RESUMEN
The Serine Protease Inhibitor (serpin) protein has been suggested to play a key role in the interaction of bifidobacteria with the host. By inhibiting intestinal serine proteases, it might allow bifidobacteria to reside in specific gut niches. In inflammatory diseases where serine proteases contribute to the innate defense mechanism of the host, serpin may dampen the damaging effects of inflammation. In view of the beneficial roles of this protein, it is important to understand how its production is regulated. Here we demonstrate that Bifidobacterium longum NCC 2705 serpin production is tightly regulated by carbohydrates. Galactose and fructose increase the production of this protein while glucose prevents it, suggesting the involvement of catabolite repression. We identified that di- and oligosaccharides containing galactose (GOS) and fructose (FOS) moieties, including the human milk oligosaccharide Lacto-N-tetraose (LNT), are able to activate serpin production. Moreover, we show that the carbohydrate mediated regulation is conserved within B. longum subsp. longum strains but not in other bifidobacterial taxons harboring the serpin coding gene, highlighting that the serpin regulation circuits are not only species- but also subspecies- specific. Our work demonstrates that environmental conditions can modulate expression of an important effector molecule of B. longum, having potential important implications for probiotic manufacturing and supporting the postulated role of serpin in the ability of bifidobacteria to colonize the intestinal tract.
Asunto(s)
Proteínas Bacterianas/biosíntesis , Bifidobacterium/metabolismo , Fructosa/farmacología , Galactosa/farmacología , Oligosacáridos/farmacología , Serpinas/biosíntesis , Proteínas Bacterianas/genética , Bifidobacterium/genética , Serpinas/genéticaRESUMEN
The influence of pH on the adhesion of two Lactobacillus strains to Caco-2 human intestinal cells was investigated. One strain, Lactobacillus johnsonii La1, was adherent at any pH between 4 and 7. The other one, L. acidophilus La10, did not attach to this cell line under the same experimental conditions. On the basis of these results, we used the monoclonal antibody technique as a tool to determine differences on the surface of these bacteria and to identify a factor for adhesion. Mice were immunized with live La1, and the hybridomas produced by fusion of spleen cells with ONS1 cells were screened for the production of antibodies specific for L. johnsonii La1. A set of these monoclonal antibodies was directed against a nonproteinaceous component of the L. johnsonii La1 surface. It was identified as lipoteichoic acid (LTA). This molecule was isolated, chemically characterized, and tested in adhesion experiments in the same system. The adhesion of L. johnsonii La1 to Caco-2 cells was inhibited in a concentration-dependent way by purified LTA as well as by L. johnsonii La1 culture supernatant that contained LTA. These results showed that the mechanism of adhesion of L. johnsonii La1 to human Caco-2 cells involves LTA.
Asunto(s)
Adhesión Bacteriana , Lactobacillus/fisiología , Lipopolisacáridos/metabolismo , Ácidos Teicoicos/metabolismo , Animales , Anticuerpos Monoclonales/biosíntesis , Anticuerpos Monoclonales/inmunología , Antígenos de Superficie/inmunología , Células CACO-2 , Humanos , Concentración de Iones de Hidrógeno , Inmunización , Inmunohistoquímica , Lactobacillus/química , Lactobacillus acidophilus/química , Lactobacillus acidophilus/fisiología , Lipopolisacáridos/análisis , Proteínas de la Membrana/inmunología , Ratones , Ácidos Teicoicos/análisisRESUMEN
The protective effects of milk and milk products against dental caries have been demonstrated in many animal studies. We have shown that this effect was mediated by micellar casein or caseinopeptide derivatives. A reduction in the Streptococcus sobrinus population in the oral microbiota of animals fed diets supplemented with these milk components was consistently observed. A possible explanation for these findings is that milk components are incorporated into the salivary pellicle, thereby reducing the adherence of S. sobrinus. This hypothesis was tested in vitro by the incubation of bovine enamel discs with unstimulated saliva. The resulting pellicle was washed and incubated with caseinoglycomacropeptide (CGMP) and/or caseinophosphopeptide (CPP) labeled with 17- and 12-nm gold particles. All samples were prepared for electron microscopy by high-pressure freezing followed by freeze-substitution. It was demonstrated by high-resolution scanning electron microscopy with back-scattered electron imaging, as well as by transmission electron microscopy, that both peptides were incorporated into the pellicle in exchange for albumin, confirming previous findings. This protein was identified with a mouse anti-human serum albumin followed by goat anti-mouse IgG labeled with 25-nm gold particles. Incorporation of CGMP and/or CPP into salivary pellicles reduced the adherence of both S. sobrinus and S. mutans significantly. It is suggested that the calcium and phosphate-rich micellar casein or caseinopeptides are incorporated into the pellicle. The resulting ecological shifts, together with the increased remineralization potential of this biofilm, may explain its modified cariogenic potential.
