Characterization of the rainbow trout (Oncorhynchus mykiss) mucosal glycosphingolipid repertoire and Aeromonas salmonicida binding to neutral glycosphingolipids.

Infections pose a challenge for the fast growing aquaculture sector. Glycosphingolipids are cell membrane components that pathogens utilize for attachment to the host to initiate infection. Here, we characterized rainbow trout glycosphingolipids from five mucosal tissues using mass spectrometry and nuclear magnetic resonance and investigated binding of radiolabeled Aeromonas salmonicida to the glycosphingolipids on thin-layer chromatograms. 12 neutral and 14 acidic glycosphingolipids were identified. The glycosphingolipids isolated from the stomach and intestine were mainly neutral, whereas glycosphingolipids isolated from the skin, gills and pyloric caeca were largely acidic. Many of the acidic structures were poly-sialylated with shorter glycan structures in the skin compared to the other tissues. The sialic acids found were Neu5Ac and Neu5Gc. Most of the glycosphingolipids had isoglobo and ganglio core chains, or a combination of these. The epitopes on the rainbow trout glycosphingolipid glycans differed between epithelial sites leading to differences in pathogen binding. A major terminal epitope was fucose, that occurred attached to GalNAc in a α1-3 linkage but also in the form of HexNAc-(Fuc-)HexNAc-R. A. salmonicida were shown to bind to neutral glycosphingolipids from the gill and intestine. This study is the first to do a comprehensive investigation of the rainbow trout glycosphingolipids and analyze binding of A. salmonicida to glycosphingolipids. The structural information paves the way for identification of ways of interfering in pathogen colonization processes to protect against infections in aquaculture and contributes towards understanding A. salmonicida infection mechanisms.

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.

Muc2-dependent microbial colonization of the jejunal mucus layer is diet sensitive and confers local resistance to enteric pathogen infection.

Intestinal mucus barriers normally prevent microbial infections but are sensitive to diet-dependent changes in the luminal environment. Here we demonstrate that mice fed a Western-style diet (WSD) suffer regiospecific failure of the mucus barrier in the small intestinal jejunum caused by diet-induced mucus aggregation. Mucus barrier disruption due to either WSD exposure or chromosomal Muc2 deletion results in collapse of the commensal jejunal microbiota, which in turn sensitizes mice to atypical jejunal colonization by the enteric pathogen Citrobacter rodentium. We illustrate the jejunal mucus layer as a microbial habitat, and link the regiospecific mucus dependency of the microbiota to distinctive properties of the jejunal niche. Together, our data demonstrate a symbiotic mucus-microbiota relationship that normally prevents jejunal pathogen colonization, but is highly sensitive to disruption by exposure to a WSD.

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.

Aeromonas salmonicida binds α2-6 linked sialic acid, which is absent among the glycosphingolipid repertoires from skin, gill, stomach, pyloric caecum, and intestine.

Carbohydrates can both protect against infection and act as targets promoting infection. Mucins are major components of the slimy mucus layer covering the fish epithelia. Mucins can act as decoys for intimate pathogen interaction with the host afforded by binding to glycosphingolipids in the host cell membrane. We isolated and characterized glycosphingolipids from Atlantic salmon skin, gill, stomach, pyloric caeca, and intestine. We characterized the glycosphingolipids using liquid chromatography - mass spectrometry and tandem mass spectrometry and the glycan repertoire was compared with the glycan repertoire of mucins from the same epithelia. We also investigated Aeromonas salmonicida binding using chromatogram and microtiter well based binding assays. We identified 29 glycosphingolipids. All detected acid glycans were of the ganglio-series (unless shorter) and showed a high degree of polysialylation. The non-acid glycans were mostly composed of the neolacto, globo, and ganglio core structures. The glycosphingolipid repertoire differed between epithelia and the proportion of the terminal moieties of the glycosphingolipids did not reflect the terminal moieties on the mucins from the same epithelia. A. salmonicida did not bind the Atlantic salmon glycosphingolipids. Instead, we identified that A. salmonicida binding to sialic acid occurred to α2-6 Neu5Ac but not to α2-3 Neu5Ac. α2-6 Neu5Ac was present on mucins whereas mainly α2-3 Neu5Ac was found on the glycosphingolipids, explaining the difference in A. salmonicida binding ability between these host glycoconjugates. A. salmonicida´s ability to bind to Atlantic salmon mucins, but not the glycosphingolipids, is likely part of the host defence against this pathogen.

