Molecular Mechanism For Attenuated Virulence Of Brucella Abortus RfbE And VdtR Gene Deletion Mutants

Brucella is a Gram-negative facultative intracellular bacterium.According to its phenotype,it can be divided into smooth-and rough-type Brucella.Brucella has no classical virulence factors,and its virulence mainly depends on its ability to parasitize and survive in the host cell,and to escape the host immune response,inhibit apoptosis,and establish chronic infection in the host.Therefore,it is of great significance to identify Brucella virulence genes and investigate their function.It is reported that some smooth-type Brucella can spontaneously dissociate into rough-type mutants during infection,which contributes to infected macrophage death.This feature is essential for the spread and reinfection of Brucella among cells.However,the molecular mechanism of macrophage death caused by rough-type Brucella is incompletely clear.In this study,we used Brucella rfbE gene-deleted mutant(rough-type Brucella strain,named RB14 thereafter)as a model strain to explore the molecular mechanism of macrophages death caused by rough-type Brucella.Firstly,we verified that RB14-infected macrophages death is the type ? secretion system(T4SS)-dependent.To further investigate whether the macrophages death depends on Brucella T4SS components or secretion activity,we constructed two T4SS mutants with deletion of VirB4 or VirB11 ATP domain,both mutants affect only the T4SS secretion activity,but not T4SS components expression.Cytotoxicity analysis showed that RB14-induced macrophage death was dependent on T4SS secretory activity.In further studies,we used overexpression and transfection methods to evaluate the role of 15 reported T4SS effectors in cytotoxicity.The results showed that neither of the 15 effector overexpression strains were cytotoxic for macrophages.Moreover,transfection with each of the selected 10 effector or co-transfection with five of them showed no cytotoxic for macrophages either.The results suggested that the reported 15 effectors were not associated with macrophages death infected by RB14.Besides,we evaluated the roles of endoplasmic reticulum stress IRE1? pathway,Txnip-and Caspase-2 proteins in RB14-induced macrophage death.The results showed that IRE1? activation.Caspase and Caspase-2 activation were not related to macrophages death induced by RB14 infection.Meanwhile,casp2 and txnip gene knockout RAW264.7 cells showed similar cytotoxicity compared to parental RAW264.7 cells when infected by RB14.In conclusion,macrophages death infected by RB14 depends on T4SS secretory activity and is not associated with activation of IRE1?,Txnip,and Caspase-2 signal pathway.In previous study,we found a novel DeoR-family transcription regulator related to Brucella abortus virulence,designated as the vdtR gene.In this study,we successfully constructed a vdtR deleted mutant(?vdtR)and a in situ restored strain(?vdtR-Rev).The basic phenotype analysis showed that the ?vdtR mutant has no change of LPS structure and growth rate compared to the wild-type strain.Tolerance test showed that the vdtR deletion did not affect the resistance of Brucella to oxidative stress,nitriding stress,positive bactericidal peptides,nor in natural serum.The cell infection experiment demonstrated that the?vdtR mutant significantly reduced the ability of intracellular survival.The IFA analysis showed that the?vdtR mutant significantly reduced the ability of Brucella to escape lysosomal degradation and transport to the endoplasmic reticulum to form mature replicasomes.Animal infection experiment revealed that the bacterial loads in spleen of mice infected by the ?vdtR mutant was significantly lower than that of the wild-type strain,indicating that VdtR plays an important role in regulation of Brucella virulence.To explore the molecular mechanism of its regulation,in further study,we analyzed the differentially transcribed genes of wild-type strain and the ?vdtR mutant based on bacterial transcriptomics.Based on qPCR verification,we found that VdtR regulates seven target gene expressions,which are BAB_RS27025-55 gene cluster and BAB_RS27060 gene.RT-PCR analysis showed that genes in the BAB_RS27025-55 gene cluster are co-transcribed.Afterward,the analysis of promoter activity revealed that only the promoter of the BAB_RS27055 gene depends on the expression of the VdtR protein.The results suggested that the VdtR protein plays a role by regulating an operon of the BAB_RS2 7025-55 gene cluster.EMSA was performed to verify the direct regulation of VdtR,which showed that VdtR protein can directly bind to the promoter region of the BAB_RS27055 gene.During the infection process,the BAB_RS27025-55 gene cluster showed significant down-regulated expression in intracellular Brucella by qPCR analysis,and the expression of gene cluster also depends on the VdtR protein in intracellular Brucella,which showed a significant up-regulation in the ?vdtR mutant compared to the wild-type strain within macrophages.In summary,the VdtR plays an important role in regulating Brucella virulence,including escape from the degradation of lysosomes and participation in the formation of mature replicasomes,and the result also showed that the VdtR inhibited the expression of BAB_RS27025-55 gene cluster,which suggested that metabolic pathways mediated by the gene cluster may be necessary for the intracellular survival of Brucella.This study provides novel insights into Brucella pathogenesis.