/l; tricaine methane sulphonate, MS-222, Western Chemical Inc., Ferdale, WA, USA) and randomly allocated in triplicate groups (20 fish per tank) to six 700 litres flow-through fibreglass tanks, supplemented with 10 l/min of water. The average temperature during the experiment was 5.8°C ± 1.8. The experiment was carried out in compliance with laws and regulations concerning experiments with live animals overseen by the Swedish Board of Agriculture and approved by the Ethical Committee for Animal Experiments in Umeå, Sweden (license no. A62-10). Fish were fed two different fish meal based diets (Table 1) for 43 days containing either soybean concentrate and soy bean oil (control) or full-fat ground soybean (FFSB) to induce intestinal inflammation. The diets were extruded at the Finnish Game and Fisheries Research Institute (Laukaa Research Station, Finland) on a twin-screw extruder (3 mm die, BC-45 model, Clextral, Creusot Loir, France). During the extrusion process, 20% of additional moisture was added to the feed mash, which was heated to 120-130°C for 30s, dried overnight by warm air and then sprayed with lipids using a vacuum coater (Pegasus PG-10VC, Dinnissen, Sevenum, Netherlands). Fish were fed the experimental diets distributed by automatic feeders (Arvo-Tec T 2000, Huutokoski, Finland). All fish were fed a predetermined amount of feed (1.5% of total biomass in each tank) based on the theoretical energy requirements of the fish 30. This amount was observed to be close to the maximum voluntary feed intake of the fish. The feed ration was increased weekly based on a specific growth rate (SGR) of 0.7 at 6°C 31. At sampling, fish were anaesthetised in buffered MS-222 (200 mg/l) and quickly killed with a sharp blow to the head. The distal region of the intestine, posterior to the ileo-rectal region where SBM previously has been shown to induce intestinal inflammation in Atlantic

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salmon 4-6, was sampled and subjected to either histological evaluation of inflammation, intestinal

barrier function or mucin analyses described below. Due to the small size of the region, different fish were used for histology, using chamber experiments and mucin isolation. Ingredients

Diet Control 450 100 42 33 120 86 40 120 5 5

meal1

Fish Fish oil Rapeseed oil Soybean oil Wheat gluten2 Wheat meal2 Cellulose Soy protein concentrate Soybean meal3 Full fat soybean4 Mineral & vitamin premix Titanium dioxide

FFSB 450 100 30 100 70 40 200 5 5

Table 1: Feed composition (g kg-1 dry matter) of the control and full fat soybean (FFSB) diets. Icelandic low temperature capelin meal. Group, Raisio Finland. 3 Hexane extracted GMO-free soybeans, IMCOSOY®, Imcopa Food Ingredients B.V. Netherlands. 4 Ground, non-heat treated GMO free soybeans, Imcopa Food Ingredients B.V. Netherlands. 1

2 Raisio

Histological evaluation of inflammation On one set of fish (n=9/group, three fish from each of the three replicate tanks per group), the corresponding region of the distal intestine was fixed in 3.7% PFA for 24 h and stored in 70% ethanol until further processing. Tissues were dehydrated and embedded in paraffin using standard procedures. Intestinal sections (7 μm) were produced with a Shandon finesse microtome (Thermo Fisher Scientific, Waltham, MA, USA) and mounted on 3′-aminopropyltriethoxysilane (APES; Sigma-Aldrich)-coated slides, dried at 37°C for 24 h. One set of slides were stained with a combination of haematoxylin-eosin and Alcian blue 8 GX, pH 2.5 and used for assessment of inflammatory status according to the scoring system reported here

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Another set of slides were stained with periodic-acid-Schiff/Alcian blue (pH 2.5) to enumerate mucus

producing goblet cells. The sections were examined in a Nikon eclipse E1000 microscope and photographs taken with a Nikon DXM1200 camera (Nikon Instruments Europe, Amsterdam, Netherlands). In addition, morphometric assessment of mucosal fold height and width (μm) at both the widest and the narrowest part of the villi and goblet cells per mm epithelium were performed on four non-overlapping areas per intestine and fish using Biopix imaging software (Biopix AB, Gothenburg, Sweden). Histological samples were blinded before evaluation. Intestinal barrier function Intestinal barrier function was determined as transepithelial electrical resistance (TER) and apparent permeability towards mannitol (Papp) in a set of custom-made Ussing chambers

