Differentiation of Pig Gastric Primary Cells into Mucus Producing Epithelial Cells.

There is a growing interest in the development of in vitro models that mimic the intrinsic characteristics of cells in vivo to replace and/or reduce the use of experimental animals. The stomach is lined with mucus secreting epithelial cells, creating a thick mucus layer that protects the underlying epithelial cells from acid, pathogens, and other harmful agents. Mucins are a main component of the mucus layer, and their secretion is an important protective feature of epithelial cells in vivo. Here, we present a method that differentiates pig gastric primary cells into mucin secreting epithelial cells by culturing the cells on polyester membranes under semi-wet interface for 14 days, using differentiation medium containing the N-[(3,5-difluorophenyl)acetyl]-L-alanyl-2-phenyl]glycine-1,1-dimethylethyl ester (DAPT) in the basolateral compartment for the first 7 days and subsequent 7-day culture in non-differentiation medium. The in vitro mucosal surfaces created by these cells are harvested 2 weeks post confluence, and two preservation methods are described to fix the monolayers for further analysis.

Porcine intestinal glycosphingolipids recognized by Brachyspira hyodysenteriae.

Swine dysentery caused by Brachyspira hyodysenteriae is a disease present worldwide with an important economic impact on the farming business, resulting in an increased use of antibiotics. In the present study, we investigated the binding of B. hyodysenteriae to glycosphingolipids from porcine small intestinal epithelium in order to determine the glycosphingolipids involved in B. hyodysenteriae adhesion. Specific interactions between B. hyodysenteriae and two non-acid glycosphingolipids were obtained. These binding-active glycosphingolipids, were characterized by mass spectrometry as lactotetraosylceramide (Galß3GlcNAcß3Galß4Glcß1Cer) and the B5 glycosphingolipid (Galα3Galß4GlcNAcß3Galß4Glcß1Cer). Comparative binding studies using structurally related reference glycosphingolipids showed that B. hyodysenteriae binding to lactotetraosylceramide required an unsubstituted terminal Galß3GlcNAc sequence, while for binding to the B5 pentaosylceramide the terminal Galα3Galß4GlcNAc sequence is the minimum element recognized by the bacteria. Binding of Griffonia simplicifolia IB4 lectin to pig colon tissue sections from healthy control pig and B. hyodysenteriae infected pigs showed that in the healthy pigs the Galα3Gal epitope was mainly present in the lamina propria. In contrast, in four out of five pigs with swine dysentery there was an increased expression of Galα3Gal in the goblet cells and in the colonic crypts, where B. hyodysenteriae also was present. The one pig that had recovered by the time of necropsy had the Galα3Gal epitope only in the lamina propria. These data are consistent with a model where a transient increase in the carbohydrate sequence recognized by the bacteria occur in colonic mucins during B. hyodysenteriae infection, suggesting that the mucins may act as decoys contributing to clearance of the infection. These findings may lead to novel strategies for treatment of B. hyodysenteriae induced swine dysentery.

Mucin O-glycosylation and pathogen binding ability differ between rainbow trout epithelial sites.

Mucins are highly glycosylated proteins that make up the mucus covering internal and external surfaces of fish. Mucin O-glycans regulate pathogen quorum sensing, growth, virulence and attachment to the host. Knowledge on this mucosal defense system can enable alternative treatments to diseases posing a threat to productivity and welfare in aquaculture. Here, we characterize the rainbow trout (Oncorhynchus mykiss) gill, skin, pyloric ceca and distal intestinal mucin O-glycosylation and compare it to known teleost O-glycomes. We identified 54 O-glycans, consisting of up to nine monosaccharide residues. Skin glycans were most acidic, shortest on average and consisted mainly of NeuAcα2-6GalNAc. Glycans from the gills were less acidic with predominantly core 1 and 2 glycans, whereas glycans from pyloric ceca and distal intestine expressed an increased number of core 5 glycans, distinctly decorated with NeuAcα2-8NeuAc- like epitopes. When compared to Atlantic salmon and Arctic charr, trends on the core distribution, average size and overall acidity remained similar, although the epitopes varied. Rainbow trout mucins from gill and intestine bound A. salmonicida and A. hydrophila more efficiently than skin mucins. This is in line with a model where skin mucins with small glycans limit bacterial adhesion to the fish surface whereas the complex intestinal mucin glycans aid in trapping and removing pathogens from the epithelial surface.

