Immune Protection Of B.abortus S19 BvfA Deletion Strain And Interactionomics With Sertoli Cells

Brucellosis,a global zoonotic disease,is seriously affecting the health and economic development of residents in endemic areas,and the prevention and control of brucellosis is very important.At present,immune prevention is one of the main means to control brucellosis.However,there are many shortcomings in the existing animal vaccines,such as residual toxicity and interference with routine serological tests.Therefore,it is a new trend to prepare effective and safe virulence gene deletion live vaccine for brucellosis control.bvfA(Brucella virulence factor A)is a unique virulence gene of Brucella,which is necessary for proliferation in cells.bvfA gene is considered to be necessary for Brucella to establish replication niche in cells.However,the biological function mechanism of bvfA gene in strains is unclear,especially the research on the function and regulation mode of bvfA gene in Brucella infection of target cells is still blank.In this study,the gene sequence encoding BvfA protein was determined by open reading frame prediction software,and the distribution frequency of bvfA gene in clinical Brucella isolates was figured by PCR technology.B.abortus S19?bvfA strain and its complement strain were constructed using the principle of gene homologous recombination and bacterial conjugation transfer,and stress ability of B.abortus S19?bvfA strain were tested by simulating the cell environment.The proliferation ability of B.abortus S19?bvfA strain in TM4 Sertoli cells(TM4 cells)was evaluated by CCK-8 method.The damage of B.abortus S19?bvfA strain to TM4 cells was determined by LDH method.The immunogenicity and immunoreactivity of B.abortus S19?bvfA strain were evaluated by ELISA.Tissue bacteria count was used to evaluate the immune protection of B.abortus S19?bvfA strains against wild strain.HE staining was used to study the difference of tissue damage between B.abortus S19?bvfA strain and other Brucella strains.The functions of bvfA gene in B.abortus S19 and TM4 cells infecting Brucella were analyzed by using Illumina Hiseq sequencing and liquid chromatography-mass spectrometry at transcriptome,proteome and metabolome level.Results:(1)The gene sequence encoding BvfA protein was obtained,which was 336 bp.the gene was submitted to Genbank and the gene sequence number was obtained(MN651999).It was confirmed that bvfA gene was 100%preserved in clinical isolates.(2)B.abortus S19?bvfA strain grew slower than its parent strain and complement strain.Under the conditions of heat,high salt,high osmotic pressure,strong acid or antibiotics,the growth of B.abortus S19?bvfA strain was inhibited,but it grew well only under oxidative pressure(p<0.05).(3)At 12 h,24 h and 48 h,the number of colonies that B.abortus S19?bvfA invaded TM4 cells was 90%,95%and 96%of that of B.abortus S19 strain,respectively.The mortality of TM4 cells infected with B.abortus S19?bvfA strain and B.abortus S19 strain were 13.39%and 7.14%(12 h),14.97%and 12.40%(24 h),33.8%and 31.2%(48 h),respectively.(4)The antibody titers in the serum of mice immunized with B.abortus S19?bvfA strain and B.abortus S19 strain showed an upward trend in 1-3 weeks,and tended to be stable in 4 weeks.There was no significant difference in antibody levels between them(P>0.05).Serum IFN-? content increased from 1 week to 2 weeks,IL-2 increased from 1 week to 4 weeks,and IL-4 increased from 2 weeks to 3 weeks.The antibody titers of BvfA protein and mice serum reaction immunized with B.abortus S19?bvfA strain and B.abortus S19 strain were 2.16 and 4.21(2 weeks),or 2.16 and 4.52(3 weeks),respectively.The amount of spleen bacteria in B.abortus S19?bvfA strain immunized mice was 86%(1 week),93%(2 weeks)and 84%(4 weeks)of B.abortus S19 strain immunized mice,respectively.When exposed to B.melitensis 16 M,the amount of spleen bacteria of mice immune to both B.abortus S19?bvfA strain and B.abortus S19 strain was 0.03%and 2.9%(1 week),or 0.75%and 0.33%(2 weeks)of non-immune mice,respectively.Moreover,when exposed to B.abortus 2308,the amount of spleen bacteria of mice immune to both B.abortus S19?bvfA strain and B.abortus S19 strain was 9.8%and 6.5%(1 week),or 6.8%and 6.8%(2 weeks)of non-immune mice,respectively.(5)Correlation analysis between transcriptomics and proteomics showed that the expression of rpoA,rpoC and rpoB genes in RNA polymerase of B.abortus S19?bvfA strain decreased.Proteomics and metabolomics showed that the difference between B.abortus S19?bvfA strain and B.abortus S19 strain was ABC transport,purine metabolism and aminoacyl-tRNA biosynthesis.(6)Transcriptomics and proteomics showed that the difference between TM4 cells infected with B.abortus S19?bvfA strain and B.abortus S19 strain was retrograde endocannabinoid signaling pathway,mineral absorption and sphingolipid signaling pathway.Conclusions:(1)BvfA gene is involved in the proliferation of B.abortus S19 strain in host cells and its adaptation to intracellular environment.B.abortus S19?bvfA strain is more harmful to TM4 cells than other Brucella strains in the early stage(12 h).Intraperitoneal injection of B.abortus S19 ?bvfA strain can induce immune response in 1 week and it has a good immune protection effect.B.abortus S19?bvfA strain has less toxicity and pathological damage to mouse spleen and testis than B.abortus S19 strain.(2)BvfA gene mainly participates in the RNA polymerase,ABC transport,purine metabolism and aminoacyl-tRNA biosynthesis in B.abortus S19.BvfA gene can interfere with mineral absorption,sphingolipid signaling pathway and function of mitochondrial NADH ubiquinone oxidoreductase complex.The expression level of CatSper2 protein decreases in TM4 cells infecting Brucella,which suggests that CatSper channel might be the main target of infertility caused by Brucella in the complex.The expression level of P53 protein increases,which suggests that bvfA gene can inhibit the apoptosis of TM4 cells.