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using modifications as

described 33. In brief, 4 fish from each tank were randomly sampled in one quick dip netting and immediately anesthetized as previously described and killed with a sharp blow to the head. The distal intestine was sampled and placed in ice-cold Ringer solution 34. The serosa was peeled off and the intestinal segments were mounted in the chambers and were allowed 60 min of recovery for stabilization of the electrical parameters. Thereafter, the experiment was started by renewing the Ringer solution (4 ml in each halfchamber) on the serosal side and replacing the Ringer solution on the mucosal side with Ringer containing 14C-mannitol

(specific activity, 0.042 MBq ml−1). A 100 μl portion of the serosal Ringer was sampled at

time points 0, 20, 30, 60, 80 and 90 min. Radioactivity was assessed in a liquid scintillation counter (Wallac 1409 Liquid Scintillation Counter, Turku, Finland) after adding 5 ml Ultima Gold (PerkinElmer). The rate of accumulation of 14C-mannitol on the serosal side was used to assess the Papp of the intestinal segments according to Equation (1): Papp = dQ/dt x 1/ACo

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where dQ/dt is the appearance rate of

14C-mannitol

in the serosal compartment (mol s−1) of the Ussing

chamber, A is the area of intestinal surface exposed in the chamber (0.75 cm2) and Co is the initial concentration on the mucosal side (mol ml−1). Isolation and purification of mucins The intestinal mucus and mucosa were separated from the underlying muscle and serosa tissue by scraping the mucosa off using a microscope slide. The mucosal scrapings were snap-frozen in liquid nitrogen and stored at -80°C until further processing. Upon analysis, the scrapings were placed in five volumes of extraction buffer (6 M Guanidinium Hydrochloride (GuHCl), 5 mM EDTA, 10 mM sodium phosphate buffer, pH 6.5, containing 0.1 M phenylmethylsulfonyl fluoride (PMSF)), homogenized with Dounce homogenizer (four strokes with a loose pestle) and stirred slowly at 4°C, overnight. The insoluble material was removed by centrifugation at 23,000 × g for 50 min at 4°C (Beckman JA-30 rotor) and the pellet was re-extracted twice with 10 ml extraction buffer. The supernatants from these three extractions were pooled and contained the GuHCl soluble mucins used in the subsequent assays. Due to the highly O-glycosylated nature of mucins (70-90% carbohydrate content), they can be separated from other proteins based on density. Distal intestinal scrapings of Arctic charr were subjected to isopycnic density-gradient centrifugation to separate the mucins from less glycosylated proteins and lipids of lower density, as well as from nucleic acids of higher density. The samples were dialyzed twice against ten volumes of extraction buffer and filled up to 26 ml with extraction buffer. CsCl was added to the samples by gentle stirring and the samples were transferred to Quick Seal ultracentrifuge tubes (Beckman Coulter). The tubes were filled with 10 mM NaH2PO4 to give a starting density of 1.35 g/ml and samples were subjected to density gradient centrifugation at 40,000 × g for 90 h at 15°C. The fractions were collected from the bottom of the tubes with a fraction collector equipped with a drop counter. Density gradient fractions of purified mucin samples were analyzed for carbohydrates as periodate-oxidizable structures in a microtiter-based assay: Fractions diluted 1:100, 1:500 and 1:1000 in 4 M GuHCl were coated on 96-well plates (PolySorpTM, NUNC A/S, Roskilde, Denmark) and incubated overnight at 4°C. The rest of the assay was carried out at 23-24°C. After washing 11



