Involvement of human mucous saliva and salivary mucins in the aggregation of the oral bacteria Streptococcus sanguis,Streptococcus oralis,and Streptococcus rattus

The contribution of human parotid (Par) and submandibular/sublingual (SM/SL) saliva and of the human whole salivary mucin fraction (HWSM) to saliva-induced bacterial aggregation was studied for S. sanguis C476, S. oralis I581, and S. rattus HG 59. The mucous SM/SL saliva showed a much higher aggregation potency towards the S. sanguis and S. oralis strain than did the serous Par saliva. The SM/SL saliva-induced aggregation was observed after 30 min, at 60 min followed by the Par saliva-induced aggregation, and showed a 4-fold higher aggregation titer of 128 for S. sanguis, and an 8-fold higher titer of 516 for S. oralis. In contrast, the Par saliva showed a slightly higher aggregation activity than the SM/SL saliva towards S. rattus as judged by a twofold higher titer of 64. Morphologically, however, the SM/SL saliva-induced aggregation of S. rattus was far more pronounced as was also found for S. sanguis. Finally, the HWSM-induced aggregation showed a 4 to 8-fold higher titer than the originating salivary source, measuring 2048 for S. oralis and 128 for S. rattus. Moreover, no difference was observed in aggregation activity between the HWSM from whole saliva of a blood group O donor and the HWSM from SM/SL saliva of a blood group A donor. All the data point to an important, though not exclusive role of the human salivary mucin fraction in the saliva-induced aggregation of these strains.     

Aggregation of 27 oral bacteria by human whole saliva

Twenty-seven oral strains of the genera Actinomyces (5), Bacteroides (3), and Streptococcus (19) were tested for aggregation by human whole saliva, as well as the effect of culture medium, Ca-ions, and bacteria concentration thereupon. Of the media tested, GF-broth gave rise to less interference by autoaggregation or higher aggregation titers than BHI and TSB, and was used throughout this study. In most cases, Ca-ions (1 mM) only enhanced the rate of induced aggregation, whereas raising the bacteria concentration increased the rate of both induce- and autoaggregation. The final titers, ranging from 1–64, were hardly affected by these parameters, except those of S. rattus HG 59 and S. mutans HG 199, which were respectively increased and decreased by Ca-ions. Saliva-induced aggregation was observed for 21 strains of A. viscosus, A. naeslundii, A. israelii, B. gingivalis, B. intermedius, S. cricetus, S. mutans, S. rattus, S. sanguis, and S. sobrinus, mostly within 15 min to 3 h. Seventeen of these strains also showed autoaggregation, usually well after the onset of induced aggregation. Any potential induced aggregation of B. gingivalis HG 91 was always masked by autoaggregation, as well as that of the S. mutans strains under a particular set of conditions. The aggregation rate and titer varied considerably in a mutually unrelated and strain-dependent way. These microtiterplate data were matched by the 5 spectrophotometric patterns observed for saliva-bacterial interaction, which moreover, gave the better differentiation between induced and autoaggregation. In conclusion, most strains tested can show rapid saliva-induced aggregation in a strain-dependent way, yet strongly affected by the experimental conditions and interference from autoaggregation.     

Differential expression of salivary mucin bacterial aggregating activity with caries status

• 1.1. The low and high mol. wt mucin forms were isolated from saliva of caries-resistant (CR) and caries-susceptible (CS) individuals, and assessed for their bacterial aggregating potential towards S. mutans and S. sanguis, the common cariogenic microorganisms encountered in the oral cavity.
• 2.2. The high mol. wt mucin from both groups of subjects exhibited similar protein and carbohydrate content, but the level ofcovalently bound fatty acids was significantly lower in the CR group. The mucin from CR group showed only a weak inhibitory potential, and no inhibitory activity was observed with the mucin of CS group.
• 3.3. The low mol. wt mucins from both groups, while displaying compositional similarities, showed a marked variation in the bacterial aggregating activity. With both bacteria, the activity of the mucin from CR group was at least 128-fold greater than that of CS group.
• 4.4. The conversion of the high mol. wt mucin to a low mol. wt form through the action of salivary protease produced in both groups enhancement in mucin's bacterial aggregating capacity. This enhancement was, however, considerably less pronounced in the case of mucin from CS group.
• 5.5. The results for the first time demonstrate that the bacterial aggregating epitope of salivary mucins is expressed to a greater extent in CR individuals, and that this epitope is apparently more accessible to bacteria in the low mol. wt mucin form.

