Effect of trimming inhibitors on the secretion and biological activity of a murine IgE monoclonal antibody.

Since secretion of IgE antibodies is known to be blocked by tunicamycin, the first aim of the present study was to determine at which step of glycosylation or processing secretion was restored. For this purpose, murine hybridoma cells secreting an anti beta-lactoglobulin IgE were incubated either in the presence of inhibitors of glucosidase I (castanospermine or N-methyl-1-deoxynojirimycin), or of an inhibitor of Golgi mannosidase II (swainsonine). Terminal galactoses predominate on the native IgE N-linked carbohydrate chains. The action of the trimming inhibitors, which results in changes in these terminal galactose residues, was monitored through detecting binding modifications to Concanavalin A and to the lectin of Ricinus communis. The antibody activity was also evaluated by a radioimmunoassay. It was shown that neither secretion nor anti beta-lactoglobulin activity of the IgE antibody are modified in the presence of any of the trimming inhibitors, whereas secretion is blocked in the presence of tunicamycin. Other biological activities of this IgE were investigated: no difference was observed in the binding of the carbohydrate-modified IgE molecules to normal mouse mast cells, nor to RBL-1 cells, as demonstrated by passive cutaneous anaphylaxis and in vitro binding tests respectively. However, traces of unglycosylated epsilon chain (mol. wt 61,000) found in tunicamycin treated cell supernatant did not bind to RBL-1 cell Fc epsilon receptors. These findings globally suggest that secretion occurs only if the tetradecasaccharide precursor of N-linked carbohydrate chains is transferred from its lipid-carrier to the polypeptide. Further, the presence of such non-processed oligosaccharides (Glc3Man9GlcNAc2) on IgE, does not seem to modify any of the biological activities of this molecule.

The development of functional foods: lessons from the gut.

Functional foods have resulted from the gradual recognition that healthy diets result from eating nutritious foods and from the identification of the mechanisms by which foods modulate metabolism and health. After initial successes with foods that reduce blood cholesterol level, probiotic bacteria and prebiotic carbohydrates have now also demonstrated added health benefits. As ingredients become more complex, the need to stabilize such ingredients in foods become increasingly important to the success of functional foods. Modern biotechnologies such as genomics, genetic expression and biomarkers of health and performance will be applied to this increasingly visible portion of human diets.

Molecular organisation of the ice nucleation protein InaV from Pseudomonas syringae.

A new ice nucleation gene from Pseudomonas syringae was isolated and overexpressed as a fully active protein in Escherichia coli in order to gain experimental data about the structure of ice nucleation proteins. No evidence of a signal sequence or secondary glycosylation was found. Differences in the extent of aggregation were shown to modulate the ice nucleation activity. The circular dichroism spectrum of the purified protein indicated the presence of beta-sheet structure. This finding supports a recently proposed hypothetical model for the structure of ice nucleation proteins, which provides a plausible explanation for their aggregation tendency.

Carbohydrate and glycoprotein specificity of two endogenous cerebellar lectins.

Two endogenous cerebellar mannose binding lectins have been isolated in an active form by immunoaffinity chromatography employing their respective immobilized antibodies. One of them, termed cerebellar soluble lectin (CSL), was extracted in the absence of detergents, whereas the other, called Receptor 1 (R1), was soluble only in the presence of detergents. Tests of inhibition of agglutination of erythrocytes were performed with mono-, oligo and polysaccharides, as well as glycoconjugates of known structures. On the basis of agglutinating activities these 2 lectins are different from the previously reported lectins in brain, since they were not inhibited by galactosides and lactosides and were only marginally inhibited by glycosaminoglycans. CSL and R1 were better inhibited by mannose-rich glycopeptides as compared to the corresponding oligosaccharides. The different inhibition patterns obtained with glycans of known structures indicated that these lectins are very discriminative. Although CSL and R1 have similar specificities, they differed in their binding properties towards glycopeptides of ovalbumin. Both lectins showed considerable affinity for endogenous cerebellar glycopeptides, also rich in mannose. These glycopeptides belong to a few endogenous Con A-binding cerebellar glycoprotein subunits and are not present on other endogenous Con A-binding glycoproteins. In the forebrain, where CSL and R1 were also present, at least some of the glycoproteins interacting with the lectins were different from that observed in the cerebellum. Our data overall suggest that specific cell recognition in the nervous system could be invoked via the interactions between widely distributed lectins and cell-specific glycoproteins.

Effect of oligomannoside-type glycopeptides in diarrheal disease of rabbits induced by Escherichia coli strain RDEC-1.