Streptococcus oralis Employs Multiple Mechanisms of Salivary Mucin Binding That Differ Between Strains.

Streptococcus oralis is an oral commensal and opportunistic pathogen that can enter the bloodstream and cause bacteremia and infective endocarditis. Here, we investigated the mechanisms of S. oralis binding to oral mucins using clinical isolates, isogenic mutants and glycoconjugates. S. oralis bound to both MUC5B and MUC7, with a higher level of binding to MUC7. Mass spectrometry identified 128 glycans on MUC5B, MUC7 and the salivary agglutinin (SAG). MUC7/SAG contained a higher relative abundance of Lewis type structures, including Lewis b/y, sialyl-Lewis a/x and α2,3-linked sialic acid, compared to MUC5B. S. oralis subsp. oralis binding to MUC5B and MUC7/SAG was inhibited by Lewis b and Lacto-N-tetraose glycoconjugates. In addition, S. oralis binding to MUC7/SAG was inhibited by sialyl Lewis x. Binding was not inhibited by Lacto-N-fucopentaose, H type 2 and Lewis x conjugates. These data suggest that three distinct carbohydrate binding specificities are involved in S. oralis subsp. oralis binding to oral mucins and that the mechanisms of binding MUC5B and MUC7 differ. Efficient binding of S. oralis subsp. oralis to MUC5B and MUC7 required the gene encoding sortase A, suggesting that the adhesin(s) are LPXTG-containing surface protein(s). Further investigation demonstrated that one of these adhesins is the sialic acid binding protein AsaA.

Brachyspira Species Avidity to Colonic Mucins from Pigs with and without Brachyspira hyodysenteriae Infection Is Species Specific and Varies between Strains.

Brachyspira hyodysenteriae is commonly associated with swine dysentery (SD), a disease that has an economic impact on the swine industry. B. hyodysenteriae infection results in changes to the colonic mucus niche with massive mucus induction, which substantially increases the number of B. hyodysenteriae binding sites in the mucus. We previously determined that a B. hyodysenteriae strain binds to colon mucins in a manner that differs between pigs and mucin types. Here, we investigated if adhesion to mucins is a trait observed across a broad set of B. hyodysenteriae strains and isolates and furthermore at a genus level (B. innocens, B. pilosicoli, B. murdochii, B. hampsonii, and B. intermedia strains). Our results show that binding to mucins appears to be specific to B. hyodysenteriae, and within this species, the binding ability to mucins varies between strains/isolates, increases for mucins from pigs with SD, and is associated with sialic acid epitopes on mucins. Infection with B. hyodysenteriae strain 8dII results in mucin glycosylation changes in the colon, including a shift in sialic acid-containing structures. Thus, we demonstrate through hierarchical cluster analysis and orthogonal projections to latent structures discriminant analysis (OPLS-DA) models of the relative abundances of sialic acid-containing glycans that sialic acid-containing structures in the mucin O-glycome are good predictors of B. hyodysenteriae strain 8dII infection in pigs. The results emphasize the role of sialic acids in governing B. hyodysenteriae interactions with its host, which may open perspectives for therapeutic strategies.

Role of Sialic Acid in Brachyspira hyodysenteriae Adhesion to Pig Colonic Mucins.

