Study On Relationship Between Single Enzyme Activity Of Peroxiredoxin6 Of Host And Brucella Infection

Peroxiredoxin 6(Prdx6)is a member of the peroxidase family and protects the body from oxidative stress.Prdx6 keeps both peroxidase activity and phospholipase A2 activity.The motif of peroxidase is located at position 47 Cysteine,and the phospholipase A2 motif is located at position 32 and the two activities can exist independently.Moreover,the peroxidase activity and its phospholipase A2 activity of Prdx6 can be inhibited by of inhibitors of Mercaptosuccinate and MJ33 respectively.Brucellosis is a widespread epidemic of human and animal diseases,which poses a serious threat to human health and causes huge economic losses of animal husbandry.There are more than 60 types of animals infected,and the symptoms of sheep,cattle and pigs are the most serious.The pathogen of brucellosis is Brucella,which mainly parasitizes in macrophages,thereby escaping the humoral immune process of the body and relying on the nutrients of the host cells to survive and multiply.When the reproduction of Brucella comes to a certain amount,the host cell dies,releasing Brucella into the blood or lymph,causing the body to produce an inflammatory diseases accompanied by elevated body temperature,and the Brucella that migrates with the circulatory system can adhere to the surface of the new macrophage,and multiple cycles form a "wave heat".By now,the immune evasion mechanism of Brucella is still not clear.Therefore,the prevention and treatment of Brucella is necessary to let the host recognize Brucella and eliminate it in the immune response.The study of Brucella-associated host-reactive proteins is particularly critical.Our previous studies have shown that Prdx6 can enhance the intracellular survival ability of Brucella.Therefore,this experiment was designed to clarify which function of Prdx6 plays a major role in the parasitic process of Brucella.First,the peroxidase activity center of Prdx6 and the phospholipase A2 motif were mutated by SOE-PCR,and the obtained mutant gene was used to construct a single peroxidase motif and a single phospholipase A2 of Prdx6 eukaryotic expression vector.The activity of single peroxidase and phospholipase A2 active central protein was verified by substrate catalysis.Different plasmids were transfected into Raw264.7 mouse macrophages and then different inhibitors were added.Brucella S2 infects differently treated Raw264.7 cell,then we counted the number of live Brucella in macrophages at different time points,after that we analyzed the effect of different activities of Prdx6 on intracellular survival ability of Brucella.The cytokine chip was used to screen the differential expression cytokines of the host during the infection by Brucella,and the differentially expressed molecules were verified by real-time PCR.The experimental results showed that the peroxidase activity center and the phospholipase A2 motif of Prdx6 were mutated successfully.The Prdx6 single peroxidase motif and the single phospholipase A2 motif eukaryotic expression vector were constructed successfully.The verification of protein activity showed that the peroxidase activity of Prdx6 mutated successfully and the mutation had no effect on the phospholipase A2 activity;the phospholipase A2 activity of Prdx6 mutated successfully and the mutation had no effect on the peroxidase activity.Inhibitor analysis experiments showed that the inhibitors MJ33 and Mercaptosuccinate had no effect on the growth of Brucella.In the Raw264.7 cell treated with overexpressing different Prdx6 enzymatic activities proteins and adding different inhibitors of Prdx6,the MJ33 inhibitor group and the both inhibition group showed a significant decrease in parasitic number,while in Mercaptosuccinate inhibitor and the non-inhibitor group,the number of Brucella changes little.It is indicated that the PLA2 activity of Prdx6 plays a more important role than the NSGPx activity during intracellular parasites of Brucella.A total number of 14 differential expressed cytokines were screened out by cytokine chip combined with Prdx6 activity analysis.The differential expressed proteins were mainly concentrated in the Cytokine-Cytokine Receptor Interaction signaling pathway,phospholipases A2 activity of Prdx6 is related to MIP-1b,ICAM-1,Proranulin and DLL4.JAM-A and MCSF are related to the peroxidase activity of Prdx6.Prolatin is related to the process of Brucella infection.This study confirmed that the phospholipase A2 of Prdx6 has a stronger ability to promote the intracellular survival ability of Brucella compared with peroxidase activity of Prdx6,and a upstream and downstream signaling pathway molecule of single functional Prdx6 were found.This will provide an important theoretical clue for us to study the immune evasion mechanism of Brucella and the prevention and treatment of brucellosis.