Nestlé Research Center, Vers-chez-les-Blanc, CH-1000 Lausanne 26, Switzerland, 2CJF 94.07 INSERM, UFR de Pharmacie, Université Paris XI, F-92296 Châtenay-Malabry, France, and 3Department of Medical Biochemistry, Göteborg University, S-413 90 Göteborg, Sweden
Received on March 13, 2000; Revised on June 21, 2000; accepted on June 21, 2000.
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Abstract |
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Key words: glycolipids/mannoprotein/intestinal epithelial cells/Lactobacillus johnsonii La1/probiotic bacterial adhesion
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Introduction |
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Regarding now the molecular species involved in the adherence of lactic acid bacteria to the intestinal mucosa, the topic remains poorly documented. The importance of a glycolipid class, the lipoteichoic acids (Ofek et al., 1975; Beachey and Ofek, 1976
; Pinto et al., 1983
; Sherman and Savage, 1986
; Teti et al., 1987a
,b) and/or cell surface proteins (Toba et al., 1995
) as mediators of adhesion of Lactobacillus spp. or other Gram-positive bacteria to human epithelial cells has been demonstrated. For L.johnsonii La1, the first hypothesis of an adhesion mechanism mediated by a bacterial "bridging" protein (Bernet et al., 1994
) has been revised, after having established the key role played by the cell surface-associated La1 lipotechoic acid for the La1 attachment to Caco-2 cells (Granato et al., 1999
). On the other hand, the nature of the receptors present on the surface of mammalian intestinal cells has been even less documented. The identification of certain glycolipids of the rat intestinal mucosa as putative receptors for a rat Lactobacillus casei strain (Yamamoto et al., 1996
), and the necessary presence of carbohydrate determinants on the surfaces of cultured human intestinal cells (HT-29) for the binding of two Lactobacillus plantarum strains (Adlerberth et al., 1996
) have suggested the occurrence of adhesion mechanisms involving complex carbohydrates. In this context, our first aim was here to identify the carbohydrate-binding specificities of L.johnsonii La1 by testing soluble oligosaccharides and glycoconjugates as adhesion inhibitors. As Caco-2 cells display typical features of an enterocytic differentiation (Pinto et al., 1983
), bacterial binding to these cells was studied in the presence of such potential inhibitors. It should be noted that the adhesion properties of L.johnsonii La1 onto Caco-2 intestinal cells have previously been shown to be dependent of the state of enterocytic differentiation of the cells (Bernet et al., 1994
). Thus, these cells were cultured accordingly, in order to assure the expression of the required receptor(s) for La1. In parallel, we examined the attachment of L.johnsonii La1 to glycolipids extracted from different sources and compared them to those already known to be specifically recognized by enteropathogens (Karlsson, 1989
; Sporsem Oro et al., 1990
; Jagannatha et al., 1991
; Wenneras et al., 1995
). As a result, we identified here two major carbohydrate-binding specificities of L.johnsonii La1, a first one mannose-specific, and a second one revealed by a strong affinity for the gangliotetraosylceramide (GA1 = asialo-GM1) glycolipid. These findings put a new light on the way by which probiotic bacteria can provide protection to the gut against microbial pathogens, since both these specificities are equally shared by several enteropathogens, the latter using them for adhering to the host gut mucosa.
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Results and discussion |
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All other compounds tested (see Table I) were ineffective, which suggested a binding of La1 to Caco-2 cells inhibited either by specific glycolipids or by selected oligomannosides.
Binding of L.johnsonii La1 on thin-layer chromatograms
In a second step, we focused our attention on the binding of metabolically labeled L.johnsonii La1 to glycolipids extracted from different sources and separated on TLC plates according to the method of Karlsson (1987).