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three times with washing solution (5 mM Tris-HCl, 0.15 M NaCl, 0.05% Tween 20, 0.02% NaN3, pH 7.75), the carbohydrates were oxidized by adding 25 mM sodium metaperiodate in 0.1 M sodium acetate buffer, pH 5.5, for 20 min. The plates were washed again and the wells were blocked with DELFIA blocking solution (50 mM Tris-HCl, 0.15 M NaCl, 90 mM CaCl2, 4 mM EDTA, 0.02% NaN3, 0.1% BSA, pH 7.75) for 1 h. After further washing steps, the samples were incubated for 1 h with 2.5 mM biotin hydrazide in 0.1 M sodium acetate buffer, pH 5.5, followed by another washing step. Europium-labeled streptavidin was diluted 1:1,000 in Delfia assay buffer (PerkinElmer; 50 mM Tris-HCl, 0.15 M NaCl, 20 mM DTPA, 0.01% Tween 20, 0.02% NaN3, 1.5% BSA, pH 7.75) and was added to the wells. After 1h incubation, the plates were washed six times and then incubated with Delfia enhancement solution (PerkinElmer; 0.05 M NaOH, 0.1 M phthalate, 0.1% Triton X-100, 50 mM TOPO, 15 mM b-NTA) for 5 min at 120 rpm on an orbital shaker. Fluorescence (λexcitation=340 and λemission=615) was measured using a Wallac 1420 VICTOR2 plate reader with the Europium label protocol (PerkinElmer, Waltham, MA, USA). Density measurements were performed using a Carlsberg pipette as a pycnometer: 300 µl of sample was aspirated into the pipette, weighed and density was calculated as g/ml. Preparation of mucin samples Gradient fractions containing mucins were pooled together to obtain one sample for each gradient. Mucin concentrations in pooled samples were determined based on their carbohydrate content: serial dilutions of the samples were compared with a standard curve prepared from a fusion protein, constructed from MUC1, 16TR and IgG2a Fc. The standard curve started at a concentration of 20 mg/ml and followed by seven 1:2 serial dilutions. The carbohydrates were detected as periodate-oxidizable structures in a microtiter-based assay as described above. Release of O-glycans from purified mucins Approximately 100 µg of mucins purified from differentially fed Arctic charr intestine were dot blotted on PVDF membrane (Millipore) and stained using Alcian blue 8GX solution in acetic acid, excised and

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transferred to an Eppendorf tube. Subsequently, the Alcian blue (Sigma-Aldrich) stained spots were subjected to reductive β-elimination with 0.5 M sodium borohydride in 50 mM sodium hydroxide for 16 h at 50°C to release the O-glycans. The reduction reaction was quenched by the addition of glacial acetic acid to the mixtures and desalted using strong cation exchange resin packed on top of C18 ziptip column (Millipore). 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 × 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). Oglycans 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). Using this approach, mucin O-glycan structures were characterized with high confidence based on their molecular mass, monosaccharide composition, MS/MS fragmentation pattern and their retention time on PGC column. In addition to that, partial linkage information between monosaccharide residues was also obtained, and any assumptions in linkage information are mentioned where applicable. Finally, using LCMS the relative molar abundances of each of the identified O-glycans were assessed with good accuracy and the relative abundances were used for statistical analysis comparing the different datasets. The observed

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O-glycan structures were depicted using the Consortium for Functional Glycomics symbol nomenclature (www.functionalglycomics .org) and the structures were drawn using Glyco Workbench software. A. salmonicida binding to mucins Mucins were diluted in 4 M GuHCl/PBS (140 mM NaCl, 2.7 mM KCl, 10 mM Phosphate buffer, pH 7.4) and coated on 96-well polysorp plates overnight at 4°C. The plates were washed 3 times with washing buffer (PBS containing 0.05 % Tween 20) and the wells were blocked for 1 hour with blocking buffer (0.5% BSA in washing buffer). After discarding the blocking buffer, A. salmonicida cultured in the log phase of growth were washed gently (two times at 3,000 × g, resulting in a soft, easily resuspendable pellet) and diluted in blocking buffer to OD600 of 0.1 and then further diluted 1:10 in blocking buffer. The bacterial suspension was added to the plates, which then were incubated on an orbital shaker (100 rpm) at 23-24 °C for 2 hours. The plates were washed 3 times and then incubated for 1 hour at RT with blocking buffer containing antiA. salmonicida IgG monoclonal antibody (clone 3B11/G5; Austral biological, San Ramon) diluted 1:5,000). After washing, horse radish peroxidase (HRP) conjugated anti-mouse IgG (Jackson ImmunoResearch Laboratories, Inc., West Grove, PA, USA) diluted 1:10,000 in blocking buffer was added. After further washing steps, tetramethylbenzidine (TMB) substrate (Sigma-Aldrich co.) was added and the plates were incubated for 20 min. The reaction was stopped with an equivalent amount of 0.5 M H2SO4 and the plates were read in a microplate reader at 450 nm after color stabilization. The binding was analyzed at four mucin concentrations (two-fold dilution steps) with results that were similar when the binding signal was expressed per unit of carbohydrate signal for at least three of the concentrations (i.e. the assay was performed within a linear range), including a glycan value of 40,000 Europium count (approximately corresponding to 8 µg/ml). For every analysis, parallel microtiter plates were coated for glycan detection analysis to ensure that small differences in binding were not due to coating differences. The binding values shown are normalized for a glycan value of 40,000 Europium count. A. salmonicida culture conditions and proliferation assay

