Adhesive Mechanism Of Lactobacillus Salivarius FDB86 To Intestinal Epithelial Cells

The adhesion of lactobacillus to intestinal epithelial cells has been a research hotspot for playing an important role in colonizing the intestinal tract and health benefit to host.Lactobacillus salivarius FDB86（L.salivarius FDB86）was shown to adhere to intestinal tract,but its adhesins on the surface of cells and receptor on intestinal epithelial cells were unclear.L.salivarius FDB86 was took as research object in this study,to screen and identify the adhesins on cell surface,and to validate the adhesive ability and cell surface localization of S-layer proteins,furthermore,to screen the key adhesion protein and the adhesion receptors.The results of the study provided direct scientific evidence for adhesive mechanism of L.salivarius S-layer proteins,and the relationship between the lactobacillus and intestinal cells.To validate the ability of FDB86 to adhere to intestinal epithelial cells,and screen and identify the adhesins on the cell surface,high adhesive ability of FDB86 to HT-29 cells and mucin was proved.After treatment with sodium periodate and bovine serum albumin,there was no significant decrease of bacterial adherence,which indicated that exopolysaccharides and lipoteichoic acid were not adhesins of FDB86.After treatment with proteases and LiCl,bacterial adherence significantly reduced compared to non-treated strains,which indicated that S-layer proteins were the main adherence factors of FDB86.The S-layer proteins were extracted using guanidine hydrochloride,and there were two S-layer proteins on the cell surface with approximate molecular masses of 35 kDa and 50 kDa.They were named SlpA and SlpB,respectively,which were also able to bond with mucin.Then,SlpA and SlpB were sequenced using MALDI-TOF-MS,which showed that SlpA was nearly identical to peptidase,specifically the M23 family,and SlpB was identical to the N-acetylmuramoyl-L-alanine amidase,and their locations were DGL₆54 and DGL₆83 in FDB86 genome.To further study the adhesion function and cell surface localization of S-layer proteins,SlpA and SlpB expressed as His6 fusions in E.coli were used to produce SlpA-and SlpB-specific antibodies in rabbits.In antibody-mediated inhibition assays,adherence of FDB86 to HT-29 cells was significantly inhibited by addition of the anti-SlpA antiserum and anti-SlpB antiserum in a dose-dependent manner,which showed that SlpA and SlpB took part in adhesion.Localization of SlpA and SlpB on the cell surface of FDB86 was demonstrated by indirect immunofluorescence assay.Green fluorescence was detected when FDB86 bound anti-SlpA antiserum and anti-SlpB antiserum.The preimmune serum did not give any signal in immunofluorescent staining.After treated with LiCl,both anti-SlpA and anti-SlpB antisera gave weak signals in immunofluorescent staining.These results indicated that both SlpA and SlpB were present on FDB86 cell surface.To find the key adhesive proteins,△slpA and AslpB mutants were constructed by pORI system with helper plasmid pTRK669,and their adhesive ability to HT-29 cells and mucin was compared.The△slpA mutant exhibited a 62%reduction in adhesion to HT-29 cells,as compared to wild type（WT）cells,which was significantly greater than the 16%reduction the AslpB mutant exhibited in binding to HT-29 cells as compared to the WT cells.In addition,a greater reduction was observed in the binding of△slpA mutants to mucin than △slpB mutants.These results indicated that SlpA was the key adherence factor of FDB86.Adhesion was a process of SlpA binding receptor on the surface of intestinal epithelial cells.A receptor protein of SlpA on HT-29 cells was detected in a pull-down assay,with the molecular masse of approximate 35 kDa.The receptor was sequenced using MALDI-TOF-MS,which showed that receptor was nearly identical to enolase（ENO1）.The interaction between SlpA and ENO1 was confirmed by Western blot and Co-immunoprecipitation assay.In antibody-mediated inhibition assay,adherence of FDB86 to HT-29 cells was significantly inhibitable by addition of anti-ENO1,which further confirmed that ENO1 was the receptor of SlpA.