Up-regulation of MUC2 and IL-1β expression in human colonic epithelial cells by Shigella and its interaction with mucins

Background

The entire gastrointestinal tract is protected by a mucous layer, which contains complex glycoproteins called mucins. MUC2 is one such mucin that protects the colonic mucosa from invading microbes. The initial interaction between microbes and mucins is an important step for microbial pathogenesis. Hence, it was of interest to investigate the relationship between host (mucin) and pathogen interaction, including Shigella induced expression of MUC2 and IL-1β during shigellosis.

Methods

The mucin-Shigella interaction was revealed by an in vitro mucin-binding assay. Invasion of Shigella dysenteriae into HT-29 cells was analyzed by Transmission electron microscopy. Shigella induced mucin and IL-1β expression were analyzed by RT-PCR and Immunofluorescence.

Results

The clinical isolates of Shigella were found to be virulent by a congo-red binding assay. The in vitro mucin-binding assay revealed both Shigella dysenteriae and Shigella flexneri have binding affinity in the increasing order of: guinea pig small intestinal mucinShigella dysenteriae into HT-29 cells occurs within 2 hours. Interestingly, in Shigella dysenteriae infected conditions, significant increases in mRNA expression of MUC2 and IL-1β were observed in a time dependent manner. Further, immunofluorescence analysis of MUC2 shows more positive cells in Shigella dysenteriae treated cells than untreated cells.

Conclusions

Our study concludes that the Shigella species specifically binds to guinea pig colonic mucin, but not to guinea pig small intestinal mucin. The guinea pig colonic mucin showed a greater binding parameter (R), and more saturable binding, suggesting the presence of a finite number of receptor binding sites in the colonic mucin of the host. In addition, modification of mucins with TFMS and sodium metaperiodate significantly reduced mucin-bacterial binding; suggesting that the mucin-Shigella interaction occurs through carbohydrate epitopes on the mucin backbones. Overproduction of MUC2 may alter adherence and invasion of Shigella dysenteriae into human colonic epithelial cells.     

Adherence of Shigella dysenteriae 1 to Human Colonic Mucin

The pathogenic potential of Shigella is correlated with the ability of the organism to invade and multiply within the cells of colonic epithelium. Although invasion is the ultimate event, a preceding step is adherence. Shigella dysenteriae 1 preferentially adhered to colonic mucin and not to small intestinal mucin. The pathogen showed a very strong adherence pattern to human colonic mucin when compared with guinea pig and rat mucin. The adherence pattern of S. dysenteriae 1 was not altered on preincubation with monosaccharides present in mucins, suggesting that the receptor for the pathogen is not a simple sugar. Binding of S. dysenteriae 1 to human colonic mucin was not by weak hydrophobic forces. The bacterium also adhered to glycolipids, emphasizing the role of glycoconjugates as receptors for S. dysenteriae 1. Received: 10 August 2000 / Accepted: 30 October 2000     

Reactivity of mucin-specific lectin from Sambucus sieboldiana with simple sugars, normal mucins and tumor-associated mucins. Comparison with other lectins.

Mucin-specific lectin from Sambucus sieboldiana (SSA-M) reacts in Western blotting and ELISA with mucins from porcine stomach, bovine and ovine submaxillary glands, the human milk fat globule membrane, in vitro human ovarian, breast and colonic tumor cell lines, and mucins produced in vivo in the ascites of patients with endometrial and ovarian tumors, but not with fetal bovine fetuin or human transferrin. Sialidase treatment of these mucins led to an increase in the binding of SSA-M, suggesting that sialic acid is not part of the binding site for this lectin. Furthermore, sialic acid did not inhibit lectin binding. Treatment of asialomucin with O-glycanase decreased the binding of SSA-M, confirming the reactivity of the lectin with an O-linked carbohydrate. Treatment of mucins with trifluoromethanesulfonic acid, which removes all but core carbohydrate, led to an increase in the binding of SSA-M, suggesting that the lectin reacts with O-linked core glycans. Indeed, the increased reactivity after sialidase treatment of ovine submaxillary mucin suggests the lectin reacts with peptide-linked N-acetylgalactosamine (GalNAc), since more than 98% of the glycan chains attached to this mucin are sialylated GalNAc. The binding of SSA-M to sialidase-treated porcine mucin was inhibited strongly by GalNAc and disaccharides containing galactose (lactose, melibiose, and N-acetyllactosamine) but not by free galactose (Gal), suggesting that the glycan for optimum binding is Gal beta(1-3)GalNAc. This pattern of inhibition was different to other core glycan-reactive lectins tested, indicating that SSA-M is distinct, and should be of use in the isolation and characterisation of mucins and O-linked glycans.     

