Implication of an Outer Surface Lipoprotein in Adhesion of Bifidobacterium bifidum to Caco-2 Cells

Bacterial strains and culture conditions.Table 1 lists the bacterial strains used in this study. Bifidobacterium strains were grown overnight anaerobically at 37°C in MRS broth (Difco, Detroit, MI) supplemented with 0.05% l-cysteine hydrochloride (cMRS). Lactobacillus rhamnosus and Lactobacillus helveticus were cultivated overnight in MRS broth at 37°C and 42°C, respectively. Streptococcus thermophilus was cultivated overnight in M17 medium (Difco) at 37°C. Listeria monocytogenes ATCC 15313 was grown in brain hearth infusion broth (Difco) at 37°C under agitation.

Bacterial adhesion to Caco-2 cells.Caco-2 cells were routinely grown in 3-cm petri plates on microscopy cover glasses in Dulbecco's modified Eagle's medium supplemented with 10% (vol/vol) heat-inactivated (30 min at 56°C) fetal calf serum, 100 U ml⁻¹ penicillin, 100 mg ml⁻¹ streptomycin, 0.1 mM nonessential amino acids, and 2 mM l-glutamine and incubated at 37°C in a water-jacketed incubator in an atmosphere of 95% air and 5% carbon dioxide. The culture medium was changed twice weekly. For adhesion assays, cells were used 15 days after confluence (fully differentiated cells). Cell monolayers were carefully washed twice with phosphate-buffered saline (PBS) (pH 7.3) before bacterial cells were added. The bacterial cell concentration of a culture grown overnight was determined microscopically after DAPI (4′,6′-diamidino-2-phenylindole) staining. Approximately 2 × 10⁸ cells of each strain resuspended in PBS (pH 7.3) were incubated with a monolayer of fully differentiated Caco-2 cells. After 1 h at 37°C in anaerobic conditions, all monolayers were washed three times with PBS to release unbound bacteria. Cells were then fixed with 3 ml of methanol and incubated for 8 min at room temperature. After methanol was removed, cells were stained with 3 ml of Giemsa stain solution (1:20) (Carlo Erba, Milan, Italy) and left for 30 min at room temperature. Wells were then washed until no color was observed in the washing solution and dried in an incubator for 1 h. Microscopy cover glasses were then removed from the petri plate and examined microscopically (magnification, ×100), immersed in oil. Adherent bacteria in 20 randomly selected microscopic fields were counted and averaged. An unpaired Student t test was run for statistically significant differences.

Preparation of bacterial cell wall extract.Bacterial cells from 0.2 liters of liquid culture were harvested by centrifugation and processed according to methods described previously by Mattarelli et al. (36), with a modified use of the French press (12,000 lb/in²) for breaking cells.

SDS-PAGE and N-terminal sequence analysis.Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) was performed as described previously by Laemmli (32), using a 4% stacking gel and a 10% resolving gel. For BopA sequence analysis, proteins were electrotransferred onto a polyvinylidene difluoride membrane (ProBlott; Applied Biosystems, Foster City, CA). The membrane was first stained with Coomassie blue, and the band corresponding to BopA was then excised. The sample was desalted using a ProSpin cartridge (Applied Biosystems) and placed onto a Polybrene-coated and precycled glass fiber filter, followed by sequencing with an Applied Biosystems model 477A protein sequencer equipped with an online model 120A PTH amino acid analyzer. For internal sequencing, the sample was subjected to digestion with trypsin (sequencing grade; Promega, Milan, Italy) as described previously by Fernandez et al. (16). The peptides were separated with a high-performance liquid chromatograph equipped with a Vydac C₁₈ column (2.1 mm by 150 mm). One of the tryptic peptides was applied onto glass fiber filters and sequenced as described above.

Partial purification of BopA.An equal volume of 5 M LiCl was added to the bacterial cell wall preparations, and after extensive vortexing, the suspensions were incubated for 2 h at 37°C. After centrifugation, the supernatant was recovered, and the buffer was exchanged to 50 mM Tris-HCl buffer (pH 8.0) using an Amicon Ultra (Millipore, Billerica, MA) centrifugal filter device (5,000-molecular-weight membrane cutoff). Aliquots of 200 μl of the resulting solution were applied to a MonoQ 5/50 GL column (0.46 cm by 25 cm) (GE Healthcare, Milan, Italy) equilibrated in 50 mM Tris-HCl buffer (pH 8.0) using an high-performance liquid chromatography apparatus (Water) at a flow rate of 1 ml min⁻¹. The unbound fraction containing the BopA protein was collected and concentrated 15 times by ultrafiltration using the same device and conditions described above. The protein fraction retained by the chromatographic column was eluted with the equilibration buffer containing 1 M NaCl and then discarded. Protein purity was determined by SDS-PAGE analysis.