As seen in Table III, there was a consistent binding to gangliotriosyl- and gangliotetraosylceramide (also fucosylated gangliotetraosylceramide) and to lactotetraosylceramide (occurring in human meconium) and lactosylceramide. Some glycolipids ending in N-acetyllactosamine showed occasional binding and a number of glycolipids were consistently negative. Binding to asialo-GM1 (gangliotetraosylceramide) was very strong and has also been observed for a rat L.casei strain, another lactic acid bacteria (Yamamoto et al., 1996), and for some species of Bifidobacteria by Fujiwara et al. (1997)
. When dealing with pathogenic strains, Sporsem Oro et al. (1990)
showed by immuno-thin layer chromatography that asialo-GM1 was a binding structure for E.coli colonization factor antigens CFA/II and CFA/IV. Later, Wenneras et al. (1995)
demonstrated by blotting experiments that the coli surface 3 (CS3) subcomponent of the colonization factor antigen II of enterotoxigenic E.coli was binding to electrophoretically separated and transblotted rabbit intestinal proteins, but that the binding could be inhibited by asialo-GM1 as well as by GM1 and GM2, but not GM3. This observation implied that the critical CS3 epitope consisted of the carbohydrate sequence GalNAcß14Gal and that this sequence or a sequence having the same tertiary configuration was found in the blotted protein recognized by CS3. This sequence has also been implicated as a binding structure for enteropathogenic E.coli (Jagannatha et al., 1991
). In the case of La1, the highest binding to TLC plates was found with this sequence, but was not restrictive as can be seen with the lactosylceramides.
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Materials and methods |
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For adhesion assays, metabolically labeled bacteria were washed three times with PBS and resuspended at a concentration of 108 cfu/ml in 0.05 M acetate buffer pH 5 containing 0.15 M NaCl (a solution 0.05 M sodium acetate and 0.15 M sodium chloride was adjusted to pH 5.0 with glacial acetic acid); 1 ml of the bacterial suspension was incubated at 37°C for 1 h onto Caco-2 cells (6 wells plates) which had been previously washed with acetate buffer. Afterwards, supernatants were removed and cells were washed three times with the same buffer. Cell monolayers were disrupted by addition of 1 ml of 1 M NaOH. The lysate and a 0.5-ml wash were transferred in a counting vial containing 1 ml of benzethonium hydroxide (Sigma). Vials were put in an incubator at 60°C for 1 h, cooled, and counted after the addition of 10 ml of Ultima Gold (Packard). Each experiment was performed in triplicate and mean values were calculated.
For adhesion inhibition assays, the compounds to be tested were dissolved in acetate buffer, then mixed with the bacterial suspension and incubated on Caco-2 cells as described above. Experiments were repeated twice or three times (always involving triplicates) for each test compound. The logit transformation was chosen as the best tool for statistical calculations (better than the use of a mean or a median), since it takes into account the limitation of the inhibition rate between 0 and 100% only.
Human newborn meconium glycolipids and glycoproteins were separated as described by Hounsell et al. (1985). The preparation of the samples to be used for testing concentrations of 1 mg/ml and 3 mg/ml of this glycolipid mixture in acetate buffer yielded clear solutions, whereas the most concentrated suspensions (10 mg/ml) required a centrifugation step before mixing with the bacteria. Saccharomyces cerevisiae mnn 9 mutant was a kind gift of C.E.Ballou (Department of Biochemistry, University of California, Berkeley). The isolation of the cell wall mannoprotein was done according to Frevert and Ballou (1985)
. Briefly, anion-exchange chromatography on DEAE-Sephadex A-50 using a 0.3 M sodium chloride solution as an eluent yielded a large retained fraction which was collected and further homogenized to purity on a hydroxyapatite gel. The release of the N-linked sugar chains was performed by a treatment with endo-ß-N-acetylglucosaminidase-H (Endo-H), as described by Tsai et al. (1986)
. This enzymatic treatment was repeated twice and the removal of N-glycans was followed by high-performance thin layer chromatography: under such conditions, no more N-glycan chains were released at the end of the second incubation period. The resulting mannoprotein was finally purified by liquid chromatography on a Sephadex G-50 column (Pharmacia, Sweden). The other tested compounds were from Sigma Chemical Co. (St. Louis, MO).