Changes in colonic mucins of germfree rats in response to the introduction of a "normal" rat microbial flora. Rat colonic mucin.

In order to determine the influence of bacterial colonization on amount and composition of colonic mucins, germfree male AS/Ztm rats were colonized with a rat specific intestinal flora for different times (2, 7, 14, 21, 28, 35, 120 days). The amount of colonic mucins was determined by gel filtration on Sepharose CL-4B; the relative amount of acidic mucins was calculated after ion exchange chromatography. In addition, cecal weight and dry matter of feces were monitored. While germfree and SPF rats revealed similar amounts of colonic mucins (7.0 vs. 7.2 mg mucin/300 g body weight), the initial phase of association was characterized by considerably decreasing values. After four weeks of association, the total amount of colonic mucins had almost equalized in the two groups. The amount of acidic mucins, having decreased during the first three weeks of colonization, rendered values comparable to the SPF mucins after four months of adaptation. Cecomegaly in germfree rats disappeared within the first two days, while solidification of the intestinal content occurred within four months. Mucin losses during initial phase of association are attributed 1. to the disappearance of the cecal mucin pool, and 2. to the mucin degrading activity of some bacterial strains known to be present in the intestinal flora. Further development is conducted by a stimulation of mucin secretion, described to follow the colonization. The initially increased secretion of neutral mucins is attributed to a pronounced release of immature mucin glycoproteins, while the shift to more acidic mucins is considered to result from stimulated secretion as well as from a selective bacterial degradation of neutral mucin components.     

Functional Heterogeneity of Colonic Adenocarcinoma Mucins for Inhibition of Entamoeba histolytica Adherence to Target Cells1

Mucins secreted from the gastrointestinal epithelium form the basis of the adherent mucus layer which is the host's first line of defense against invasion by Entamoeba histolytica. Galactose and N-acetyl-D-galactosamine residues of mucins specifically inhibit binding of the amebic 170 kDa heavy subunit Gal-lectin to target cells, an absolute prerequisite for pathogenesis. Herein we characterized the secretory mucins isolated from the human colon and from three human colonic adenocarcinoma cell lines: two with goblet cell-like (LS174T and T84) and one with absorptive cell-like morphology (Caco-2). By Northern blot analysis the intestinal mucin genes MUC2 and MUC3 were constitutively expressed by confluent LS174T and Caco-2 cells, whereas T84 cells only transcribed MUC2 and not MUC3 mRNA. ³H-glucosamine and ³H-threonine metabolically labeled proteins separated as high M_r mucins in the void (V_o > 10⁶ Da) of Sepharose-4B column chromatography and remained in the stacking gel of SDS-PAGE as depicted by fluorography. All mucin preparations contained high amounts of N-acetyl-glucosamine, galactose, N-acetyl-galactosamine, fucose and sialic acid, saccharides typical of the O-linked carbohydrate side chains. Mucin samples from the human colon and from LS174T and Caco-2 cells inhibited E. histolytica adherence to Chinese hamster ovary cells, whereas mucins from T84 cells did not. These results suggest that genetic heterogeneity and/or posttranslational modification in glycosylation of colonic mucins can affect specific epithelial barrier function against intestinal pathogens.     