     

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 break-down 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.     

Regulation of buccal mucosal calcium channel activity by salivary mucins

• 1.1. The effect salivary mucins on the activity of calcium channel isolated from buccal mucosal cell membranes was investigated. The uptake of ⁴⁵Ca²⁺while only moderately (15%) affected by the intact low and high molecular weight mucin forms, was significantly inhibited, by the acidic low and high molecular weight salivary mucins which evoked 64 and 60% inhibition, respectively.
• 2.2. The inhibitory effect of salivary mucins was associated with the sialic acid and sulfate ester groups of the carbohydrate chains, as the removal of either group caused partial loss in the glycoproteins inhibition, and the complete loss in the inhibitory effect occurred following desialylation and desulfation.
• 3.3. The channel in the presence of epidermal growth factor (EGF) and ATP responded by an increase in tyrosine phosphorylation of 55 and 170 kDa proteins, and the phosphorylated channels showed a 46% increase in ⁴⁵Ca²⁺ uptake. The phosphorylation and the calcium uptake were susceptible to inhibition by a specific tyrosine kinase inhibitor, genistein.
• 4.4. The binding of EGF to calcium channel receptor protein was inhibited by the low and high molecular weight acidic mucins, causing 41.2 and 36.1% reduction, respectively. This reduction in binding was dependent upon the presence of sulfate ester and sialic acid groups, as evidenced by the loss of the glycoproteins' inhibitory capacity following removal of these groups.
• 5.5. The results for the first time demonstrate that salivary mucins actively participate in the modulation of the EGF-controlled buccal mucosal calcium channel activity expression, a process of importance to the preservation of oral tissue integrity.

     

Adsorption to hydroxyapatite of partially deglycosylated human salivary mucins in competition with phosvitin and phytate.

The effect of phosvitin and phytate on the binding of native as well as partially deglycosylated human whole salivary mucins (HWSM) to hydroxyapatite was studied. Native HWSM preadsorbed onto hydroxyapatite was completely desorbed in the presence of greater than 500 micrograms/ml phosvitin. In contrast, in similar experiments, asialo-HWSM was desorbed approximately 10%. Desorption of preadsorbed asialo-afuco-HWSM in the presence of 1 mg/ml phosvitin was approximately 20%. Further deglycosylation of HWSM resulted in preparations which, after preadsorption to hydroxyapatite, were not desorbed upon subsequent incubation with phosvitin. With phytate, a less effective competitor of HWSM for the hydroxyapatite surface, essentially the same results were obtained, i.e. increase in deglycosylation of HWSM was concomitant with decrease in desorption by phytate. Using other incubation conditions (preadsorption of a phosphocompound, and simultaneous incubation of HWSM and phosphocompounds) essentially the same conclusion was obtained. The data indicate that the ability of salivary mucins to absorb to hydroxyapatite in competition with phosphocompounds appears to be enhanced by deglycosylation.     

Human carcinoma-associated and salivary mucins detected by anti-bovine submaxillary mucin antibodies