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A. salmonicida strain VI-88/09/03175 (culture collection, Central Veterinary Laboratory, Oslo, Norway) was cultured in Brain Heart Infusion broth (BHI) at 19°C and stocks were stored in BHI/glycerol 1:1 at 80°C. Before proliferation experiments, A. salmonicida was cultured overnight in BHI broth on an orbital shaker at 120 rpm and washed three times with Phosphate Buffered Saline (PBS, 140 mM NaCl, 2.7 mM KCl, 10 mM phosphate buffer, pH 7.4) at 3,000 × g, 5 min. In proliferation assays A. salmonicida, at a concentration of OD600=0.1, were cultured in defined medium (DM). Purified mucin samples in 4 M GuHCl were dialyzed against PBS eight times and diluted in sterile PBS to a concentration of 100 µg/ml. The GuHCl containing isolation buffer was dialyzed and diluted in parallel, and used as a reference of normal growth (Dialysis control, DC). Bacteria were cultured in 96 well Nunc-Delta surface flat bottom plates (NUNC A/S, Roskilde, Denmark) in 8 replicates at 23°C and 120 rpm in the presence of Alamar blue (Invitrogen) to monitor reduction of the dye. Statistical analysis Statistical analysis was performed using GraphPad Prism version 7.02 software (La Jolla, CA). Comparisons of qualitative frequency data were performed using Fisher’s exact test (two-tailed), and relative quantitative glycan data are expressed as the median ± interquartile range and analyzed with the Mann-Whitney U-test or Kruskal-Wallis with Dunn´s post hoc test. Intestinal inflammatory score was analyzed using MannWhitney U-test and the morphometric assessment and intestinal barrier function was analyzed using twotailed t-test. P values ≤ 0.05 were considered as statistically significant.

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Results: Intestinal inflammation and barrier function The initial and final weight (g) of the fish after an experimental period of 42 days were 194.6 g ± 7.4 and 216.8 g ± 9.2 (mean ± SEM, n = 59), respectively for control group and 172.3g ± 6.6 and 198.4g ± 8.4, respectively for FFSB diet and growth was thus similar in the cohorts. The FFSB group displayed increased numbers of goblet cells (Figure 1a,b; P = 0.004) and more eosinophilic granular cells (EGC) in the lamina propria and sub-mucosa (P = 0.03, Table 2). The FFSB group also showed wider villi compared to control (P = 0.02), while no difference could be observed in villi length (Table 2). Ussing chamber analysis revealed a reduced TER in the FFSB group (233.8 ± 34.3 Ω*cm2) compared to control (361.6 ± 48.4 Ω*cm2, P = 0.04, Figure 1c). No difference was observed in the apparent permeability coefficient (Papp) for mannitol between the dietary groups (control: 5.95*10-7 ± 1.46*10-7 cm/s and FFSB: 8.52*10-7 ± 4.1*10-7 cm/s).

Figure 1: Goblet cell abundance and intestinal barrier function in control and FFSB fed Arctic charr intestine. a) AB/PAS stained histosections. b) Goblet cell enumeration based on PAS/AB stained tissue sections. c) Transepithelial electrical resistance (TER) reflecting intestinal barrier function. Data were analyzed using independent two-tailed t-test and * represents P < 0.05 and ** represents P < 0.01. 16