Binding of L.johnsonii La1 on thin-layer chromatograms
Briefly, total acid and nonacid glycolipid fractions, from the sources given in Table III, were isolated as described (Karlsson, 1987). The pure glycolipids used in the binding studies were isolated by repeated chromatography of native glycolipids, or acetylated derivatives, on silicic acid columns (Iatrobeads 6RS-8060, Iatron Laboratories Inc., Tokyo, Japan) or by HPLC on silicic acid columns. The isolated glycolipids were characterized by mass spectrometry (Samuelsson et al., 1990
), proton NMR spectroscopy (Falk et al., 1979a
c; Koerner et al., 1983
), and degradation studies (Yang and Hakomori, 1971
; Stellner et al., 1973
).
Binding of 35S-labeled L.johnsonii La1 to glycolipids on thin-layer chromatograms was examined as follows: L.johnsonii bacteria were cultured in MRA broth supplemented with 50 µCi 35S-methionine/10 ml (Amersham, UK) at 37°C overnight under anaerobic conditions. The bacteria were collected by centrifugation, and washed three times with phosphate-buffered saline (PBS), pH 7.3. The bacteria were thereafter suspended in PBS to approximately 1 x 108 CFU/ml. The specific activities of the suspensions were approximately 1 c.p.m. per 100 bacterial cells. The chromatogram binding assay was done as described previously (Hansson et al., 1985). Glycolipid mixtures (2040 µg/lane), or pure glycolipids (14 µg/lane), were separated on glass- or aluminum-backed silica gel 60 HPTLC plates (Merck, Darmstadt, Germany), using chloroform/methanol/water (60:35:8, by volume) as solvent system. The dried chromatograms were dipped in 0.30.5% (w/v) polyisobutylmethacrylate (Plexigum P28, Röhm, GmbH, Darmstadt, Germany) in diethylether/n-hexane (1:5, by volume) for 1 min, and air-dried. Blocking of unspecific binding sites was done by immersing the plates in PBS containing 2% bovine serum albumin (w/v) and 0.1% Tween 20 (w/v) for 2 h at room temperature. Thereafter, suspensions of radiolabeled bacteria (diluted in PBS to 1 x 108 CFU/ml and 15 x 106 c.p.m./ml) were gently sprinkled over the chromatograms, and incubated for 2 h at room temperature. After washing six times with PBS, and drying, the thin-layer plates were autoradiographed for 1248 h using XAR-5 x-ray films (Eastman Kodak, Rochester, NY).
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Acknowledgments |
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Footnotes |
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References |
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Ballou,C.E. (1982) Yeast cell wall and cell surface, In Strathern,J.N., Jones,E.W. and Broach,J.R. (eds.), The Molecular Biology of the Yeast Saccharomyces. Metabolism and Gene Expression. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, pp. 335360.