Infection with Brachyspira hyodysenteriae results in mucoid hemorrhagic diarrhea. This pathogen is associated with the colonic mucus layer, mainly composed of mucins. Infection regulates mucin O-glycosylation in the colon and increases mucin secretion as well as B. hyodysenteriae binding sites on mucins. Here, we analyzed potential mucin epitopes for B. hyodysenteriae adhesion in the colon, as well as the effect of colonic mucins on bacterial growth. Associations between B. hyodysenteriae binding to pig colonic mucins and mucin glycan data showed that B. hyodysenteriae binding was associated with the presence of N-glycolylneuraminic acid (NeuGc) on mucins. The role of sialic acid in B. hyodysenteriae adhesion was analyzed after the removal of sialic acid residues on the mucins by enzymatic treatment with sialidase A, which decreased bacterial binding to the mucins. The effect of pig colonic mucins on B. hyodysenteriae growth was determined in carbohydrate-free medium. B. hyodysenteriae growth increased in the presence of mucins from two out of five infected pigs, suggesting utilization of mucins as a carbon source for growth. Additionally, bacterial growth was enhanced by free sialic acid and N-acetylglucosamine. The results highlight a role of sialic acid as an adhesion epitope for B. hyodysenteriae interaction with colonic mucins. Furthermore, the mucin response and glycosylation changes exerted in the colon during B. hyodysenteriae infection result in a potentially favorable environment for pathogen growth in the intestinal mucus layer.

BabA-mediated adherence of pediatric ulcerogenic H. pylori strains to gastric mucins at neutral and acidic pH.

Helicobacter pylori infection can result in non-ulcer dyspepsia (NUD), peptic ulcer disease (PUD), adenocarcinoma, and gastric lymphoma. H. pylori reside within the gastric mucus layer, mainly composed of mucins carrying an array of glycan structures that can serve as bacterial adhesion epitopes. The aim of the present study was to characterize the binding ability, adhesion modes, and growth of H. pylori strains from pediatric patients with NUD and PUD to gastric mucins. Our results showed an increased adhesion capacity of pediatric PUD H. pylori strains to human and rhesus monkey gastric mucins compared to the NUD strains both at neutral and acidic pH, regardless if the mucins were positive for Lewis b (Leb), Sialyl-Lewis x (SLex) or LacdiNAc. In addition to babA positive strains being more common among PUD associated strains, H. pylori babA positive strains bound more avidly to gastric mucins than NUD babA positive strains at acidic pH. Binding to Leb was higher among babA positive PUD H. pylori strains compared to NUD strains at neutral, but not acidic, pH. PUD derived babA-knockout mutants had attenuated binding to mucins and Leb at acidic and neutral pH, and to SLex and DNA at acidic pH. The results highlight the role of BabA-mediated adherence of pediatric ulcerogenic H. pylori strains, and points to a role for BabA in adhesion to charged structures at acidic pH, separate from its specific blood group binding activity.

Differentiation of Gastrointestinal Cell Lines by Culture in Semi-wet Interface.

Epithelial cells grown in vitro provide opportunities to elucidate cellular mechanisms in response to chemical, viral, or bacterial agents in isolation from the effects of other bodily functions, such as hormonal and immune responses. However, cells that do not form a tight epithelium, polarize or secrete mucins lack some of the important protection mechanisms intrinsic to epithelial cells in vivo, increasing their susceptibility to external agents, and exposing basolateral targets for interactions that may not occur in vivo. Here, we present a method that transforms some epithelial cell lines into mucin secreting polarized epithelial surfaces with high transepithelial resistance: the cells are cultured on semi-permeable membranes in differentiation medium for the first 6 days, followed by culture under semi-wet interface with mechanical stimulation for 22 days. The procedure can be performed using standard laboratory reagents and equipment. A description on how to fix and paraffin embed these in vitro mucosal membranes for histology purposes is also included.

Mucus-Pathogen Interactions in the Gastrointestinal Tract of Farmed Animals.

