The composition of the gut microbiota shapes the colon mucus barrier

Two C57BL/6 mice colonies maintained in two rooms of the same specific pathogen-free (SPF) facility were found to have different gut microbiota and a mucus phenotype that was specific for each colony. The thickness and growth of the colon mucus were similar in the two colonies. However, one colony had mucus that was impenetrable to bacteria or beads the size of bacteria—which is comparable to what we observed in free-living wild mice—whereas the other colony had an inner mucus layer penetrable to bacteria and beads. The different properties of the mucus depended on the microbiota, as they were transmissible by transfer of caecal microbiota to germ-free mice. Mice with an impenetrable mucus layer had increased amounts of Erysipelotrichi, whereas mice with a penetrable mucus layer had higher levels of Proteobacteria and TM7 bacteria in the distal colon mucus. Thus, our study shows that bacteria and their community structure affect mucus barrier properties in ways that can have implications for health and disease. It also highlights that genetically identical animals housed in the same facility can have rather distinct microbiotas and barrier structures.     

Evidence for the presence of calmodulin in fish mucus

Partly purified mucus collected from the skin of three species of fish contains a protein that, on sodium dodecyl sulphate/polyacrylamide gel electrophoresis, comigrates with bovine brain calmodulin and shows the same calcium-dependent shift in electrophoretic mobility as calmodulin. Fish mucus contains a heat-stable activator of cyclic nucleotide phosphodiesterase; activation is concentration dependent and sensitive to the specific calmodulin inhibitor calmidazolium (R 24571). The presence of calmodulin in fish mucus is further indicated by means of a specific radioimmunoassay. A drop in the calcium concentration of the water induces an increase in the immunoassayable calmodulin concentration of mucus, which indicates that the function of calmodulin in mucus is related to control of permeability of the skin epithelium to water and ions.     

Design of a specific colonic mucus marker using a human commensal bacterium cell surface domain

Imaging living cells and organs requires innovative, specific, efficient, and well tolerated fluorescent markers targeting cellular components. Such tools will allow proceeding to the dynamic analysis of cells and the adaptation of tissues to environmental cues. In this study, we have identified and synthesized a novel non-toxic fluorescent marker allowing a specific fluorescent staining of the human colonic mucus. Our strategy to identify a molecule able to specifically bind to the human colonic mucus was on the basis of the mucus adhesion properties of commensal bacteria. We identified and characterized the mucus-binding property of a 70-amino acid domain (MUB(70)) expressed on the surface of Lactobacillus strains. The chemical synthesis of MUB(70) was achieved using the human commensal bacterium Lactobacillus reuteri AF120104 protein as a template. The synthesized Cy5-conjugated MUB(70) marker specifically stained the colonic mucus on fixed human, rabbit, and guinea pig tissues. Interestingly, murine tissue was not stained, suggesting significant differences in the composition of the murine colonic mucus. In addition, this marker stained the mucus of living cultured human colonic cells (HT29-MTX) and human colonic tissue explants. Using a biotinylated derivative of MUB(70), we demonstrated that this peptide binds specifically to Muc2, the most abundant secreted mucin, through its glycosylated moieties. Hence, Cy5-MUB(70) is a novel and specific fluorescent marker for mammalian colonic mucus. It may be used for live imaging analysis but also, as demonstrated in this study, as a marker for the diagnosis and the prognosis of colonic mucinous carcinomas.     

The gastric mucus of domestic animals

The gastric mucus of different domestic mammals shows a certain pattern of reactions which is typical for the species but not for the respective gland. There is no "mucoid" as an uniform specific mucus substance and a greater plurality in the composition of the secretions as it is reflected by the classification in acid and neutral mucins. A classification of the glands as to the secretion is problematical and further consideration is necessary.     

MUCUS PRODUCTION AND PHYSIOLOGICAL ENERGETICS IN PATELLA VULGATA L.

