Metagenomics-based Analysis Of Gut Microbiota Structure And Function In Patients With Type 2 Diabetes Mellitus

Objective:Type 2 diabetes(T2D)is a common metabolic disease cha racterized by insulin resistance and hyperglycemia.Previous studies have f ound that the occurrence and development of T2D is closely related to int estinal flora.Based on previous 16S high-throughput sequencing,this study further explored the structure and function changes of intestinal microflor a in patients with T2D through metagenomic technology.Methods:Propensity Score Matching(PSM)was used to screen 19 samples out of 133 samples in total,including 11 samples nondiabetic(ND)and 8 T2D samples for metagenomic sequencing.Species annotation infor mation of species abundance was obtained through species analysis of the high quality sequences obtained by sequencing.Then Kraken and Calypso were used to visualize species abundance distribution and evolutionary rela tionship.Based on species ? diversity,? diversity and enterotypes analyze d,the DESeq2 packet of R language and LEfSe analysis were used for s pecies difference analysis to screen out alternative biomarkers.Then Bayesi an network was constructed through R language bnlearn package,igraph p ackage and Cytoscape software for correlation analysis,and the T2D biom arkers were screened out.On the other hand,IDBA-UD software was use d to assemble high-quality data,CD-HIT software was used to construct n on-redundant gene set,and SOAPalign software was used to compare the high-quality sequences with the constructed non-redundant gene set to obta in gene abundance information.BLASTP software was used to compare ge ne abundance information with NR and Swissprot databases to obtain prot ein abundance annotation information;COG database was used to obtain p rotein cluster abundance information;KEGG database was used to obtain metabolic pathway information.At last,Blast2GO was used to convert the NR database annotation results into GO annotation results to obtain gene function information.The differences of protein abundance,gene function and metabolic pathway between the two groups were compared by using the software of STAMP.Results:A total of 329,796,471 Clean Reads were obtained from the initial data of the samples,with an average of 17,357,709 Clean Reads pe r sample,the minimum number of Reads was 14,397,776,and the maxim um number of Reads was 20,839,579.There are 1,568 species in ND gro up and 1,532 species in T2D group.There were 1,456 common species in the two groups,112 endemic species in ND group and 76 endemic speci es in T2D group.In general,Bacteroidetes and Firmicutes are dominant sp ecies at phylum level;Bacteroides,Eubacterium and Escherichia are domi nant species at genus level;Bacteroides_vulgatus?Bacteroides_xylanisol vens and Bacteroides fragilis are the dominant species at species level.T he results of ? diversity analysis showed that there was no significant dif ference between T2D group and ND group,while there was significant dif ference in ? diversity analysis between the two groups.Enterotypes analysi s showed that the two groups of samples were enriched in ET B and et F.At the genus level,there were 15 species with significant differences among groups,among which 6 species were significantly increase in T2D group,namely viillonella,Escherichia,Salmonella,Cronobacter,Roseburia and Shigella,and 5 species were significantly decrease in T2D group,na mely Megamonas,Eggerthella,Riemerella,Prevotella and Haemophilus.Th ere were 20 biomarkers of bacterial flora in species level.Among them,t he biomarkers of T2D group were Veillonella_parvula?Candidatus_Protochl amydia_amoebophila?Shewanella_sp MR 4?Acidiphilium_multivorum?Sulf urospirillum_deleyianum?Helicobacter_bizzozeronii?Candidatus_Hamiltonell a_defensa?Leptospira_borgpetersenii?Mycobacterium_sp_JLS?Haloquadrat um_walsbyi,Mycoplasma_parvum?Pseudomonas_savastanoi.The biomarkers of ND group were Nitrosomonas_sp_AL212?Blattabacterium_punctulatus?R iemerella_anatipestifer?Eggerthella_enta?Burkholderia_mallei?Prevotella_ruminicola?Megamonas_hypermegale?Eubacterium_rectale.Through Baye sian network correlation analysis,we finally determined that there are six biomarkers most closely related to T2D.Sulfurospirillum_deleyianum?Lept ospira_borgpetersenii?My cobacterium_sp_JLS was increase in T2D group;Nitrosomonas_sp_AL212?Megamonas_hypermegale?Eubacterium_rectale wa s decrease in T2D group.In total,1,327,642 Contigs were produced by Clean Reads assembly,with a total length of 3,004,864,358 bp.According to 90%similarity and 90%coverage,the redundant genes were removed,and a non-redundant ge ne set with a total number of 1,441,752 genes and a total length of 1,103,955,95 1bp was finally obtained.There were 1,243,823 genes in ND group and 1,229,591 genes in T2D group.There were 1,035,521 common genes in the two groups,208,302 specific genes in ND group and 194,070 spe cific genes in T2D group.In general,the relatively high abundant proteins in the samples are TonB-dependent receptor SusC,Beta-galactosidase,Tyr osine recombinase XerC,and Sensor histidine kinase TodS.The most abun dant COG function are[L]Replication,recombination and repair,[G]Carb ohydrate transport and metabolism.The main GO function are ATP bindin g,DNA binding and cytoplasmic synthesis.The main primary metabolic p athways are Metabolism and Genetic Information Processing.The main sec ondary metabolic pathways are Biosynthesis of amino acids,Carbon metab olism and ABC transporters.There were 10 different proteins among the g roups,including Fatty acid synthase subunit beta,Transcriptional regulatory protein SrrA,Uncharacterized protein MJ0165,Xylulose kinase and 30S r ibosomal protein S8,Mu-like prophage FluMu protein C,Hypoxanthine-gu anine phosphoribosyltransferase,SPBc2 prophage-derived uncharacterized pr otein YomE,Glycosyltransferase Ag1J,which are increased significantly in T2D group,while the abundance of Omega-amidase NIT2-B decreased sig nificantly.There are 10 different functional genes,including lipoprotein bio synthetic process,dipeptidase activity,dipeptidyl-peptidase activity,phosphat idylglycerol-prolipoprotein diacylglyceryl transferase activity,L,L-diaminopi melate aminotransferase activity,aspartate carbamoyltransferase complex,pr otein lipoylation,(R)-2-methylmalate dehydratase activity,ATP-dependent he licase activity,which were significantly down regulated in T2D group,and the phosphoglucosemutase activity was significantly up-regulated.There wa s one differential metabolic pathway,ribosome metabolic pathway,which was significantly up-regulated in T2D group.Conclusion:The intestinal flora of T2D patients and ND population a re different in species diversity,species composition,protein abundance,ge ne function and metabolic pathway,and the intestinal flora of T2D patient s shows a certain degree of ecological imbalance.This study confirmed th e correlation between the occurrence and development of T2D and the cha nges of intestinal flora,clarified the differences of intestinal flora of T2D,and provided some enlightenment and basis for the study on the pathoge nesis of T2D and the treatment of T2D with intestinal flora as the target.