The Function Of Cu/Zn SOD In Brucella Abortus Intracellular Survival

The genus Brucella is small gram-negative,facultative intracellular bacteria of zoonotic importance.These bacteria are causative agents of brucellosis,a chronic disease of animals and humans.In animals,this disease often results in abortions,stillbirth and infertility leading to severe economic losses to livestock producers.Moreover,brucellosis causes undulant fever and debilitating disorders in acute infection of human;labor lost and orchitis are more commonly seen in subacute brucellosis of human.Brucellosis causes huge economic loss in animal husbandry and grave threat in public health.The Brucella Cu/Zn superoxide dismutase(Cu/Zn SOD)is an 18.5 kDa periplasmic protein encoded by Brucella sodC gene.Cu/Zn SOD showed huge potential not only as a DNA vaccine but also as a vector-delivered Brucella vaccine.More importantly,overexpression of Cu/Zn SOD in a RB51 vaccine strain(RB51SOD strain)significantly increases its vaccine efficacy against B.abortus challenge and decreases its survival rate in macrophages.However,the mechanism by which overexpressed Cu/Zn SOD mediates bacterial intracellular replication remains unknown.Therefore,this study was designed for determining the role of Cu/Zn SOD in regulating Brucella intracellular growth.In order to obtain the antiserum against Cu/Zn SOD protein,sodC was amplified by PCR and subcloned into pET-32 a for expression in E.coli.Then the antiserum was prepared in rabbits by immunization of the purified recombinant protein.The result of indirect ELISA showed that the antiserum has high antibody titer and appropriate specificity.To make Cu/Zn SOD overexpressed in B.abortus 2308,a constitutive Cu/Zn SOD expression system pBBR-trc-sod and an inducible Cu/Zn SOD expression system pBBR-lacPtrc-sod were constructed by genetic engineering in this research.The level of Cu/Zn SOD expression in B.abortus 2308 was analyzed by SDS-PAGE.Our data indicated that Cu/Zn SOD which expressed through the inducible protein expression system pBBR-lacPtrc-sod had high expression level and could react with antiserum specifically.To determine the role of Cu/Zn SOD in regulating Brucella intracellular growth,bone marrow derived macrophages(BMDMs)and BALB/c mice were infected with wild type,Cu/Zn SOD mutant strains and overexpressing Cu/Zn SOD.Our data indicate that overexpression of Cu/Zn SOD in Brucella can suppress bacterial replication within host cells.The last experiments focused on the mechanism by which overexpressed Cu-Zn SOD mediated bacterial intracellular replication.Immunoprecipitation and immunoblotting studies revealed that Cu/Zn SOD interacted with Sar1 but not with Rab2.Our data also demonstrated that Brucella infection-induced increase of Sar1 expression in host cells was not modulated by the overexpression of Cu/Zn SOD.Finally we found that overexpressed Cu/Zn SOD inhibits B.abortus intracellular growth via suppressing the activity of the small GTPase protein Sar1.In conclusion,these data demonstrated that overexpression of Cu/Zn SOD in B.abortus inhibits bacterial intracellular growth by abolishing Sar1 activity.Moreover,our data not only provide an importantly theoretical significance on designing a safe vaccine in the future,but help further elucidate the molecular mechanism of Brucella intracellular growth.