Most energy budgets for marine gastropods lack a measured mucusterm. This is a major omission since mucus is widespread inmolluscan physiological processes. In this paper we outlinemethods for estimating mucus production in the intertidal gastropod,Patella vulgata L. This species uses mucus primarily for adhesionand locomotion. Pedal mucus production rates were measured inaerial environments of 70% relative humidity (RH) and 100% RHand in seawater. Animals in 70% RH did not move, while thosein 100% RH and seawater were mobile. Animals in 100% RH showedthe highest mucus production rate. Those in seawater secretedabout 75% of the mucus produced in 100% RH and those in 70%RH only about 40% of that produced in 100% RH. Significant logarithmicrelationships between mucus production rate and whole dry weightof animal, and mucus production rate and shell length were foundin all cases. The mucus term in the energy budget was calculated as 23% ofingestion both for individuals and for a specific population.This is much higher than the estimate of 4% of Wright &Hartnoll (1981). We have recalculated an energy budget for Patellavulgata to include the substantial mucus term. The recalculatedbudget is structurally different from the original budget. Thissuggests conclusions drawn from budgets not including a measuredmucus term should be regarded with caution. (Received 12 September 1989; accepted 13 December 1989)     

A comparative study on innate immune parameters in the epidermal mucus of various fish species

Fish epidermal mucus and its components provide the first line of defense against pathogens. Little is known about the role of epidermal mucus enzymes in the innate immune system of fish species such as Arctic char (Salvelinus alpinus), brook trout (S. fontinalis), koi carp(Cyprinus carpio), striped bass (Morone saxatilis), haddock, (Melanogrammus aeglefinus), Atlantic cod (Gadus morhua) and hagfish (Myxine glutinosa). The epidermal mucus samples from these fish were analysed for the specific activities of various hydrolytic enzymes including lysozyme, alkaline phosphatase, cathepsin B and proteases and the enzyme levels were compared among the fish species. Of all the species hagfish mucus showed a high activity for lysozyme and proteases and koi carp mucus had the highest levels of alkaline phosphatase and cathepsin B. A wide variation in enzyme activities was observed among the seven species and also between species of same family such as Arctic char and brook trout (salmonidae), haddock and cod (gadidae). Only lysozyme levels showed a marked variation with salinity where seawater fish showed approximately two times higher lysozyme activity than freshwater-reared fish species. Characterization of proteases with specific inhibitors showed Arctic char, brook trout, haddock and cod having higher levels of serine over metalloproteases whereas koi carp and striped bass had higher levels of metalloproteases over serine proteases. In contrast, hagfish had almost equal proportion of both serine and metalloproteases. This study demonstrates variation in the level of hydrolytic enzymes in the epidermal mucus of fish. These results provide preliminary information for a better understanding of the role of epidermal mucus and its components in the fish innate immune system.     

Mucus Properties and Goblet Cell Quantification in Mouse,Rat and Human Ileal Peyer's Patches

Peyer''s patches (PPs) are collections of lymphoid follicles in the small intestine, responsible for scanning the intestinal content for foreign antigens such as soluble molecules, particulate matter as well as intact bacteria and viruses. The immune cells of the patch are separated from the intestinal lumen by a single layer of epithelial cells, the follicle-associated epithelium (FAE). This epithelium covers the dome of the follicle and contains enterocyte-like cells and M cells, which are particularly specialized in taking up antigens from the gut. However, the presence and number of goblet cells as well as the presence of mucus on top of the FAE is controversial. When mouse ileal PPs were mounted in a horizontal Ussing-type chamber, we could observe a continuous mucus layer at mounting and new, easily removable mucus was released from the villi on the patch upon stimulation. Confocal imaging using fluorescent beads revealed a penetrable mucus layer covering the domes. Furthermore, immunostaining of FAE from mice, rats and humans with a specific antibody against the main component of intestinal mucus, the MUC2 mucin, clearly identify mucin-containing goblet cells. Transmission electron micrographs further support the identification of mucus releasing goblet cells on the domes of PPs in these species.     

