The Molecular Mechanism Of Intracellular Survival Regulated By The Type IV Secretion System In Brucella Melitensis

Brucellosis,which is caused by Brucella,is one of the most important bacterial zoonoses endemic in many countries,especially in developing countries.Brucella is an intracellular bacterium,and the virulence of Brucella depends upon its ability to survive and replicate within host cells.TypeⅣsecretion system(T4SS)is a recently described family of multi-protein complex which serve to secrete macromolecules.More recently, the typeⅣsecretion system encoded by the virB operon(virB)was identified in Brucella. The virB region is essential for intracellular survival and multiplication in Brucella, nevertheless the molecular mechanisms underlying regulation remain unclear.In the present study,homologous recombination was used to construct inactivation mutant of the virB operon,and then,the intracellular survival related phenotypes of wild-type and mutant were compared to define the ones affected by the virB operon.And the proteome of the mutant was compared to the wild-type strain to find the differentially expressed proteins,which may be regulated by the virB operon.By combining the function information of the proteins and differences in expression level,the alterations of the phenotypes in the mutant were putatively explained.Thereafter,the transcription profiles of virB and the genes regulated by virB were analyzed in Brucella under conditions simulating infection.With this information,the putative molecular mechanism of intracellular survival regulated by the T4SS in Brucella was elucidated.In order to analyze the T4SS related phenotypes,the virB mutant and complementary strains were to be firstly constructed.The 11 sequential ORFs encoding TypeⅣsecretion machinery comprise virB,which made virB inactivation possible by promoter deletion.By using the suicide plasmid pUC19-SacB,we successfully constructed the mutant of virB operon,and then,the 11kb virB operon of Brucella was cloned by plasmid rescue and subcloned into pBBR1MCS-5 capable of replication in Brucella to construct the complementary plasmid,which was then successfully used to construct the complementary strain.Semi-quantitative RT-PCR results showed that transcription of the virB genes was detected in the wild-type and complementary strain,but not virB mutant,indicating that virB was successfully inactivated in the mutant and restored in the complementary strain.Then,the related intracellular survival phenotypes of wild-type,mutant and complementary strain were compared in order to define the phenotypes affected by the virB operon,especially those related with Brucella intracellular survival.Results from cell adherence and invasion assays showed that the virB mutant displayed increased attachment to cells,but the internalization was not changed,indicating surface modifications. Compared with the wild-type strain,the virB mutant had a decreased survival under the stress conditions and in the macrophages.We also found that the disruption of virB led to a drastic reduction in spleen or liver colonization in mice,and the virB mutant was unable to establish chronic infection,implying that T4SS is essential for intracellular survival and chronic infection of Brucella.Meanwhile,the virB mutant showed reduced virulence and induced miner histopathogenic lesions in mice.The virB mutant was more sensitive to high salt,osmotic stress and polymyxin B,thereby indicating that virB operon is important for membrane integrity,and resistance to hostile environments.The virB mutant was also sensitive,to oxidative and acidic stress,which simulating intracellular environment, indicating its role in adaptation to these harsh environments.The virB mutant produced exopolysaccherides(EPS)and aggregated at high cell density,a key characteristic of biofilm,implicating that Brucella can form biofilm and the virB play some roles in this process.In addition to its induced expression,the virB operon of B.melitensis is also constitutively expressed.In the phenotype assays,we found that the complementary strain, where the virB was highly transcribed,showed reduced survival in macrophages and mice, implying the overexpression of virB is harmful for intracellular survival.The overexpression of virB induced cytotoxicity in macrophages,as well as the cytotoxicity observed with wild-type strains at extreme multiplicities of infection(＞1000).However, the virB mutant did not show cytotoxicity,which is presented that proper induction of virB is essential for Brucella intracellular survival,either inactivation or overexpression of it is harmful for the survival.After the elucidation of the intracellular survival phenotypes,a comparative proteome analysis was used in the present study to define the proteins regulated by T4SS,with