The T84 human colonic adenocarcinoma cell line produces mucin in culture and releases it in response to various secretagogues.

The T84 colonic adenocarcinoma cell line, which has been used extensively as a model for studies of epithelial chloride secretion, also produces mucin and secretes it in culture. Electron microscopy of fixed sections of cultured cells, along with Immunogold labelling with an antibody to human small intestine (SI) mucin, revealed the presence of goblet-like cells with mucin-containing secretory granules. The mucin was of high molecular mass, had an amino acid composition similar to that of purified human SI and colonic mucins, and competed effectively with SI mucin for binding to the anti-(SI mucin) antibody. A sensitive solid-phase immunoassay specific for intestinal mucins was developed and used to measure mucin secretion by T84 cells. Cultures were treated for 30 min at 37 degrees C with a number of agents known to cause chloride secretion by T84 cell monolayers and the amount of mucin appearing in the medium was measured. Carbachol (1 mM), A23187 (10 microM), prostaglandin E1 (PGE1) (1 microM) and vasoactive intestinal polypeptide (VIP) (0.1 microM) all stimulated mucin release, but histamine (1 mM) had no effect. Whereas VIP is reported to stimulate chloride secretion more strongly than carbachol, it was less effective than carbachol in stimulating mucin secretion. Phorbol 12-myristate 13-acetate (PMA) (0.1-10 microM) also stimulated mucin release strongly, implicating a responsive protein-kinase C-dependent pathway. Additive secretory responses were obtained with combined stimulation by VIP (10 nM-1 microM) and carbachol (1 mM). Responses to stimulation with A23187 (1-10 microM) together with PMA (10 nM-10 microM) suggest that cytosolic Ca2+ concentration is a modulator of PMA activity.     

Variation of mucin distribution in the rat intestine, caecum and colon: effect of the bacterial flora.

The influence of the intestinal microflora on mucin types was studied in the small intestine, caecum and colon of conventional (CV) rats as compared to germ-free (GF) rats. A colorimetric method was used on purified water-soluble mucin extracted from mucosal scrapings and contents. Variations occurred between the three anatomical sites both in the mucosas and intestinal contents of GF rats. In CV rats, the presence of the bacterial flora led to different effects depending on the intestinal site: in the small intestinal mucosa, neutral and sulphomucins values were higher whereas sialomucin was much lower. Conversely, sialomucin was higher in the caecal and colonic mucosas and contents whereas sulphated mucins were decreased significantly in caecal contents and caecal and colonic mucosas. These variations in the contents may reflect the bacterial mucolytic activity and the effect of bacterial metabolites on the mucosa.     

Novel goblet cell gene related to IgGFcgammaBP is regulated in adapting gut after small bowel resection

The loss of functional small bowel surface area leads to a well-described adaptive response in the remnant intestine. To elucidate its molecular regulation, a cohort of cDNAs were cloned using a rat gut resection model and subtractive/differential hybridization cloning techniques. This study reports a novel cDNA termed "ileal remnant repressed" (IRR)-219, which shares 80% nucleotide identity with the 3'end of a human intestinal IgG Fc binding protein (IgGFcgammaBP) and is homologous to human and rat mucins. IRR-219 mRNA is expressed in intestine and colon only. At 48 h after 70% intestinal resection, mRNA levels decreased two- to fivefold in the adaptive small bowel but increased two- to threefold in the colon. Expression of IRR-219 was suppressed in adaptive small bowel as late as 1 wk after resection. IRR-219 expression is also regulated during gut ontogeny. In situ hybridization revealed IRR-219 expression in small intestinal and colonic goblet cells only. Its unique patterns of expression during ontogeny and after small bowel resection suggest distinctive roles in small bowel and colonic adaptation.     

