Study On The Role And Mechanism Of BrbI In Brucella

Brucella is Gram-negative,facultative intracellular pathogen and can infect human,cow,goat,pig,and dog.Brucellosis caused by Brucella is a serious and global zoonotic infectious disease,which threaten humans' health and the development of animal husbandry.However,the pathogenesis of Brucella is still need to be investigated and the available brucellosis vaccines interfere with serological diagnoses and retain enough residual virulence to infect humans,which seriously hinder the prevention and control of brucellosis.BAX inhibitor 1(BI-1)is a transmembrane protein conserved in eukaryotes,and can suppress cell death induced by multiple stimuli.In prokaryotes,the homologs of BI-1 also exist extensively,including Escherichia coli YccA and Bacillus subtilis YetJ,but their biological functions and molecular mechanism are still unclear.Brucella also conserved a homolog of BI-1,we refer to this Brucella homolog of BI-1 as BrBIIn order to explore the biological function of brbI,we constructed a Brucella brbI deletion mutant strain through homologous recombination method and constructed its complemented strain through expressing brbl exogenously in the mutant strain.Then,using scanning electron microscopy,biochemical tests,stress tests and other biological methods,as well as proteomics and transcriptomics to investigate the biological function of brbI in Brucella and its molecular basis and mechanism.Moreover,we explored the role of brbl in the virulence and immunogenicity of Brucella at both the in vitro and in vivo levels through ELISA,HE staining and liquid-phase microarray.The main results are as follows:(1)The Brucella brbI deletion mutant strain AbrbI was constructed by homologous recombination.An expression plasmid pBBARpc that can replicate and express exogenous gene in Brucella and contains an ampicillin resistance gene and the promotor for gentamicin-resistant gene was constructed.The complemented strain ?AbrbI pBBARbrbI was constructed through expressing brbl exogenously in the mutant strain using the plasmid pBBARpc(2)The brbl deletion mutant strain showed a significantly reduced growth rate in both solid and liquid media,but the biochemical test results showed that the brbI deletion had no significant effect on the physiological metabolism of Brucella.The results of scanning electron microscopy and propidium iodide staining showed that the cell division and cell viability of the brbl deletion mutant strain were significantly disrupted,and the brbl deletion significantly inhibited the resistance of Brucella to low pH,hydrogen peroxide,polymyxin B,and lincomycin.Besides,the membrane properties of Brucella were also significantly altered because of the deletion of brbI.These results indicate that brbI has a global biological functions in Brucella.(3)The deletion of brbI significantly inhibited the early and late survival of Brucella in RAW264.7 murine macrophages,and significantly promoted the secretion of IL-1? and IL-10.In the mouse model,the virulence of the brbl deletion mutant strain was also significantly attenuated,which was mainly manifested as the significant reduced splenic bacterial load,no splenomegaly,and slight pathological changes in the spleen.In addition,although the deletion of brbl has no significant effect on the inflammatory response in mice but significantly improved the humoral immunity induced by Brucella.(4)The results of proteomics and transcriptomics showed that a total of 363 differentially expressed proteins and 677 differentially expressed mRNAs were respectively obtained in the brbl deletion mutant strain,which were mainly associated with membrane proteins(such as Hf1K,Hf1C,BamA,OMP2a,OMP2b,OMP22,OMP25b,OMP31),cell division proteins(FtsQ,FtsB,FtsL,FtsI),transcription factors or regulatory factors,peptidases/protease,transporter,and transferase,which may reveal the molecular basis of the biological roles of brbI in Brucella.As HfIK and Hf1C are negative regulators for FtsH,a metalloproteinase located in membrane,we integrated proteomic and transcriptomic analyses to speculate a possible mechanism of brbI in the deletion of brbl up-regulated HflK and HflC,suppressing the proteinase activity of FtsH,which further affected the expression of multiple genes directly or indirectly.To sum up,this study revealed the biological function of the Brucella homologue of BI-1,BrBI,and explored its molecular basis and molecular mechanism via integrated proteomic and transcriptomic analysis,which expanded the study of prokaryotic homologs of BI-1.In addition,this study revealed the important role of brbI on the virulence and immunogenicity of Brucella,which suggests brbl to be an ideal target for developing new brucellosis vaccines.