| Enterococcus faecalis,a symbiotic bacteria of the gastrointestinal tract of humans,it can not only affect the colonization of other flora in the gut and the function of the intestinal barrier,but also play a particularly important role in the development and maturity of the body's immune system.As a kind of lactic acid bacteria,some strains of E.faecalis are used in food.At the moment there are few comprehensive studies on its function produced in the gastrointestinal tract.Therefore,this study combined genomics analysis with in vitro cell tests,in vivo mice test to study the function characteristics and the immunomodulatory effects of an E.faecalis strain isolated from gastrointestinal tract of healthy infant feces,to provide a theoretical reference for further exploration of the functional effects of E.faecalis on human health.This study was aimed at an E.faecalis strain preserved by laboratory which isolated from breast-feeding and healthy infant feces.After the identification and morphological observation of the strain,gene prediction and functional annotation were performed by whole genome sequencing technology to carry out genome components and functional genes.Took further research of immunity-related genes at the genetic level.Evaluated growth characteristics,antibiotic susceptibility and hemolysis,tolerance to the gastrointestinal tract environment and adhesion ability of the strain by biological tests.The expression of cytokine genes in the host cells was studied by using real-time RT-PCR technology,and the immune-related signaling pathways in the host cells were predicted and analyzed by transcriptome sequencing technology.Finally,immunomodulatory effects of the strain was verified by mice experiments.The findings of this experiment are as follows:(1)The genomic characteristics of E.faecalis JY32 and immune-related genes were clarified.The whole genome sequencing was performed on E.faecalis JY32,the genomic characteristics of the strain were analyzed and the immune function genes were annotated and analyzed to predict their immune effects.The total length of E.faecalis JY32 genome was 2,933,718 bp.A total of2788 coding genes have been predicted,including 62 t RNA,12 r RNA,19 gene islands(GIs).The strain contains no plasmid.Genes related to immune function dlt X,tag B1,tag B2,tag H and so on were found in the genome of E.faecalis JY32,which was genetically explained that the strain has a certain immune regulatory effect.(2)E.faecalis JY32 was sensitive to many common clinical antibiotics and was not hemolytic.The drug-sensitive paper diffusion method was used to evaluate the sensitivity of E.faecalis JY32 to commonly used antibiotics in the clinic.The results showed that it was sensitive to penicillin,ampicillin,amoxicillin,imipenem,chloramphenicol,vancomycin,and rifampicin.Hemolysis test showed that E.faecalis JY32 has no hemolytic ability.The results further verified the safety of the strain.(2)E.faecalis JY32 could tolerate the environment of gastrointestinal tract,and had good tolerance to acids,bile salts and digestive enzymes.The tolerance of the strains was evaluated by simulating the environment of artificial gastrointestinal tract.After 3 h treatment of p H 2.0 and 3.0environment,survival rate of E.faecalis JY32 were 69.15%and 94.34%,respectively.After being treated with 0.3,0.5,and 1.0 g/100 m L of medium for 4 h,the survival rates were 93.36%,82.36%,and 79.60%.E.faecalis JY32 were firstly treated with simulated artificial gastric fluid for 3 h and secondly treated with artificial intestinal fluid and cultured for 8 h,the rate can still be maintained at 79.20%.The adhesion rate of E.faecalis JY32 to Caco-2 cells was 77.14%,which had strong adhesion ability.The strain has a strong tolerance to acids,bile salts,and gastrointestinal fluids,which can survive and colonize in the gastrointestinal tract.(3)E.faecalis JY32 could activate immune-related factors of the host cells,which hasd a certain immune regulating effect.By co-culturing JY32 with Caco-2 cells and using real-time RT-PCR technology to study the expression of inflammatory cytokine genes in Caco-2 cells,it was found that this strain can significantly activate IL-8,IL-1?,IL-4.IL-10,IFN-?,TNF-?levels.Further transcriptomics was used to analyze genes related to host cell immunity,and it was found that genes related to immune response were mainly concentrated in signal pathways such as MAPK signaling pathway.(4)E.faecalis JY32 could enhance the immunity of mice,and its immune effect has been verified.E.faecalis JY32 was selected at 10~7,10~8,and 10~9 cfu/m L as low-,medium-and high-dose group,and the mice were sacrificed for 28 days,the organ index,clearance index,cytokines and spleen lymphocyte activity were measured.It was found that compared with the blank control group,the,the thymus index of the experimental group increased significantly,but the spleen index did not increase significantly.The clearance index and splenic lymphocyte activity were significantly improved(p<0.05).The cytokines IL-8,IL-1?,TNF-?and IFN-?content increased significantly,which proved that E.faecalis JY32 could enhance the immunity of mice to a certain degree. |
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