Polyclonal rabbit anti-bovine submaxillary mucin antibodies, anti-BSM IgG, were analyzed by autoradiography and densitometry (after SDS electrophoresis and blotting), ELISA, and IRMA assays for reactivity against native antigen BSM, deglycosylated and desialylated BSM, and human salivary and carcinoma-associated mucins. Known human tumor marker CA19-9 antigen reacted with rabbit anti-BSM IgG under different conditions, either soluble or immobilized. As soluble antigen, in IRMA it reacted with anti-BSM antibodies used both as a catcher and a tracer, as well as in combination with monoclonal anti-CA19-9 antibodies as a catcher. MUC1 mucin is a carrier of this carbohydrate antigen, CA19-9, or sialyl-Lewis^a, as well as of CA15-3 antigen, a known breast tumor marker. Autoradiography and densitometry demonstrated binding of anti-BSM IgG to intact MUC1 in a sample of commercial standard preparation of CA19-9 antigen. The same method and analysis demonstrated binding of anti-BSM IgG to MUC1 and to smaller antigens of 85–120 kD in samples containing CA15-3 antigen: commercial standard preparation, human breast tissue, and human milk. In a sample of whole human saliva, reactions of both isoforms of MUC7 were detected by autoradiography, as bands of 85 and 115 kD, and densitogram analysis also demonstrated reaction with MUC5B. Chemical modifications performed as periodate oxidation and desialylation of the BSM demonstrated carbohydrate (i.e., sialic acid) epitope sensitivity for anti-BSM IgG. The results presented in this work indicate that polyclonal anti-BSM antibodies are specific for sialylated carbohydrate structure on mucins and could serve as a tool for investigation of human carcinoma-associated and salivary mucins.     

Human carcinoma-associated and salivary mucins detected by anti-bovine submaxillary mucin antibodies

Polyclonal rabbit anti-bovine submaxillary mucin antibodies, anti-BSM IgG, were analyzed by autoradiography and densitometry (after SDS electrophoresis and blotting), ELISA, and IRMA assays for reactivity against native antigen BSM, deglycosylated and desialylated BSM, and human salivary and carcinoma-associated mucins. Known human tumor marker CA19-9 antigen reacted with rabbit anti-BSM IgG under different conditions, either soluble or immobilized. As soluble antigen, in IRMA it reacted with anti-BSM antibodies used both as a catcher and a tracer, as well as in combination with monoclonal anti-CA19-9 antibodies as a catcher. MUC1 mucin is a carrier of this carbohydrate antigen, CA19-9, or sialyl-Lewis(a), as well as of CA15-3 antigen, a known breast tumor marker. Autoradiography and densitometry demonstrated binding of anti-BSM IgG to intact MUC1 in a sample of commercial standard preparation of CA19-9 antigen. The same method and analysis demonstrated binding of anti-BSM IgG to MUC1 and to smaller antigens of 85-120 kD in samples containing CA15-3 antigen: commercial standard preparation, human breast tissue, and human milk. In a sample of whole human saliva, reactions of both isoforms of MUC7 were detected by autoradiography, as bands of 85 and 115 kD, and densitogram analysis also demonstrated reaction with MUC5B. Chemical modifications performed as periodate oxidation and desialylation of the BSM demonstrated carbohydrate (i.e., sialic acid) epitope sensitivity for anti-BSM IgG. The results presented in this work indicate that polyclonal anti-BSM antibodies are specific for sialylated carbohydrate structure on mucins and could serve as a tool for investigation of human carcinoma-associated and salivary mucins.     

Colonic mucins in ulcerative colitis: evidence for loss of sulfation

Colonic tissue obtained at surgery from control individuals and patients with ulcerative colitis was used to isolate mucins and to prepare mucin glycopolypeptides by pronase digestion. These were compared with mucins labelled with [³⁵S] sulfate and [³H]-glucosamine after organ culture tissue samples from the same patients. A significant loss of mucin sulfation was detected in the colitis patients by both metabolic labelling and chemical analysis of the glycopolypeptides. A change in the size distribution of purified mucin oligosaccharides fractionated on BioGel P6 after release by -elimination was seen in both radiolabelled and non-labelled colitis mucins compared with controls. Amino acid analysis of the glycopolypeptides showed a close similarity to the expected ratio of serine:threonine:proline for MUC2 and did not vary between control and colitis groups. Analysis of the mucins confirmed >90% purity in the labelling experiments, characteristic behaviour on density gradient centrifugation and agarose gel electrophoresis in control and ulcerative colitis groups and differences in sulfation and turnover at various sites in the normal colon.Abbreviations WGA wheat germ agglutinin - UC ulcerative colitis - HRP horseradish peroxidase     

Evaluation of bifidobacterial adhesion to acidic sugar chains of porcine colonic mucins

