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Adhesion properties of a putative polymorphic fimbrial subunit protein from Bifidobacterium longum subsp. longum.

ABSTRACT: In our previous study, we found that the open reading frame bl0675 in the genome of Bifidobacterium longum subsp. longum isolated from human feces encoded a novel putative fimbrial protein, was highly polymorphic, and had five variants (A, B, C, D, and E types). The aim of this study was to evaluate the affinity of these variants to porcine colonic mucins (PCMs). Protein-binding properties were examined using the recombinant BL0675 protein containing a C-terminal 6 × His tag (His-BL0675). Surface plasmon resonance analysis demonstrated that the His-BL0675 A type had strong affinity to PCMs (KD = 9.82 × 10(-8) M), whereas the B, C, D, and E types exhibited little or no binding. In a competitive enzyme-linked immunosorbent assay, His-BL0675 A type binding was reduced by addition of mucin oligosaccharides, suggesting that the binding occurs via carbohydrate chains of PCMs. The localization of BL0675 to the B. longum subsp. longum cell surface was confirmed by western blot analysis using A type polyclonal antibodies. Bacterial adhesion of B. longum subsp. longum to PCMs was also blocked by A type-specific antibodies; however, its adhesion properties were strain specific. Our results suggest that the BL0675 variants significantly contribute to the adhesion of B. longum subsp. longum strains. The expression and the adhesive properties of this protein are affected by genetic polymorphisms and are specific for B. longum subsp. longum strains. However, further studies are required on the properties of binding of these putative fimbrial proteins to the human gastrointestinal tract.

SUBMITTER: Suzuki K

PROVIDER: S-EPMC4735030 | biostudies-literature | 2016

REPOSITORIES: biostudies-literature

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Project description:Adhesion to the gastrointestinal tract is considered to be important for bifidobacteria to colonize the human gut and exert their probiotic effects. Some cell surface proteins of bifidobacteria, known as adhesins, play critical roles in the binding to host cells or the extracellular matrix (ECM). To elucidate the mechanisms associated with the adhesion of Bifidobacterium longum BBMN68, a centenarian originated potential probiotic, PSORTdb was employed to identify putative extracellular localized proteins in the B. longum BBMN68. Of the 560 predicted extracellular proteins, 21 were further identified as putative adhesion proteins using the conserved domain database of NCBI, and four were successfully overexpressed in the heterologous host, Lactococcus lactis NZ9000. Notably, a recombinant strain expressing FimM showed a significantly increased adhesive affinity for both HT-29 and mucus-secreting LS174T goblet cells (2.2- and 5.4-fold higher than that of the control strain, respectively). Amino acid sequence alignment showed that FimM is a major pilin subunit protein containing a Cna-B type domain and a C-terminal LPKTG sequence. However, in silico analysis of the fimM-coding cluster revealed that BBMN68_RS10200, encoding a pilus-specific class C sortase, was a pseudogene, indicating that FimM may function as a surface adhesin that cannot polymerize into a pili-like structure. Immunogold electron microscopy results further confirmed that FimM localized to the surface of L. lactis NZfimM and B. longum BBMN68 but did not assemble into pilus filaments. Moreover, the adhesive affinity of L. lactis NZfimM to fibronectin, fibrinogen, and mucin were 3.8-, 2.1-, and 3.1-fold higher than that of the control. The affinity of FimM for its attachment receptors was further verified through an inhibition assay using anti-FimM antibodies. In addition, homologs of FimM were found in Bifidobacterium bifidum 85B, Bifidobacterium gallinarum CACC 514, and 23 other B. longum strains by sequence similarity analysis using BLASTP. Our results suggested that FimM is a novel surface adhesin that is mainly present in B. longum strains.

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Adhesion properties of a putative polymorphic fimbrial subunit protein from Bifidobacterium longum subsp. longum.

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