Somatostatin stimulates colonic MUC2 expression through SSTR5-Notch-Hes1 signaling pathway

Colonic mucus barrier is regarded as the first defense line against bacteria and antigens from directly attaching to the epithelium, which would further lead to intestinal inflammation activation and pathological conditions. As MUC2 mucin is the predominant component of the mucus, understanding the regulatory mechanisms of MUC2 is important for mucus barrier protection. Somatostatin (SST) has been found to play a role in colon protection through various manners. However, whether SST involves in colonic mucus barrier regulation is still unclear. The aim of this study is to investigate the effects and potential mechanisms of SST on colonic MUC2 expression and mucus secretion. In vivo study, exogenous somatostatin (octreotide) administration effectively stimulated mice colonic MUC2 expression and mucus secretion. In human goblet-like cell LS174T cells, SST exposure also significantly stimulated MUC2 expression and mucus secretion. Further studies indicated that SST receptor 5 (SSTR5) was significantly activated by SST, whereas specific SSTR5 siRNA transfection of LS174T cells significantly blocked SST-induced increase in MUC2 expression and mucus secretion. In addition, SSTR5 agonist L817,818 also upregulated MUC2 expression and mucus secretion in LS174T cells. Mechanistic studies further demonstrated that SST/SSTR5-mediated MUC2 upregulation was dependent on Notch-Hes1 pathway suppression by detecting notch intracellular domain (NICD) and Hes1 proteins. Taken together, our findings suggested that SST could participate in colonic mucus barrier regulation through SSTR5-Notch-Hes1-MUC2 signaling pathway. These findings provide a deep insight into the role of SST on colonic mucus regulation under physiological conditions.     

乳杆菌表面粘附蛋白的提取、鉴定及粘附特异性的初步研究

以从健康牙鲆肠道中分离筛选的乳杆菌L15(Lactobacillussp.L15)和嗜酸乳杆菌ATCC4356为实验材料,应用5mol/L LiCl提取其表面蛋白,利用蛋白印迹法鉴定出在L15表面蛋白中分子量为61.8kDa和54.6kDa的蛋白质分别参与对牙鲆和鲤鱼粘液的粘附过程,为新发现的粘附蛋白种类,将其命名为MAPPpo1和MAPPcc。ATCC4356中分子量分别为43.0kDa和63.3kDa的两个表面蛋白参与对牙鲆粘液的粘附,而分子量为43.0kDa的蛋白参与对鲤鱼粘液的粘附。同时,蛋白质印迹法显示,L15和ATCC4356在牙鲆和鲤鱼肠粘液中均具有相同的粘附受体,在牙鲆肠粘液中是分子量为29.7kDa和30.3kDa的两种蛋白质,而在鲤鱼肠粘液中只有分子量为26.2kDa的蛋白作为受体参与L15和ATCC4356的粘附过程。结果显示,乳杆菌对肠粘液的粘附不但具有菌种的特异性,而且也有宿主的特异性。     

Toward the modeling of mucus draining from the human lung: role of the geometry of the airway tree

Mucociliary clearance and cough are the two main natural mucus draining methods in the bronchial tree. If they are affected by a pathology, they can become insufficient or even ineffective, then therapeutic draining of mucus plays a critical role to keep mucus levels in the lungs acceptable. The manipulations of physical therapists are known to be very efficient clinically but they are mostly empirical since the biophysical mechanisms involved in these manipulations have never been studied. We develop in this work a model of mucus clearance in idealized rigid human bronchial trees and focus our study on the interaction between (1) tree geometry, (2) mucus physical properties and (3) amplitude of flow rate in the tree. The mucus is considered as a Bingham fluid (gel-like) which is moved upward in the tree thanks to its viscous interaction with air flow. Our studies point out the important roles played both by the geometry and by the physical properties of mucus (yield stress and viscosity). More particularly, the yield stress has to be overcome to make mucus flow. Air flow rate and yield stress determine the maximal possible mucus thickness in each branch of the tree at equilibrium. This forms a specific distribution of mucus in the tree whose characteristics are strongly related to the multi-scaled structure of the tree. The behavior of any mucus distribution is then dependent on this distribution. Finally, our results indicate that increasing air flow rates ought to be more efficient to drain mucus out of the bronchial tree while minimizing patient discomfort.     