Quantitation of lectin binding sites in human colon mucins by use of peanut and wheat germ agglutinins

We have developed a novel method for quantitation of lectin binding sites in mucins derived from colon tissues. Binding of peanut agglutinin and wheat germ agglutinin was measured in extracts from normal and malignant human colon epithelium. Binding of wheat germ agglutinin was used as an estimate of the total mucin present in the tissue extract. Peanut agglutinin was found to bind to mucin from normal colon, but at levels that may be difficult to appreciate by fluorescence microscopy. The yield of mucin extracted from colon cancer was more variable than that from normal colon, and the binding ratio of peanut agglutinin to wheat germ agglutinin was greater in extracts from tumors than in normal tissues. Our findings confirm the histological observation that peanut agglutinin binds more avidly to mucins from colon cancer than to those from normal colon. The finding of peanut agglutinin binding sites in mucins front normal colon was not expected. The quantitative technique may have detected small numbers of binding sites not readily appreciable by fluorescence microscopy. Alternatively, the chromatographic method for measuring lectin binding may be sufficiently sensitive to detect nonspecific binding of the lectin to terminal galactose residues other than the Thomsen-Friedenreich antigen.     

The human commensal Bacteroides fragilis binds intestinal mucin

The mammalian gastrointestinal tract harbors a vast microbial ecosystem, known as the microbiota, which benefits host biology. Bacteroides fragilis is an important anaerobic gut commensal of humans that prevents and cures intestinal inflammation. We wished to elucidate aspects of gut colonization employed by B. fragilis. Fluorescence in situ hybridization was performed on colonic tissue sections from B. fragilis and Escherichia coli dual-colonized gnotobiotic mice. Epifluorescence imaging reveals that both E. coli and B. fragilis are found in the lumen of the colon, but only B. fragilis is found in the mucosal layer. This observation suggests that physical association with intestinal mucus could be a possible mechanism of gut colonization by B. fragilis. We investigated this potential interaction using an in vitro mucus binding assay and show here that B. fragilis binds to murine colonic mucus. We further demonstrate that B. fragilis specifically and quantitatively binds to highly purified mucins (the major constituent in intestinal mucus) using flow cytometry analysis of fluorescently labeled purified murine and porcine mucins. These results suggest that interactions between B. fragilis and intestinal mucin may play a critical role during host-bacterial symbiosis.     

The contribution of tandem repeat number to the O-glycosylation of mucins

The serine- and threonine-rich tandem repeat (TR) units that make up the characteristic feature of mucin glycoproteins are often polymorphic with substantial genetic variation in TR number. The precise effect of TR number on O-glycosylation is not fully understood, although the TR number of several mucins may be associated with apparent susceptibility to certain human diseases. To evaluate the contribution of TR number to O-glycosylation, we generated a series of chimeric mucins carrying increasing numbers of TR units from the MUC5B mucin in the context of an epitope-tagged MUC1 mucin backbone. These mucins were expressed in Caco2 colon carcinoma cell clones and purified by immunoprecipitation. O-Glycosylation was investigated by western blotting with antibodies to known carbohydrate structures and by fast atom bombardment-mass spectrometry. Additional carbohydrate epitopes were detected with antibodies on chimeric mucins with a higher TR number in comparison to those with fewer TRs. Using mass spectrometry, higher-molecular-weight glycans were detected more frequently on the mucins with extended TRs compared to those with fewer TRs. However no novel carbohydrate structures were seen, suggesting that TR number does not affect the specificity of O-glycosylation.     

The putative ''link'' glycopeptide associated with mucus glycoproteins. Composition and properties of preparations from the gastrointestinal tracts of several mammals.

