| Akkermansia muciniphila is a mucin-degrading bacterium commonly found in the intestinal tract of mammals,which has been reported to have important probiotic properties in alleviating metabolic diseases and regulating intestinal function,and is considered as a candidate for "next generation probiotics".A large number of studies has shown that one of the key mechanism for the probiotic function of A.muciniphila is based on its regulation of the host intestinal barrier,thereby inhibiting the entry of intestinal bacteria-borne endotoxin into the blood and relieving the chronic inflammation of the body.At present,studies on A.muciniphila are mainly focused on the model strain ATCC BAA-835,but a few reports have already shown that different strains of A.muciniphila have significant variations in their probiotic functions.Previous studies indicated that the differences in the physiological functions of probiotics may be related to their specific functional genes,surface substances and metabolites.Therefore,this study intends to explore the strain specificity of A.muciniphila strains for their regulation of intestinal barrier function,to analyze the involved action modes of different strains and the functional genes related to the gut barrier regulation function of the strains,and to further reveal the specific cellular components or metabolites responsible for the the intestinal barrier regulation function of A.muciniphila.The main results of the study are as follows:First,six strains with different genomic characteristics were randomly selected and the gut barrier regulation function of each strain was evaluated in an intestinal barrier impaired mouse model with induced by sodium dextran sulfate(DSS).The results showed a marked strain specifity of the intestinal barrier regulation function of A.muciniphila.Compared with the mice receiving DSS only,the administration of A.muciniphila JL50 significantly improved the physiological status of mice,reduced intestinal permeability,increased the expression of colonic tight junction protein,and prevented the pathological damage of colonic tissue caused by DSS exposure.An other A.muciniphila strain,GD49,did not exhibited the the identical protective effects to A.muciniphila JL50,and even down-regulated the m RNA expression of tight junction proteins such as ZO-1 and Occludin in the colon.An in vitro monolayer model of intestinal cells based on Transwell technology also demonstrated the strain specificity of intestinal barrier regulation of by different strains.The recovery degree of A.muciniphila JL50 on trans-epithelial resistance was close to 95%,while the that of the other strains ranged from 0% to 85%.Based on the above results,we further analyzed the differences in the action modes of different strains in regulating intestinal barrier.The effect of A.muciniphila on the viability of intestinal epithelial cells was evaluated by the method of CCK-8,and the results showed that all tested strains could significantly restore the viability of epithelial cells inhibited by DSS without a strain specificity.Using Real-time polymerase chain reaction and Western Blot approaches,it is observed that A.muciniphila JL50 could restore the expression of tight junections of the epithelial cells including ZO-1 and Claudin-1,while A.muciniphila GD49 did not show the same significant effect,which was consistent with the results of the intestinal barrier regulation abilities of different strains in animal models and cell models.These resulted suggested that the specificity of A.muciniphila for intestinal barrier regulation functions may be related to their different regulative abilities on the rexpression intestinal tight junction protein.We further compared the effects of living and dead A.muciniphila strains on gut barrier regulation and found out the strain viability did not significantlt affect the regulation effects,indicating that the surface components of the strains may play a more important role in gut barrier regulation than the metabolites.Based on these findings,the genomic characteristics of A.muciniphila JL50 and A.muciniphila GD49 strains with significant differences in intestinal barrier regulation abilities were analyzed by comparative genomic techniques.Firstly,the proportion of core functional genes in different strains was compared based on the database of Clusters of Orthologous Groups of proteins database and it was found that A.muciniphila JL50 and A.muciniphila GD49 had no significant differences in all core functions,including carbohydrate transport and metabolism,amino acid transport and metabolism,and inorganic ion transport.The average nucleotide identity of two A.muciniphila strains was 98.17%,indicating that they belong to the same genus and the genomic similarity was high.In addition,the genes realted to the surface structure of the strain were predicted and analyzed by subcellular localization.Compared with the negative strain A.muciniphila GD49,A.muciniphila JL50,which could significantly enhance the intestinal barrier,possessed 16 special genes including COG0438,COG2244,etc.Some related genes were proved to be related to the protein synthesis for the surface components of the strain by gene functional annotations.Based on the results of comparative genomics,another A.muciniphila strains with above mentioned differential functional genes,A.muciniphila GS9,was selected to verify the role of these genes in gut barrier regulation.The results from both animal and cell experiments proved that compared with the negative strains of A.muciniphila GD49,the A.muciniphila GS9 exhibit siginicant protective effects on gut barrier function,which was consistent with A.muciniphila JL50.When the surface proteins of the two positive strains,A.muciniphila JL50 and A.muciniphila GS9,were removed by protease treatment,their intestinal barrier regulation abilities were significantly inhibited.We further isolated the surface proteins of the above strains and their regulatory effects on tight junction proteins were verified in cell models.In conclusion,this study demonstrated the strain specific effects of A.muciniphila strains on intestinal barrier regulation in both animal and cell models.This phenomenon may be explained bydifferent abilities of strains to affect the expression of tight junction proteins of intestinal epithelial cells.Comparative genomic analysis proved that the ability of A.muciniphila to regulate intestinal barrier was related to the functional genes related to the synthesis of cellular surface proteins.The role of surface proteins of A.muciniphila in intestinal barrier regulation was further verified by strain-comparative experiments in animal and cell models,and surface protein-removal/extraction trials.This study provides a new perspective for resource collecting and function analysis of the next generation of probiotics A.muciniphila,and also provides relevant gene and protein targets for the screening of functional probiotic strains with significant intestinal barrier regulation abilities. |
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