Mucin Degradation Research Articles

Influence of Antibiotics on Some Intestinal Microflora Associated Characteristics

Intestinal functions related to the presence of microbes in host organisms are normally heavily influenced by administration of antimicrobial drugs. We have investigated the effect of several antibiotics in man and rat, on some MACs (Microflora Associated Characteristics).A MAC is defined as the recording of any anatomical structure, biochemical or physiological function in the host organism which is influenced by microflora. When functional, active microbes are absent as in germfree animals, healthy newborns, or in relation to antimicrobial therapies, a MAC defined as a GAC (Germfree Animal Characteristic).Faecal samples have been collected prior to, during and up to several weeks after the antimicrobial administration in order to investigate different MAC/GAC patterns. Microbial conversion of cholesterol to coprostanol, bilirubin to urobilinogen and 7-alpha-dehydroxylation of cholic acid have been investigated to evaluate hepatic/intestinal co-functions, and degradation of intestinal mucin in order to evaluate the integrity in the intestinal mucosa. Furthermore, degradation of the dietary derived β-aspartylglycine, the level of faecal trypsin and production of short chain fatty acids were investigated to evaluate dietary/intestinal co-functions.It is concluded that each antimicrobial drug creates its own profile, both with regard to duration and function.

Bacterial glycosulphatases and sulphomucin degradation.

The presence of a high bacterial population in a region of the gastrointestinal tract is usually associated with the secretion of sulphomucins into the mucus gel covering that region. The term 'sulphomucin' is a histochemical description of the staining properties of mucin. At present this term can only be qualitatively related to the percentage of sulphate in the mucin molecule, which makes the term difficult to use in a biochemical and functional sense. Sulphomucins are thought to carry out the normal functions attributed to mucins; in addition, heavy sulphation rate-limits the degradation of mucins by bacterial mucin-degrading glycosidases. A number of mucin-specific glycosulphatases have been reported in bacteria, although only two such enzymes have been purified. These enzymes remove part of the sulphate content from sulphomucins and make them more susceptible to further enzymic degradation. The variety of chain locations and sugar attachment sites of sulphate esters on the mucin oligosaccharides, taken together with the data on the enzymes, suggest there will be a spectrum of bacterial glycosulphatases, with different properties, cellular locations and substrate specificities. Bacterial glycosulphatases have the potential to modify sulphated glycoconjugates at mucosal surfaces and should prove useful as biochemical tools for the study of sulphated glycoconjugates.

Lysosomal enzyme activities in gallbladder mucosa of gallstone-free subjects and patients with gallstones

Background/Aims: Gallstone patients have a reduced cellular lysosome content in the gallbladder mucosa cells compared with gallstone-free subjects. The purpose of the study was to further evaluate the possible role of lysosomes in the pathogenesis of cholesterol gallstone formation in humans. Methods: Lysosomal enzyme activities were assayed in gallbladder mucosa and for comparison in liver specimens of 19 gallstone-free subjects and 24 gallstone patients undergoing cholecystectomy. Results: Gallstone patients had 25–50% lower activities of the lysosomal proteases cathepsin B, D and L in their gallbladder mucosa compared with gallstone-free subjects. The activity of acid phosphatase also tended to be decreased in gallstone patients. The liver lysosomal enzyme activities were not significantly different between the two groups. Conclusions: The results show that gallstone patients have diminished lysosomal enzyme activities in the gallbladder mucosa, a finding which may be related to decreased intracellular degradation of proteins and/or mucin in the mucosal cells. This may lead to a higher concentration of mucin in gallbladder bile and thus an increased risk of precipitation of cholesterol crystals and gallstone formation.

Evidence for degradation of gastrointestinal mucin by Candida albicans secretory aspartyl proteinase.