Rainbow trout gastrointestinal mucus, mucin production, mucin glycosylation and response to lipopolysaccharide.

Mucus, whereof the highly glycosylated mucins are a major component, protects the epithelial mucosal surfaces. The aim of this study was to characterize the rainbow trout (Oncorhynchus mykiss) gastrointestinal mucus barrier function, mucin production, glycosylation and response to lipopolysaccharide. Both gastric and intestinal mucus was thick and impenetrable to bacteria-sized beads ex vivo. The secreted mucus covering the gastric epithelium predominantly contained sialylated mucins. Plume-like structures emerging from the gastric pits were both sialylated and fucosylated, indicating heterogeneity in gastric mucus secreted by the surface mucus cells and gland secretory cells, whereas intestinal mucus appeared more homogenous. In vivo metabolic mucin labelling revealed regional differences in mucin production and basal to apical transport, while lipopolysaccharide stimulation increased the rate of mucin production and basal to apical transport in both stomach and intestine. Using mass spectrometry, 34 mucin O-glycans were identified, with ∼70% of the relative abundance being sialylated, ∼40% di-sialylated and 20-25% fucosylated. No effects of lipopolysaccharide treatment were apparent regarding O-glycan repertoires, relative abundance of components, size distribution or core structures. Thus, the mucus production and organization differ between epithelial sites but provide a barrier to bacteria in both stomach and intestine. Furthermore, mucin production and basal to apical transport was stimulated by lipopolysaccharide in all regions, suggesting a mechanism to combat infections.

Gill Mucus and Gill Mucin O-glycosylation in Healthy and Amebic Gill Disease-Affected Atlantic Salmon.

Amoebic gill disease (AGD) causes poor performance and death in salmonids. Mucins are mainly comprised by carbohydrates and are main components of the mucus covering the gill. Since glycans regulate pathogen binding and growth, glycosylation changes may affect susceptibility to primary and secondary infections. We investigated gill mucin O-glycosylation from Atlantic salmon with and without AGD using liquid chromatography-mass spectrometry. Gill mucin glycans were larger and more complex, diverse and fucosylated than skin mucins. Confocal microscopy revealed that fucosylated mucus coated sialylated mucus strands in ex vivo gill mucus. Terminal HexNAcs were more abundant among O-glycans from AGD-affected Atlantic salmon, whereas core 1 structures and structures with acidic moieties such as N-acetylneuraminic acid (NeuAc) and sulfate groups were less abundant compared to non-infected fish. The fucosylated and NeuAc-containing O-glycans were inversely proportional, with infected fish on the lower scale of NeuAc abundance and high on fucosylated structures. The fucosylated epitopes were of three types: Fuc-HexNAc-R, Gal-[Fuc-]HexNAc-R and HexNAc-[Fuc-]HexNAc-R. These blood group-like structures could be an avenue to diversify the glycan repertoire to limit infection in the exposed gills. Furthermore, care must be taken when using skin mucus as proxy for gill mucus, as gill mucins are distinctly different from skin mucins.

Colonic levels of vasoactive intestinal peptide decrease during infection and exogenous VIP protects epithelial mitochondria against the negative effects of IFNγ and TNFα induced during Citrobacter rodentium infection.