Beachey,E. and Ofek,I. (1976) Epithelial cell binding of group A Streptococci by lipoteichoic acid on fimbriae denuded M protein. J. Exp. Med., 143, 759771.[Abstract]
Berg,R.D. (1996) The indigenous gastrointestinal microflora. Trends Microbiol., 4, 430435.[ISI][Medline]
Bernet,M-F., Brassart,D., Neeser,J.-R. and Servin,A.L. (1994) Lactobacillus acidophilus LA 1 binds to cultured human intestinal cell lines and inhibits cell attachment and cell invasion by enterovirulent bacteria. Gut, 35, 483489.[Abstract]
Bernet-Camard,M-F., Liévin,V., Brassart,D., Neeser,J.-R., Servin,A.L. and Hudault,S. (1997) The human Lactobacillus acidophilus strain La1 secretes a non bacteriocin antibacterial substance active in vitro and in vivo. Appl. Environ. Microbiol., 63, 27472753.[Abstract]
Brassart,D. and Schiffrin,E.J. (1997) Probiotics for improved gut health. Trends Food Sci. Technol., 8, 321326.[ISI]
Canil,C., Rosenshine,I., Ruschkowski,S., Donnenberg,M.S., Kaper,J.B. and Finlay,B.B. (1993) Enteropathogenic Escherichia coli decreases the transepithelial electrical resistance of polarized epithelial monolayers. Infect. Immun., 61, 27552762.[Abstract]
Falk,K.-E., Karlsson,K.-A. and Samuelsson,B.E. (1979a) Proton nuclear magnetic resonance analysis of anomeric structure of glycolipids. The globoseries (one to five sugars). Arch. Biochem. Biophys., 192, 164176.[ISI][Medline]
Falk,K.-E., Karlsson,K.-A. and Samuelsson,B.E. (1979b) Proton nuclear magnetic resonance analysis of anomeric structure of glycolipids. Blood group ABH-active substances. Arch. Biochem. Biophys., 192, 177190.[ISI][Medline]
Falk,K.-E., Karlsson,K.-A. and Samuelsson,B.E. (1979c) Proton nuclear magnetic resonance analysis of anomeric structure of glycolipids. Lewis-active and Lewis-like substances. Arch. Biochem. Biophys., 192, 91202.
Finley,B.B. and Cossart,P. (1997) Exploitation of mammalian host cell functions by bacterial pathogens. Science, 276, 718725.
Finlay,B.B. and Falkow,S. (1990) Salmonella interactions with polarized human intestinal Caco-2 epithelial cells. J. Infect. Dis., 162, 10961106.[ISI][Medline]
Finlay,B.B. and Siebers,A. (1995) Mechanisms of mucosal colonization and penetration by bacterial pathogens. In Roth,J.A., Bolin,C.A., Brogden,K.A., Minion,F.C. and Wannemuhler,M.O. (eds.), Virulence Mechanisms of Bacterial Pathogens, 2nd ed. American Society for Microbiology, Washington, DC, pp. 3345.
Firon,N., Ofek,I. and Sharon,N. (1983) Carbohydrate specificity of the surface lectins of Escherichia coli, Klebsiella pneumoniae and Salmonella typhimurium. Carbohydr. Res., 120, 235249.[ISI][Medline]
Frevert,J. and Ballou,C.E. (1985) Saccharomyces cerevisiae structural cell wall mannoprotein. Biochemistry, 24, 753759.[ISI][Medline]
Fujiwara,S., Hashiba,H., Hirota,T. and Forsner,J.F. (1997) Proteinaceous factor (s) in culture supernatant fluids of Bifidobacteria which prevents the binding of enterotoxigenic Escherichia coli to gangliotetraosylceramide. Appl. Environ. Microbiol., 63, 506512.[Abstract]
Glegg,S. and Swenson,D.L. (1994) Salmonella fimbriae. In Klemm,P. (ed.), Fimbriae: Adhesion, Genetics, Biogenesis and Vaccines. CRC Press, Boca Raton, FL, pp. 105112.
Granato,D., Perotti,F., Masserey,I., Rouvet,M., Golliard,M., Servin,A. and Brassart,D. (1999) Cell surface-associated lipoteichoic acid acts as an adhesion factor for attachment of Lactobacillus johnsonii La1 to human enterocyte-like Caco-2 cells. Appl. Environ. Microbiol., 65, 10711077.
Haller,D., Blum,S., Bode, Ch., Hammes,W.P. and Schiffrin,E.J. (2000) Activation of human PBMC by non-pathogenic bacteria in vitro: evidence of NK cells as primary targets. Infect. Immun., 68, 752759.