Gastrointestinal infections cause significant challenges and economic losses in animal husbandry. As pathogens becoming resistant to antibiotics are a growing concern worldwide, alternative strategies to treat infections in farmed animals are necessary in order to decrease the risk to human health and increase animal health and productivity. Mucosal surfaces are the most common route used by pathogens to enter the body. The mucosal surface that lines the gastrointestinal tract is covered by a continuously secreted mucus layer that protects the epithelial surface. The mucus layer is the first barrier the pathogen must overcome for successful colonization, and is mainly composed of densely glycosylated proteins called mucins. The vast array of carbohydrate structures present on the mucins provide an important setting for host-pathogen interactions. This review summarizes the current knowledge on gastrointestinal mucins and their role during infections in farmed animals. We examine the interactions between mucins and animal pathogens, with a focus on how pathogenic bacteria can modify the mucin environment in the gut, and how this in turn affects pathogen adhesion and growth. Finally, we discuss analytical challenges and complexities of the mucus-based defense, as well as its potential to control infections in farmed animals.

Neutrophil Elastase and Interleukin 17 Expressed in the Pig Colon during Brachyspira hyodysenteriae Infection Synergistically with the Pathogen Induce Increased Mucus Transport Speed and Production via Mitogen-Activated Protein Kinase 3.

Brachyspira hyodysenteriae colonizes the pig colon, resulting in mucoid hemorrhagic diarrhea and mucus layer changes. These changes are characterized by a disorganized mucus structure and massive mucus induction with de novo expression of MUC5AC and increased production of MUC2. To investigate the mechanisms behind this altered mucin environment, we quantified the mRNA levels of mucin pathway genes and factors from the immune system in the colons of infected and control pigs and observed upregulation of neutrophil elastase, SPDEF, FOXA3, MAPK3/ERK1, IL-17A, IL-1ß, IL-6, and IL-8 expression. In vitro, colonic mucus-producing mucosal surfaces were treated with these factors along with B. hyodysenteriae infection and analyzed for their effect on mucin production. Neutrophil elastase and infection synergistically induced mucus production and transport speed, and interleukin 17A (IL-17A) also had similar effects, in both the presence and absence of infection. A mitogen-activated protein kinase 3 (MAPK3)/extracellular signal-regulated kinase 1 (ERK1) inhibitor suppressed these effects. Therefore, we suggest that the SPDEF, FOXA3, and MAPK3/ERK1 signaling pathways are behind the transcriptional program regulating mucin biosynthesis in the colon during B. hyodysenteriae infection. In addition to furthering the knowledge on this economically important disease, this mechanism may be useful for the development of therapies aimed at conditions where enhancing mucus production may be beneficial, such as chronic inflammatory disorders of the colon.

Brachyspira hyodysenteriae Infection Regulates Mucin Glycosylation Synthesis Inducing an Increased Expression of Core-2 O-Glycans in Porcine Colon.

Brachyspira hyodysenteriae causes swine dysentery (SD), leading to global financial losses to the pig industry. Infection with this pathogen results in an increase in B. hyodysenteriae binding sites on mucins, along with increased colonic mucin secretion. We 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 noninfected 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 interindividual variation, although a larger array of glycan structures was 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 toward 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 or predominantly contained NeuGc instead of NeuAc, whereas they had a lower abundance of structures that were fucosylated, acidic, or sulfated than those from noninfected 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.

Helicobacter pylori Adapts to Chronic Infection and Gastric Disease via pH-Responsive BabA-Mediated Adherence.