We examined the effects of a soluble receptor-like complex carbohydrate (an oligomannoside-type glycopeptide) on adherence of a type 1 fimbriated rabbit enteropathogen, Escherichia coli strain RDEC-1. Oligomannoside-type glycopeptide, but not a non-glycosylated peptide mixture used as control, inhibited adherence of type 1 fimbriated RDEC-1 to both rabbit ileal brush borders and guinea pig erythrocytes in vitro. In contrast, during RDEC-1 infection of rabbits the receptor-like oligomannoside did not affect the presence and severity of diarrhea, fecal shedding of organisms, luminal colonization by RDEC-1, or enteroadherence of organisms.

Incorporation of caseinoglycomacropeptide and caseinophosphopeptide into the salivary pellicle inhibits adherence of mutans streptococci.

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.

Routine o-glycan characterization in nutritional supplements--a comparison of analytical methods for the monitoring of the bovine kappa-casein macropeptide glycosylation.

Analytical procedures, including capillary isoelectric focusing (CIEF), high-performance anion-exchange chromatography coupled to amperometric detection (HPAEC-PAD) and normal-phase chromatography with fluorescence detection are presented for the characterization of a highly O-glycosylated caseinomacropeptide (CGMP) and the detection of subtle glycosylation differences between CGMP Batches obtained with two different preparation procedures. Modified two-step CIEF allowed monitoring of glycopeptide heterogeneity and determination of the isoelectric points of acidic glycoforms. The mixture of wide and narrow pH range ampholytes was optimized to improve glycoform resolution. The pI of the different CGMP glycoforms was evaluated with pI internal standards and found to range between 3.08 and 3.58, which indicates a very acidic glycopeptide. Moreover, the monosaccharide composition was determined with HPAEC-PAD after neutral and amino sugars release by using adequate acidic hydrolysis of CGMP. Results indicated a similar composition for Batches I and II, but the monosaccharide percentages were 3-4 fold higher in Batch I, particularly for galactose and glucose. This likely reflects a higher content in lactose in the case of Batch I. Finally, O-linked oligosaccharides were released with an automated hydrazinolysis and derivatized with a sensitive labelling reagent, 2-aminobenzamide. The derivatives were then analyzed by normal-phase HPLC coupled with fluorescence detection, and separated on the basis of hydrophilic interaction, which allowed oligosaccharide mapping of the two CGMP. It appeared that the two CGMP preparations had an almost identical O-glycan population, but CGMP Batch I was more glycosylated than Batch II. Additionally, the sizes of the separated glycans, expressed as the number of glucose units, were tentatively assigned using calibration with a partial hydrolysate of dextran. In conclusion, a combination of electrophoretic and chromatographic techniques was found powerful in studying glycoprotein heterogeneity and assessing batch-to-batch consistency.

Development of a capillary zone electrophoresis method for caseinoglycomacropeptide determination.

Caseinoglycomacropeptide (CGMP) is a polypeptide of 64 amino acid residues, derived from the C-terminal part of bovine kappa-casein. A sensitive and selective capillary zone electrophoresis method has been developed and validated for the analysis and quantitation of CGMP. Separation is carried out at 30 kV, using an uncoated fused-silica capillary and 20 mM sodium citrate buffer at acidic pH 3.5. The described method allows the separation of various CGMP subcomponents. The validation data proves that the method has the requisite selectivity, sensitivity, reproducibility and linearity for CGMP assay and for quality control during CGMP manufacturing (batch-to-batch reproducibility).

G.l.c. of O-methyloxime and alditol acetate derivatives of neutral sugars, hexosamines, and sialic acids: "one-pot" quantitative determination of the carbohydrate constituents of glycoproteins and a study of the selectivity of alkaline borohydride reductions.

A new procedure for the quantification by g.l.c. of the carbohydrate constituents of glycoproteins is proposed which involves (a) simultaneous action of neuraminidase and neuraminic acid aldolase, (b) hydrolysis with 4M trifluoroacetic acid at 125 degrees for 1 h, and (c) conversion of the products into O-methyloxime acetates and g.l.c. The procedure has been successfully tested on fetuin, transferrin, alpha 1-acid glycoprotein, and mucin. The g.l.c. conditions used also enabled the complete separation of O-methyloxime and alditol acetate derivatives in one run, so that the release of carbohydrate chains from glycoproteins by treatment with alkaline borohydride can be investigated conveniently. There was complete release of O-linked oligosaccharides from fetuin on treatment with 0.1M NaOH/0.8M NaBH4 (68 h, 37 degrees) or 0.05M KOH/M KBH4 (24 h, 45 degrees) and also release of approximately 75% and 35-40%, respectively, of N-asparagine-linked chains. Reduced oligosaccharides were formed only from O-linked chains; the mechanism by which N-linked chains were released is still not clear.