Asunto(s)
Adhesión Bacteriana , Caseínas/metabolismo , Caries Dental/etiología , Esmalte Dental/microbiología , Fragmentos de Péptidos/metabolismo , Fosfopéptidos/metabolismo , Saliva/metabolismo , Proteínas y Péptidos Salivales/metabolismo , Streptococcus mutans/fisiología , Streptococcus sobrinus/fisiología , Adulto , Animales , Bovinos , Interpretación Estadística de Datos , Caries Dental/prevención & control , Depósitos Dentarios/microbiología , Película Dental , Durapatita , Congelación , Humanos , Técnicas In Vitro , Ratones , Microscopía Electrónica , Microscopía Electrónica de Rastreo , Microtomía , Persona de Mediana Edad , LecheRESUMEN
Bovine caseinate, derivatives of its glycosylated moiety [caseinoglycomacropeptide (CGP)], and caseinophosphopeptides were evaluated as inhibitors of adhesion of oral bacteria to saliva-coated hydroxyapatite beads (S-HA). All milk casein-derived components behaved as potent inhibitors of Streptococcus sanguis OMZ 9 and Streptococcus sobrinus OMZ 176 adhesion to S-HA, whereas neither bovine serum albumin nor polyethyleneglycol were able to interfere with the adhesion of these strains. By contrast, none of the molecular species tested was able to inhibit the attachment of Actinomyces viscosus Ny 1 to S-HA. On the other hand, casein derivatives were shown to displace human serum albumin from S-HA beads. They were also able to bind to the bacterial cell surface of all strains examined. Collectively, these findings suggest that interactions between acidic casein-derived milk components and the biological surfaces involved in bacterial adhesion to S-HA result in an inhibitory effect that is selective for the oral streptococci examined.
Asunto(s)
Adhesión Bacteriana/efectos de los fármacos , Proteínas de la Membrana Bacteriana Externa/metabolismo , Caseínas/metabolismo , Streptococcus/fisiología , Actinomyces viscosus/química , Actinomyces viscosus/efectos de los fármacos , Actinomyces viscosus/fisiología , Análisis de Varianza , Animales , Adhesión Bacteriana/fisiología , Unión Competitiva , Cariostáticos/metabolismo , Cariostáticos/farmacología , Caseínas/farmacología , Bovinos , Cromatografía de Afinidad , Durapatita , Humanos , Boca/microbiología , Unión Proteica , Saliva/química , Saliva/fisiología , Proteínas y Péptidos Salivales/metabolismo , Albúmina Sérica/metabolismo , Streptococcus/química , Streptococcus/efectos de los fármacos , Streptococcus sanguis/química , Streptococcus sanguis/efectos de los fármacos , Streptococcus sanguis/fisiología , Streptococcus sobrinus/química , Streptococcus sobrinus/efectos de los fármacos , Streptococcus sobrinus/fisiologíaRESUMEN
Quantitative determination of all structural families of complex carbohydrate micronutrients was performed on bovine milk samples, milk-based infant formulas, and whey-based manufacturing raw materials. Differences found between formulas depended mainly on their whey: casein ratios. A solvent separation procedure was required for quantitative estimation of the gangliosides and neutral glycolipids within the fat fraction. All infant formulas except one contained slightly more gangliosides than bovine milk. Complex carbohydrates were consistently higher in the nonfat fraction. By gel permeation chromatography, an oligosaccharide subfraction was separated from a glycopeptide one. Oligosaccharide content of infant formulas increased as a function of the whey:casein ratio, and glycopeptides were found only in formulas made with whey components. Neuraminic acids from infant formulas were associated primarily with the glycoprotein fraction, except in hydrolysate-based preparations in which "precipitable" glycoproteins were converted into "soluble" glycopeptides by trypsin treatment. Because whey-based raw materials are very rich in all bovine milk glycoconjugates and oligosaccharides their increased use will result in high contents of these micronutrients in modern formulas.