Competitive adhesion assay.The effect of BopA on bacterial adhesion was examined by incubating a Caco-2 cell monolayer with purified BopA (37.5 and 375 mg ml⁻¹ in PBS as final concentrations) at 37°C for 1 h; 1 mg ml⁻¹ bovine serum albumin (BSA) was used as a control. Subsequently, a bacterial adhesion assay was run as described above. A Student t test was run for statistically significant differences.

Stimulation of Caco-2 monolayers and enzyme-linked immunosorbent assay measurement of cytokine production.For all assays, a known number of Caco-2 cells were seeded into 24-well plates and grown as described above. Probiotic bacteria (final concentration of 10⁸ bacterial cells ml⁻¹) and purified BopA protein (final concentrations of 0.1, 10, and 20 μg ml⁻¹) were added to monolayers of Caco-2 cells in 0.5 ml of fresh antibiotic-free Dulbecco's modified Eagle's medium and incubated overnight at 37°C in 5% CO₂. Cytokine IL-1β (1 ng ml⁻¹), casein (10 μg ml⁻¹), and BSA (10 μg ml⁻¹) were used as controls. To test the effect of Listeria monocytogenes, Caco-2 cells were incubated with 10⁸ bacterial cells ml⁻¹ at 37°C for 1 h. After that, microtiter plates were kept at −70°C for 4 h to completely disrupt the Caco-2 cells. The supernatant and disrupted Caco-2 cells were then collected, and phenylmethylsulfonyl fluoride was added (20 mM final concentration). After centrifugation at a relative centrifugal force of 14,000 for 1 min, levels of IL-6 and IL-8 in the supernatants were determined by using RayBio Human IL enzyme-linked immunosorbent assay kits (RayBiotech, Inc., Norcross, GA) as instructed by the manufacturer. Each sample was processed in duplicate in two independent experiments.

PCR amplification of the DNA region coding for BopA.Each 25-μl reaction mixture contained 200 μM of deoxynucleoside triphosphate, 2.5 μl of 10× reaction buffer (Fermentas, Vilnius, Lithuania), 2.5 mM of MgCl₂, each primer at a concentration of 0.5 μM, 0.5 U of Taq polymerase (Fermentas), and 1 μl of bacterial DNA solution (containing about 100 ng of DNA). The initial denaturation step at 95°C for 2 min was followed by 35 cycles of denaturation at 94°C for 45 s and annealing at 54°C to 62°C for 45 s, depending on the primer, with an extension step at 72°C for 1 min. The final cycle was followed by an additional 7-min elongation period at 72°C. The primers employed were as follows: InvXf1 (5′-GTGTGTACACGCTGACCA-3′), InvXrS (5′-TCTTCTCGGTCCATTCGAT-3′), InvXr2 (5′-GGAAGGTGGAGAACAGCT-3′), InvXfD (5′-CCTCGACGTGTCGATTCA-3′), and InvXr1 (5′-GCCACCCTGCTTCAGCT-3′).

Sequence determination of the DNA region coding for BopA.Starting from the N-terminal amino acid sequence of the whole BopA and its trypsin fragment, we designed a pair of degenerated primers according the codon use of Bifidobacterium longum NCC2705. The primers used were BIFOPf (5′-GGCAACTCCAACATIGGCWSSGCSGGCAA-3′) and BIFOPr (5′-CTTYTCGTAGTAGCCGTCSSWGTT-3′). PCR experiments run with BIFOP primers yielded a 1,191-bp fragment that was cloned into the pGEM-T vector (Promega) and sequenced. Subsequent self-ligation and inverse PCR experiments were run as follows. For self-ligation experiments, 5 μg of chromosomal DNA was digested overnight with 30 U of BamHI or SalI restriction enzyme (Fermentas). Digested DNA was purified by phenol-chloroform extraction and used in an overnight ligation reaction at 15°C in a volume of 0.5 ml with 5 U of T4 DNA ligase (Fermentas). Different amounts of self-ligated DNA were used as a template in inverse PCRs with DyNAzyme Ext DNA polymerase (Finnzymes, Helsinki, Finland).