The existence of a discrete 'link' peptide in epithelial mucins has been debated for many years. There is evidence that at least some mucins contain a specific 'link' peptide (or glycopeptide) that enhances mucin polymerization by forming disulphide bridges to large mucin glycoprotein subunits. A major difficulty has been to know whether the reported differences in putative 'link' components represent artifacts generated by inter-laboratory differences in technical procedures used in mucin purification. The present paper outlines the results of a collaborative study involving five laboratories and 53 samples of purified gastrointestinal mucins (including salivary, gastric, small-intestinal and colonic mucins) prepared by five techniques from four different animal species. An early step in mucin purification in all cases was the addition of proteinase inhibitors. Representative mucins were analysed for their composition, electrophoretic mobility in SDS/polyacrylamide-gel electrophoresis before and after disulphide-bond reduction, and for their reactivity with monospecific antibodies developed against the 118 kDa putative 'link' glycopeptide isolated from either rat or human small-intestinal mucins. Our results indicate that, despite differences in laboratory techniques, preparative procedures, organs and species, each of the purified mucins contained a 'link' component that was released by disulphide-bond reduction and produced a band on SDS/polyacrylamide-gel electrophoresis at a position of approx. 118 kDa. After electroelution and analyses, the 118 kDa bands from the different mucins were found to have similar amino acid profiles and to contain carbohydrate. It would appear therefore that a 'link' glycopeptide of molecular mass approx. 118 kDa is common to all of the gastrointestinal mucins studied.     

Leptin modulates the expression of secreted and membrane-associated mucins in colonic epithelial cells by targeting PKC, PI3K, and MAPK pathways

Mucins play an essential role in the protection and repair of gastrointestinal mucosa. We recently showed that luminal leptin strongly stimulated mucin secretion in vivo in rat colon. In the present study, we challenged the hypothesis that leptin may act directly on goblet cells to induce mucin expression in rat and human intestinal mucin-producing cells (DHE and HT29-MTX). The endoluminal effect of leptin was also studied in vivo in rat perfused colon model. The presence of leptin receptors was demonstrated in the two cell lines by Western blot and RT-PCR. In rat DHE cells, leptin (0.01-10 nmol/l, 60 min) dose dependently increased the secretion of mucins (210 +/- 3% of controls) and the expression of Muc2, Muc3, and Muc4 (twofold basal level) but not of Muc1 and Muc5AC. Luminal perfusion of leptin (60 min, 0.1-100 nmol/l) in rat colon also increased the mRNA level of Muc2, Muc3, and Muc4 but not of Muc1. In human HT29-MTX cells, leptin (0.01-10 nmol/l, 60 min) dose dependently enhanced MUC2, MUC5AC, and MUC4 mRNA levels. These effects were prevented by pretreatment of cells with the leptin mutein L39A/D40A/F41A, which acts as a receptor antagonist. Finally, pathway inhibition experiments suggest that leptin increased mucin expression by activating PKC-, phosphatidyl inositol 3-kinase-, and MAPK-dependent pathways but not the JAK/STAT pathway. In conclusion, leptin may contribute significantly to membrane-associated and secreted mucin production via a direct stimulation of colonic epithelial cells and the activation of leptin receptors. These data are consistent with a role for leptin in regulation of the intestinal barrier function.     

Biochemical and histochemical effects of perorally applied endotoxin on intestinal mucin glycoproteins of the common carp Cyprinus carpio

Mucins are high molecular weight glycoproteins produced by goblet cells and secreted on mucosal surfaces. We investigated biochemical and histochemical properties of intestinal mucins of virus- and parasite-free common carp Cyprinus carpio in response to a single peroral application of endotoxin (lipopolysaccharide = LPS). Intracellular mucins were quantified histochemically by their carbohydrate content and characterized by specific, lectin-based methods. In addition, secreted epithelial (intracellular) and luminal (extracellular) mucins were isolated and separated by downward gel filtration. Carbohydrate and protein content were determined photometrically. Subsequently, terminal glycosylation was characterized by a lectin-binding assay. A peroral endotoxin application altered intestinal secretion and composition of intestinal mucin glycoproteins in common carp. A statistically significant decrease in mature luminal mucins was demonstrated, linked to a new biosynthesis of intracellular mucin glycoproteins. Simultaneous changes in the glycosylation pattern of isolated mucins were found. The intestinal mucosal system is purported to provide a removal mechanism for bacterial noxes by increasing secretion of mucins inducing a flushing-out effect, in combination with altered glycosylation patterns that change adhesion properties. Consequently, pseudofaeces of fish, which are a common sign of intestinal parasitical infections, may also be interpreted as an elimination mechanism for strong bacterial noxes.     

