The Effect Of OxyR On The Growth Resistance And Intracellular Survival Of Brucella

Brucellosis is a severe zoonotic disease caused by Brucella,causing a major threat to human health and stockbreeding.However,the vaccines and diagnostic methods used for the prevention and control of Brucella still need to be optimized.Therefore,the need of understanding the pathogenic mechanism of Brucella has become urgent.Studies have shown that high levels of reactive oxygen species(ROS)can cause abnormal metabolism of bacteria,which can be killed because ROS exceeds the physiological regulation level.Brucella has to deal with the endogenous ROS produced by the respiratory metabolism process and the exogenous ROS caused by the phagocytes.Brucella uses superoxide dismutase,catalase,and peroxidase to resist internal ROS and exogenous oxidative damage.It is known that OxyR is a ubiquitous hydrogen peroxide-inducible genes activator in bacteria,which can regulate the expression of bacterial antioxidant-related enzymes to reduce bacterial damage caused by ROS.At present,the mechanism of OxyR in Escherichia coli and Pseudomonas aeruginosa research more in-depth and wide-ranging,but the antioxidant effect of OxyR in the survival and infection of Brucella has not been intensively studied.Therefore,based on the construction of the OxyR deletion and complementary strain in Brucella,this study investigated the effect of OxyR on the growth and stress of Brucella and its effect on the cellular immune activity in the infection of macrophages by Brucella.The research content and results are as follows:1.Using Brucella S2(B.suis S2)as the parent strain,the OxyR deletion strain(?OxyR)was constructed by the principle of homologous recombination,and then the OxyR complementary strain(C-?OxyR)was obtained by transferring the expression vector into the deletion strain.2.Aligning the amino acid sequence of Brucella OxyR with Clustal and drawing a phylogenetic tree,which have shown that OxyR is highly conserved in Brucella,and OxyR has low homology with Escherichia coli and Pseudomonas aeruginosa compared to Brucella3.Cultivation and stress tests have shown that the loss of OxyR slows down the growth of B.suis S2 in vitro and increases sensitivity to acid stress,polymyxin B stress and oxidative stress.When ?OxyR under oxidative stress,the level of peroxidase decreased sharply.4.Through the B.suis S2??OxyR and C-?OxyR infection RAW264.7 assay,the results showed that the intracellular proliferation has no significant different between the strains;the NO content measurement results showed that the loss of OxyR caused NO secretion significant decrease at 12 h and 24 h;RT-PCR results showed that OxyR deletion caused a significant decrease in the gene transcription of IL-6 and IL-1? in macrophages.The results indicate that OxyR is highly conserved in Brucella;the absence of OxyR slows down the growth of B.suis S2 in vitro,but does not affect its proliferation in macrophages;it reduces the ability of B.suis S2 to resist oxidative stress.Moreover,the transcription of inducible nitric oxide synthase and pro-inflammatory factors IL-6 and IL-1?induced by mouse macrophages was reduced.It will lay the foundation for further research on the function of OxyR.