The aim of this study was to assess the adhesion of Bifidobacterium strains to acidic carbohydrate moieties of porcine colonic mucin. Mucins were extracted and purified via gel filtration chromatography followed by density-gradient ultracentrifugation. The presence of sulfated and sialylated carbohydrates in mucins was shown by enzyme-linked immunosorbent assays using PGM34 and HMC31 monoclonal antibodies (mAbs), respectively. Adhesion of Bifidobacterium strains to mucin preparations was markedly affected by the degree of purification. In eight of 22 strains, we observed increased adhesion to mucin preparations purified by ultracentrifugation. Moreover, in some of these eight strains, adhesion to mucin was reduced by pretreatment with sulfatase and/or sialidase, and competitively inhibited by pretreatment with PGM34 and/or HCM31 mAbs. Our results showed that some Bifidobacterium strains adhered to sulfo- and/or sialomucin and were able to recognize carbohydrate structures of the mAbs epitopes.     

Inhibition of Helicobacter pylori haemogglutination activity by human salivary mucins

Abstract Thirty isolates of Helicobacter pylori from gastric biopsies agglutinated human erthyrocyte suspensions. Crude mucin preparation derived from saliva of 20 different donors were examined for their ability to inhibit haemagglutination. All mucin preparations exhibited strong inhibitory activity. Removal of sialic residues from mucin preparations by treatment with neuraminidase resulted in a substantial reduction of their inhibitory activity. The mucin prepations had no bactericidal or aggregation activity for H. pylori . These results are discussed in the context of the role of mucins in colonization of the gastric mucosa by H. pylori     

Inhibition of Helicobacter pylori haemagglutination activity by human salivary mucins

Thirty isolates of Helicobacter pylori from gastric biopsies agglutinated human erythrocyte suspensions. Crude mucin preparations derived from saliva of 20 different donors were examined for their ability to inhibit haemagglutination. All mucin preparations exhibited strong inhibitory activity. Removal of sialic residues from mucin preparations by treatment with neuraminidase resulted in a substantial reduction of their inhibitory activity. The mucin preparations had no bactericidal or aggregation activity for H. pylori. These results are discussed in the context of the role of mucins in colonization of the gastric mucosa by H. pylori.     

Particle tracking microrheology of purified gastrointestinal mucins

The rheological characteristics of gastric and duodenal mucin solutions, the building blocks of the mucus layer that covers the epithelia of the two organs, were investigated using particle tracking microrheology. We used biochemically well characterized purified porcine mucins (MUC5AC and MUC2) as models for human mucins, to probe their viscoelasticity as a function of mucin concentration and pH. Furthermore, we used both reducing (dithiothreitol, DTT) and chaotropic agents (guanidinium chloride and urea) to probe the mesoscopic forces that mediate the integrity of the polymer network. At neutral pH both gastric and duodenal mucins formed self‐assembled semi‐dilute networks above a certain critical mucin concentration (c*) with the viscosity (η) scaling as for MUC5AC and for MUC2, where c is the mucin concentration. Above an even higher mucin concentration threshold (c_e, the entanglement concentration) reptation occurs and there is a dramatic increase in the viscosity scaling, for MUC5AC and for MUC2. The dynamics of the self‐assembled comb polymers is examined in terms of a scaling model for flexible polyelectrolyte combs. Both duodenum and gastric mucin are found to be pH switchable gels, gelation occurring at low pHs. There is a hundred‐fold increase in the elastic shear modulus once the pH is decreased. The addition of DTT, guanidinium chloride and urea disassembles both the semi‐dilute and gel structures causing a large increase in the compliance (decrease in their shear moduli). Addition of the polyphenol EGCG has a reverse effect on mucin viscoelasticity, that is, it triggers a sol–gel transition in semi‐dilute mucin solutions at neutral pH. © 2013 The Authors. Biopolymers published by Wiley Periodicals, Inc. Biopolymers 101: 366–377, 2014.     

