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. 2008 Aug;74(15):4695-702.
doi: 10.1128/AEM.00124-08. Epub 2008 Jun 6.

Implication of an outer surface lipoprotein in adhesion of Bifidobacterium bifidum to Caco-2 cells

Simone Guglielmetti  1 , Isabella Tamagnini, Diego Mora, Mario Minuzzo, Alessio Scarafoni, Stefania Arioli, Jukka Hellman, Matti Karp, Carlo Parini
Affiliations

Implication of an outer surface lipoprotein in adhesion of Bifidobacterium bifidum to Caco-2 cells

Simone Guglielmetti et al. Appl Environ Microbiol. 2008 Aug.

Abstract

We found that the human intestinal isolate Bifidobacterium bifidum MIMBb75 strongly adhered to Caco-2 cells. Proteinase K and lithium chloride treatments showed that proteins play a key role in MIMBb75 adhesion to Caco-2 cells. By studying the cell wall-associated proteins, we identified a surface protein, which we labeled BopA. We purified the protein chromatographically and found that it functioned as an adhesion promoter on Caco-2 cells. In silico analysis of the gene coding for this protein and globomycin experiments showed that BopA is a cysteine-anchored lipoprotein expressed as a precursor polypeptide. A database search indicated that BopA appears to function biologically as an oligopeptide/tripeptide-solute-binding protein in the ABC transport system. We discovered a protein corresponding to BopA and its gene in eight other highly adherent B. bifidum strains. Finally, we found that B. bifidum MIMBb75 and BopA affected the production of interleukin-8 in Caco-2 epithelial cells. BopA is the first protein described to date to be directly involved in the adhesion of bifidobacteria to Caco-2 cells and to show immunomodulatory activity.

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Figures

FIG. 1.
FIG. 1.
Adherence of bacterial strains to a Caco-2 cell monolayer. (a) Adhesion of Bifidobacterium bifidum MIMBb75 (a and c) and Bifidobacterium pseudocatenulatum NAP32 (b) to a Caco-2 cell monolayer as observed with Giemsa staining under a light microscope. MIMBb75 adhesion was specific to Caco-2 cells: no adhesion was detected on the cover glass underlying Caco-2 cells (c, left). Bars, 5 μm in a and b and 10 μm in c. (b) Quantification of adhesion ability. The numbers above each column refer to the adhesion index (bacterial cells adhered to 100 Caco-2 cells). The data represent the means of at least two independent experiments conducted in duplicate. The vertical bars indicate standard deviations. Bifidobacterium lactis stands for Bifidobacterium animalis subsp. lactis.
FIG. 2.
FIG. 2.
Role of proteins in B. bifidum MIMBb75 adhesion to Caco-2 cells. (a) Percent changes in adhesion after incubation of MIMBb75 cells in a proteinase K (Prot. K) solution and after extraction of surface proteins with 5 M LiCl. *, statistically significant difference compared to untreated cells (P < 0.05). (b) SDS-PAGE analysis of MIMBb75 cell wall-associated proteins grown under standard conditions (lane A) and after treatment with 5 M LiCl (lane B). The molecular masses (in kDa) of the standard proteins are indicated at right.
FIG. 3.
FIG. 3.
(A) SDS-PAGE analysis of B. bifidum MIMBb75 cell wall-associated proteins grown under standard conditions. (B and C) Purified BopA (B) and cell wall proteins after growth in 50 μg ml−1 globomycin (C) are also shown. The molecular masses (in kDa) of the standard proteins are indicated at left.
FIG. 4.
FIG. 4.
Percent changes in adhesion to Caco-2 cells of B. bifidum MIMBb75 cells in competition with BSA or BopA proteins and after growth of bacterial cells in 50 μg ml−1 globomycin (Gbm). The data represent the means of at least two independent experiments conducted in duplicate. The vertical bars indicate standard deviations. *, statistically significant difference compared to an adhesion assay carried out under standard conditions (P < 0.05).
FIG. 5.
FIG. 5.
(A) Schematic representation of the genome region including bopA in Bifidobacterium bifidum MIMBb75. The number in parentheses after the putative promoter refers to the Neural Network Promoter Prediction probability. The white open arrows refer to the target of the primers indicated below. (B) Putative domain architecture of BopA from B. bifidum MIMBb75 shown with the first 30 amino acids of BopA and containing the signal peptide and the lipobox (underlined). The cleavage site for thioacylation, between Ala-25 and Cys-26, is indicated with a white triangle. The number in parentheses refers to the SignalP-HMM probability of the signal peptide. TM, transmembrane region. A BLAST search was used to assign putative biological roles to the genes or domains.
FIG. 6.
FIG. 6.
Production of interleukins by Caco-2 cells induced by various treatments with probiotic bacteria (overnight treatment), proteins (overnight treatment), or Listeria monocytogenes (1-h treatment). (a) IL-6 production. (b) IL-8 production. Numerical results are given in the arithmetic means ± standard deviations. B. lactis stands for Bifidobacterium animalis subsp. lactis. *, statistically significant difference compared to untreated Caco-2 cells (P < 0.05).
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