A zone of extracellular digestion of the mucin layer around Candida albicans blastoconidia was observed by transmission electron microscopy in the jejunum of mice inoculated intragastrically (G. T. Cole, K. R. Seshan, L. M. Pope, and R. J. Yancey, J. Med. Vet. Mycol. 26:173-185, 1988). This observation prompted the hypothesis that a putative mucinolytic enzyme(s) may contribute to the virulence of C. albicans by facilitating penetration of the mucus barrier and subsequent adherence to and invasion of epithelial cells. Mucinolytic activity was observed as zones of clearing around colonies of C. albicans LAM-1 grown on agarose containing yeast nitrogen base, glucose, and hog gastric mucin. In addition, concentrated culture filtrate obtained after growth for 24 h in yeast nitrogen base, supplemented with glucose and mucin as the sole nitrogen source, contained proteolytic activity against biotin-labelled mucin which was inhibited by pepstatin A. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the culture filtrate revealed two components of 42 and 45 kDa, with pIs of 4.1 and 5.3, respectively. A zymogram showed that mucin was degraded only by the 42-kDa component, which was also recognized by immunoblotting with an anti-secretory aspartyl proteinase (anti-Sap) 2p monoclonal antibody. The N-terminal sequence of the first 20 amino acids matched that reported for Sap2p. These results demonstrate that Sap2p is responsible for proteolysis of mucin by C. albicans in vitro and may be involved as a virulence factor in the breakdown of mucus and penetration of the mucin barrier by C. albicans.

Synthesis and Intracellular Trafficking of Muc-1 and Mucins by Polarized Mouse Uterine Epithelial Cells

Mucins function as a protective layer rendering the apical surface of epithelial cells nonadhesive to a variety of microorganisms and macromolecules. Muc-1 is a transmembrane mucin expressed at the apical cell surface of mouse uterine epithelial cells (UEC) that disappears as UEC become receptive for embryo implantation (Surveyor, G. A., Gendler, S. J., Pemberton, L., Das, S. K., Chakraborty, I., Julian, J., Pimental, R. A., Wegner, C. W., Dey, S. K., and Carson, D. D. (1995) Endocrinology 136, 3639-3647). In the present study, the kinetics of Muc-1 assembly, cell surface expression, release, and degradation were examined in polarized mouse UEC in vitro. Mucins were identified as the predominant glycoconjugates synthesized, apically expressed, and vectorially released in both wild-type and Muc-1 null mice. When mucins were released, greater than 95% were directed to the apical compartment. Approximately half of the cell-associated mucins lost during a 24-h period were found in the apical compartment. Vectorial biotinylation detected apically disposed, cell-surface mucin and indicated that at least 34% of these mucins are released apically within 24 h. This suggests that release of mucin ectodomains is part of the mechanism of mucin removal from the apical cell surface of UEC. The half-lives of total cell-associated mucins and Muc-1 were 19.5 +/- 1 and 16.5 +/- 0.8 h, respectively. Muc-1 represented approximately 10% of the [3H]glucosamine-labeled, cell-associated mucins. Studies of the kinetics of intracellular transport of Muc-1 indicated transit times of 21 +/- 15 min from the rough endoplasmic reticulum to Golgi apparatus and 111 +/- 28 min from the Golgi apparatus to the cell surface. Collectively, these studies provide the first comprehensive description of Muc-1 and mucin maturation, metabolism, and release by polarized cells, as well as defining a major metabolic fate for mucins expressed by UEC. Normal metabolic processing appears to be sufficient to account for the removal of Muc-1 protein during the transition of UEC to a receptive state.

Effect of Olestra Upon Intestinal Microecology as Reflected by Five Microflora Associated Characteristics in Man

To determine the effect of olestra on microbial ecology of the gut five microflora associated characteristics (MACs) were assessed in 93 subjects in a 36 d parallel, placebo-controlled, double-blind study. Degradation of tryptic activity, β-aspartylglycine and mucin, and conversion of cholesterol to coprostanol and bilirubin to urobilinogen were determined in faecal samples collected at the end of an 8 d low fibre baseline period and 28 d treatment period. The test meal consisted of either a moderate (7 g) or high (24 g) level of fibre and olestra (24 g) or an equivalent amount of conventional fat. Olestra had no effect on degradation of tryptic activity or β-aspartylglycine, and no consistent effect on mucin degradation. Olestra reduced significantly the conversion of cholesterol to coprostanol and urobilin to urobilinogen although the individual values were all within the range of healthy population after treatment. The lipophilic nature of cholesterol and urobilin is the most likely reason for the find...