Hansson,G.C., Karlsson,K.-A., Larson,G., Strömberg,N. and Thurin,J. (1985) Carbohydrate-specific adhesion of bacteria to thin-layer chromatograms: a rationalized approach to the study of host cell glycolipid receptors. Anal. Biochem., 146, 158163.[ISI][Medline]
Hounsell,E.F., Lawson,A.M., Feeney,J., Gooi,H.C., Pickering,N.J., Stoll,M.S., Lui,S.C. and Feizi,T. (1985) Structural analysis of the O-glycosidically linked core-region oligosaccharides of human meconium glycoproteins which express oncofetal antigens. Eur. J. Biochem., 148, 367377.[Abstract]
Jagannatha,H.M., Sharma,U.K., Ramaseshan,T., Surolia,A. and Balganesh,T.S. (1991) Identification of carbohydrate structures as receptors for localized adherent enteropathogenic Escherichia coli. Microbial Pathogenesis, 11, 259268.[ISI][Medline]
Karlsson,K.-A. (1987) Preparation of total non-acid glycolipids for overlay analysis of receptors for bacteria and viruses and for other studies. Methods Enzymol., 138, 212220.[ISI][Medline]
Karlsson,K-A., (1989) Animal glycosphingolipids as membrane attachment sites for bacteria. Annu. Rev. Biochem., 58, 309350.[ISI][Medline]
Koerner,T.A.W.,Jr., Prestegard,J.H., Demou,P.C. and Yu,R.K. (1983) High-resolution proton NMR studies of gangliosides. 1. Use of homonuclear spin-echo J-correlated spectroscopy for determination of residue composition and anomeric configurations. Biochemistry, 22, 26762687.[ISI][Medline]
Krivan,H.C., Roberts D.D. and Ginsburg V. (1988) Many pulmonary pathogenic bacteria bind specifically to the carbohydrate sequence GalNAcß14Gal found in some glycolipids. Proc. Natl. Acad. Sci. USA, 85, 61576161.[Abstract]
Lee,K.K., Sheth,H.B., Wong,W.Y., Sherburne,R., Paranchych,W., Hodges,R.S., Lingwood,C.A., Krivan,H. and Irvin,R.T. (1994) The binding of Pseudomonas aeruginosa pili to glycosphingolipids is a tip-associated event involving the C-terminal region of the structural pilin subunit. Mol. Microbiol., 11, 705713.[ISI][Medline]
Lee,Y.-K. and Salminen,S. (1995) The coming of age of probiotics. Trends Food Sci. Technol., 6, 241245.[ISI]
Link-Amster,H., Rochat,F., Saudan,K.Y., Mignot,O. and Aeschlimann,J.-M. (1994) Modulation of a specific humoral immune response and changes in intestinal flora mediated through fermented milk intake. FEMS Immunol. Med. Microbiol., 10, 5564.[ISI][Medline]
Neeser,J.-R., Koellreutter,B. and Wuersch,P. (1986) Oligomannoside-type glycopeptides inhibiting adhesion of Escherichia coli strains mediated by type 1 pili: preparation of potent inhibitors from plant glycoproteins. Infect. Immun., 52, 428436.[ISI][Medline]
Neeser,J.-R., Chambaz,A., Golliard,M., Link-Amster,H., Fryder,V. and Kolodziejczyk,E. (1989) Adhesion of colonization factor antigen II-positive enterotoxigenic Escherichia coli strains to human enterocyte-like differentiated HT-29 cells: a basis for hostpathogen interactions in the gut. Infect. Immun. 57, 372734.[ISI][Medline]
Ofek,I., Beachey,E.H., Jefferson,W. and Campbell,G.L. (1975) Cell membrane binding properties of group A streptococcal lipoteichoic acid. J. Exp. Med. 141, 9901003.[Abstract]
Pinto,M., Robine-Leon,S., Appay,M.D., Kedinger,M., Triadou,N., Dussaulx,E., Lacroix,B., Simon-Assmann,P., Haffen,K., Fogh,J. and Zweibaum,A. (1983) Enterocyte-like differentiation and polarization of the human colon carcinoma cell line Caco-2 in culture. Biol. Cell., 47, 323330.[ISI]
Samuelsson,B.E., Pimlott,W. and Karlsson,K.-A. (1990) Mass spectrometry of mixtures of intact glycosphingolipids. Methods Enzymol., 193, 623646[Medline]
Schiffrin,E.J., Rochat,F., Link-Amster,H., Aeschlimann,J.-M. and Donnet-Hughes,A. (1995) Immunomodulation of human blood cells following the ingestion of lactic acid bacteria. J. Dairy Sci., 78, 491497.
