Mucin Breakdown Research Articles

How microbial glycosyl hydrolase activity in the gut mucosa initiates microbial cross-feeding

The intestinal epithelium is protected from direct contact with gut microbes by a mucus layer. This mucus layer consists of secreted mucin glycoproteins. The outer mucus layer in the large intestine forms a niche that attracts specific gut microbiota members of which several gut commensals can degrade mucin. Mucin glycan degradation is a complex process that requires a broad range of glycan degrading enzymes, as mucin glycans are intricate and diverse molecules. Consequently, it is hypothesized that microbial mucin breakdown requires concerted action of various enzymes in a network of multiple resident microbes in the gut mucosa. This review investigates the evolutionary relationships of microbial carbohydrate-active enzymes that are potentially involved in mucin glycan degradation and focuses on the role that microbial enzymes play in the degradation of gut mucin glycans in microbial cross-feeding and syntrophic interactions.

Prominent members of the human gut microbiota express endo-acting O-glycanases to initiate mucin breakdown

The thick mucus layer of the gut provides a barrier to infiltration of the underlying epithelia by both the normal microbiota and enteric pathogens. Some members of the microbiota utilise mucin glycoproteins as a nutrient source, but a detailed understanding of the mechanisms used to breakdown these complex macromolecules is lacking. Here we describe the discovery and characterisation of endo-acting enzymes from prominent mucin-degrading bacteria that target the polyLacNAc structures within oligosaccharide side chains of both animal and human mucins. These O-glycanases are part of the large and diverse glycoside hydrolase 16 (GH16) family and are often lipoproteins, indicating that they are surface located and thus likely involved in the initial step in mucin breakdown. These data provide a significant advance in our knowledge of the mechanism of mucin breakdown by the normal microbiota. Furthermore, we also demonstrate the potential use of these enzymes as tools to explore changes in O-glycan structure in a number of intestinal disease states.

Gut Barrier Function Is Improved in Infants Colonized by Bifidobacterium Longum Subsp. Infantis EVC001 (FS04-05-19)

ObjectivesThe gut epithelium is single-celled barrier that employs many different mechanisms that together provide the first line of defense to physically separate the gut epithelium from our gut microbiome. Notably, the epithelial barrier is protected by a mucin layer providing a physical barrier limiting pathogen access to the epithelial monolayer. We sought to assess how changes in the gut microbiome resulting from colonization by a single strain of Bifidobacterium longum subsp. infantis EVC001 could alter gut barrier function. MethodsFecal samples from this trial were assessed for: (1) endotoxin (lipopolysaccharide) concentration; (2) functional contributions to the gut microbiome by shotgun metagenome sequencing; and (3) fecal glycan profiles by mass spectrometry to assess gut epithelial barrier integrity via breakdown of colonic mucin glycoproteins. ResultsColonization with Bifidobacterium, including B. infantis EVC001, showed a significant four-fold reduction in fecal endoxtoxin levels and reductions in fecal inflammatory markers (P < 0.05). Shotgun metagenomics identified LPS-producing Enterobacteriaceae as the most significant contributor of virulence factors in the infant gut metagenome. These bacteria (primarily E. coli and Klebsiella spp.) were also significantly correlated with both mucolytic bacteria (e.g., Bacteroides) and the signatures of mucin breakdown, as assessed by mass spectrometric quantification of colonic mucin-derived glycans. Five different colonic-mucin specific glycans (3_1_1_0, 2_1_2_0, 2_1_1_1, 2_1_1_0, and 1_1_0_1) were significantly associated with microbiome composition (P < 0.05). Overall mucin glycans were inversely correlated with Bifidobacteriaceae abundance (Spearman’s rho −0.66, FDR-corrected P value 0.04). ConclusionsComplex interactions between the degradation of gut barrier function (e.g., mucin), the production of pro-inflammatory endotoxins, and the risk of infection by these bacteria coming in close contact with the gut epithelium suggest that B. infantis EVC001 can play a role in reducing these combined risks for neonates. Funding SourcesThis work was funded by Evolve Biosystems, Inc.

Breakdown of Mucin as Barrier to Digestive Enzymes in the Ischemic Rat Small Intestine

