Molecular Mechanism Of BvrA Regulating Brucella Abortus Virulence

Brucella is an important zoonotic bacterial pathogen with no classical virulence factors,and its virulence is mainly reflected in its ability to invade host cells and proliferate intracellularly.After invading cells,Brucella rapidly adjusts the expression of a series of genes,alters metabolic pathways to adapt to the intracellular environment,and modifies pathogen-associated molecular patterns to escape the host immune response.Therefore,functional research of regulatory genes is important for elucidating Brucella pathogenicity.The LysR-type transcriptional regulators can directly bind to the promoter regions of target genes and repress or activate their transcription,thus having a significant impact on Brucella survival,replication and establishment of chronic infection in the host.In this study,we identified a LysR-type transcriptional regulator involved in the regulation of Brucella abortus pathogenesis,and named it BvrA.Around BvrA,this experiment explored the molecular mechanism of BvrA regulation of Brucella abortus pathogenesis.Firstly,the mutant strainΔbvrA and the complement strainΔbvrA-Com of bvrA gene were constructed using Brucella abortus S2308 as the parental strain,and the tolerance of different strains to bactericidal factors was determined.The results suggested thatΔbvrA showed enhanced sensitivity to the positive bactericidal peptide polymyxin B and anionic surfactant SDS,but no change in sensitivity to H₂O₂.The cell infection assay revealed that the ability ofΔbvrA to invade macrophages and to proliferate intracellularly was not significantly different compared with the parental strain.The mouse infection assay revealed thatΔbvrA showed a significant decrease in virulence after 4 weeks of infection in mice,and the deficient strain partially reverted to pathogenicity after reverting to bvrA,the results suggested that BvrA was involved in regulating Brucella pathogenicity.Secondly,we screened and validated the key target genes regulated by BvrA by comparative transcriptome and fluorescence quantitative PCR,and identified the target genes bab＿RS29675,bab＿RS33975,bab＿RS33205,bab＿RS30460 and bab＿RS30465directly regulated by BvrA by Electropphoretic mobility shift assay.Finally,this study further explored the relationship between BvrA regulatory target genes and Brucella virulence.The results of tolerance assay showed that the mutant of bab＿RS33975 and bab＿RS33205 attenuated the ability of Brucella to tolerate polymyxin B.The mutant of opp A1 attenuated the transport and utilization of oligopeptides by Brucella and significantly attenuated the ability of bacteria to tolerate SDS.The results of cell infection assay showed that the mutant of bab＿RS33975 and bab＿RS33205 significantly attenuated the ability of Brucella to invade cells and proliferate intracellularly.Finally,the mouse infection assay confirmed the involvement of BvrA in regulating Brucella pathogenicity at least through bab＿RS33975 expression.In conclusion,this study revealed that BvrA plays an important role in regulating Brucella pathogenicity.BvrA affects Brucella peptide transport and utilization,resistance to positive bactericidal peptide and surfactant killing,intracellular survival and pathogenicity to mice by regulating expression of several genes,providing a reference for the study of Brucella pathogenesis.