Comparative proteomics analyses reveal the virB of B. melitensis affects expression of intracellular survival related proteins

Abstract

Background: Brucella melitensis is a facultative, intracellular, pathogenic bacterium that replicates within macrophages. The type IV secretion system encoded by the virB operon (virB) is involved in Brucella intracellular survival. However, the underlying molecular mechanisms, especially the target proteins affected by the virB, remain largely unclear.

Methodology/principal findings: In order to define the proteins affected by virB, the proteomes of wild-type and the virB mutant were compared under in vitro conditions where virB was highly activated. The differentially expressed proteins were identified by MALDI-TOF-MS. Forty-four down-regulated and eighteen up-regulated proteins which exhibited a 2-fold or greater change were identified. These proteins included those involved in amino acid transport and metabolism, lipid metabolism, energy production, cell membrane biogenesis, translation, post-translational modifications and protein turnover, as well as unknown proteins. Interestingly, several important virulence related proteins involved in intracellular survival, including VjbR, DnaK, HtrA, Omp25, and GntR, were down-regulated in the virB mutant. Transcription analysis of virB and vjbR at different growth phase showed that virB positively affect transcription of vjbR in a growth phase dependent manner. Quantitative RT-PCR showed that transcription of these genes was also affected by virB during macrophage cell infection, consistent with the observed decreased survival of the virB mutant in macrophage.

Conclusions/significance: These data indicated that the virB operon may control the intracellular survival of Brucella by affecting the expression of relevant proteins.

Figure 1. Construction and confirmation of BMΔvirB and BM-IVGT strains.

A) The promoter was amplified from BM and BM-IVGT, but not from BMΔvirB, showing that the promoter was deleted in BMΔvirB and complemented in BM-IVGT. B) RT-PCR amplification of virB1 and virB8 showed that the two genes could be amplified from BM and BM-IVGT, but not BMΔvirB, indicating that transcription of virB genes was inactivated in BMΔvirB and restored in BM-IVGT.

Figure 2. Determination of in vitro induction conditions of virB.

A: Transcription of virB under different in vitro condition. BM was firstly cultured in TSB to logarithmic phase and then subjected to different stresses. RNA was isolated and transcription of virB was quantified by qRT-PCR. virB was greatly activated under GEM4.0. B: Transcription of virB at different incubation time in GEM4.0. BM was subjected to different incubation time in GEM4.0 and then transcription of virB was quantified. The virB was greatly activated at 3 h.

Figure 3. Proteomes of B. melitensis strains BM and BMΔvirB in the pH range of 4.0 to 7.0.

BM (A) and BMΔvirB (B) were firstly cultured in TSB to logarithmic phase and then transferred into GEM4.0 for 3 h. Protein extracts (800 µg) of each strain were focused with IPG strips and run on 12% SDS-PAGE gels. The gels were stained with Coomassie Brilliant Blue R-350 and subjected to 2 DE analyses. The gels of BM and BMΔvirB were scanned and compared with ImageMaster™ 2D Platinum software. The labeled protein spots were the ones whose expressions were changed over 2 folds.

Figure 4. Confirmation of comparative proteome by semi-quantitative RT-PCR.

A: Spot distribution of the selected virulence related protein on gel of BM. Several protein spots with different MW and pI of omp25 were identified. B: Relative transcription of virulence related genes in BM and BMΔvirB. BM and BMΔvirB were firstly cultured in TSB to logarithmic phase and then transferred into GEM4.0 for 3 h. RNA was isolated and relative transcription of virulence related genes was quantified by normalization with 16S rRNA. These genes were transcribed at a lower level in BMΔvirB than in BM.

Figure 5. Transcriptional profile of virB during host cell infection.

Macrophage like cell J774A.1 were infected with BM, BMΔvirB and BM-IVGT. At different time (0, 12, 24 and 48 h) post the infection, RNA was isolated from the infection mixtures and reverse transcribed into cDNA. Transcription of virB genes were then quantified by qRT-PCR. A: Relative transcription of virB1 and virB8. The virB1 and virB8 was identically transcribed. B: Relative transcription of virB8 in BM, BMΔvirB and BM-IVGT. Transcription of virB8 peaked at 12 h and then decreased in BM and BM-IVGT. No transcription of virB8 was detected in BMΔvirB and uninfected macrophage cells.

Figure 6. Transcription of vjbR, dnaK, htrA, omp25, and gntR during host cell infection (⧫— BM; ▪ — BMΔvirB; ▴ — BM-IVGT; •—BLANK).

Macrophage cells were infected with BM, BMΔvirB and BM-IVGT. At different time (0, 12, 24 and 48 h) post the infection, RNA was isolated from the infection mixtures and reverse transcribed into cDNA. Transcription of selected genes was then quantified by qRT-PCR.

Figure 7. virB positively regulates vjbR.

A) Transcription of vjbR and virB8 during the early logarithmic (EL), mid-logarithmic (ML), late logarithmic (LL), and stationary phases (SP) demonstrate that vjbR and virB8 are greatly transcribed in EL and ML. B) Transcription of vjbR in BM, BMΔvirB, and BM-IVGT at EL, ML, LL, and SP show that vjbR is down-regulated at EL in BMΔvirB. C) BMΔvirB demonstrated higher growth rate than BM and BM-IVGT. D) virB and vjbR positively regulate each other and form a positive regulation circuit.