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

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.

SUBMITTER: Guglielmetti S

PROVIDER: S-EPMC2519326 | biostudies-literature | 2008 Aug

REPOSITORIES: biostudies-literature

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

Guglielmetti Simone S Tamagnini Isabella I Mora Diego D Minuzzo Mario M Scarafoni Alessio A Arioli Stefania S Hellman Jukka J Karp Matti M Parini Carlo C

Applied and environmental microbiology 20080606 15

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 ...[more]

PMID: 18539800

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Project description:Bifidobacterium is a natural inhabitant of the human gastrointestinal (GI) tract. We studied the role of the extracellular sialidase (SiaBb2, 835 amino acids [aa]) from Bifidobacterium bifidum ATCC 15696 in mucosal surface adhesion and carbohydrate catabolism. Human milk oligosaccharides (HMOs) or porcine mucin oligosaccharides as the sole carbon source enhanced B. bifidum growth. This was impaired in a B. bifidum ATCC 15696 strain harboring a mutation in the siabb2 gene. Mutant cells in early to late exponential growth phase also showed decreased adhesion to human epithelial cells and porcine mucin relative to the wild-type strain. These results indicate that SiaBb2 removes sialic acid from HMOs and mucin for metabolic purposes and may promote bifidobacterial adhesion to the mucosal surface. To further characterize SiaBb2-mediated bacterial adhesion, we examined the binding of His-tagged recombinant SiaBb2 peptide to colonic mucins and found that His-SiaBb2 as well as a conserved sialidase domain peptide (aa 187 to 553, His-Sia) bound to porcine mucin and murine colonic sections. A glycoarray assay revealed that His-Sia bound to the α2,6-linked but not to the α2,3-linked sialic acid on sialyloligosaccharide and blood type A antigen [GalNAcα1-3(Fucα1-2)Galβ] at the nonreducing termini of sugar chains. These results suggest that the sialidase domain of SiaBb2 is responsible for this interaction and that the protein recognizes two distinct carbohydrate structures. Thus, SiaBb2 may be involved in Bifidobacterium-mucosal surface interactions as well as in the assimilation of a variety of sialylated carbohydrates.IMPORTANCE Adhesion to the host mucosal surface and carbohydrate assimilation are important for bifidobacterium colonization and survival in the host gastrointestinal tract. In this study, we investigated the mechanistic basis for B. bifidum extracellular sialidase (SiaBb2)-mediated adhesion. SiaBb2 cleaved sialyl-human milk oligosaccharides and mucin glycans to produce oligosaccharides that supported B. bifidum growth. Moreover, SiaBb2 enhanced B. bifidum adhesion to mucosal surfaces via specific interactions with the α2,6 linkage of sialyloligosaccharide and blood type A antigen on mucin carbohydrates. These findings provide insight into the bifunctional role of SiaBb2 and the adhesion properties of B. bifidum strains.

Project description:BackgroundBifidobacteria belong to one of the predominant bacterial groups in the intestinal microbiota of infants and adults. Several beneficial effects on the health status of their human hosts have been demonstrated making bifidobacteria interesting candidates for probiotic applications. Adhesion of probiotics to the intestinal epithelium is discussed as a prerequisite for colonisation of and persistence in the gastrointestinal tract.ResultsIn the present study, 15 different strains of bifidobacteria were tested for adhesion. B. bifidum was identified as the species showing highest adhesion to all tested intestinal epithelial cell (IEC) lines. Adhesion of B. bifidum S17 to IECs was strongly reduced after treatment of bacteria with pronase. These results strongly indicate that a proteinaceous cell surface component mediates adhesion of B. bifidum S17 to IECs. In silico analysis of the currently accessible Bifidobacterium genomes identified bopA encoding a lipoprotein as a B. bifidum-specific gene previously shown to function as an adhesin of B. bifidum MIMBb75. The in silico results were confirmed by Southern Blot analysis. Furthermore, Northern Blot analysis demonstrated that bopA is expressed in all B. bifidum strains tested under conditions used to cultivate bacteria for adhesion assays. The BopA gene was successfully expressed in E. coli and purified by Ni-NTA affinity chromatography as a C-terminal His6-fusion. Purified BopA had an inhibitory effect on adhesion of B. bifidum S17 to IECs. Moreover, bopA was successfully expressed in B. bifidum S17 and B. longum/infantis E18. Strains overexpressing bopA showed enhanced adhesion to IECs, clearly demonstrating a role of BopA in adhesion of B. bifidum strains.ConclusionsBopA was identified as a B. bifidum-specific protein involved in adhesion to IECs. Bifidobacterium strains expressing bopA show enhanced adhesion. Our results represent the first report on recombinant bifidobacteria with improved adhesive properties.