Future aspects on inflammatory bowel disease.

Inflammatory bowel diseases (IBD) are probably multifactorial diseases caused by an interplay of the external and internal environment. External environmental factors revealed in the literature so far can be grouped into four major groups or hypotheses: (a) the sheltered child, (b) infectious agents, (c) dietary factors, and (d) vascular factors. Internal environmental factors mainly concern genetics which have been studied in families or twins. At present, HLA class II antigens are most in focus but also genetic markers such as pANCA. However, the internal environment can be changed by external factors changing the balance of the faecal or mucosa related flora, such as infectious agents or antibiotic treatment. This in turn may upset the building up and degradation of colonic mucin, making the mucosa more vulnerable. The increasing knowledge of external and internal environmental factors might affect our way of treatment in the future. So also may our increasing knowledge of immunology which will probably lead to targeted treatment directed against individual components within the inflammatory cascade.

Morphometric analysis of intestinal mucins under different dietary conditions and gut flora in rats

Elucidation of the mechanisms that alter the biosynthesis, turnover, and degradation of intestinal mucins is relevant to the understanding of both the normal gut ecosystem and various intestinal diseases. In this study image analysis was used to quantify the effects of diet and microbial flora on the mucin composition of goblet and deep crypt cells, the number and volume density of mucin-containing cells, and the staining density of their stored mucins in the small and large intestine of germ-free and conventionally maintained rats fed two different diets. One was a coarsely ground commercial rodent diet containing crude fiber of cereal origin and the other a purified diet composed of finely powdered ingredients, including cellulose as a source of fiber. The changes in mucin production were also analyzed in germ-free rats colonized with a human flora. Feeding a commercial diet reduced the volume density of cells containing neutral and sulfomucins in the jejunum of conventional rats and the staining density of neutral and acidic mucins in the germ-free rats. Both rat and human floras reduced the number of cells containing acidic and sulfomucins and the staining density of neutral mucins in the small intestine of animals fed on a purified diet. However, inoculation of human flora increased the staining density of stored neutral and sulfated mucins in the cells of the large intestine. The results demonstrate that the dietary changes are influential in modifying the amount and proportion of mucins in the small intestine and the microbial flora in the large intestine.

Pouchitis: result of microbial imbalance?

To elucidate the role of microbiological factors in pouchitis, this study investigated the composition of ileal reservoir microflora, the mucus degrading capacity of bacterial enzymes as well as the pH and the proteolytic activity of pouch effluent. Stool samples were collected from five patients with pouchitis and nine patients without pouchitis. The flora of patients with pouchitis had an increased number of aerobes, a decreased ratio anaerobes to aerobes, less bifidobacteria and anaerobic lactobacilli, more Clostridium perfringens, and several species that were not found in control patients (for example, fungi). Furthermore the pH was significantly higher in patients with pouchitis (median value 6.5) than in control patients (5.4). To find out if the pH might influence the breakdown of intestinal mucus glycoproteins, the activity of glycosidases and proteases, and the degradation of hog gastric mucin by the pouch flora was tested at pH 5.2-7.6. Some glycosidases were inhibited, others were stimulated by a low pH, however, in each sample the proteolytic activity was inhibited for 75% at pH 5.2 compared with pH 6.8 and 7.6. Degradation of hog gastric mucin by the pouch flora was an active process at pH 7.2: within two to four hours of incubation more than half of the mucin was degraded. At pH 5.2 it took twice as long. It is concluded that pouchitis possibly results from instability of the flora in the pouch, which causes homeostasis to disappear (dysbiosis), and the protection of the pouch epithelium by the mucus layer becomes affected by increased activity of bacterial and host derived enzymes.

