The Regulation Mechanism Of Intracellular Survival Regulated By Quorum Sensing Regulator VjbR In Brucella Melitensis

Brucellosis, which is caused by Brucella, is one of the most important bacterial zoonoses endemic in the world, especially in developing countries. These pathogens can affect a broad range of mammals and cause serious economic losses. The unique pathogenicity is the research focus on this pathogen. Brucella is an intracellular bacterium, and the virulence depends upon its ability to survive and replicate within host cells. Quorum sensing is a cell-density dependent global regulation system, which is used for cell to cell communication mediated by the signal molecule, N-acyl-homoserine lactone (AHL). However, the molecular mechanisms underlying regulation remain unclear.In the present study, homologous recombination was used to construct the vjbR deletion mutant, and then, the survival in macrophages and in mice of wild-type and mutant were compared to define related phenotypes affected by vjbR. The transcriptome of the mutant was compared to the wild-type strain to define the differentially transcribed genes, which may be regulated by vjbR. By combining the function information of the differentially transcribed genes and the alterations of the phenotypes in the mutant, the regulation mechanism was putatively explained.To define the vjbR related phenotypes and the target genes regulated by vjbR, the vjbR mutant and its complementary strain were to be constructed. Firstly, the kanamycin gene of pBBR1MCS-2 was amplified by PCR method and cloned at the multicloning site of pUC19 to generate a new suicide plasmid pUC19K, which was then used to construct the deletion mutant strain of 16M to generate16MΔvjbR. And then, the ORF of vjbR gene was cloned into pMD18-T vector to construct the complementary plasmid, which was then transformed into 16MΔvjbR to generate the complementary strain 16MΔvjbR-C. Semi-quantitative reverse transcription PCR results showed that transcription of the vjbR genes was detected in the wild-type and complementary strain, but not in the vjbR mutant, indicating that vjbR 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. Growth curve assays in vitro showed that the wild-type virulent strain 16M displayed higher growth rate than the vjbR mutant and the complementary strain at logarithmic phase. This implied that QS positively regulate growth of Brucella by regulating other related genes. Survival in macrophages and in mice showed that the mutant could invade the macrophages but the disruption of vjbR led to a decreased survival in macrophages and a drastic reduction in spleen or liver colonization in mice, implying that vjbR is essential for intracellular survival and chronic infection of Brucella. Survival under stress conditions showed that the vjbR mutant was sensitive to the stress conditions which simulated intracellular environments, including high salt, high osmosis, low pH, heat shock and oxidative stress. The vjbR mutant was more sensitive to high salt, osmotic stress, polymyxin B and sodium deoxycholate, indicating that vjbR is important for membrane integrity, and resistance to hostile environments. The vjbR mutant was also sensitive to oxidative and acidic stress, which simulated intracellular environment, indicating its role in adaptation to these harsh environments.After the elucidation of the intracellular survival phenotypes, a comparative transcriptome analysis was used to define the genes regulated by vjbR, with the aims to explain the possible regulation mechanisms of vjbR at transcription level. As a tool to perform the comparative transcriptome analysis, the non-redundant whole-genome DNA microarrays were developed based on the genomic sequences of B. melitensis strain 16M. A total number of 3968 genes was amplified by PCR and printed in duplicate onto glass slides. Fluorescently labeled probes were prepared by priming of genomic DNAs or cDNAs with random hexamers and extension with Klenow. Labelled DNAs or cDNAs were hybridized to the microarrays by methods of two-fluorescence comparative hybridization. The results were validated with semi-quantitative reverse transcription PCR.With the DNA microarray, we compared the transcription profiles of the vjbR mutant to that of 16M. Firstly, quantitative RT-PCR was used to determine the stress condition under which the transcription of vjbR was greatly activated. The results showed that the vjbR was greatly induced under acidified GEM pH4.0 for 2h. Then, comparative transcription analysis was performed under this condition. By a 2 fold change criteria, sixty three genes, including those encoding the type IV secetion system, energey/lipid/amino acid/carbohydrate/ion metabolism, cell envelop and hepothetical protein, were differentially transcribed in vjbR mutant. The results above indicated that the vjbR mutant showed several phenotype changes, reduced survival in the macrophage and mice, and further comfirmed its role in intracellular survival by affecting related phenotypes. Results from comparative transcription profiles showed that the vjbR may contribute the adaptation of Brucella to hostile environments and survival in the host cell by regulating the expression of related genes. These results expand our knowledge about roles of vjbR and molecular mechanism of Brucella intracellular survival.