Loss of integrity of the epithelial/mucosal barrier in the small intestine has been associated with different pathologies that originate and/or develop in the gastrointestinal tract. We showed recently that mucin, the main protein in the mucus layer, is disrupted during early periods of intestinal ischemia. This event is accompanied by entry of pancreatic digestive enzymes into the intestinal wall. We hypothesize that the mucin-containing mucus layer is the main barrier preventing digestive enzymes from contacting the epithelium. Mucin breakdown may render the epithelium accessible to pancreatic enzymes, causing its disruption and increased permeability. The objective of this study was to investigate the role of mucin as a protection for epithelial integrity and function. A rat model of 30 min splanchnic arterial occlusion (SAO) was used to study the degradation of two mucin isoforms (mucin 2 and 13) and two epithelial membrane proteins (E-cadherin and toll-like receptor 4, TLR4). In addition, the role of digestive enzymes in mucin breakdown was assessed in this model by luminal inhibition with acarbose, tranexamic acid, or nafamostat mesilate. Furthermore, the protective effect of the mucin layer against trypsin-mediated disruption of the intestinal epithelium was studied in vitro. Rats after SAO showed degradation of mucin 2 and fragmentation of mucin 13, which was not prevented by protease inhibition. Mucin breakdown was accompanied by increased intestinal permeability to FITC-dextran as well as degradation of E-cadherin and TLR4. Addition of mucin to intestinal epithelial cells in vitro protected against trypsin-mediated degradation of E-cadherin and TLR4 and reduced permeability of FITC-dextran across the monolayer. These results indicate that mucin plays an important role in the preservation of the mucosal barrier and that ischemia but not digestive enzymes disturbs mucin integrity, while digestive enzymes actively mediate epithelial cell disruption.

Genetic strategies for mucin metabolism in Bifidobacterium bifidum PRL2010: An example of possible human-microbe co-evolution

Recently, we decoded the genome sequence of Bifidobacterium bifidum PRL2010, an isolate originally recovered from a stool sample of a three month old infant. In silico analysis revealed that the the 2,214,650 bp genome of B. bifidum PRL2010 encoded a rich gene repertoire dedicated to the metabolism of host-derived glycans, such as those present in mucin. Whole proteome profiling as well as transcriptomic investigations were used to identify the genetic players and mechanisms responsible for mucin breakdown, revealing a set of chromosomal loci that allow degradation of various O-linked glycans in mucin (such as core 1, core 2, core 3 and core 4 O-glycans). Comparative genomic analyses involving different B. bifidum genomes demonstrated the ubiquitous presence of these genetic regions in genomes of members of this bifidobacterial taxon, and suggest specific B. bifidum metabolic traits that underpin this case of host-microbe co-evolution.

Biochemical Functions ofBacillus licheniformisin Gnotobiotic Mice

A probiotic bacterial strain, Bacillus licheniformis NCTC 13123, has been mono-associated with germ-free mice. After colonization, the following biochemical microflora-associated characteristics (MACs) were analyzed in the large intestinal contents: excretion of short-chain fatty acids (SCFAs), inactivation of tryptic activity, degradation of β -aspartylglycine, breakdown of mucin, conversion of cholesterol to coprostanol and transformation of bilirubin to urobilins. An additional MAC, b-glucuronidase activity, was tested in vitro. β -Glucuronidase activity was not expressed by the bacterium. The amount of total SCFAs was lower than that previously found in germ-free mice. Utilization of individual SCFAs rather than production by the strain may be assumed. The other MACs remained similar to the basal values in germfree mice. We conclude that B. licheniformis NCTC 13123 is not able to alter any of the microbial functions investigated in vitro or in the gastrointestinal tract of germ-free mice. Whether and to what extent the strain has the capability to interfere with these functions in the presence of other micro-organisms remains to be investigated. Key wordis : Bacillus licheniforms , probiotics, microflora-associated characteristics, germ-free mice.

Glycosidases in mucin-dwelling protozoans.

A range of protozoans were tested for the presence of glycosidases using p-nitrophenyl sugars as substrates. Some of the organisms were mucin dwellers whereas others were blood borne parasites. It had been hypothesized that glycosidase production would be significantly higher in the mucin dwellers. The results obtained demonstrated that the urogenital protozoans Tritrichomonas foetus and Trichomonas vaginalis produced a vast range of glycosidases which included those required for mucin breakdown. The gut dwelling protozoans Giardia lamblia and Entamoeba histolytica both produced beta-N-acetylglucosaminidase. G.lamblia also had detectable beta N-acetylgalactosaminidase activity, and small amounts of beta mannosidase were found in the extracts from E. histolytica. In contrast, little or no glycosidase activity was detected under the same experimental conditions in Leishmania donovani, Trypanosoma brucei or T. cruzi. The mucin dwelling protozoans all produce beta-N-acetylglucosaminidase but only the Trichomonads produced the range of enzymes required for complete breakdown of mucin. This seems to suggest that mucin breakdown is not a characteristic of all mucin dwelling protozoans.