Lactobacillus helveticus Lh59 secretes an exopolysaccharide that is identical to the one produced by Lactobacillus helveticus TN-4, a presumed spontaneous mutant of Lactobacillus helveticus TY1--2.

Lactobacillus helveticus Lh59 produces a high-molecular-mass exopolysaccharide (> or = 2 x 10(6) Da) when cultured in skimmed milk. Compositional analysis, methylation analysis and NMR experiments (1H and 13C) recorded from the native polysaccharide as well as from oligosaccharides released by partial acid hydrolysis, allowed the complete structural determination of this polysaccharide, which consists of the following hexasaccharide repeating unit: [symbol: see text] This structure is identical to the one of an EPS produced by L. helveticus TN-4, which was claimed to be a spontaneous mutant of strain TY1-2.

A quantitative determination by capillary gas-liquid chromatography of neutral and amino sugars (as O-methyloxime acetates), and a study on hydrolytic conditions for glycoproteins and polysaccharides in order to increase sugar recoveries.

Complete gas-liquid chromatographic separation of O-methyloxime acetates (syn and anti isomers) prepared from eight neutral sugars, three hexosamines, and muramic acid has been obtained, using a fused-silica Carbowax 20M capillary column. A single hydrolytic step for carbohydrate-containing biological material (less than or equal to 2.5 X 10(-3) M sugar solution in 4 N trifluoroacetic acid at 125 degrees C for 1 h) has been developed, and results have been compared with those obtained with standard hydrolytic conditions in order to ensure complete release of amino sugars from glycoproteins, together with minimum losses of neutral sugars. The combination of this acid hydrolysis with the above improved derivatization procedure for the gas-liquid chromatographic analysis has led to a simple, rapid, and sensitive analytical method, which has been successfully tested on three glycoproteins (fetuin, mucin, and peroxidase) and two plant cell-wall polysaccharide fractions (soluble fibers from carrots and soybeans).

Binding of the fibrillar CS3 adhesin of enterotoxigenic Escherichia coli to rabbit intestinal glycoproteins is competitively prevented by GalNAc beta 1-4Gal-containing glycoconjugates.

We have attempted to characterize the binding specificity of the coli surface 3 (CS3) subcomponent of colonization factor antigen II of enterotoxigenic Escherichia coli, by means of an immunoblot method in which the binding of fimbriated bacteria to sodium dodecyl sulfate-polyacrylamide gel electrophoresis-separated rabbit intestinal cell membranes was evaluated. Isolated CS3 fibrillae as well as bacteria expressing CS3 on their surface bound to several intestinal cell membrane structures, i.e., structures present in the electrophoretic front and in the 30- to 35-kDa range and, most prominently, 120- to 140-kDa structures. Delipidization and protein digestion of the rabbit brush borders revealed that CS3 bound to structures of a proteinaceous nature. Sodium meta-periodate oxidation of the intestinal cell membranes abolished all their CS3 binding activity, indicating that CS3 bound to carbohydrate moieties of glycoproteins. The binding of CS3 to the separated intestinal proteins could also be inhibited by preincubation with the lectin derived from Maackia amurensis, indicating that CS3 bound to galactoproteins in the rabbit intestine. Inhibition experiments using equimolar amounts of various gangliosides demonstrated that GM1, asialo-GM1, and GM2 inhibited the binding of CS3 equally well, whereas GM3 was not as effective. These results suggested that the critical CS3 binding epitope consisted of the carbohydrate sequence GalNAc beta 1-4Gal. This was supported by electron microscopic experiments showing that this disaccharide, O linked to bovine serum albumin via a spacer, localized around CS3-positive bacteria but not at all around corresponding CS3-negative mutants. Furthermore, CS3-expressing bacteria recognized this neoglycoprotein when it was immobilized on nitrocellulose. The GalNAc beta 1-4Gal disaccharide has also been implicated as a binding structure for other pathogenic bacteria such as enteropathogenic E. coli and Pseudomonas aeruginosa.

Identification and characterization of the eps (Exopolysaccharide) gene cluster from Streptococcus thermophilus Sfi6.