Project description:Over the past decade, a variety of lactic acid bacteria have been commercially available to and steadily used by consumers. However, recent studies have shown that some lactic acid bacteria produce toxic substances and display properties of virulence. To establish safety guidelines for lactic acid bacteria, the Food and Agriculture Organization of the United Nations (FAO)/World Health Organization (WHO) has suggested that lactic acid bacteria be characterized and proven safe for consumers’ health via multiple experiments (e.g., antibiotic resistance, metabolic activity, toxin production, hemolytic activity, infectivity in immune-compromised animal species, human side effects, and adverse-outcome analyses). Among the lactic acid bacteria, Bifidobacterium and Lactobacillus species are probiotic strains that are most commonly commercially produced and actively studied. Bifidobacterium bifidum BGN4 and Bifidobacterium longum BORI have been used in global functional food markets (e.g., China, Germany, Jordan, Korea, Lithuania, New Zealand, Poland, Singapore, Thailand, Turkey, and Vietnam) as nutraceutical ingredients for decades, without any adverse events. However, given that the safety of some newly screened probiotic species has recently been debated, it is crucial that the consumer safety of each commercially utilized strain be confirmed. Accordingly, this paper details a safety assessment of B. bifidum BGN4 and B. longum BORI via the assessment of ammonia production, hemolysis of blood cells, biogenic amine production, antimicrobial susceptibility pattern, antibiotic resistance gene transferability, PCR data on antibiotic resistance genes, mucin degradation, genome stability, and possession of virulence factors. These probiotic strains showed neither hemolytic activity nor mucin degradation activity, and they did not produce ammonia or biogenic amines (i.e., cadaverine, histamine or tyramine). B. bifidum BGN4 and B. longum BORI produced a small amount of putrescine, commonly found in living cells, at levels similar to or lower than that found in other foods (e.g., spinach, ketchup, green pea, sauerkraut, and sausage). B. bifidum BGN4 showed higher resistance to gentamicin than the European Food Safety Authority (EFSA) cut-off. However, this paper shows the gentamicin resistance of B. bifidum BGN4 was not transferred via conjugation with L. acidophilus ATCC 4356, the latter of which is highly susceptible to gentamicin. The entire genomic sequence of B. bifidum BGN4 has been published in GenBank (accession no.: CP001361.1), documenting the lack of retention of plasmids capable of transferring an antibiotic-resistant gene. Moreover, there was little genetic mutation between the first and 25th generations of B. bifidum BGN4. Tetracycline-resistant genes are prevalent among B. longum strains; B. longum BORI has a tet(W) gene on its chromosome DNA and has also shown resistance to tetracycline. However, this research shows that its tetracycline resistance was not transferred via conjugation with L. fermentum AGBG1, the latter of which is highly sensitive to tetracycline. These findings support the continuous use of B. bifidum BGN4 and B. longum BORI as probiotics, both of which have been reported as safe by several clinical studies, and have been used in food supplements for many years.

Project description:BackgroundBifidobacterium longum 105-A produces markedly high amounts of capsular polysaccharides (CPS) and exopolysaccharides (EPS) that should play distinct roles in bacterial-host interactions. To identify the biological function of B. longum 105-A CPS/EPS, we carried out an informatics survey of the genome and identified the EPS-encoding genetic locus of B. longum 105-A that is responsible for the production of CPS/EPS. The role of CPS/EPS in the adaptation to gut tract environment and bacteria-gut cell interactions was investigated using the ΔcpsD mutant.ResultsA putative B. longum 105-A CPS/EPS gene cluster was shown to consist of 24 putative genes encoding a priming glycosyltransferase (cpsD), 7 glycosyltransferases, 4 CPS/EPS synthesis machinery proteins, and 3 dTDP-L-rhamnose synthesis enzymes. These enzymes should form a complex system that is involved in the biogenesis of CPS and/or EPS. To confirm this, we constructed a knockout mutant (ΔcpsD) by a double cross-over homologous recombination. Compared to wild-type, the ∆cpsD mutant showed a similar growth rate. However, it showed quicker sedimentation and formation of cell clusters in liquid culture. EPS was secreted by the ∆cpsD mutant, but had altered monosaccharide composition and molecular weight. Comparison of the morphology of B. longum 105-A wild-type and ∆cpsD by negative staining in light and electron microscopy revealed that the formation of fimbriae is drastically enhanced in the ∆cpsD mutant while the B. longum 105-A wild-type was coated by a thick capsule. The fimbriae expression in the ∆cpsD was closely associated with the disappearance of the CPS layer. The wild-type showed low pH tolerance, adaptation, and bile salt tolerance, but the ∆cpsD mutant had lost this survivability in gastric and duodenal environments. The ∆cpsD mutant was extensively able to bind to the human colon carcinoma Caco-2 cell line and was phagocytosed by murine macrophage RAW 264.7, whereas the wild-type did not bind to epithelial cells and totally resisted internalization by macrophages.ConclusionsOur results suggest that CPS/EPS production and fimbriae formation are negatively correlated and play key roles in the survival, attachment, and colonization of B. longum 105-A in the gut.

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

Publications

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

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