Screening Of Adhesive Activity Of Lactobacilli And Preliminary Study On Potential Mechanisms Of Binding To Enterocyte-like HT-29 Cells

Probiotics play an important role in controlling undesirable microflora in the gastrointestinal tract of humans and animals.They have been shown to possess inhibitory activity toward the growth of pathogenic bacteria.The characteristics of adhesion of probiotics to the intestinal mucosal surface are a critical prerequisite for exerting beneficial effect to their host organisms.But there have some difficulties involved in studying bacterial adhesion in vivo,especially in humans.To date,there have no a proper evaluation method for adhesion in vitro,and little is known about the mechanisms by which is might occour in the human gastrointestinal tract and these bacteria attach to the intestinal mucosa. Therefore,which have led to the development of in vitro model systems for the preliminary selection of potentially adherent strains,and to gain further understanding of the mechanisms by which lactobacilli adhere to human intestinal cells and investigate intestinal mucosal barrier to prevent the infection of the enteropathogenic bacteria.Methods:1.In vitro model of HT-29 cell culture was employed to investigate the influence of the pH,buffer,growth phase,incubation time and bacteria concentration on adherence of Lactobacillus.A critical validation of in vitro adhesion model was used to screen and assess adhesive properties of 11 strains of probiotics.Methods of radioactivity and ELISA of mucin were compared as complement.Anti-JCM1081 polyclonal antibodies were prepared by immunizing rabbit to investigate colonization of Lactobacillus JCM1081 on intestinal mucosa through frozen section and immunohistochemistry.2.Different adhesive properties of Lactobacillus strains were compared to investigate the beneficial effect on protection of intestinal epithelial cells.Antibiotics decontaminated and EPEC infected mice model was established and HE stained slide,gut microflora analysis and bacteria translocation were observed for intestinal mucosal changes.3.Lactobacillus with spent culture supernatant were subjected to various treatments including heat shock,trypsin, pronase and chemical products to study on factors involved in adherence.Extraction of the cell wall surface proteins with lysozyme and mutanolysin were performed and spent culture supernatant was fractionated by ammonium sulfate precipitation.The proteins which associated with adhesion of Lactobacillus to HT-29 and mucin were dectected in the fractions by SDS-PAGE and Western blotting with horseradish peroxidase labeled mucin and NHS-Biotin labeled HT-29 cells and the adhesin of Lactobacilli were dectected in Western blotting with Anti-JCM1081 polyclonal antibodies.The proteins which asscoiated with adhesion of Lactobacillus to HT-29 cells were purified to mass spectrographic analysis.Results:1.An evaluation method for adhesion was optimized.The pH,bacteria concentration and incubation time,growth phase of Lactobacilli have influence on adhesion of Lactobacilli to HT-29 cells.In general,the adhesion approach was optimized,and the procedure is that the HT-29 monolayers which cultured at post confluence after 15d were washed twice with PBS,1 ml 1～2×10⁸ bacteria(with spent culture supernatant) suspension was mixed with DMEM and then incubated at 37℃in 10%CO₂/90%air.After incubation, the monolayers were washed five times with sterile PBS,fixed with methanol, Gram-stained,and examined microscopically.Eleven strains were examined for their ability to adhere to cultured HT-29 cells by Gram stain and radioactivity methods.The ability of the Lactobacilli and Bifidobacteria to adhere to the HT-29 cells in vitro varied considerably between different strains.High attachment was observed with the chicken intestinal tract isolate Lactobacillus reuteri JCM1081(495.07±80.03)while low attachment showed in Lactobacillus acidphilus 1.1878(135.43±13.93)and Bifidobacterium longum B050102-14(47.17±5.84).Lactobacillus reuteri JCM1081 was appeared on the intestinal mucosa surface diffusely.2.High adhesiveness strain Lactobacillus reuteri JCM1081 inhibit enteropathogenic Escherichia coli adhere to enterocyte-like cells and also reduce the invasiveness and enhance the integrity of the cells.The spent culture supernatant of the Lactobacillus acidphilus 1.1878 produces an antibacterial activity against enteropathogenic Escherichia coli.In vitro,no significant changes of the morphology,structure and function of the HT-29 cells were found after being treated with Lactobacilli for 3 hours,especially cellular LDH level,activity and permeability.Lactobacillus reuteri JCM1081 did not alter cell integrity and prevented the increase in permeability