Plasmid Mini Kit We (Omega Bio-tek Inc., Doraville, GA, United States). NZfimM and BBMN68 but did not assemble into pilus filaments. Moreover, the adhesive affinity of 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 85B, CACC 514, and 23 additional strains by sequence similarity analysis using BLASTP. Our results suggested that FimM is definitely a novel surface adhesin that is mainly present in strains. 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 NCC2705 to human being plasminogen and Caco-2 cells (Wei et al., 2014); BL0155, a large extracellular transmembrane protein isolated from VMKB44, is definitely important for its binding to HT-29 epithelial cells (Shkoporov et al., 2008); ATCC 15696 utilizes a sialidase to mediate its adhesion to mucus (Nishiyama et al., 2017); in JCM1217, endo-strains (Gonzalez-Rodriguez et al., 2012); and, in BBMN68 was isolated from a healthy centenarian in Bama Region of Guangxi province, China, which is DIAPH2 known for having a high life expectancy. BBMN68 has been reported to exert several potential probiotic functions, such as enhancing both innate and acquired immunity, alleviating allergic reactions, and improving intestinal function (Yang et al., 2009, 2015). Because adhesion to the sponsor is important for bifidobacteria to exert their health-promoting effects, in this study, we Vc-seco-DUBA investigated the mechanisms that mediate the adhesion of BBMN68 to epithelial cells. For this, Vc-seco-DUBA 9 of 21 expected BBMN68 surface adhesion proteins were expressed inside a heterologous sponsor, NZ9000. A novel pilin subunit protein C BBMN68_RS02235, designated FimM C was identified as a putative surface adhesion protein that mediates the adhesion of BBMN68 to mucin, fibronectin, and fibrinogen. Materials and Methods Bacterial Strains and Growth Conditions The bacterial strains and plasmids used in this study are outlined in Supplementary Table S1. BBMN68 cells (GenBank accession no. “type”:”entrez-nucleotide”,”attrs”:”text”:”NC_014656.1″,”term_id”:”312132275″NC_014656.1) were grown anaerobically at 37C in de Man-Rogosa-Sharpe (MRS) broth supplemented with 1% (NZ9000 was grown at 30C in M17 medium (Oxoid, Unipath, Basingstoke, United Kingdom) containing 0.5% (strains were grown aerobically at 37C in Luria-Bertani (LB) medium with shaking (220 rpm). When required, media were supplemented with the relevant antibiotics at the following concentrations: 100 g ml?1 ampicillin and 10 g ml?1 chloramphenicol for NZ9000 Genomic DNA was extracted from BBMN68 using a TIANamp Bacteria DNA Kit according to the manufacturers instructions (TianGen, Beijing, China). Nine genes encoding expected surface adhesion proteins were amplified from genomic DNA using the primer pairs outlined in Supplementary Table S2. Vc-seco-DUBA The PCR products digested with NZ9000 by electroporation using Bio-Rad Gene Pulser Xcell (Bio-Rad, Richmond, CA, United States) as previously explained (DeRuyter et al., 1996). The producing recombinant plasmids were isolated using the E.Z.N.A. Plasmid Mini Kit I (Omega Bio-tek Inc., Doraville, GA, United States). The recombinant strains were confirmed by plasmid sequencing and further analyzed with the DNAMAN software package. In the mean time, NZCK harboring the bare pNZ81481 vector was used as the control strain. Overnight cultures of the recombinant strains Vc-seco-DUBA were inoculated (1% inocula) into 10 ml of new GM17 medium comprising 5 g ml?1 chloramphenicol. To induce gene manifestation, when the cell denseness experienced reached an OD600 nm of 0.2~0.3, Vc-seco-DUBA the ethnicities were supplemented with 10 ng ml?1 nisin (Sigma-Aldrich, Milwaukee, WI, United States) and incubated for an additional 2 h at 30C. The cell pellets were then collected after centrifugation at 6,000 for 5 min at 4C for subsequent SDSCpolyacrylamide gel electrophoresis (SDSCPAGE) analysis and adhesion assays. Bacterial Adhesion to HT-29 and LS174T Cells The human being colon adenocarcinoma cell collection HT-29 and the goblet cell-derived cell collection LS174T were from the China Infrastructure of Cell Collection Source. The cells were cultured at 37C inside a humidified atmosphere with 5% CO2. The HT-29 cells were cultivated in Dulbeccos high-glucose revised Eagles medium (DMEM, Thermo Fisher Scientific, Rockford, IL, United States) supplemented with 10% (strains were washed twice with PBS and resuspended in DMEM or.
Plasmid Mini Kit We (Omega Bio-tek Inc
