The Regulation Of Genome-wide DNA Methylation And Functional Verification Of Candidate Genes For Bovine Staphylococcus Aureus Mastitis Resistance

Staphylococcus aureus(S.aureus)is one of the most prevalent contagious bacterial pathogen responsible for bovine subclinical mastitis.Bovine mastitis caused by S.aureus(bovine S.aureus mastitis)is characterized by longer latency,persistent infection,highly infectious and resistance to antibiotics.Now we have no effective vaccine to prevent this disease,which results in great losses to the dairy industry worldwide.Bovine S.aureus mastitis affected by the environment and pathogens has low heritability.Therefore,it is difficult to carry on the effective prevention and control only from the pathogens or genetics perspective,but the role of epigenetics might be in consideration to provide a new strategy for studying bovine S.aureus mastitis.DNA methylation is relatively stable and heritable epigenetic modification accountable for suppression of gene expression and could be used as an entry point to study complex disease such as bovine S.aureus mastitis.The study was designed to reveal the difference of DNA methylation and the pattern of regulating between S.aureus subclinical mastitis cows and healthy cows,to establish the model of S.aureus infection to Mac-T cells in vitro,to verify the function of S.aureus mastitis candidate genes in cellular level and to investigate the molecular mechanisms of folic acid resistance to bovine mastitis induced by S.aureus.This study will provide the genetic and epigenetic basis for preventing bovine S.aureus mastitis and its molecular breeding for disease resistance.Experiment 1.A total of 6,three each from S.aureus subclinical mastitis and healthy cows were chosen to investigate the genome-wide promoter DNA methylation pattern and modification difference of peripheral blood lymphocyte.The MeDIP-chip technology was adopted to profile the host differentially methylated genes.The comparison of S.aureus subclinical mastitis cows and healthy cows showed a total of 1078 genes to be significantly methylation altered.Out of these,527 were up-regulated and 551 were down-regulated methylated genes.The DNA methylation pattern showed that methylation level in subtelomeric regions on chromosomes significantly higher compared with non-subtelomeric regions,DNA methylation levels dramatically peaked at the TSS,sharply dropped and plateaued after the TTS and the negative regulation of DNA methylation and gene expression was observed in the 1 kb region upstream of TSS in S.aureus subclinical mastitis cows.By integrating DNA methylation and transcriptome data,58 differentially methylated genes were shared with differently expressed genes,in which NRG1,MST1 and NAT9 genes were strongly correlated with the progression of bovine S.aureus subclinical mastitis and could be used as potential candidate gene for resistance to bovine mastitis.Experiment 2.Bovine mammary gland epithelial cell line Mac-T is an immortalized cell line with normal morphology and characteristics of the mammary epithelial cell,which was used increasingly to characterize epithelial cell interactions with mastitis pathogen.Quantitative real-time PCR was applied to quantification of S.aureus adhesion and invasion to Mac-T cells.The results showed that the adhesion capacity of S.aureu and MRSA were most strong at 90 min and 10 min,respectively.The invasion capacity of S.aureu and MRSA were mainly strong at 6 h and 8 h,respectively.And the adhesion and invasion capacity of MRSA were stronger than S.aureu.We established the model of S.aureu and MRSA infection to Mac-T cells that MOI = 10:1 for 6 h.Experiment 3.TRAPPC9 and JAK2 were selected as candidate genes for bovine S.aureus mastitis.With the RNA intervention technique,we silenced candidate genes expression and studied the molecular functions of candidate genes in response of Mac-T cells to infection with S.aureus.The result showed that the mRNA expression level of TRAPPC9 and JAK2 genes were significantly decreased during S.aureus and MRSA infection and the knockdown of TRAPPC9 gene decreased the mRNA expression level of NRG1,MST1 and NAT9.However,the knockdown of TRAPPC9 and JAK2 genes in Mac-T cells both contributed to the expression of IL6,IL1? and IL8 mRNA in response to MRSA or S.aureus infection.Our results suggested the anti-inflammatory activity of TRAPPC9 and JAK2 genes in Mac-T cells,which can be major candidate genes resistant to bovine S.aureus mastitis.Experiment 4.In vitro effect of folic acid on bovine S.aureus mastitis was studied in cell model.The results demonstrated that folic acid accelerate candidate genes expression of bovine S.aureus mastitis and inhibited inflammation cytokines accumulation resistant to S.aureus invasion,which exerted positive and anti-inflammatory effect during MRSA or S.aureus infection.And the optimal treated dose of 5 ?g/mL folic acid in a dose-dependent manner.Finally,we presented a hypothesis that folic acid could provide potent protection against mastitis inflammation induced by MRSA or S.aureus via inhibiting the expression of major components(IL8 and IL6)of the NF-?B and JAK-STAT signaling pathway-linked in Mac-T cells.The data identified deeply candidate genes for bovine S.aureus mastitis,validated the gene function from epigenome molecular genetics aspects and explored the molecular mechanisms of folic acid resistance to bovine S.aureus mastitis,which provide the preliminary groundwork for epigenetic regulation and breeding for disease resistance of bovine S.aureus mastitis.