induced by enteropathogenic Escherichia coli infection.The distribution of F-actin was also not altered.In contrary,after HT-29 cells being infected by enteropathogenic Escherichia coli in vitro for 3 hours,the cellular activity decreased,the permeability of single layer cell increased and the distribution of F-actin were sparse or dot dome.The group of co-incubation showed that the degree of cell injury was less than the group of enteropathogenic Escherchia coli infection.The results showed that after de-contaminated and EPEC infected,Enterobacteria increased significantly,but anaerobic bacterial such as Bifidobacteria and Lactobacilli decreased and the ratio of anaerobes to aerobes declined,which lead to dysbiosis.The degree of dysbiosis in Lactobacillus feeding group was much less than that in non-feeding group,as well as the rate of organ bacteria count.3.Adherence of Lactobacillus reuteri JCM1081 and Lactobacillus acidphilus 1.1878 cells were significantly reduced by treatment with trypsin and protease.Which indicated that the adhesion of Lactobacillus to HT-29 cells appeared to be mediated by a cell surface component,and the cell surface determinant of the strain appeared to be proteinaceous. Extraction of the cell surface proteins with different solutions,were analyzed on SDS-PAGE and Western blotting with HRP-mucin and NHS-biotin labeled HT-29 cells. The strongest-reacting band that bound mucin and HT-29 cells yielded 14kD and 29kD subunit.The proteins which extracted from cell surface of Lactobacillus reuteri JCM1081 were performed to adhere to HT-29 cells,and elutes were analyzed on SDS-PAGE and Western blotting with anti-JCM1081 polyclonal antibodies.There have only one strongest-reacting band which yielded 29kD submit.The 29kD protein was purified and analyzed by mass spectrography,the results indicated that high degree similarities between 29kD protein from Lactobacillus reuteri JCM1081 and the protein lr0793 of Lacobacillus reuteri ATCC55730 strain(approx 71.1%identity) were confirmed.Which belong to the putative ATP-Binding Cassette transportor proteins superfamily. A highly significant loss of adhesion was observed when the spent culture supernatant of Lactobacillus reuteri JCM1081 was discarded or when it was replaced by fresh MRS culture medium.These results strongly suggest that a secretory component in the spent culture supernatant was involved in adhesion of Lactobacillus reuteri JCM1081.Treatment of the spent culture supernatant with trypsin or protease almost totally abolished the adhesiveness of Lactobacillus reuteri JCM1081.The SCS of Lactobacillus reuteri JCM1081 has no adhesion promoting effect on Lactobacillus acidphilus 1.1878.Extraction of promoting adhesion protein in spent culture supernatant of Lactobacillus reuteri JCM1081 with ammonium sulfate precipitation.These fractions were analyzed by SDS-PAGE and Western blotting with HRP-mucin.The reacting 29kD protein was found in them.Conclusion:1.An optimized Gram-stained method could be regarded as effective method to evaluate the adhesive ability of probiotics in vitro.2.The ability of the probiotics to adhere to the HT-29 cells in vitro varied considerably between strains and genus.Lactobacillus reuteri JCM1081 have a good ability to adhere to HT-29 cells and could colonize in intestinal mucosa of mice.3.Lactobacillus reuteri JCM1081 could protect HT-29 cells by its adhesive abilities which relates to its effectiveness.Lactobacillus reuteri JCM1081 interact with intestinal epithelial cell receptor to Competitively inhibit the adhesion and invasion of enteropathogenic Escherichia coli to HT-29 cells by incubation.Lactobacillus reuteri JCM1081 have ability to adhere and colonize to intestinal mucosa and form biologic barrier to enhance host resistance against enteropathogenic Escherichia coli infection.4.The adhesion of Lactobacillus reuteri JCM1081 to HT-29 cells appeared to be mediated by two components:a cell surface component and extracellular factor.The cell surface determinant of Lactobacillus reuteri JCM1081 appeared to be proteinaceous,and relative molecular mass which are 14kD and 29kD might be involved in adhesion of Lactobacillus to HT-29 cells.29kD protein could be adhesin of Lactobacillus reuteri JCM1081,which have high degree similarities with the protein lr0793 of Lacobacillus reuteri ATCC55730 strain(approx 71.1%identity) and belong to the putative ATP-Binding Cassette transportor proteins superfamily.A secretory proteinaceous component in the spent culture supernatant with relative molecular mass 29kD was considered to involve in adhesion of the Lactobacillus reutri JCM1081 too.The adhesion of strain JCM1081 was mediated by 29kD protein of bacteria secreted in the spent culture supernatant.