Mucin networks: Dynamic structural assemblies controlling mucus function

Contrary to first appearances, mucus structural biology is not an oxymoron. Though mucus hydrogels derive their characteristics largely from intrinsically disordered, heavily glycosylated polypeptide segments, the secreted mucin glycoproteins that constitute mucus undergo an orderly assembly process controlled by folded domains at their termini. Recent structural studies revealed how mucin complexes promote disulphide-mediated polymerization to produce the mucus gel scaffold. Additional protein–protein and protein-glycan interactions likely tune the mesoscale properties, stability, and activities of mucins. Evidence is emerging that even intrinsically disordered glycosylated segments have specific structural roles in the production and properties of mucus. Though soft-matter biophysical approaches to understanding mucus remain highly relevant, high-resolution structural studies of mucins and other mucus components are providing new perspectives on these vital, protective hydrogels.     

The infrastructure of the orthochromatic mucous cells of some British lumbricids (Annelida)

The infrastructure of the orthochromatic mucous cells of 11 British lumbricids is described. The prominent secretory globules show species variation with standard fixation.
In four genera the core material exhibits consistent and characteristic electron dense ultrastructural patterning, the four types being described, and species within these genera show specific differences. Such patterned globules are termed heterogeneous, in contrast to the homogeneous globules of the other two genera. The generic and specific ultrastructural patterns line up well with, and support, the modern, revised lumbricid taxonomy.
The electron dense heterogeneity is first visualized at the mature Golgi face and the patterning is eliminated after proteolytic enzyme treatment, suggesting that the dense areas within the globule reflect the protein sites within the mucus, the final elaboration of which takes place within the polarized Golgi.
No mucus extrusion mechanisms were observed and it is suggested, from the positioning of the supporting cell tonofilaments and the body wall musculature, that extrusion accompanies locomotory activities.
A suggestion as to the possible function of a type of mucus with clear specific differences, as evidenced by the ultrastructure, is advanced.     

Comparison of antimicrobial activity in the epidermal mucus extracts of fish

The mucus layer on the surface of fish consists of several antimicrobial agents that provide a first line of defense against invading pathogens. To date, little is known about the antimicrobial properties of the mucus of Arctic char (Salvelinus alpinus), brook trout (S. fontinalis), koi carp (Cyprinus carpio sub sp. koi), striped bass (Morone saxatilis), haddock (Melanogrammus aeglefinus) and hagfish (Myxine glutinosa). The epidermal mucus samples from these fish were extracted with acidic, organic and aqueous solvents to identify potential antimicrobial agents including basic peptides, secondary metabolites, aqueous and acid soluble compounds. Initial screening of the mucus extracts against a susceptible strain of Salmonella enterica C610, showed a significant variation in antimicrobial activity among the fish species examined. The acidic mucus extracts of brook trout, haddock and hagfish exhibited bactericidal activity. The organic mucus extracts of brook trout, striped bass and koi carp showed bacteriostatic activity. There was no detectable activity in the aqueous mucus extracts. Further investigations of the activity of the acidic mucus extracts of brook trout, haddock and hagfish showed that these fish species had specific activity for fish and human pathogens, demonstrating the role of fish mucus in antimicrobial protection. In comparison to brook trout and haddock, the minimum bactericidal concentrations of hagfish acidic mucus extracts were found to be approximately 1.5 to 3.0 times lower against fish pathogens and approximately 1.6 to 6.6 folds lower for human pathogens. This preliminary information suggests that the mucus from these fish species may be a source of novel antimicrobial agents for fish and human health related applications.     

Comparison of the biochemical composition of normal epidermal mucus and extruded slime of hagfish (Myxine glutinosa L.)

