Association Study Between Single Nucleotide Mutation Of Gut Microbe Bacteroides Coprocola And Type 2 Diabetes

The number of people with diabetes has increased rapidly over the past few decades.The prevalence of the Chinese population is about 11.6%,of which type 2diabetes（T2D）accounts for 90%.In 2015,diabetes became the sixth major disability.Type 2 diabetes is characterized by a relative deficiency of insulin caused by islet beta cell dysfunction and insulin resistance.Although more and more risk factors for type 2diabetes and evidence to prevent their progression are revealed,the incidence of type 2diabetes is still growing.The human intestinal microbes and the host form a mutually beneficial symbiotic relationship in the long-term co-evolution process.The dietary habits and physiological phenotype of the host affect the structure of the intestinal flora,and the intestinal flora also affects the health of the host.The function of human intestinal microbes on the human body mainly includes nutrient metabolism and immune defense.In recent years,several studies have shown that intestinal flora disorders are associated with more than50 diseases,including diabetes,colorectal cancer,cirrhosis,hypertension,and autism.With the continuous study of intestinal microbes,the role of intestinal flora in the development and treatment of type 2 diabetes is becoming more and more important.Studies have found that the development of type 2 diabetes is accompanied by a decrease in short-chain fatty acid-producing bacteria and an increase in opportunistic pathogens.Another study using Chinese herbal compound Gegen Qinlian Decoction in patients with type 2 diabetes found that the intestinal flora of patients with type 2diabetes has significant changes,and the changes in the flora are earlier than the improvement of symptoms.This result suggests that Gegen Qinlian Decoction may be effective in treating type 2 diabetes by altering the intestinal flora.Although the association between type 2 diabetes and intestinal microbes has been widely reported,current research remains focused on the study of intestinal microbial structure and abundance.It has not yet penetrated into the gene level of the strain,and has not studied the function of the protein brought about by the difference in gene function of the strain.In addition,there is currently no method for directly amplifying a gene of interest from the fecal genome.Therefore,this study first established a set of methods for directly amplifying the gene of interest from the fecal genome,and analyzed the difference in the target genes between type 2 diabetes and healthy people,and analyzed the difference in protein function caused by gene mutations.First,a local primer comparison database was established.The database contains sequencing data for all bacterial genes contained in the NCBI database prior to June2017.Primer design uses NCBI-PRIMER online primer design software to design primers,which requires that the designed primers must be completely specific to the target gene,and use BLAST tools to screen primers that can only match the target gene.Secondly,the PCR amplification method was selected.Since the number of gut metagenome is very large,it is very difficult to directly amplify the target gene from the intestinal microbiome by a single PCR method.Therefore,the target gene was amplified by nested PCR.The amplified products were subjected to Sanger sequencing,and the sequencing results were subjected to phylogenetic tree analysis and mutation site statistics,and amino acid annotations were made.Phylogenetic analysis revealed significant differences in EDU99824.1 of intestinal microbial B.coprocola in patients with type 2 diabetes and healthy people.Sequence analysis reveals that mutations occur dominantly in the adjacent loci 879^th（151 of 329 samples）and 880^th（123 of 329samples）.The two mutations are not linked and are both biallelic sites(879^th A>G,880^th C>T).Interestingly,the 879^th mutation occurs more frequently in HCs than T2D patients,whereas the 880^th mutation dominantly occurs in T2D patients.Further amino acid annotation of the base mutation results revealed that the 879^th mutation in healthy humans belongs to the synonymous mutation encoding glutamate,whereas 880^thh mutation in type 2 diabetes patients is a missense mutation,whose encoded amino acid(the 294^th amino acid)is changed from proline to serine.In the analysis of the gene sequence,we found that the 879^th and 880^th polyclonal strains of B.coprocola were present in healthy people and type 2 diabetes patients,i.e.,both wild type and mutant strains were present in one sample.To further investigate the effect of gene mutations on the function of the protein encoded by EDU99824.1,we performed an in vitro activity assay for the glycosidase encoded by the EDU99824.1 gene.First,a prokaryotic expression vector was constructed,and the wild type and mutant strain of EDU99824.1 gene were connected to a plasmid vector,and then transformed into E.coli BL21 competent cells for expression.Finally,the purification column was used to purify the target protein,and the Western blot analysis showed that the target protein was successfully purified.Detection of EDU99824.1 glycosidase activity using p-nitrophenyl-β-D-glucoside（pNPG）and geniposide as substrates.The enzyme activity test showed that the purified protein was active on both substrates.The wild type and the 879^th mutant had no statistical difference in pNPG activity,while the 880^th mutant had much higher enzyme activity on pNPG than wild type and the 879^th mutant.The enzyme activity using geniposide as a substrate was found enzyme activity gradually increased in the order of wild type,the 879^th mutant strain and the 880^th mutant strain.The results show that EDU99824.1 glycosidase of B.coprocola produces more glucose than healthy people in type 2 diabetes patients,and we suspect that this mutation may be involved in the development and progression of type 2 diabetes.In order to deeply analyze the causes of the effects of gene mutations on enzyme activity,we performed a three-dimensional structure simulation of the protein in Expasy SWISS-MODEL.The simulation results show that the amino acid at 294^th is located at the outermost periphery of the three-dimensional structure of the protein.It is speculated that the change of amino acid from non-polar proline to polar serine may increase the affinity of the enzyme for the substrate,thereby increasing the enzyme activity.Our research work is the first to reveal in the metagenomics field that type 2 diabetes is associated with a single nucleotide mutation in the EDU99824.1 gene of the intestinal microbial B.coprocola.The above conclusions reveal the relationship between gut microbes and diseases from the genetic level,providing a new perspective for metagenomic research.