Microbial adhesins to gastrointestinal mucus

The gastrointestinal tract (GIT) is lined by a layer of mucus formed by mucin glycoproteins. This layer constitutes a physical and chemical barrier between the intestinal contents and the underlying epithelia. In addition to this protective role, mucins harbor glycan-rich domains that provide preferential binding sites for pathogens and commensal bacteria. Although mucus-microbial interactions in the GIT play a crucial role in determining the outcome of relationships of both commensal and pathogens with the host, the adhesins and ligands involved in the interaction are poorly delineated. This review focuses on the current knowledge of microbial adhesins to gastrointestinal mucus and mucus components.     

Characterization of rat gastric mucins using a monoclonal antibody,RGM23, recognizing surface mucous cell-type mucins

A novel anti-mucin monoclonal antibody (mAb), designated RGM23, was developed against mucin purified from rat gastric mucosa. RGM23 reacted with the mucin attached to the ELISA well. The reactivity was lost by trypsin treatment, but not by periodate oxidation, indicating that RGM23 recognizes the peptide moiety of the mucin molecule. Histochemical study showed that RGM23 stained the corpus and antral surface mucosa of rat stomach, but not their glandular mucosa, nor duodenal, small intestinal or large intestinal mucosa. The area stained with RGM23 was coincident with that stained with 45M1, a mAb reacting with MUC5AC mucin. Examination of the mucin subunits extracted from rat stomach by Sepharose CL-4B and Q-Sepharose chromatography and CsTFA equilibrium centrifugation showed that RGM23 reacted with the surface mucous cell-type mucins that were stained with periodate-Schiff (PAS) and reacted with mAb RGM21. The gastric gland-type mucin, which reacted with mAb HIK1083, did not react with RGM23. On Q-Sepharose chromatography, a part of the RGM21-reactive mucins was only faintly stained with PAS and did not react with RGM23. The results together indicated that RGM23 probably reacted with the rat MUC5AC (rMuc5AC) mucin present in the surface mucosa of the stomach, and that the surface mucosal cells in rat stomach may contain mucin bearing non-rMuc5AC core protein in addition to rMuc5AC mucins.     

The StcE metalloprotease of enterohaemorrhagic Escherichia coli reduces the inner mucus layer and promotes adherence to human colonic epithelium ex vivo

Enterohaemorrhagic Escherichia coli (EHEC) is a major foodborne pathogen and tightly adheres to human colonic epithelium by forming attaching/effacing lesions. To reach the epithelial surface, EHEC must penetrate the thick mucus layer protecting the colonic epithelium. In this study, we investigated how EHEC interacts with the intestinal mucus layer using mucin‐producing LS174T colon carcinoma cells and human colonic mucosal biopsies. The level of EHEC binding and attaching/effacing lesion formation in LS174T cells was higher compared to mucin‐deficient colon carcinoma cell lines, and initial adherence was independent of the presence of flagellin, Escherichia coli common pilus, or long polar fimbriae. Although EHEC infection did not affect gene expression of secreted mucins, it resulted in reduced MUC2 glycoprotein levels. This effect was dependent on the catalytic activity of the secreted metalloprotease StcE, which reduced the inner mucus layer and thereby promoted EHEC access and binding to the epithelium in vitro and ex vivo. Given the lack of efficient therapies against EHEC infection, StcE may represent a suitable target for future treatment and prevention strategies.