Effect Of Mucin On Metabolism Of Akkermansia Muciniphila

Akkermansia muciniphila is considered a probiotic and has attracted much attention.In gut,A.muciniphila could use mucin as the sole source of nitrogen and carbon for growth,meanwhile,it showed a positive regulatory effect on mucus layer thickness and intestinal barrier integrity.However,the underlying mechanisms of interactions between A.muciniphila and mucin remains unclear.The environment in the intestine is complex,and the macroscopic changes of the mucus layer may be related to many factors such as diet,intestinal microbes,host,etc.It is difficult to establish a direct connection between A.muciniphila and mucin.Therefore,there is certain limitations on the mechanism study of mucin degradation by A.muciniphila.To further determine the specific mechanism of interactions between A.mucinphila and mucin,this research systematically studied how A.muciniphila responds to the addition of mucin and the corresponding metabolic characteristics.Genomic DNA sequencing was applied to get the whole genome of A.mucinphila,and the molecular mechanism of A.muciniphila in response to different concentrations of mucin and changes in metabolites were analyzed by transcriptomics and metabolomics.The main research contents and results are shown as follows:1.Whole genomic sequencing analysis of Akkermansia muciniphila DSM22959The whole genome sequencing results of Akkermansia muciniphila DSM22959 showed that the total length of the genome was 2664043 bp,the GC content was 55%,and the coding genes were 2568,including 61 glycoside hydrolases,44 glycosyl transferases,9carbohydrate-binding modules,4 carbohydrate esterases and 1 auxiliary activities.There are53 copies of t RNA,9 copies of r RNA and no s RNA.At the same time,comparative genomics analysis with three different strains of A.muciniphila and the same strain ATCC-BAA835 showed that 1708 common gene sets were obtained,and compared with ATCC-BAA835,Akkermansia muciniphila DSM22959 has 490 unique gene sets,and its functions are mainly focused on bacterial metabolism,including DNA,RNA,translation,membrane protein,ATPase,sulfate hydrolase,β-galactosidase and oxidoreductase and so on.The whole genome sequencing of A.muciniphila provides a basic reference gene sequence data for the subsequent studies.2.Effects of different mucin concentrations on growth and gene expression of A.muciniphilaTo investigate the response of A.muciniphila to the mucin content in the medium,referring to the previously reported in the literature on the mucin dose effect of A.muciniphila culture,BHI medium was supplemented with 0%,0.05%,0.2% and 0.5% mucin,respectively for A.muciniphila cultivation.The growth curve and scanning electron microscope were used to reflect the phenotypic changes of A.muciniphila,basic on which,some of specific enzyme activities and gene expressions were determined for verification.The results showed that as the concentration increases,it took less time for A.muciniphila to reach the logarithmic telophase,the overall shape of the external structure of the bacteria became smaller,and the internal cytoplasmic structure became clearer.At the transcriptional level,0.05% and 0.2%mucin slightly affected the gene expression of A.muciniphila,only 2 and 31 differentially expressed genes were found,respectively.However,0.5% of mucins induced 1191 differential expressed genes,which were mainly involved in the oxidation-reduction process and the O-glycosyl compound hydrolase activity process in the GO database.These results indicated that supplementation with varied amount of mucin can differentially affect the growth characteristics and gene expression levels of A.muciniphila.3.Effects of mucin concentration on metabolic characteristics of A.muciniphilaBased on the results of transcriptomics,the mucin concentrations of 0.05% and 0.5%with obvious dose effects are selected as the test groups in this chapter.The non-targeted metabolome GC-MS was applied to determine the changes in the content of the main energy substances(glucose and fructose)of A.muciniphila during the growth process.Finally,combined with the co-culture experiment of A.muciniphila and fluorescently labeled mucin,the mechanism between A.muciniphila and mucin was directly approved.Results of metabolomics analysis showed that 161 compounds were identified by GC-MS,of which 49 compounds were differential metabolites.Compared to the 0.05% mucin group,the relative contents of sorbose,mannose,2,7-anhydro-beta-sedoheptulose,fructose and galactinol were significantly reduced in the 0.5% mucin group,while the relative contents of Nacetylneuraminic acid were increased significantly.Compared with the BHI group,the addition of mucin could overall inhibited the metabolism of phenylalanine,threonine,lysine,ornithine,asparagine and glutamic acid.However,0.5% mucin could promote the metabolism of norleucine and norvaline,while the difference between the 0.05% mucin group and the BHI group was not observed.when A.muciniphila grows to 9-18 h,the addition of mucin will accelerate the degradation of glucose,which is the main energy substance for bacteria growth.Co-cultivation experiments of A.muciniphila and fluorescently labeled mucin showed that when mucins are present in the BHI medium,A.muciniphila will respond rapidly and form a localization on the cell membrane surface,stimulating A.muciniphila to produce a series of enzymes to degrade them.Mucins,which are degraded into small molecules of-NH3 group,will further enter the cell and play a role.Therefore,at the level of metabolites,the addition of mucin mainly affects the production of different metabolites such as sugars and amino acids in A.muciniphila and these substances cause changes in the metabolic characteristics of A.muciniphila by mucin.These results provide a reference for further exploring mechanisms that exert probiotic effect of A.muciniphila in the intestine.