We report the identification and characterization of the eps gene cluster of Streptococcus thermophilus Sfi6 required for exopolysaccharide (EPS) synthesis. This report is the first genetic work concerning EPS production in a food microorganism. The EPS secreted by this strain consists of the following tetrasaccharide repeating unit:-->3)-beta-D-Galp-(1-->3)-[alpha-D-Galp-(1-->6)]-beta-D- D-Galp-(1-->3)-alpha-D-Galp-D-GalpNAc-(1-->. The genetic locus The genetic locus was identified by Tn916 mutagenesis in combination with a plate assay to identify Eps mutants. Sequence analysis of the gene region, which was obtained from subclones of a genomic library of Sfi6, revealed a 15.25-kb region encoding 15 open reading frames. EPS expression in the non-EPS-producing heterologous host, Lactococcus lactis MG1363, showed that within the 15.25-kb region, a region with a size of 14.52 kb encoding the 13 genes epsA to epsM was capable of directing EPS synthesis and secretion in this host. Homology searches of the predicted proteins in the Swiss-Prot database revealed high homology (40 to 68% identity) for epsA, B, C, D, and E and the genes involved in capsule synthesis in Streptococcus pneumoniae and Streptococcus agalactiae. Moderate to low homology (37 to 18% identity) was detected for epsB, D, F, and H and the genes involved in capsule synthesis in Staphylococcus aureus for epsC, D, and E and the genes involved in exopolysaccharide I (EPSI) synthesis in Rhizobium meliloti for epsC to epsJ and the genes involved in lipopolysaccharide synthesis in members of the Enterobacteriaceae, and finally for eps K and lipB of Neisseria meningitidis. Genes (epsJ, epsL, and epsM) for which the predicted proteins showed little or no homology with proteins in the Swiss-Prot database were shown to be involved in EPS synthesis by single-crossover gene disruption experiments.

In vitro inhibition of adhesion of Candida albicans clinical isolates to human buccal epithelial cells by Fuc alpha 1----2Gal beta-bearing complex carbohydrates.

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.

Quantitative determination of complex carbohydrates in bovine milk and in milk-based infant formulas.

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.

Oligomannoside-type glycopeptides inhibiting adhesion of Escherichia coli strains mediated by type 1 pili: preparation of potent inhibitors from plant glycoproteins.

Various structurally defined glycopeptides of natural origin were tested as inhibitors of guinea pig erythrocyte agglutination by enteropathogenic Escherichia coli strains expressing type 1 pili. Besides hybrid-type glycoasparagines from ovalbumin which were not active, large oligomannoside-type carbohydrate chains from legume storage glycoproteins moderately inhibited hemagglutinations, whereas the short oligomannoside-type glycoasparagine from ovalbumin Man alpha(1----6) [Man alpha(1----3)]Man alpha(1----6)[Man alpha(1----3)] Man beta(1----4)GlcNAc beta(1----4)GlcNAc beta(1----N)Asn exhibited a potent activity. These results strongly suggested that the nonsubstitution of the alpha(1----3)-linked mannosyl residue from the N-linked glycopeptide core structure is the key determinant in the minimal structural requirement specific to this fimbrial lectin. Such Man5GlcNAc2-containing glycopeptides were obtained from larger N-linked carbohydrate chains, occurring abundantly in natural sources. The ability of jack bean alpha-mannosidase to cleave the alpha(1----2)-linked mannoses more rapidly than the others allowed the controlled digestion of large oligomannoside-type glycopeptides from legume storage glycoproteins. Such shortened glycopeptides of plant origin were prepared which strongly inhibited guinea pig erythrocyte agglutinations as well as bacterial adhesion on human buccal cells, thus confirming their similarity (if not identity) with the receptor of type 1 pili on mammalian cells. The importance of this preparation of a receptorlike compound that inhibits bacterial adhesion with regard to the research on the role of type 1 pili in E. coli pathogenicity is discussed.

Unraveling the function of glycosyltransferases in Streptococcus thermophilus Sfi6.