Galectin-1 binds mucin in human trophoblast

Mucins are multifunctional highly glycosylated proteins expressed by the female reproductive tract. Differential expression of MUC1 and MUC15 has been shown in trophoblast. This study was undertaken to establish the distribution of mucin(s) in cytotrophoblast cell cultures using anti-bovine submaxillary mucin (BSM) and to investigate the possibility of MUC1/mucin(s) being a binding partner of trophoblast galectin-1. MUC1 is demonstrated here using immunocytochemistry on isolated cytotrophoblast and the HTR-8/SVneo extravillous trophoblast cell line but detection of additional trophoblast mucins cannot be excluded. Western blot analysis showed similar bands ranging from 30 to >200 kDa with anti-BSM and the well-known mucin antibodies HMFG1 and B72.3. Immunocytochemistry and cell-based ELISA data were found to support that all of the antibodies used are reactive with BSM, suggesting the presence of shared epitopes between BSM and trophoblast mucin(s). Binding of galectin-1 to trophoblast MUC1/mucin(s) was analyzed using a solid-phase assay and co-immunoprecipitation. Recombinant galectin-1 binding to isolated trophoblast mucin in solid-phase assay was sensitive to lactose, a carbohydrate inhibitor of galectin binding. In whole HTR-8/SVneo lysates, ~200 kDa mucin was detected in galectin-1 immunoprecipitates, while endogenous galectin-1 was present in BSM-immunoprecipitates. Furthermore, double fluorescence immunocytochemistry showed overlap of galectin-1 and trophoblast mucins at the plasma membrane of HTR-8/SVneo cells. These results suggest that trophoblast mucin(s) could act as binding partners of galectin-1, in a carbohydrate-dependent manner.     

Binding properties of Clostridium botulinum type C progenitor toxin to mucins

It has been reported that Clostridium botulinum type C 16S progenitor toxin (C16S toxin) first binds to the sialic acid on the cell surface of mucin before invading cells [A. Nishikawa, N. Uotsu, H. Arimitsu, J.C. Lee, Y. Miura, Y. Fujinaga, H. Nakada, T. Watanabe, T. Ohyama, Y. Sakano, K. Oguma, The receptor and transporter for internalization of Clostridium botulinum type C progenitor toxin into HT-29 cells, Biochem. Biophys. Res. Commun. 319 (2004) 327–333]. In this study we investigated the binding properties of the C16S toxin to glycoproteins. Although the toxin bound to membrane blotted mucin derived from the bovine submaxillary gland (BSM), which contains a lot of sialyl oligosaccharides, it did not bind to neuraminidase-treated BSM. The binding of the toxin to BSM was inhibited by N-acetylneuraminic acid, N-glycolylneuraminic acid, and sialyl oligosaccharides strongly, but was not inhibited by neutral oligosaccharides. Both sialyl α2–3 lactose and sialyl α2–6 lactose prevented binding similarly. On the other hand, the toxin also bound well to porcine gastric mucin. In this case, neutral oligosaccharides might play an important role as ligand, since galactose and lactose inhibited binding. These results suggest that the toxin is capable of recognizing a wide variety of oligosaccharide structures.     

A dual staining method for identifying mucins of different gastric epithelial mucous cells

Summary A dual staining method has been developed to identify two types of mucous secreting cells in the gastric mucosa of human and rat in one and the same tissue section. Sections were stained first using the galactose oxidase-cold thionin Schiff (GOCTS) procedure and then with paradoxical Concanavalin A staining (PCS). Surface mucous cell mucin stained blue with GOCTS, whereas gland mucous cell mucin stained brown with PCS. This method enabled us to differentiate these two types of mucins not only in gastric epithelial cell cytoplasm but also in the extracellular space. Sugar residues detected by GOCTS were explored by employing four species of lectins, which were peanut andAllomyrina dichotoma agglutinins for -galactose andVicia villosa andWistaria floribunda agglutinins for -N-acetylgalactosamine. The effect of oxidation with galactose oxidase was also examined on the affinities of reactive sites for these lectins. The results indicated that, in the human stomach, the sugar residues responsible for this reactivity were most likely -N-acetylgalactosamine and -galactose in specimens lacking secretion of blood group determinants and -N-acetylgalactosamine in those showing the secretion. In the rat stomach, on the other hand, sugar residues responsible for GOCTS were not elucidated by these lectins.     