Establishment of a Mucin‐Degrading Intestinal Microflora During the First Two Years of Human Life

The establishment of a mucin‐degrading intestinal microflora was followed in 30 healthy Swedish children by agar gel electrophoresis of fecal samples taken at 0, 1, 3, 6, 9, 12, 15, 18, 21, and 24 months of age. At 3 months, 18 children showed a limited degree of mucin degradation, while 12 children showed no degradation. In children exclusively breast‐fed for at least 4 months, mucin degradation was initiated significantly later than in children who received formula, with or without breast milk, by 1 month of age. Complete degradation of mucin was seen in 21 children after 1 year of age and in all children at 2 years. A positive correlation between increasing age and increased degradation of mucin was found between birth and 1 month and between 6 and 9 months, after standardization for the period of exposure to foods other than breast milk.

Establishment of a mucin-degrading intestinal microflora during the first two years of human life.

The establishment of a mucin-degrading intestinal microflora was followed in 30 healthy Swedish children by agar gel electrophoresis of fecal samples taken at 0, 1, 3, 6, 9, 12, 15, 18, 21, and 24 months of age. At 3 months, 18 children showed a limited degree of mucin degradation, while 12 children showed no degradation. In children exclusively breast-fed for at least 4 months, mucin degradation was initiated significantly later than in children who received formula, with or without breast milk, by 1 month of age. Complete degradation of mucin was seen in 21 children after 1 year of age and in all children at 2 years. A positive correlation between increasing age and increased degradation of mucin was found between birth and 1 month and between 6 and 9 months, after standardization for the period of exposure to foods other than breast milk.

Colonic cell growth and mucin degradation in rats fed diets containing various levels of beta-carotene with and without dietary agar

1. 1. To either an agar-containing diet or an agar-free diet, 0, 0.3 and 2.0 mg/100g of beta-carotene were incorporated and fed to groups of five rats for 28 days. 2. 2. Weight gain and food consumption of rats fed different dietary groups did not show a significant difference ( P > 0.05). 3. 3. Colon weight, colonie mucosal DNA and RNA were generally higher in rats fed agar diets than rats fed agar-free diets at either beta-carotene supplementation level. 4. 4. Mucinase activity was higher ( P < 0.05) in rats fed the agar diet than in rats fed an agar-free diet without beta-carotene. However, the difference was not observed ( P > 0.05) when beta-carotene was incorporated. 5. 5. These data suggest that colonie mucin degradation in rats fed an agar diet decreased when the dietary beta-carotene inclusion level increased.

Mucin degradation in the human gastrointestinal tract and its significance to enteric microbial ecology

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The roles of enteric bacterial sialidase, sialateO-acetyl esterase and glycosulfatase in the degradation of human colonic mucin

Sialidase activity in normal faecal extracts showed a preference for mucin-related glycoprotein and oligosaccharide substrates, but the presence of two or more O-acetyl esters at positions C7-C9 on the sialic acids retarded the rate of hydrolysis. A specific sialate O-acetyl esterase was detected with a lower total activity relative to sialidase with mucin substrates and having a pH optimum of 7.8 and a KM of approximately 1 mM sialate O-acetyl ester. A specific glycosulfatase activity was found in faecal extracts using the substrate lactit-[3H]ol 6-O-sulfate with a pH optimum of pH 5.0 and a KM of approximately 1 mM. Faecal extracts from ulcerative colitis (UC) patients had higher sialate O-acetyl esterase and glycosulfatase activity, while mucin sialidase activity was unchanged. Metabolically labelled mucin isolated from UC patients contained less sulfate and had lower sialic acid O-acetylation compared with normal mucin. Colonic mucin was degraded more efficiently by faecal extracts from UC patients compared with normal extracts. The UC mucin was degraded more rapidly than the normal mucin by faecal enzyme extracts from both normal and UC subjects.

Mucin degradation in the human colon: production of sialidase, sialate O-acetylesterase, N-acetylneuraminate lyase, arylesterase, and glycosulfatase activities by strains of fecal bacteria.

