Effect Of Transcriptional Regulator ArsR6 On Regulation Of Virulence In Brucella

Brucella is a facultative intracellular bacteria and it is a ball rods or short rods.Brucella can infect people and domestic animals,including cattle pigs and sheep,and cause brucellosis,which seriously threatens human and animal health.The prevention and control of brucellosis is still extremely serious due to Brucella attenuated live vaccines defects such as interference with diagnostic tests,pathogenicity for humans,potential to cause abortion in pregnant animals,among others.Therefore,it is very important to explore the pathogenesis of Brucella virulence factors for the research and development of brucellosis vaccine and the prevention and control of brucellosis.In recent years,it has been found that a large number of transcription regulators,including Ars R transcription regulators,are involved in the pathogenicity of bacteria.Ars R proteins are implicated in numerous biological processes,such as metal ion homeostasis,stress response,biofilm formation,primary and secondary metabolism and virulence.Brucella has 6 Ars R family members,among which 3 members are located on chromosome I and 3 members are located on chromosome II.The functions of Ars R family members in Brucella are not clear.In order to explore the role and function of Brucella BSS2＿I1540（Ars R6）,a member of Ars R family,ars R6 gene deletion strain（Δars R6）and ars R6 gene complementing strain（CΔars R6）was constructed.The effects of Ars R6 on Brucella virulence were explored by growth detection,stress resistance assays,intracellular survival detection in macrophages and mouse infection model experiment.The target genes of Ars R6 were identified by RNA-seq,Chip-seq EMSA and DNase I footprint assay.At the same time,the biological functions of related target genes were explored by Ultraviolet light analysis,stress resistance assays and mouse infection model experiment.The results are as follows:（1）The ars R6 gene deletion strain（Δars R6）and ars R6 gene complementing strain（CΔars R6）was constructed by homologous recombination and plasmid complementation.The growth curve ofΔars R6 was similar to that of the B.suis S2 in TSB,indicating that Ars R6 was not affected Brucella growth in these condition.The survival rate ofΔars R6was decreased in heat（p<0.05）,oxidative（p<0.05）and osmotic stress conditions（p<0.05）compared to the B.suis S2,but was restored partly or fully in CΔars R6.The spleen weight decreased at a higher rate than in mice infected with the B.suis S2 inΔars R6-infected mice（p<0.01）.Furthermore,the bacterial load in the spleen was decreased significantly inΔars R6-infected mice compared to B.suis S2-infected mice（p<0.05）.In addition,infection withΔars R6 impacted the pathological characteristics of the spleen less severely with a modest decrease in the white-to-red pulp ratio and slight white pulp expansion.（2）RNA-seq analysis revealed 438 differentially expressed genes between B.suis S2andΔars R6,including 158 up-regulated genes and 280 down-regulated genes,which were mainly involved in a variety of important biological processes,including“ribosome”,“ABC transport system”,“related metabolic pathways”and“related biosynthetic pathways”.ChIP-seq analysis revealed 453 peaks,of which 344 peaks were located on chromosome I and 109 peaks were located on chromosome II.The 453 peaks were mainly involved in“ABC transport system”,“two-component regulatory system”and“related biosynthetic and metabolic pathways”.The 34 potential target genes of Ars R6 were identified by RNA-seq and ChIP-seq data combined analysis,of which 8 genes were up-regulated and 26 genes down-regulated,including Brucella virulence factor Omps.（3）RNA-seq and ChIP-seq combined analysis showed that Ars R6 could be have a self-regulatory function.The EMSA,DNase I footprinting andβ-galactosidase activity assays showed that Ars R6 can bind to the cognate promoter region and Ars R6autorepresses the activity of its cognate promoter at the transcriptional level.Meanwhile,The EMSA assays showed Ni²⁺,Cd²⁺and Cu²⁺specifically impair the DNA-binding ability of Ars R6.The Ultraviolet light analysis showed that Ars R6 is a Ni²⁺,Cd²⁺and Cu²⁺-binding protein.The growth ofΔars R6 was inhibited adding Cu²⁺and Ni²⁺compared to B.suis S2,but fully restored in CΔars R6.The survival ofΔars R6 was decreased in RAW264.7 macrophages adding Cu²⁺compared to B.suis S2,but fully restored in CΔars R6（p<0.01）.The results demonstrate that Ars R6 is involved in maintaining Ni²⁺and Cu²⁺homeostatic levels in Brucella.（4）RNA-seq and ChIP-seq combined analysis showed that outer membrane protein25D（omp25D）was a potential target gene of Ars R6.The EMSA,DNase I footprinting and q RT-PCR showed that Ars R6 can bind to the omp25D promoter region to enhance omp25D expression（p<0.05）.The growth pattern ofΔomp25D was similar to that of the B.suis S2in standard medium,indicating that Omp25D was not affect Brucella growth in normal medium.However,the survival rate of theΔomp25D strain decreased under osmotic stress（p<0.05）,but had no effect on the survival rate under heat,oxidative,acid and polymyxin stress.This impairment was reversed fully in CΔomp25D.Omp25D was not affected the intracellular survival of Brucella in RAW264.7 macrophages.However,the spleen weight was decreased inΔomp25D-infected mice compared to that were infected with the B.suis S2（p<0.01）.The bacterial load in the spleen was decreased significantly inΔomp25D-infected mice compared to infection with B.suis S2（p<0.05）.In addition,infection withΔomp25D reduced the pathological characteristics of the spleen with a modest decrease in the white-to-red pulp ratio and slight white pulp expansion compared to spleens from mice infected with B.suis S2.In conclusion,Ars R family member Ars R6 autoregulates its expression and also modulates Omp25D expression to maintain metal ion homeostasis and virulence in Brucella,laying a foundation for further improving the pathogenesis of Brucella and the research and developing Brucella genetically engineered.