Sequence analysis.We used the Basic Local Alignment Search Tool (BLAST) for searching for similarities against the GenBank and EMBL sequence databases and predicted the promoter by using Neural Network Promoter Prediction (http://www.fruitfly.org/seq_tools/promoter.html ). A putative signal peptide sequence was found with SignalP software (http://www.cbs.dtu.dk/services/SignalP-2.0/ ). We also used DOLOP database software (http://www.mrc-lmb.cam.ac.uk/genomes/dolop/analysis.shtml ) to predict bacterial lipoproteins.

Nucleotide sequence accession number.The sequence data for the 3,419-bp DNA fragment from Bifidobacterium bifidum MIMBb75 have been deposited in the EMBL database under accession number AM710395.

Bifidobacterium bifidum MIMBb75 adheres to the Caco-2 human colonic cell line.The human fecal isolate Bifidobacterium bifidum MIMBb75 was tested and found to be well adherent to Caco-2 human colonic cells (Fig. 1a). After extensive washing with PBS, a significant proportion of cells of this bacterial strain remained attached to the Caco-2 monolayer, providing evidence that the adhesion was not only nonspecific physical entrapment. In particular, about 55 bacterial cells adhered to a 1-mm² epithelial cell monolayer, resulting in an adhesion index (bacterial cells/100 Caco-2 cells) of 3,874 (Fig. 1b), which is statistically the highest among the strains tested (P < 0.026). Also, Bifidobacterium longum NCC2705, Lactobacillus helveticus MIMLh22, and Lactobacillus rhamnosus GG adhered well to Caco-2 cells, resulting in adhesions of about 25, 11, and 9 bacterial cells per mm², respectively (adhesion indexes of 2,327, 629, and 648, respectively). The remaining strains, Bifidobacterium adolescentis NAA38, Bifidobacterium longum NAL8, Bifidobacterium pseudocatenulatum NAP32, and Streptococcus thermophilus DSM20617^T, adhered to Caco-2 cell surfaces with less than 2 cells per mm² (Fig. 1b).

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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.

Proteins are involved in the adhesion of B. bifidum MIMBb75 to Caco-2 cells.We tested adhesion after incubating B. bifidum MIMBb75 with proteinase K (0.5 mg ml⁻¹) for 30 min at 37°C. The bacterial strain was now dramatically less adherent to Caco-2 cells (83% less adherent), suggesting that surface proteinaceous compounds may promote the strain's binding to epithelial cells (Fig. 2a).

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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.

The cell wall-associated proteins of B. bifidum MIMBb75 were extracted and separated through SDS-PAGE. Their electrophoretic profiles revealed the presence of a predominant protein with an apparent molecular mass of about 60 kDa (Fig. 2b, lane A). When the cell surface proteins were removed by extraction with 5 M LiCl, the band corresponding to the dominant protein appeared drastically reduced by SDS-PAGE (Fig. 2b, lane B), confirming the protein's outside location. At the same time, treatment with LiCl significantly reduced the adhesion of B. bifidum MIMBb75 (58% reduced adhesion) (Fig. 2a). According to data reported previously by Mattarelli et al. (36), we named the protein BopA (bifidobacterial outer protein).

BopA competes with MIMBb75 adhesion.BopA was chromatographically purified from bacterial cell wall extracts to apparent homogeneity as observed by SDS-PAGE (Fig. 3). Purified BopA was then used in competition experiments through a Caco-2 adhesion assay. Caco-2 cells were incubated with 375 μg of purified BopA for 1 h at 37°C in anaerobic condition. After this treatment, B. bifidum MIMBb75 was markedly less adherent (70% less adherent) (Fig. 4), suggesting that BopA may have interfered with the strain's adhesion mechanism with regard to Caco-2 cells. On the other hands, its adhesion was not significantly affected by BSA or 37.5 μg of BopA (Fig. 4).

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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.

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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).

Identification and characterization of the gene coding for BopA.We determined the N-terminal amino acids of BopA (X-G-N-S-N-N-G-S-A-G-N [X refers to an undetermined amino acid]) and a trypsin-generated BopA peptide (N-S-D-G-Y-Y-E-K). Starting from these sequences, via PCR experiments, we determined a 3,419-bp genomic region (Fig. 5a). This nucleotidic sequence contains the gene of BopA and a putative ribosomal binding site in a purine-rich region (AGGAGAGGAA) upstream of the translational start codon ATG. The deduced polypeptide sequence of BopA starts with an N-terminal signal sequence of 25 amino acids, which meets all the requirements of a transmembrane helix, and contains the lipobox motif (L-X-Z-C-Y-Z). This motif functions as a recognition signal for lipid modification by thioacylation made on the conserved and essential cysteine residue. The lipid residue covalently linked to the cysteine moiety is thought to allow for the anchoring of proteins in a plasma membrane (49).