Oligosaccharide side chains of human colonic mucins contain O-acetylated sialic acids and glycosulfate esters. Although these substituents are considered to protect the chains against degradation by bacterial glycosidases, sialate O-acetylesterase, N-acetylneuraminate lyase, and glycosulfatase activities have been found in fecal extracts. To better define the source of these activities, we measured extracellular and cell-bound sialidase, sialate O-acetylesterase, N-acetylneuraminate lyase, arylesterase, and glycosulfatase activities produced by 23 isolates of human fecal bacteria grown anaerobically in a hog gastric mucin culture medium; these represented dominant populations of fecal anaerobes, facultative anaerobes, and the subset of mucin oligosaccharide-degrading bacteria. Every strain produced sialidase and high levels of arylesterase, and all but five facultative anaerobes produced sialate O-acetylesterase. Sialic acids containing 2 mol or more of O-acetyl ester per mol of sialic acid were cleaved from mucin glycoproteins more slowly by sialidases of mucin oligosaccharide-degrading stains than were sialic acids containing 1 or 0 mol, and only N-acetyl- and mono-O-acetylated sialic acids were recovered from enzyme digests of a mucin containing di-O-acetylated sialic acids. No detectable N-acetylneuraminate lyase activity was produced by any strain, but low activity was induced by increasing the glycoprotein-bound sialic acid concentration in the culture medium of six Escherichia coli strains. Using lactitol-6-sulfate as a substrate, we found weak glycosulfatase activity in the partially purified, concentrated enzyme mixture in the culture supernatants of four mucin oligosaccharide-degrading strains but in none of the unconcentrated culture fractions. We conclude that the presence of two or more O-acetyl groups on sialic acids inhibits enteric bacterial sialidases but that production of sialate O-acetylesterases by several populations of enteric bacteria lessens the likelihood that mucin oligosaccharide chains terminating in O-acetylated sialic acids are protected from degradation. Sialate O-acetylesterases have a role in bacterial degradation of mucin glycoproteins in the human colon.

The structure of tracheobronchial mucins from cystic fibrosis and control patients.

Tracheobronchial mucin samples from control and cystic fibrosis patients were purified by gel filtration chromatography on Sephacryl S-1000 and by density gradient centrifugation. Normal secretions contained high molecular weight (approximately 10(7] mucins, whereas the cystic fibrosis secretions contained relatively small amounts of high molecular weight mucin together with larger quantities of lower molecular weight mucin fragments. These probably represent products of protease digestion. Reducing the disulfide bonds in either the control or cystic fibrosis high molecular weight mucin fractions released subunits of approximately 2000 kDa. Treating these subunits with trypsin released glycopeptides of 300 kDa. Trypsin treatment of unreduced mucin also released fragments of 2000 kDa that could be converted into 300-kDa glycopeptides upon disulfide bond reduction. Thus, protease-susceptible linkages within these mucins must be cross-linked by disulfide bonds so that the full effects of proteolytic degradation of mucins remain cryptic until disulfide bonds are reduced. Since various combinations of protease treatment and disulfide bond reduction release either 2000- or 300-kDa fragments, these fragments must represent important elements of mucin structure. The high molecular weight fractions of cystic fibrosis mucins appear to be indistinguishable from control mucins. Their amino acid compositions are the same, and various combinations of disulfide bond reduction and protease treatment release products of identical size and amino acid composition. Sulfate and carbohydrate compositions did vary considerably from sample to sample, but the limited number of samples tested did not demonstrate a cystic fibrosis-specific pattern. Thus, tracheobronchial mucins from cystic fibrosis and control patients are very similar, and both share the same generalized structure previously determined for salivary, cervical, and intestinal mucins.