The BabA adhesin mediates high-affinity binding of Helicobacter pylori to the ABO blood group antigen-glycosylated gastric mucosa. Here we show that BabA is acid responsive-binding is reduced at low pH and restored by acid neutralization. Acid responsiveness differs among strains; often correlates with different intragastric regions and evolves during chronic infection and disease progression; and depends on pH sensor sequences in BabA and on pH reversible formation of high-affinity binding BabA multimers. We propose that BabA's extraordinary reversible acid responsiveness enables tight mucosal bacterial adherence while also allowing an effective escape from epithelial cells and mucus that are shed into the acidic bactericidal lumen and that bio-selection and changes in BabA binding properties through mutation and recombination with babA-related genes are selected by differences among individuals and by changes in gastric acidity over time. These processes generate diverse H. pylori subpopulations, in which BabA's adaptive evolution contributes to H. pylori persistence and overt gastric disease.

BabA dependent binding of Helicobacter pylori to human gastric mucins cause aggregation that inhibits proliferation and is regulated via ArsS.

Mucins in the gastric mucus layer carry a range of glycan structures, which vary between individuals, can have antimicrobial effect or act as ligands for Helicobacter pylori. Mucins from various individuals and disease states modulate H. pylori proliferation and adhesin gene expression differently. Here we investigate the relationship between adhesin mediated binding, aggregation, proliferation and adhesin gene expression using human gastric mucins and synthetic adhesin ligand conjugates. By combining measurements of optical density, bacterial metabolic activity and live/dead stains, we could distinguish bacterial aggregation from viability changes, enabling elucidation of mechanisms behind the anti-prolific effects that mucins can have. Binding of H. pylori to Leb-glycoconjugates inhibited the proliferation of the bacteria in a BabA dependent manner, similarly to the effect of mucins carrying Leb. Furthermore, deletion of arsS lead to a decrease in binding to Leb-glycoconjugates and Leb-decorated mucins, accompanied by decreased aggregation and absence of anti-prolific effect of mucins and Leb-glycoconjugates. Inhibition of proliferation caused by adhesin dependent binding to mucins, and the subsequent aggregation suggests a new role of mucins in the host defense against H. pylori. This aggregating trait of mucins may be useful to incorporate into the design of adhesin inhibitors and other disease intervention molecules.

The levels of Brachyspira hyodysenteriae binding to porcine colonic mucins differ between individuals, and binding is increased to mucins from infected pigs with de novo MUC5AC synthesis.

Brachyspira hyodysenteriae colonizes the pig colon, resulting in mucohemorrhagic diarrhea and growth retardation. Fecal mucus is a characteristic feature of swine dysentery; therefore, we investigated how the mucin environment changes in the colon during infection with B. hyodysenteriae and how these changes affect this bacterium's interaction with mucins. We isolated and characterized mucins, the main component of mucus, from the colon of experimentally inoculated and control pigs and investigated B. hyodysenteriae binding to these mucins. Fluorescence microscopy revealed a massive mucus induction and disorganized mucus structure in the colon of pigs with swine dysentery. Quantitative PCR (qPCR) and antibody detection demonstrated that the mucus composition of pigs with swine dysentery was characterized by de novo expression of MUC5AC and increased expression of MUC2 in the colon. Mucins from the colon of inoculated and control pigs were isolated by two steps of isopycnic density gradient centrifugation. The mucin densities of control and inoculated pigs were similar, whereas the mucin quantity was 5-fold higher during infection. The level of B. hyodysenteriae binding to mucins differed between pigs, and there was increased binding to soluble mucins isolated from pigs with swine dysentery. The ability of B. hyodysenteriae to bind, measured in relation to the total mucin contents of mucus in sick versus healthy pigs, increased 7-fold during infection. Together, the results indicate that B. hyodysenteriae binds to carbohydrate structures on the mucins as these differ between individuals. Furthermore, B. hyodysenteriae infection induces changes to the mucus niche which substantially increase the amount of B. hyodysenteriae binding sites in the mucus.

Phylogenetic analysis reveals common antimicrobial resistant Campylobacter coli population in antimicrobial-free (ABF) and commercial swine systems.