Citrobacter rodentium infection is a model for infection with attaching and effacing pathogens, such as enteropathogenic Escherichia coli. The vasoactive intestinal peptide (VIP) has emerged as an anti-inflammatory agent, documented to inhibit Th1 immune responses and successfully treat animal models of inflammation. VIP is also a mucus secretagogue. Here, we found that colonic levels of VIP decrease during murine C. rodentium infection with a similar time dependency as measurements reflecting mitochondrial function and epithelial integrity. The decrease in VIP appears mainly driven by changes in the cytokine environment, as no changes in VIP levels were detected in infected mice lacking interferon gamma (IFNγ). VIP supplementation alleviated the reduction of activity and levels of mitochondrial respiratory complexes I and IV, mitochondrial phosphorylation capacity, transmembrane potential and ATP generation caused by IFNγ, TNFα and C. rodentium infection, in an in vitro mucosal surface. Similarly, VIP treatment regimens that included the day 5-10 post infection period alleviated decreases in enzyme complexes I and IV, phosphorylation capacity, mitochondrial transmembrane potential and ATP generation as well as increased apoptosis levels during murine infection with C. rodentium. However, VIP treatment failed to alleviate colitis, although there was a tendency to decreased pathogen density in contact with the epithelium and in the spleen. Both in vivo and in vitro, NO generation increased during C. rodentium infection, which was alleviated by VIP. Thus, therapeutic VIP administration to restore the decreased levels during infection had beneficial effects on epithelial cells and their mitochondria, but not on the overall infection outcome.

Influence of the viscosity of healthy and diseased human mucins on the motility of Helicobacter pylori.

We present particle tracking microrheology results on human mucins, isolated from normal surface and gland mucosa and one tumor sample, and examine the motility of Helicobacter pylori in these mucins. At 1.5% concentration human mucin solutions are purely viscous, with viscosity η (gland mucin) > η (surface mucin) > η (tumor mucin). In the presence of motile H. pylori bacteria, particle diffusion is enhanced, with diffusivity D+bac(tumor mucin) > D+bac(gland mucin) > D+bac(surface mucin). The surface and tumor mucin solutions exhibit an elastic response in the presence of bacteria. Taken together these results imply that particle diffusion and active swimming are coupled and impact the rheology of mucin solutions. Both J99 wild type (WT) and its isogenic ΔbabA/ΔsabA mutant swam well in broth or PGM solutions. However, the human mucins affected their motility differently, rendering them immotile in certain instances. The distribution of swimming speeds in human mucin solutions was broader with a large fraction of fast swimmers compared to PGM and broth. The bacteria swam fastest in the tumor mucin solution correlating with it having the lowest viscosity of all mucin solutions. Overall, these results suggest that mucins from different tissue locations and disease status differ in their microrheological properties and their effect on H. pylori motility.

IL-4 Protects the Mitochondria Against TNFα and IFNγ Induced Insult During Clearance of Infection with Citrobacter rodentium and Escherichia coli.

Citrobacter rodentium is a murine pathogen that serves as a model for enteropathogenic Escherichia coli. C. rodentium infection reduced the quantity and activity of mitochondrial respiratory complexes I and IV, as well as phosphorylation capacity, mitochondrial transmembrane potential and ATP generation at day 10, 14 and 19 post infection. Cytokine mRNA quantification showed increased levels of IFNγ, TNFα, IL-4, IL-6, and IL-12 during infection. The effects of adding these cytokines, C. rodentium and E. coli were hence elucidated using an in vitro colonic mucosa. Both infection and TNFα, individually and combined with IFNγ, decreased complex I and IV enzyme levels and mitochondrial function. However, IL-4 reversed these effects, and IL-6 protected against loss of complex IV. Both in vivo and in vitro, the dysfunction appeared caused by nitric oxide-generation, and was alleviated by an antioxidant targeting mitochondria. IFNγ -/- mice, containing a similar pathogen burden but higher IL-4 and IL-6, displayed no loss of any of the four complexes. Thus, the cytokine environment appears to be a more important determinant of mitochondrial function than direct actions of the pathogen. As IFNγ and TNFα levels increase during clearance of infection, the concomitant increase in IL-4 and IL-6 protects mitochondrial function.