Hagfish (Myxine glutinosa) secrete normal epidermal mucus and extruded slime. The epidermal mucus is produced continuously to prevent pathogen adherence while the extruded slime is observed predominantly during feeding, provocation or stress. To date little is known about the involvement of extruded slime in the physiological functions of hagfish. In this preliminary study, innate immune enzymes and the protein composition of hagfish normal epidermal mucus and extruded slime were analysed and compared. The lysozyme specific activity of hagfish was observed approximately two-fold higher in extruded slime than that of epidermal mucus. The extruded slime had approximately 3.5-5.0 fold increased levels of alkaline phosphatase, cathepsin B and proteases in comparison to epidermal mucus. Protease characterization using specific inhibitors showed that the extruded slime had higher levels of serine trypsin-like proteases compared to metalloproteases whereas epidermal mucus showed equal proportion of both serine and metalloproteases. SDS-PAGE analysis showed high levels of three proteins with molecular masses in the range of 13-16kDa in the extruded slime. The LC/MS/MS analysis of protein bands 1, 2 and 3 showed closest matches to hemoglobulin-3, histone H3 and H2B proteins, respectively. The observation of elevated levels of innate immune parameters in the extruded slime suggested that the extruded slime has a significant role in innate immunity of hagfish against infectious pathogens.     

Specific plasmid patterns and high rates of bacterial co‐occurrence within the coral holobiont

Despite the importance of coral microbiomes for holobiont persistence, the interactions among these are not well understood. In particular, knowledge of the co‐occurrence and taxonomic importance of specific members of the microbial core, as well as patterns of specific mobile genetic elements (MGEs), is lacking. We used seawater and mucus samples collected from Mussismilia hispida colonies on two reefs located in Bahia, Brazil, to disentangle their associated bacterial communities, intertaxa correlations, and plasmid patterns. Proxies for two broad‐host‐range (BHR) plasmid groups, IncP‐1β and PromA, were screened. Both groups were significantly (up to 252 and 100%, respectively) more abundant in coral mucus than in seawater. Notably, the PromA plasmid group was detected only in coral mucus samples. The core bacteriome of M. hispida mucus was composed primarily of members of the Proteobacteria, followed by those of Firmicutes. Significant host specificity and co‐occurrences among different groups of the dominant phyla (e.g., Bacillaceae and Pseudoalteromonadaceae and the genera Pseudomonas, Bacillus, and Vibrio) were detected. These relationships were observed for both the most abundant phyla and the bacteriome core, in which most of the operational taxonomic units showed intertaxa correlations. The observed evidence of host‐specific bacteriome and co‐occurrence (and potential symbioses or niche space co‐dominance) among the most dominant members indicates a taxonomic selection of members of the stable bacterial community. In parallel, host‐specific plasmid patterns could also be, independently, related to the assembly of members of the coral microbiome.     

Kinetics of adhesion of selected fish-pathogenic Vibrio strains of skin mucus of gilt-head sea bream (Sparus aurata L.).

The kinetics of adhesion of Vibrio strains isolated from diseased fish to skin mucus of gilt-head sea bream was studied. A modified Langmuir adsorption isotherm was calculated, and the results obtained indicate that the strains tested (Vibrio alginolyticus DP1HE4 and Vibrio anguillarum-like DC12R8 and DC12R9) showed a saturation kinetics except for V. alginolyticus (CAN), which showed a proportional adsorption kinetics. The adhesive capability for skin mucus does not seem to be an essential virulence factor of pathogenic strains of Vibrio, since this specific interaction depended on several environmental factors, temperature and salinity being the most important. However, the absence of an inhibitory effect of mucus on the pathogenic microorganisms, and the capability of the Vibrio strains to utilize mucus as a carbon source, could favor their settlement on the skin with a potential for infection of cultured, stressed fish.     