Sherman,L.A. and Savage,D.C. (1986) Lipoteichoic acids in Lactobacillus strains that colonize the mouse gastric epithelium. Appl. Environ. Microbiol., 52, 302304.[ISI][Medline]
Sporsem Oro,H., Kolsto, A-B., Wenneras,C. and Svennerholm, A-M. (1990) Identification of asialo GM1 as a binding structure for Escherichia coli colonization factor antigens. FEMS Microbiol. Lett., 72, 289292.[ISI]
Stellner,K., Saito,H. and Hakomori,S.-I. (1973) Determination of aminosugar linkages in glycolipids by methylation. Aminosugar linkages of ceramide pentasaccharides of rabbit erythrocytes and of Forssman antigen. Arch. Biochem. Biophys., 155, 464472.[ISI][Medline]
Tancrede,C. (1992) Role of human microflora in health and disease. Eur. J. Clin. Microbiol. Infect. Dis., 11, 10121015.[ISI][Medline]
Teti,G., Chiofalo,M.S., Tomasello,F., Orefici,G. and Mastroeni,P. (1987a) Mediation of Staphylococcus saprophytus adherence to uroepithelial cells by lipoteichoic acid. Infect. Immun., 55, 839842.[ISI][Medline]
Teti,G., Tomasello,F., Chiofalo,M.S., Orefici,G. and Mastroeni,P. (1987b) Adherence of group B Streptococci to adult and neonatal epithelial cells is mediated by lipoteichoic acid. Infect. Immun., 55, 30573064.[ISI][Medline]
Toba,T., Virkola,R., Westerlund,B., Björkman,Y., Sillanpää,J., Vartio,T., Kalkkinen,N. and Korhonen,T.K. (1995) A collagen-binding S-layer protein n Lactobacillus crispatus. Appl. Environ. Microbiol., 61, 24672471.[Abstract]
Tsai,P-K., Dell,A. and Ballou,C.E. (1986) Characterization of acetylated and acetolyzed glycoprotein high-mannose core oligosaccharides by fast-atom-bombardment mass spectrometry. Proc. Natl. Acad. Sci. USA, 82, 41194123.
Wenneras,C., Neeser,J.-R. and Svennerholm,A.-M. (1995) Binding of the fibrillar CS3 adhesin of enterotoxigenic Escherichia coli to rabbit intestinal glycoproteins is competitively prevented by GalNAcß14Gal-containing glycoconjugates. Infect. Immun., 63, 640646.[Abstract]
Yamamoto,K., Miwa,T., Taniguchi,H., Nagano,T., Shimamura,K., Tanaka,T. and Kumagai,H. (1996) Binding specificity of Lactobacillus to glycolipids. Biochem. Biophys. Res. Commun., 228, 148152.[ISI][Medline]
Yang,H.-J. and Hakomori,S.-I. (1971) A sphingolipid having a novel ceramide and lacto-N-fucopentose III. J. Biol. Chem., 246, 11921200.
Yu,Y.Y., Kummar,V. and Bennett,M. (1992) Murine natural killer and marrow graft rejection. Annu. Rev. Immunol., 10, 189213.[ISI][Medline]