Abstract
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.
Highlights
Bifidobacteria are common inhabitants of the human gastrointestinal tract (GIT), constituting approximately 60–90% of the total gut microbiome in early life (Odamaki et al, 2016)
Because adhesion to the host is important for bifidobacteria to exert their health-promoting effects, in this study, we investigated the mechanisms that mediate the adhesion of B. longum BBMN68 to epithelial cells
Using PSORTdb, a subcellular localization database for bacteria and archaea, 560 extracellular proteins were predicted to be present in the B. longum BBMN68 genome (Peabody et al, 2016)
Summary
Bifidobacteria are common inhabitants of the human gastrointestinal tract (GIT), constituting approximately 60–90% of the total gut microbiome in early life (Odamaki et al, 2016). In Bifidobacterium bifidum PRL2010, two sortase-dependent pili bind to Caco-2 cells and extracellular matrix (ECM) proteins such as fibronectin, plasminogen, and laminin (Turroni et al, 2013); the moonlighting proteins EF-Tu and enolase serve as surface adhesins for the binding of Bifidobacterium longum NCC2705 to human plasminogen and Caco-2 cells (Wei et al, 2014); BL0155, a large extracellular transmembrane protein isolated from B. longum VMKB44, is important for its binding to HT-29 epithelial cells in vitro (Shkoporov et al, 2008); B. bifidum ATCC 15696 employs a sialidase to mediate its adhesion to mucus (Nishiyama et al, 2017); in B. longum JCM1217, endo-α-N-acetylgalactosaminidase was reported to bind mucin (Suzuki et al, 2009); transaldolase was reported to act as a surface mucin-binding protein in several B. bifidum strains (Gonzalez-Rodriguez et al, 2012); and, in Bifidobacterium animalis, enolase, the chaperone protein DnaK, and the heat-shock protein GroEL were shown to bind plasminogen or Caco-2 cells (Candela et al, 2009, 2010; Sun et al, 2016). A novel pilin subunit protein – BBMN68_RS02235, designated FimM – was identified as a putative surface adhesion protein that mediates the adhesion of B. longum BBMN68 to mucin, fibronectin, and fibrinogen
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