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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.

The mature BopA has a calculated molecular mass of 62 kDa and an isoelectric point of 5.03. The molecular mass correlates well with that estimated by SDS-PAGE.

A database search with the BopA protein sequence revealed significant homology with the solute-binding protein of the ABC transport system from high-GC gram-positive bacteria (Arthrobacter, Propionibacterium, Mycobacterium, Streptomyces, Nocardia, and Brevibacterium) (Fig. 5b). The closest similarity occurred with genes encoding the core domain of the ABC-type tripeptide/oligopeptide binding protein (CDD accession numbers COG0747 and COG4166) of Arthrobacter sp. strain FB24 (GenBank accession number ABK05389) (identities in 187/544 isolates [34%], with 283/544 [52%] positive results and 43/544 [7%] gaps) and Arthrobacter aurescens TC1 (accession number ABM09237) (I = 192/602 [31%]; P = 287/602 [47%]; G = 64/602 [10%]).

Computational analysis of the region encompassing bopA revealed, upstream of this gene, the 3′ terminus of a putative gene showing close similarity with the ATP-binding protein of the oligopeptide/dipeptide ABC transporter (CDD accession number cd03257). Moreover, downstream of bopA and on the opposite reading frame, we identified the 3′ terminus of a putative gene showing close similarity with bacterial aminopeptidase C (pepC) (CDD accession number cd00585) (Fig. 5a).

Experiments with globomycin.To confirm that BopA is a lipoprotein, we grew B. bifidum MIMBb75 with globomycin, a potent and specific inhibitor of lipoprotein signal peptidases, resulting in an accumulation of prolipopetide (24, 49). When B. bifidum MIMBb75 was grown in cMRS containing 50 μg ml⁻¹ of globomycin, its growth was only slightly inhibited, and its cell morphology changed, showing unusual bulges at the extremities of many cells (data not shown). The SDS-PAGE profile of cell wall proteins after growth in globomycin revealed a radical reduction of BopA (Fig. 3). Furthermore, the bacterial cells grown in globomycin were markedly less capable of adhering to Caco-2 cells (63% lower adherence) (Fig. 4).

Adhesion and presence of BopA in Bifidobacterium bifidum strains.We ran adhesion assays on eight other strains ascribed to the species Bifidobacterium bifidum (Table 1). All the strains adhered to Caco-2 cells without a significant difference from B. bifidum MIMBb75 (data not shown). Moreover, a band corresponding to BopA appeared on the SDS-PAGE profile of all the B. bifidum strains (data not shown), a finding agreeing with our PCR experiments. Targeting bopA and the flanking genes, we used the following sets of primers: InvXf1-InvXrS, InvXf1-InvXr2, and InvXfD-InvXr1 (Fig. 5a). We found the presence of the bopA gene in all the strains (data not shown). Finally, no amplification bands were detected when the primers were used in PCR experiments with the other bifidobacterial strains not belonging to the B. bifidum species listed in Table 1.

B. bifidum MIMBb75 and BopA induce production of IL-8.Overnight incubation of a Caco-2 monolayer with probiotic bacteria resulted in significantly increased levels of IL-8 production (P < 0.05) (Fig. 6). The bifidobacterial strains Bb12 and MIMBb75 especially enhanced the production of IL-8 from 30 pg ml⁻¹ to 676 and 574 pg ml⁻¹, respectively. Lactobacillus rhamnosus GG and Bifidobacterium longum NCC2705 produced 213 and 233 pg ml⁻¹, respectively. When the purified protein BopA was used instead of whole bacterial probiotic cells, IL-8 production increased depending on the protein's concentration (Fig. 6b). In contrast, the IL-6 concentration showed no marked change. One-hour incubation of Caco-2 cells with Listeria monocytogenes was sufficient to obtain a marked increase in levels of IL-6 and IL-8 (Fig. 6). Casein and BSA, included in the experiment as controls, affected neither IL-6 nor IL-8 production.

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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).