Roles of Vibrio fischeri and nonsymbiotic bacteria in the dynamics of mucus secretion during symbiont colonization of the Euprymna scolopes light organ

During light organ colonization of the squid Euprymna scolopes by Vibrio fischeri, host-derived mucus provides a surface upon which environmental V. fischeri forms a biofilm and aggregates prior to colonization. In this study we defined the temporal and spatial characteristics of this process. Although permanent colonization is specific to certain strains of V. fischeri, confocal microscopy analyses revealed that light organ crypt spaces took up nonspecific bacteria and particles that were less than 2 micro m in diameter during the first hour after hatching. However, within 2 h after inoculation, these cells or particles were not detectable, and further entry by nonspecific bacteria or particles appeared to be blocked. Exposure to environmental gram-negative or -positive bacteria or bacterial peptidoglycan caused the cells of the organ's superficial ciliated epithelium to release dense mucin stores at 1 to 2 h after hatching that were used to form the substrate upon which V. fischeri formed a biofilm and aggregated. Whereas the uncolonized organ surface continued to shed mucus, within 48 h of symbiont colonization mucus shedding ceased and the formation of bacterial aggregations was no longer observed. Eliminating the symbiont from the crypts with antibiotics restored the ability of the ciliated fields to secrete mucus and aggregate bacteria. While colonization by V. fischeri inhibited mucus secretion by the surface epithelium, secretion of host-derived mucus was induced in the crypt spaces. Together, these data indicate that although initiation of mucus secretion from the superficial epithelium is nonspecific, the inhibition of mucus secretion in these cells and the concomitant induction of secretion in the crypt cells are specific to natural colonization by V. fischeri.     

Muc5b is the major polymeric mucin in mucus from thoroughbred horses with and without airway mucus accumulation

Mucus accumulation is a feature of inflammatory airway disease in the horse and has been associated with reduced performance in racehorses. In this study, we have analysed the two major airways gel-forming mucins Muc5b and Muc5ac in respect of their site of synthesis, their biochemical properties, and their amounts in mucus from healthy horses and from horses with signs of airway mucus accumulation. Polyclonal antisera directed against equine Muc5b and Muc5ac were raised and characterised. Immunohistochemical staining of normal equine trachea showed that Muc5ac and Muc5b are produced by cells in the submucosal glands, as well as surface epithelial goblet cells. Western blotting after agarose gel electrophoresis of airway mucus from healthy horses, and horses with mucus accumulation, was used to determine the amounts of these two mucins in tracheal wash samples. The results showed that in healthy horses Muc5b was the predominant mucin with small amounts of Muc5ac. The amounts of Muc5b and Muc5ac were both dramatically increased in samples collected from horses with high mucus scores as determined visually at the time of endoscopy and that this increase also correlated with increase number of bacteria present in the sample. The change in amount of Muc5b and Muc5ac indicates that Muc5b remains the most abundant mucin in mucus. In summary, we have developed mucin specific polyclonal antibodies, which have allowed us to show that there is a significant increase in Muc5b and Muc5ac in mucus accumulated in equine airways and these increases correlated with the numbers of bacteria.     

Evaluation of the ability of Bifidobacterium longum to metabolize human intestinal mucus

The ability of Bifidobacterium longum to use intestinal mucus as a metabolizable source was characterized. Bifidobacterium longum biotype longum NCIMB8809 was grown in a chemically semi-defined medium supplemented with human intestinal mucus, and the cytoplasmic protein profiles and several glycosyl hydrolase activities were analysed and compared with those obtained from the same bacterium grown in the absence of mucus. We were able to identify 22 different proteins in the cytoplasmic fraction, of which nine displayed a different concentration in the presence of mucus. Among the proteins whose concentrations varied, we found specific enzymes that are involved in the response to different environmental conditions, and also proteins that mediate interaction with mucus in bacteria. Significant changes in some glycoside-hydrolysing activities were also detected. In addition, stable isotope labelling of amino acids in cell culture demonstrated that B. longum incorporates leucine from the glycoprotein matrix of mucin within its proteins. This study provides the first proteomic data regarding the interaction of B. longum with intestinal mucus, and contributes to the understanding of the behaviour of this intestinal species in its natural ecological niche.