Impact of Hfq on Global Gene Expression and Intracellular Survival in <i>Brucella melitensis</i>

<div><p><i>Brucella melitensis</i> is a facultative intracellular bacterium that replicates within macrophages. The ability of brucellae to survive and multiply in the hostile environment of host macrophages is essential to its virulence. The RNA-binding protein Hfq is a global regulator that is involved in stress resistance and pathogenicity. Here we demonstrate that Hfq is essential for stress adaptation and intracellular survival in <i>B. melitensis</i>. A <i>B. melitensis hfq</i> deletion mutant exhibits reduced survival under environmental stresses and is attenuated in cultured macrophages and mice. Microarray-based transcriptome analyses revealed that 359 genes involved in numerous cellular processes were dysregulated in the <i>hfq</i> mutant. From these same samples the proteins were also prepared for proteomic analysis to directly identify Hfq-regulated proteins. Fifty-five proteins with significantly affected expression were identified in the <i>hfq</i> mutant. Our results demonstrate that Hfq regulates many genes and/or proteins involved in metabolism, virulence, and stress responses, including those potentially involved in the adaptation of <i>Brucella</i> to the oxidative, acid, heat stress, and antibacterial peptides encountered within the host. The dysregulation of such genes and/or proteins could contribute to the attenuated <i>hfq</i> mutant phenotype. These findings highlight the involvement of Hfq as a key regulator of <i>Brucella</i> gene expression and facilitate our understanding of the role of Hfq in environmental stress adaptation and intracellular survival of <i>B. melitensis</i>.</p></div

Proteomes of <i>B. melitensis</i> strains BM and BMΔvirB in the pH range of 4.0 to 7.0.

<p>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.</p

Primers used in this study.

<p>Primers used in this study.</p

Hfq upregulated the expression of Omp25/Omp31 and the Sigma factors.

<p>A. Fold changes in the transcript abundances of <i>omp25</i>, <i>omp25b</i>, <i>omp25c</i>, and <i>omp31</i> genes were detected by microarray and qRT-PCR in 16 MΔhfq, relative to 16 M. B, C. Transcript abundances of <i>rpoH1</i> (B) and <i>rpoE1</i> (C) were detected in the 16 M, 16 MΔhfq, and 16 MΔhfq-C during early logarithmic (EL), mid-logarithmic (ML), and stationary phases (SP). Significant differences between the transcription abundances of <i>rpoH1</i> and <i>rpoE1</i> in the mutant and parent strain are indicated as follows: *, P<0.001.</p

IFN-γ secretion by splenocytes stimulated with the recombinant <i>B. abortus</i> proteins.

<p>Spleen cells from mice immunized with S19 or PBS were stimulated in vitro with recombinant proteins, ConA (positive control), or complete medium (negative control). Supernatants were collected and IFN-γ was determined by using an ELISA Quantikine Mouse kit (R&D Systems). All assays were performed in triplicate and the concentrations for IFN-γ in the culture supernatants were calculated by using the quantification formula. Significant differences between S19-immunized mice and PBS-immunized mice are indicated as follows: *, P<0.001.</p

Humoral immune response induced by CobB, AsnC, and Cu-Zn SOD.

<p>BALB/c mice (n = 5) were inoculated intraperitoneally with purified recombinant proteins plus adjuvant. At 0, 15, 30, and 45 days post the immunization, serum samples were collected and IgG antibody titers were determined by ELISA. The values are mean titers ± SD of antibodies from the five mice.</p

SDS-PAGE of the recombinant proteins.

<p>The expression of recombinant fusion proteins were analyzed by SDS-PAGE. M, molecular size protein markers; lane 1–12, the purified fusion proteins: BAB1_0063, BAB1_0116, BAB1_0381, BAB1_0512, BAB1_0553, BAB1_0560, BAB1_0597, BAB1_0722, BAB1_0812, BAB1_0917, BAB1_1108, BAB1_1124.</p

Differentially expressed transcripts (upper graphs) and proteins (lower graphs) in the <i>B. melitensis</i> Δhfq mutant strain.

<p>Histograms show the number of differentially expressed genes and their distributions in the <i>B. melitensis</i> chromosome. The functional categories according to the <i>B. melitensis</i> 16 M genome sequence annotation and the KEGG database is shown to the right in circle charts. The number of genes belonging to each category are shown in brackets.</p

Proteins that induced strong T-cell responses in mice immunized with S19.

<p>Proteins that induced strong T-cell responses in mice immunized with S19.</p

Protection against <i>B. abortus</i> 544 infection induced by recombinant <i>B. abortus</i> proteins immunization.

a<p>Units of protection were obtained by subtracting the mean log₁₀CFU/spleen of the vaccinated group from the mean log₁₀CFU/spleen of the control (PBS) immunized group.</p>*<p>p value<0.05.</p