Up-regulation of sortase-dependent pili in Bifidobacterium longum BBMN68 in response to bile stress enhances its adhesion to HT-29 cells.

Adhesion to gastrointestinal tract is crucial for bifidobacteria to exert their probiotic effects. Our previous work found that bile salts significantly enhance the adhesion ability of Bifidobacterium longum BBMN68 to HT-29 cells. In this study, trypsin-shaving and LC-MS/MS-based surface proteomics were employed to identify surface proteins involved in bile stress response. Among the 829 differentially expressed proteins, 56 up-regulated proteins with a fold change >1.5 were subjected to further analysis. Notably, the minor pilin subunit FimB was 4.98-fold up-regulated in response to bile stress. In silico analysis and RT-PCR confirmed that gene fimB, fimA and srtC were co-transcribed and contributed to the biosynthesis of sortase-dependent pili Pil1. Moreover, scanning electron microscopy and immunogold electron microscopy assays showed increased abundance and length of Pil1 on BBMN68 under bile stress. As the major pilin subunit FimA serves as adhesion component of Pil1, an inhibition assay using anti-FimA antibodies further confirmed the critical role of Pil1 in mediating the adhesion of BBMN68 to HT-29 cells under bile stress. Our findings suggest that the up-regulation of Pil1 in response to bile stress enhances the adhesion of BBMN68 to intestinal epithelial cells, highlighting a novel mechanism of gut persistence in B. longum strains.

A Novel Major Pilin Subunit Protein FimM Is Involved in Adhesion of Bifidobacterium longum BBMN68 to Intestinal Epithelial Cells.

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.