Asunto(s)
Carbohidratos/análisis , Alimentos Infantiles/análisis , Leche Humana/química , Leche/análisis , Animales , Bovinos , Gangliósidos/química , Glucolípidos/química , Glicopéptidos/análisis , Glicoproteínas/análisis , Humanos , Lactante , Oligosacáridos/análisisRESUMEN
The role of cell surface glycoconjugates as possible adhesion receptors for Candida albicans yeasts on human buccal epithelial cells was investigated by using a quantitative radiometric assay involving 14C-metabolically labeled microorganisms. Various structurally defined soluble glycopeptides and oligosaccharides were tested at a low concentration (1 mg/ml) for their ability to competitively inhibit yeast adhesion to such exfoliated cells. Comparisons were also made with various molecular species previously proposed to act as adhesion molecules. A preparation of glycopeptides derived from pooled human newborn meconiums inhibited the attachment (up to 55%) of all three clinical isolates examined. The mild hydrolysis of fucosyl residues from the above mixture totally abolished its inhibitory potency. By using human milk oligosaccharide probes, the minimal structural requirement for activity was found to be the Fuc alpha 1----2Gal beta determinant (the H sugar sequence found on all blood group substances of the ABO [H] system). By contrast, the fucosylated determinants of the Lewis blood group system were found to be totally inactive. Total adhesion inhibitions were never obtained in the present experiments, suggesting that H disaccharide-bearing cell surface glycoconjugates could act as host receptors for C. albicans on human buccal epithelial cells as a part of a mechanism involving multireceptor specificities.
Asunto(s)
Candida albicans/patogenicidad , Carbohidratos/farmacología , Mucosa Bucal/microbiología , Sistema del Grupo Sanguíneo ABO , Adhesividad , Secuencia de Carbohidratos , Glicoconjugados/farmacología , Humanos , Antígenos del Grupo Sanguíneo de Lewis , Manosa/farmacología , Datos de Secuencia Molecular , Oligosacáridos/farmacología , Fragmentos de Péptidos/farmacología , Relación Estructura-ActividadRESUMEN
Enterotoxigenic Escherichia coli are the most common cause of travelers' and infant diarrhea in less-developed countries. In the present work, among several metabolically labeled human diarrheagenic E. coli strains, enterotoxigenic strains expressing colonization factor antigen II were shown to bind to HT-29 intestinal cell monolayers when these cells were grown in conditions promoting their enterocytic differentiation. Indirect immunofluorescence with fimbrial antisera revealed that pathogen attachment was associated with the production of a specific bacterial adhesin, the E. coli surface antigen CS3. Scanning and transmission electron micrographs showed an apical pattern of colonization, characteristic of enterotoxigenic E. coli infections. The above data were consistent with all observations previously made with human enterocytes obtained from intestinal biopsies. The lectin-carbohydrate nature of this cell-cell recognition mechanism was also established. Bacterial binding to differentiated HT-29 cells was inhibited by a mixture of newborn meconium glycopeptides. By coating the cell layers with the plant agglutinin from Evonymus europaea, pathogen attachment was also prevented. Binding of 125I-labeled CS3 adhesin and E. europaea agglutinin to brush border membrane proteins separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred to nitrocellulose revealed three bands of about 30, 20, and 13 kilodaltons, which acted as receptors for both bacterial and plant lectins. These data suggest that the sugar units to which the bacterial colonization factor CS3 binds are synthesized as carbohydrate chains of three brush border membrane glycoproteins in HT-29 cells by a differentiation-specific pathway.