The objective of this study was to compare the population biology of antimicrobial resistant (AR) Campylobacter coli isolated from swine reared in the conventional and antimicrobial-free (ABF) swine production systems at farm, slaughter and environment. A total of 200 C. coli isolates selected from fecal, environmental, and carcass samples of ABF (n = 100) and conventional (n = 100) swine production systems were typed by multilocus sequence typing (MLST). Sequence data from seven housekeeping genes was analyzed for the identification of allelic profiles, sequence types (STs) and clonal complex determination. Phylogenetic trees were generated to establish the relationships between the genotyped isolates. A total of 51 STs were detected including two novel alleles (glnA 424 and glyA 464) and 14 novel STs reported for the first time. The majority of the C. coli isolates belonged to ST-854 (ABF: 31, conventional: 17), and were grouped in clonal complex ST-828 (ABF: 68%, conventional: 66%). The mean genetic diversity (H) for the ABF (0.3963+/-0.0806) and conventional (0.4655+/-0.0714) systems were similar. The index of association (I(A)(S)) for the ABF (I(A)(S)= 0.1513) and conventional (I(A)(S) = 0.0991) C. coli populations were close to linkage equilibrium, indicative of a freely recombining population. Identical STs were detected between the pigs and their environment both at farm and slaughter. A minimum spanning tree revealed the close clustering of C. coli STs that originated from swine and carcass with those from the environment. In conclusion, our study reveals a genotypic diverse C. coli population that shares a common ancestry in the conventional and ABF swine production systems. This could potentially explain the high prevalence of antimicrobial resistant C. coli in the ABF system in the absence of antimicrobial selection pressure.

Longitudinal study of the persistence of antimicrobial-resistant campylobacter strains in distinct Swine production systems on farms, at slaughter, and in the environment.

The objectives of this study were to compare and characterize the prevalence of antimicrobial-resistant (AR) Campylobacter in conventional and antimicrobial-free (ABF) production systems on farms, at slaughter, and in the environment. Fecal and environmental samples were collected from ABF farms (pigs, 1,239; environment, 797) and conventional farms (pigs, 1,650; environment, 1,325). At slaughter, we collected samples from carcasses, including postevisceration swabs, postchill swabs, and mesenteric lymph nodes from ABF systems (postevisceration swabs, 182; postchill swabs, 199; mesenteric lymph nodes, 184) and conventional systems (postevisceration swabs, 272; postchill swabs, 271; mesenteric lymph nodes, 255) at separate processing facilities. We also sampled the processing plant environment, including truck and lairage floor swab samples (ABF, 115; conventional, 90). Overall, a total of 2,908 Campylobacter isolates, including Campylobacter coli (farm, 2,557, 99.8%; slaughter, 341, 98.3%) and Campylobacter jejuni (farm, 4, 0.2%; slaughter, 6, 1.7%), were isolated in the study. There was no significant difference in the prevalence of Campylobacter between ABF and conventionally raised pigs (farrowing, P = 0.20; nursery, P = 0.06; finishing, P = 0.24) and the environment (P = 0.37). At slaughter, Campylobacter was isolated from all of the stages, including postchill. The highest frequencies of resistance were exhibited against tetracycline (ABF, 48.2%; conventional, 88.3%). Ciprofloxacin-resistant C. coli isolates were observed in conventionally raised (17.1%) and ABF (1.2%) pigs (P = 0.11). Antimicrobial use data from conventional farms indicated significant associations between oxytetracycline use and tetracycline resistance in the nursery pigs (P = 0.01), between tiamulin exposure and azithromycin and erythromycin resistance in nursery (P < 0.01) and finishing (P < 0.01) pigs, and between enrofloxacin exposure and ciprofloxacin and nalidixic acid resistance in farrowing (P < 0.01) and nursery (P < 0.01) pigs. Identical antimicrobial resistance profiles were observed in the pigs and their environments on farms and at slaughter. In summary, our results highlight the persistence and dissemination of AR Campylobacter from farm to slaughter in ABF and conventionally raised pigs and their environments.