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Mucin isolation and density Isopycnic density-gradient centrifugation revealed a distinct peak of periodate-oxidizable (carbohydrate containing) material at 1.29-1.39 g/ml, representing the mucins. Ultraviolet-absorbing material was found as a sharp peak at 1.53- 1.56 g/ml, demonstrating that the mucins were successfully separated from the DNA. The mucin containing gradient fractions were pooled to give one mucin sample for each individual and body site and provided the basis for the remainder of the analyses. The mucin density was higher in the FFSB group compared to the control group (mean = 1.36 vs. 1.33 g/ml, respectively; P = 0.03, Table 2). Mucin density reflects glycosylation and we therefore pursued inflammation induced glycosylation differences between the control group and FFSB group. Table 2: Overview of mucosal characteristics of the fish specimens used in this study

Fish and diet

Arctic charr (control group) mean±SEM n=9

Arctic charr (FFSB group) mean±SEM n=9

P-value

Morphometric analyses£ Goblet cells (magenta) 57 ± 3.8 100 ± 11.1** 0.002 mm-1 epithelium Goblet cells (blue) 45 ± 5.9 98 ± 16.6** 0.005 mm-1 epithelium Villi width 136 ± 4.4 169 ± 11.6* 0.017 widest (µm) Villi width 99 ± 3.3 114 ± 7.1 0.07 narrowest (µm) Villi height (µm) 413 ± 25.9 434 ± 37.5 0.65 Inflammation score n=8 n=7 SNV 2.2 ± 0.16 3.6 ± 0.23 0.12 EGC 1.9 ± 0.12 2.8 ± 0.08* 0.03 SE 2.0 ± 0.06 2.5 ± 0.15 0.35 Mean score 2.0 ± 0.3 2.9 ± 0.4 0.07 No. of fish with score >3 1 5 Mucin analyses No. of mucus samples 5 4 analyzed with MS Mucin density 1.334 ± 0.0065 1.353 ± 0.006* 0.03 (g/ml) SNV (supra nuclear vacuolization), EGC (eosinophilic granular cells) and SE (sub-epithelium) *represents statistical difference in Arctic charr FFSB group compared to control group 17



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score of 2 sialic acid residues) and only disialylated residues (m/z 601) were observed, confirming the absence of polysialylation. In conclusion, this is the first study characterizing the Arctic charr intestinal mucin O-glycans, thereby expanding the teleost species’ O-glycosylation database. Although both being from the Salmonidae family, Arctic charr and Atlantic salmon differ in mucin glycosylation in core and terminal structures, confirming species-specific variation between the two. The high level of sialylation was common for both species and potentially play roles in mucosal defense against pathogenic colonization. One of the prominent differences is the higher level of key disialic acid (and novel mixed disialic) epitopes identified in Arctic charr. Interactions with A. salmonicida were quite similar between Arctic charr and Atlantic salmon, reflecting that although there are differences between these species, the abundance of the terminal glycan residues that A. salmonicida interact with are relatively similar between these species. In Arctic charr, intestinal inflammation appeared to result in decreased structural diversity, inter-individual variation and NeuGc levels. The loss of structural diversity and inter-individual variation has potential to be used as markers for mucosal inflammation in salmonid fish. The loss of NeuGc, on the other hand, seems to be a species specific feature, as non-inflamed Atlantic salmon lack this structure in the intestine. Furthermore, inflammation induced changes disabled A. salmonicida to utilize mucins for growth promoting purposes, which may be a defense against pathogens. Broadening the knowledge on mucin glycosylation and interactions with

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pathogens in different fish species and in response to inflammatory/infectious conditions may uncover opportunities to fight fish diseases in a growing aquaculture industry.

Figure 8: Potential pathway of disialylated (NeuAc) structure synthesis in Arctic charr intestinal mucins. di-NeuGc and mixed disialylated glycan structures identified in Arctic charr mucins are not shown here.

Acknowledgement The work was supported by the Swedish Research Council Formas, the Swedish Research Council (201605154_3), Engkvists Foundation, Wilhelm and Martina Lundgrens Foundation, NFF, SWEMARC, Kälarne research station and the Norweign Research Council (CtrlAQUA SFI 237856/O30). The mass spectrometer (LTQ) was supported by the Knut and Alice Wallenberg Foundation. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. The authors declare no competing financial interest. Supporting Information Supporting table S1. List of structurally characterized mucin O-glycans and their relative abundances identified by LC-MS/MS on five control and four FFSB fed Arctic charr intestinal mucins (XLS).

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Exploring the Arctic charr intestinal glycome: evidence for increased N

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