the aims to explain the possible regulation mechanisms of T4SS.Firstly,quantitative RT-PCR was used to determine the transcription of virB under stress conditions simulating the intracellular environment of macrophage to define the one that the virB operon was greatly activated.The results showed that the virB was greatly induced under acidified GEM for 3 h.Thenceforce,comparative proteomics analysis of whole cell proteins and outer membrane proteins were performed under in vitro conditions where virB was highly activated,and the differentially expressed proteins were identified by MALDI-TOF-MS. Proteins associated with outmembrane,stress response proteins,transcriptional regulating, lipid/energy/iron/amino acid metabolism were differentially expressed between wild-type and the virB mutant strain.The results showed that,1.The disruption of virB led to drastic surface modifications,and the permeability of outer membrane was changed,indicating its role in maintaining membrane integrity.2.Omp25 and Omp31 proteins had post-translational modifications,and these modifications may be affectd by the virB operon.3.Compared with the wild-type strain,several important virulence proteins involved in intracellular survival,including Omp25,DnaK,HtrA,VjbR and GntR,were down-regulated in the virB mutant.All these virulence proteins are essential for intracellular survival and multiplication in Brucella.4.Proteins involving proteolytic degradation,including heat shock protein,molecular chaperon and proteolytic enzymes, were down-regulated in the virB mutant.Intracellular proteolytic degradation is important in bacteria for the elimination of damaged proteins,modulation of protein levels,and maintenance of amino acid pools,thus these proteins are crucial for the intracellular survival of Brucella.5.The disruption of virB led to the alterations of proteins related to the metabolism of iron.6.Compared to the wild-type strain,the proteins associated with energy metabolisms,amino acid metabolism and proteins synthesis were down-regulated in the virB mutant,which all demonstrated the metabolism was repressed,and T4SS promotes adaptation to nutrient limitation.To sum up,all these data present that the virB operon controls the intracellular survival in Brucella by regulating the expression of relevant proteins.At last,the transcription profiles of virulence genes virB,vjbR,dnaK,htrA,omp25,gntR under in vitro stress and during host cell infection were analyzed by using quantitative RT-PCR.The results showed that the virulence genes virB,vjbR,dnaK,htrA,omp25,gntR had a high transcription level under in vitro stresses(heat and acid shock,and oxidative stress)and during macrophage infection,indicating that these virulence genes were activated under these condtions,contributing to the intracellular survival of Brucella. When compared with the wild-type strain,the transcription of vjbR,dnaK,htrA,omp25 and gntR in the virB mutant was decreased,and restored in the complementary strain, indicating that transcription of these genes are regulated by the virB operon under in vitro stresses and during macrophage infection.By regulating virulence proteins,T4SS promote the adapation of Brucella to these conditions for intracellular survival.The decreased transcription of the virulence genes was consistent with an observed decreased survival of the virB mutant under these conditions.These data indicate that the virB operon controls the intracellular survival of Brucella by regulating the expression of relevant virulence proteins.The virB operon is activated in acidified environment both in vitro acid and BCV, indicating the acidification of BCVs appears to be the activating signal of virB and these virulence genes,as they were activated following acid shock.Thus,Brucella may adapt to its intracellular environment and avoid degradation by the coordinated regulation of gene expression in the early phase of infection,vjbR is a quorum sensing regulator,a cell density dependent regulator.Results from RT-PCR indicated that the virB and vjbR positively regulate each other,and this regulation is growth phase dependent.These results indicated that the virB mutant showed several phenotype changes,reduced survival in the macrophage and mice,and further confirmed its role in intracellular survival by affecting related phenotypes.Results from comparative proteome and quantitative RT-PCR showed that the virB operon may contribute the adaptation of Brucella to hostile environments and survival in the host cell by promoting membrane integrity and regulating the expression of virulence proteins associated with the intracellular survival.These findings expand our knowledge of the T4SS,and provide various important clues for the understanding of the molecular mechanism of intracellular survival of Brucella.