A lectin from the Asian horseshoe crabTachypleus tridentatus: purification,specificity and interaction with tumour cells

A lectin from the haemolymph of the Asian horseshoe crabTachypleus tridentatus was purified to homogeneity by affinity chromatography on Sepharose 4B-boundN-acetylneuraminic acid. The specificity of this lectin was studied by haemagglutination inhibition with sialic acid analogues,N-acetylhexosamines and glycoproteins. For the interaction with the agglutinin theN-acetyl group and the glyceryl side chain ofN-acetylneuraminic acid are important, while presence of an aglycon, specially an -glycosidically linked lactose increases affinity to the lectin. The strongest glycoprotein inhibitors were ovine as well as bovine submaxillary mucin andCollocalia mucin, all beingO-chain glycoproteins but carrying completely different carbohydrate chains. The majority ofN-chain proteins were inactive. As the lectin agglutinates human erythrocytes, but not the murine lymphoma lines Eb and ESb or the human colon carcinoma HT 29, these cancer cells apparently lack the Tachypleus tridentatus agglutinin-receptor which is present on red cells andO-chain glycoproteins.Abbreviations TTA Tachypleus tridentatus agglutinin - SDS sodium dodecyl sulfate - BSM bovine sub-maxillary mucin - VCS Vibrio cholerae sialidase - OSM ovine submaxillary mucin - WGA Wheat germ agglutinin - NeuAc N-acetylneuraminic acid.     

A 23 kDa membrane glycoprotein bearing NeuNAc{alpha}2-3Gal{beta}1-3GalNAc O-linked carbohydrate chains acts as a receptor for Streptococcus sanguis OMZ 9 on human buccal epithelial cells

Streptococcus sanguis colonizes several human oral surfaces,including both hard and soft tissues. Large salivary mucin likeglycoproteins bearing sialic acid residues are known to bindvarious S.sanguis strains. However, the molecular basis forthe adhesion of S.sanguis to human buccal epithelial cells (HBEC)has not been established. The present study shows that S.sanguisOMZ 9 binds to exfoliated HBEC in a sialic acid-sensitive manner.The desialylation of such cells invariably abolhhes adhesionof S.sanguis OMZ 9 to the cell surface. A soluble glycopeptidebearing short sialylated O-linked carbohydrate chains behavesas a potent inhibitor of the attachment of S.sanguis OMZ 9 toexfoliated HBEC. The resialylation of desialylated HBEC withCMP-sialic acid and Galß1,3GalNAc     

Structure and biosynthesis of human salivary mucins

Human salivary glands secrete two types of mucins: oligomeric mucin (MG1) with molecular mass above 1 MDa and monomeric mucin (MG2) with molecular mass of 200-250 kDa. Monomers of MG1 and MG2 contain heavily O-glycosylated tandem repeats located at the central domain of the molecules. MG1 monomers are linked by disulfide bonds located at sparsely glycosylated N- and C-end. MG1 are synthesized by mucous cells and MG2 by the serous cells of human salivary glands.     

Saliva-Induced Aggregation of Oral Streptococci

Cells of several species of oral microorganisms have been shown, in earlier studies, to be aggregated by saliva. In the present study some of the basic properties of the aggregation system are examined. The observation is made that the saliva-induced aggregates of Streptococcus sanguis and S. mitis can be dissociated to stable particles which consist of about 100 cells and have a median diameter of about 4.5 μm. It is proposed that these are subunits, or core aggregates, of the large primary aggregates. Counts of the core aggregates can be taken as a precise and accurate measure of aggregation. Experiments based on this procedure show that the aggregation of S. sanguis is maximal at 10 C and at 1 meq of Ca²⁺ ions per liter and is not affected by a change in pH between 3.9 and 8.7 or by a change in the phase of growth of the microorganisms. Core aggregates diminish in number with prolonged incubation, suggesting that the aggregating factors break down with time. Formalinized cells yield stable aggregates. However, with Formalinized cell aggregation is maximal between 20 and 30 C and proceeds in the absence of calcium ions. Evidence is presented that whole saliva contains separate aggregating factors for S. sanguis and S. mitis. The factors differ in their affinity for intact cells and for hydroxyapatite and differ in their stability to dialysis. These findings suggest that many different aggregating factors exist in saliva, each of which may be capable of interacting with cells of one or several bacterial species.