Streptococcus thermophilus Sfi6 produces a texturizing exopolysaccharide (EPS) consisting of a -->3)[alpha-D-Galp-(1-->6)]-beta-D-Glcp-(1-->3)-alpha-D-GalpNAc-(1--> 3)-beta-D-Galp-(1--> repeating unit. We previously identified and analyzed a 14.5-kb gene cluster from S. thermophilus Sfi6 consisting of 13 genes responsible for its EPS production. Within this gene cluster, we found a central region of genes (epsE, epsF, epsG, and epsI) that showed similarity to glycosyltransferases. In this study, we investigated the sugar specificity of these enzymes. EpsE catalyzes the first step in the biosynthesis of the EPS repeating unit. It exhibits phosphogalactosyltransferase activity and transfers galactose onto the lipophilic carrier. The second step is fulfilled by EpsG, which transfers an alpha-N-acetylgalactosamine onto the first beta-galactoside. The activity of EpsF was determined by characterizing the EPS produced by an S. thermophilus epsF deletion mutant. This EPS consisted of the monosaccharides Gal, Glc, and GalNAc in an approximately equimolar ratio, thus suggesting that epsF codes for the branching galactosyltransferase. epsI probably codes for the beta-1,3-glucosyltransferase, since it is the only glycosyltransferase to which no gene has been assigned and it exhibits similarity to other beta-glycosyltransferases. EpsE shows the conserved features of phosphoglycosyltransferases, whereas EpsF and EpsG exhibit the primary structure of alpha-glycosyltransferases, belonging to glycosyltransferase family 4, whose members are conserved in all major phylogenetic lineages, including the Archaea and Eukaryota.

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-like glycoproteins bearing sialic acid residues are known to bind various S.sanguis strains. However, the molecular basis for the adhesion of S.sanguis to human buccal epithelial cells (HBEC) has not been established. The present study shows that S.sanguis OMZ 9 binds to exfoliated HBEC in a sialic acid-sensitive manner. The desialylation of such cells invariably abolishes adhesion of S.sanguis OMZ 9 to the cell surface. A soluble glycopeptide bearing short sialylated O-linked carbohydrate chains behaves as a potent inhibitor of the attachment of S.sanguis OMZ 9 to exfoliated HBEC. The resialylation of desialylated HBEC with CMP-sialic acid and Gal beta 1,3GalNAc alpha 2,3-sialyltransferase specific for O-glycans restores the receptor function for S.sanguis OMZ 9, whereas a similar cell resialylation with the Gal beta 1,4GlcNAc alpha 2,6-sialyltransferase specific for N-glycans is without effect. Finally, the same resialylation reaction carried out with CMP-9-fluoresceinyl-sialic acid as a substrate yields exfoliated HBEC bearing fluorescence on a single 23 kDa protein, when using the alpha 2,3-sialyltransferase as the catalyst. The latter finding demonstrates that this 23 kDa cell surface glycoprotein bears NeuNAc alpha 2-3Gal beta 1-3GalNAc O-linked sugar chains, a carbohydrate sequence which is recognized by S.sanguis OMZ 9 on exfoliated HBEC. In similar experiments carried out with a buccal carcinoma cell line termed SqCC/Y1, S.sanguis OMZ 9 did not attach in great numbers to such cultured cells, and these cells were shown to not express membrane glycoprotein bearing alpha 2,3-sialylated O-linked carbohydrate chains.

Adhesion of human bifidobacterial strains to cultured human intestinal epithelial cells and inhibition of enteropathogen-cell interactions.

Thirteen human bifidobacterial strains were tested for their abilities to adhere to human enterocyte-like Caco-2 cells in culture. The adhering strains were also tested for binding to the mucus produced by the human mucus-secreting HT29-MTX cell line in culture. A high level of calcium-independent adherence was observed for Bifidobacterium breve 4, for Bifidobacterium infantis 1, and for three fresh human isolates from adults. As observed by scanning electron microscopy, adhesion occurs to the apical brush border of the enterocytic Caco-2 cells and to the mucus secreted by the HT29-MTX mucus-secreting cells. The bacteria interacted with the well-defined apical microvilli of Caco-2 cells without cell damage. The adhesion to Caco-2 cells of bifidobacteria did not require calcium and was mediated by a proteinaceous adhesion-promoting factor which was present both in the bacterial whole cells and in the spent supernatant of bifidobacterium culture. This adhesion-promoting factor appeared species specific, as are the adhesion-promoting factors of lactobacilli. We investigated the inhibitory effect of adhering human bifidobacterial strains against intestinal cell monolayer colonization by a variety of diarrheagenic bacteria. B. breve 4, B. infantis 1, and fresh human isolates were shown to inhibit cell association of enterotoxigenic, enteropathogenic, diffusely adhering Escherichia coli and Salmonella typhimurium strains to enterocytic Caco-2 cells in a concentration-dependent manner. Moreover, B. breve 4 and B. infantis 1 strains inhibited, dose dependently, Caco-2 cell invasion by enteropathogenic E. coli, Yersinia pseudotuberculosis, and S. typhimurium strains.