Faecal Concentration of Ten Antibiotics and Influence on Some Microflora-Associated Characteristics (MACs)

Ten antimicrobial agents were given for 6 d to six or seven healthy volunteers. Ampicillin 500 mg q.i.d., bacitracin 25 000 1U q.i.d., clindamycin 150 mg q.i.d., co-trimoxazole 160/800 mg b.i.d., doxycycline 200 mg on day 1, followed by 100 mg daily, erythromycin 250 mg q.i.d., metronidazole 400 mg t.i.d., nalidixic acid 500 mg q.i.d., ofloxacin 200 mg b.i.d. or vancomycin 240 mg q.i.d. Faecal sampling was done before, during and after medication. Drug concentrations were measured in faxes and blood on day 6. All faecal samples were investigated for the presence of enteropathogenic microbes and for alterations of biochemical microflora associated characteristics (MACs): conversion of cholesterol to coprostanol, bilirubin deconjugation and formation of urobilinogen, 7α-dehydroxylation of bile acids, breakdown of mucin, presence of β-aspartylglycine, inactivation of tryptic activity and production of short chain fatty acids. Except ampicillin and metronidazole, antimicrobial activity was detected in faeces from all subjects. Intake of ampicillin induced production of β-lactamases in all but one subject. A toxin-producing strain of Clostridium difficile was found in one subject receiving clindamycin. Substances resulting in high faecal concentrations (i.e. clindamycin, bacitracin and vancomycin) resulted in the most profound alterations of the MACs studied. Such alterations in MACs may reflect severe disturbances in the intestinal ecosystem. Keywords: Ecology; intestinal microflora; Antimicrobial agents; Microflora associated characteristics; Faecal concentration of antibiotics.

Utilization of mucin by oral Streptococcus species

The ability of oral Streptococcus strains to utilize oligosaccharide chains in mucin as a source of carbohydrate was studied in batch cultures. Pig gastric mucin, as a substitute of human salivary mucin, was added to chemically defined medium containing no other carbohydrates. Strains of S. mitior attained the highest cell density, while mutans streptococci: S. mutans, S. sobrinus, S. rattus, grew very little in the medium with mucin. S. mitis, S. sanguis, and S. milleri in decreasing order, showed intermediate growth. Mucin breakdown as measured by sugar analyses indicated that oligosaccharide chains were only partially degraded. Every strain produced one or more exoglycosidases potentially involved in hydrolysis of oligosaccharide. The enzyme activities occurred mainly associated with the cells, and very little activity was found in the culture fluids. The relationships between glycosidase activities and growth, or mucin degradation were not always clear.

The Effect of a Shift from a Mixed to a Lacto-vegetarian Diet on some Intestinal Microflora Associated Characteristics

The effects of a switch to a lacto-vegetarian diet on several biochemical parameters was studied in twenty normal weight, non-smoking, healthy omnivores who were on no medical treatment. A dietary survey was performed and faecal and urinary samples collected before and three months after the diet shift. The shift resulted in no change in energy intake, a decrease in protein and fat intake ( p =0.04 and 0.009 respectively) and an increase in total carbohydrate and fibre intake ( p =0.0003 for both). Four biochemical intestinal microflora-associated characteristics (MACs) were followed. There was no significant difference in breakdown of mucin, conversion of cholesterol to coprostanol or inactivation of tryptic activity. The excretion of short chain fatty acids and faecal weight increased significantly ( p <0.01 and p <0.05, respectively). Keywords: Lacto-vegetation diet; Tryptic activity; Cholesterol; Mucin; Short chain fatty acids.

Influence of antibiotics on intestinal mucin in healthy subjects.

To determine the effect on microbial breakdown of intestinal mucin healthy volunteers were treated orally with ten different antibiotics. The most pronounced effects were seen after administration of bacitracin, clindamycin or vancomycin: the electrophoretic mucin pattern in faeces changed from a normal conventional pattern to a specific pattern similar to that found in germ-free rats. Disturbed patterns were also observed in some of the subjects treated with ampicillin, doxycycline, erythromycin, metronidazole or nalidixic acid. In most cases the electrophoretic mucin pattern normalized within five weeks after the end of treatment. There were no effects on the microbial breakdown of intestinal mucin in the groups treated with ofloxacin or trimethoprim/sulfamethoxazole. Thus, administration of antimicrobial drugs in clinically recommended doses may cause long-term disturbances in one microflora-associated characteristic, the breakdown of intestinal mucin.

Breakdown of mucin and plant polysaccharides in the human colon

To obtain an estimate of the extent to which complex carbohydrates are degraded by bacteria in the human colon, aqueous extracts of colon contents from four human subjects were separated into high and low molecular weight fractions by chromatography on a Sephadex G-100 column. The composition of these fractions was compared with the composition of similar fractions from ileal contents, i.e., from material entering the colon. In all four subjects, high molecular weight carbohydrate concentrations were lower in the colon than in the ileum, indicating that breakdown of complex carbohydrate occurs in the colon. The high molecular weight carbohydrate fraction contained sugars characteristic of plant polysaccharides (arabinose, xylose, mannose, rhamnose) as well as sugars characteristic of mucin (fucose, hexosamines, sialic acids). Concentrations of most of these sugars were uniformly lower in the colon than in the ileum. Since high molecular weight protein concentrations were lower in the colon than in the ileum of two of the four subjects tested, some degradation of protein may also occur in the colon.