Cell surface-associated lipoteichoic acid acts as an adhesion factor for attachment of Lactobacillus johnsonii La1 to human enterocyte-like Caco-2 cells - PubMed (original) (raw)

Cell surface-associated lipoteichoic acid acts as an adhesion factor for attachment of Lactobacillus johnsonii La1 to human enterocyte-like Caco-2 cells

D Granato et al. Appl Environ Microbiol. 1999 Mar.

Abstract

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.

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Figures

FIG. 1

FIG. 1

Immunogold staining of thin sections. (a) L. johnsonii La1 treated with monoclonal antibodies from the 113D2b3 clone supernatant. (b) L. acidophilus La10 incubated with the same antibodies. (c and e) L. johnsonii Lj1 incubated with the 113D2b3 clone supernatant before embedding. (d) L. johnsonii La1 incubated with hybridoma culture medium.

FIG. 1

FIG. 1

Immunogold staining of thin sections. (a) L. johnsonii La1 treated with monoclonal antibodies from the 113D2b3 clone supernatant. (b) L. acidophilus La10 incubated with the same antibodies. (c and e) L. johnsonii Lj1 incubated with the 113D2b3 clone supernatant before embedding. (d) L. johnsonii La1 incubated with hybridoma culture medium.

FIG. 2

FIG. 2

Detection of antigenic activity of L. johnsonii La1 Triton X-100 extract by chromatography on Superose HR 6. Molecular mass markers: 2,000 kDa, fraction 20; 750 kDa, fraction 30; 150 kDa, fraction 38; 67 kDa, fraction 43. OD 450, optical density at 450 nm.

FIG. 3

FIG. 3

Detection of antigenic activity of the aqueous fraction of delipidated L. johnsonii La1 after phenol extraction and nucleic acid digestion (A) or La1 culture supernatant (B) by chromatography on Superose HR 6. OD, optical density (numbers with OD are nanometers).

FIG. 4

FIG. 4

Influence of pH on the adhesion of L. johnsonii La1 and L. acidophilus La10 to Caco-2 cells. Results are the mean ± standard deviations for five experiments done in triplicate. The asterisk indicates a P value of <0.05 when data were compared to pH 7 data for each strain by a t test. For La10, standard deviations were always <0.0437.

FIG. 5

FIG. 5

Inhibition of La1 adhesion to Caco-2 cells at pH 5. Caco-2 cells were preincubated with increasing amounts of purified LTA (A) or La1 culture supernatant (B). Controls were acetate buffer (pH 5) (A) or MRS broth acidified with lactic acid (B). Each assay was done in triplicate. Error bars are standard deviations.

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