Genomic Diversity And Immune-stimulation Analysis Of Akkermansia Muciniphila

Akkermansia muciniphila,a symbiont inhabited in the gut of humans,other mammals and reptiles.It can use mucin as its sole carbon,nitrogen and energy source.Recently,it has been recognized as one of the most promising next generation of probiotics and reported to relieve obesity,both type I and II diabetes,amyotrophic lateral sclerosis(ALS)as well as to enhance the efficacy of PD-1 based immunotherapy against cancer.This research focuses on four aspects of A.muciniphila strains.Firstly,the isolation method of A.muciniphila from fecal samples was reconstructed and modified.Secondly,the strain diversity and its envotionary history was analyzed.The immune-stimulatory characteristics of A.muciniphila strains,especially,their specificities compared with other gut species,were investigated.Finally,the immune-stimualtion effect of A.muciniphila strains was verified in the in-vivo SPF and GF mice models under homestasis.The influence of A.muciniphila on disease phenotypes in different disease model was also evaluated.The present investigations were amined to provide culture resources and theoretical basis for the application of A.muciniphila in food industry and medicines.Firstly,an isolation method of A.muciniphila was re-established in our laboratory.The formula and configuration protocols of mucin-supplemented enrichment medium was optimized.Mucin-supplemented brain heart infusion(BHI)agar was used to isolate single colony.BHI liquid tubes were used to prelimianrility screen the potential positive colonies.Total of eight A.muciniphila strains were isolated from more than 250 fecal samples of Chinese individuals with a positive rate of 10% in enrichment tube and a successful isolation rate of50%.Genome sequencing and phylogenic analysis showed that eight A.muciniphila strains were belonged to Am Ia,Am IIa and Am IIIb clusters.Comparative genomic analysis of both eight newly isolated A.muciniphila strains as well as other public available Akkermansia assemblies showed that all these strains can be classified into 12 clades and 43 clusters.Among these,A.muciniphila belongs to the C10 clade and Akk41,Akk42 and Akk43 clusters.The most ancient Akkermansia species(clades C1 to C6,A.ruminant group)are mainly inhabited in the gut of ruminants and have a richer interspecies diversity.Functionally,these strains differ significantly from A.muciniphila strains in the C10 clade.A.ruminant group possesses carbohydrate binding and metabolism enzymes associated with both plant and mucin polysaccharides degradation.In addition,the ancient Akkermansia strains is also present in the gut of Fiji individuals.A.muciniphila,the youngest clade,is widely colonised in the intestines of both human and other mammalians and has evolved more diverse mucin-degrading associated glycosyl hydrolase and sulphate esterase genes,suggesting an adaptive evolution for the mucus layer niche.The immunostimulatory properties of nearly 500 commensal bacterial strains derived from the human gut including Bacteroides sp.,Bifidobacterium sp.,Lactobacillus sp.,Faecalibacterium prausnitzii,A.muciniphila and Clostridium butyricum,were compared in an in-vitro co-culture mesenteric lymph nodes(MLN)cell suspension model.The results showed that A.muciniphila had unique immunostimulatory activity.All tested A.muciniphila strains(Am I,Am II and Am III)can directly activate immune response of MLN cells.The activation of MLN by A.muciniphila was time-and dose-dependent.Besides,cell surface protein of A.muciniphila had the similar effect.Transcriptome analysis showed that T-cell-dependent B-cell activation and antibody production were the general responsive pattern of MLN cells when stimulated by commensal bacteria.Under homestasis,the proportion of CD4-positive T cells in the mesenteric lymph nodes and spleen as well as the colonic IL-10 level in specific pathogen free(SPF)and germ free(GF)mice orally administrated with Lactobacillus plantarum or A.muciniphila was determined.The results showed that oral administration of A.muciniphila AH39 and BAA835(type strain)increased the proportion of Tregs in mesenteric lymph nodes and colonic IL-10 levels in SPF mice,while there were no significant differences between the two L.plantarum gavage groups and the control group.In GF mice,oral administration of AH39 significantly increased the proportion of Tregs in the spleen,but their proportion in mesenteric lymph nodes and colonic IL-10 levels were not significantly changed when compared to the control group.In summary,gavage of A.muciniphila under steady-state regulation can affect host intestinal and systemic immunity.The functional evaluations of A.muciniphila were carried out in a series of mouse models including acute and chronic cadmium exposure,TNBS-induced intestinal damage,DSS-induced colitis under both normal condition and background microbial knock-out condition using mixed antibiotics.The results showed that oral administration of A.muciniphila influenced the disease phenotypes with a similar pattern in different models.In acute and severe experimental models,gavage of A.muciniphila increased mortality and worsened the disease phenotype,and in chronic and non-lifethreatening experimental models,gavage of A.muciniphila did not exert a significant effect on the disease phenotype.