Synergistic Degradation of Mucin by Streptococcus oralis and Streptococcus sanguis in Mixed Chemostat Cultures

Oral streptococci can grow in mucin by utilizing the oligosaccharide chains as a source of carbohydrate. The degradation of the oligosaccharides by these species is accomplished by exoglycosidase activities. In this experiment, it was investigated whether strains from different species could cooperate in the release of sugars from the mucin oligosaccharide. To this end, Streptococcus sanguis Ny 584 and Streptococcus oralis strain Ny 586 were grown continuously in a chemically-defined medium, with pig gastric mucin as the growth-limiting source of carbohydrate. In pure cultures, strain Ny 586 attained approximately three-fold-higher cell densities than did strain Ny 584 in the mucin medium. This was in accordance with the observation that S. oralis Ny 586 exhibited fucosidase activity, as indicated by the presence of fucose in the culture fluid. In contrast, strain Ny 584 has no fucosidase activity against mucin, and therefore cannot attack fucose-ending oligosaccharide chains. Stable mixed cultures of the strains were obtained. It appeared that S. sanguis Ny 584 reached significantly higher cell densities in mixed cultures with S. oralis Ny 586 than in pure culture. Stimulation of the growth of strain Ny 584 was probably due to the generation of non-fucose-ending oligosaccharide chains by fucosidase from strain Ny 586. It is concluded that the synergistic degradation of oligosaccharides in glycoproteins is a potential factor influencing the streptococcal populations in the mouth.

Establishment of Lactobacillus and Bifidobacterium species in germfree mice and their influence on some microflora-associated characteristics

Germfree mice were inoculated with both Lactobacillus acidophilus A10 and Bifidobacterium bifidum B11. Both strains were established and present in more than 10(8) cells per g of cecum and colon contents. Furthermore, L. acidophilus A10 was established in high numbers in stomach and small intestine. Contents from different parts of the intestine were investigated with regard to the following microflora-associated characteristics: degradation of mucin, beta-aspartylglycine and tryptic activity, conversion of cholesterol to coprostanol and bilirubin to urobilinogen, deconjugation of bilirubin glucuronides, and reduction of the cecum size. In spite of being established, the microbes were not able to mediate any alterations of the parameters investigated. All animals retained values as found in their germfree counterparts.

The Relationship between Proteases Activity and Glycoprotein Levels in Middle Ear Effusions from Experimental Otitis Media in Cats

The relationship between lysosomal proteases activity (elastase and cathepsin B) and levels of mucous glycoproteins in middle ear effusions (MEEs) was studied using a cat model of otitis media with effusion (OME) induced by Eustachian tube obstruction (ETO). The ratio of cathepsin B activity to total protein concentration (TPC) in MEE was 25.6 +/- 19.4 RFU/g x dl-1 at 1 week after ETO, and increased with the duration of OME. The ratio of elastase activity to TPC had a significant correlation to total leukocyte count. The ratio of fucose levels to TPC, which is one of the parameters reflecting levels of mucous glycoprotein, at 1 week after ETO was significantly higher than that at both 2 and 4 weeks after ETO. The percentage of glycoprotein levels absorbed to wheat germ lectin was highest at 1 week after ETO, and decreased with the duration of OME. In conclusion, mucous glycoproteins in cat occupy a larger portion of glycoproteins in MEE at the early stage of OME, and elastase and other lysosomal proteases may play a role in both stimulation of mucin release from goblet cells and mucin degradation. The balance of these processes seems to be a key factor determining mucin levels in MEEs.

Enteral nutrition and the function of the intestinal microflora in healthy adults

An oral feeding formula was given to 5 healthy volunteers for 8 days. Faecal samples were collected before, during and after the feeding period. The effect of enteral nutrition (EN) on the following seven intestinal microflora-associated characteristics (MACs) was studied: formation of urobilinogen, coprostanol and deoxycholic acid, degradation of mucin and β-aspartylglycine, faecal tryptic activity, and production of short-chain fatty acids (SCFAs). None of the microbial functions studied were lost during the study. The urobilinogen level increased during EN (P < 0.05) but it seems reasonable to assume that this was a concentration effect due to a decrease in stool mass. The concentration of SCFAs decreased during EN (P < 0.05) and this reflects the absence of dietary fibre in the feed used.