BtaE, an adhesin that belongs to the trimeric autotransporter family, is required for full virulence and defines a specific adhesive pole of Brucella suis

Brucella is responsible for brucellosis, one of the most common zoonoses worldwide that causes important economic losses in several countries. Increasing evidence indicates that adhesion of Brucella spp. to host cells is an important step to establish infection. We have previously shown that the BmaC unipolar monomeric autotransporter mediates the binding of Brucella suis to host cells through cell-associated fibronectin. Our genome analysis shows that the B. suis genome encodes several additional potential adhesins. In this work, we characterized a predicted trimeric autotransporter that we named BtaE. By expressing btaE in a nonadherent Escherichia coli strain and by phenotypic characterization of a B. suis ΔbtaE mutant, we showed that BtaE is involved in the binding of B. suis to hyaluronic acid. The B. suis ΔbtaE mutant exhibited a reduction in the adhesion to HeLa and A549 epithelial cells compared with the wild-type strain, and it was outcompeted by the wild-type strain in the binding to HeLa cells. The knockout btaE mutant showed an attenuated phenotype in the mouse model, indicating that BtaE is required for full virulence. BtaE was immunodetected on the bacterial surface at one cell pole. Using old and new pole markers, we observed that both the BmaC and BtaE adhesins are consistently associated with the new cell pole, suggesting that, in Brucella, the new pole is functionally differentiated for adhesion. This is consistent with the inherent polarization of this bacterium, and its role in the invasion process

Expression of VjbR under Nutrient Limitation Conditions Is Regulated at the Post-Transcriptional Level by Specific Acidic pH Values and Urocanic Acid

VjbR is a LuxR homolog that regulates transcription of many genes including important virulence determinants of the facultative intracellular pathogen Brucella abortus. This transcription factor belongs to a family of regulators that participate in a cell-cell communication process called quorum sensing, which enables bacteria to respond to changes in cell population density by monitoring concentration of self produced autoinducer molecules. Unlike almost all other LuxR-type proteins, VjbR binds to DNA and activates transcription in the absence of any autoinducer signal. To investigate the mechanisms by which Brucella induces VjbR-mediated transcriptional activation, and to determine how inappropriate spatio-temporal expression of the VjbR target genes is prevented, we focused on the study of expression of vjbR itself. By assaying different parameters related to the intracellular lifestyle of Brucella, we identified a restricted set of conditions that triggers VjbR protein expression. Such conditions required the convergence of two signals of different nature: a specific pH value of 5.5 and the presence of urocanic acid, a metabolite involved in the connection between virulence and metabolism of Brucella. In addition, we also observed an urocanic acid, pH-dependent expression of RibH2 and VirB7, two additional intracellular survival-related proteins of Brucella. Analysis of promoter activities and determination of mRNA levels demonstrated that the urocanic acid-dependent mechanisms that induced expression of VjbR, RibH2, and VirB7 act at the post-transcriptional level. Taken together, our findings support a model whereby Brucella induces VjbR-mediated transcription by modulating expression of VjbR in response to specific signals related to the changing environment encountered within the host

Analysis of expression of VjbR under standard culture conditions.

<p>(A) Western blot analysis. <i>B. abortus</i> 2308 wild type strain was grown in rich medium (TSB). At different times, optical density at 600 nm (OD₆₀₀) was determined, and samples were subjected to 10% SDS-PAGE, transferred to PVDF membranes, and developed with anti-VjbR or anti-RibH1 polyclonal antibodies. The corresponding optical density at 600 nm (OD₆₀₀) of each sample was 0.5, 0.7, 1.7, 2.6, 2.95, 3.5, and 3.6, respectively. Results from a representative of two independent experiments are shown. (B) Analysis of the <i>vjbR</i> promoter activity. Strains <i>B. abortus</i> P<i>_vjbR</i>-<i>lacZ</i> (white circles) or <i>B. abortus ΔvjbR</i> P<i>_vjbR</i>-<i>lacZ</i> (black circles) were cultured in TSB. OD₆₀₀ and β-galactosidase activities were determined along the growth curve at the indicated times. Values are means ± standard deviations of duplicate samples from a representative of two independent experiments. Dotted lines indicate the best linear fit to the data. *, <i>P</i><0.05.</p

Identification of the Quorum-Sensing Target DNA Sequence and N-Acyl Homoserine Lactone Responsiveness of the Brucella abortus virB promoter▿

VjbR is a LuxR-type quorum-sensing (QS) regulator that plays an essential role in the virulence of the intracellular facultative pathogen Brucella, the causative agent of brucellosis. It was previously described that VjbR regulates a diverse group of genes, including the virB operon. The latter codes for a type IV secretion system (T4SS) that is central for the pathogenesis of Brucella. Although the regulatory role of VjbR on the virB promoter (PvirB) was extensively studied by different groups, the VjbR-binding site had not been identified so far. Here, we identified the target DNA sequence of VjbR in PvirB by DNase I footprinting analyses. Surprisingly, we observed that VjbR specifically recognizes a sequence that is identical to a half-binding site of the QS-related regulator MrtR of Mesorhizobium tianshanense. As shown by DNase I footprinting and electrophoretic mobility shift assays, generation of a palindromic MrtR-like-binding site in PvirB increased both the affinity and the stability of the VjbR-DNA complex, which confirmed that the QS regulator of Brucella is highly related to that of M. tianshanense. The addition of N-dodecanoyl homoserine lactone dissociated VjbR from the promoter, which confirmed previous reports that indicated a negative effect of this signal on the VjbR-mediated activation of PvirB. Our results provide new molecular evidence for the structure of the virB promoter and reveal unusual features of the QS target DNA sequence of the main regulator of virulence in Brucella

Analysis of expression of VjbR at pH 5.5 between 0 and 4 hours.

<p>(A) Wild-type strain <i>B. abortus</i> 2308 was grown in TSB until OD₆₀₀≤1.5. Subsequently, cultures were centrifuged and bacteria were resuspended in medium MM1 at pH 5.5 in the absence or presence of 5 mM urocanic acid. At the indicated times, OD₆₀₀ was determined, and samples were analyzed by western blot using anti-VjbR or anti-RibH1 polyclonal antibodies. Results are representative from one of two independent experiments. (B) Analysis of <i>vjbR</i> promoter activity. Strain <i>B. abortus</i> P<i>_vjbR</i>-<i>lacZ</i> was cultured in TSB and shifted to MM1 at pH 5.5 in the absence (gray circles) or presence (black circles) of 5 mM urocanic acid. Subsequently, β-galactosidase activities were determined at the indicated times. Values are means ± standard deviations of duplicate samples from a representative of two independent experiments.</p

Quantitative real-time PCR analysis of the effect of urocanic acid on gene expression.

<p>Strain <i>B. abortus</i> 2308 was grown in TSB and shifted to MM1 at pH 5.5, in the absence (gray bars) or presence (black bars) of 5 mM urocanic acid. Subsequently, total RNA was isolated and processed for quantitative real-time PCR to analyze the levels of <i>vjbR</i>, <i>ribH2</i>, <i>virB7</i>, <i>hutC</i>, and <i>ribH1</i> transcripts. Values are expressed as relative mRNA fold differences between treatments with (black bars) or without (gray bars) 5 mM urocanic acid, indicating means ± standard deviations of duplicate samples from a representative of two independent experiments. **, <i>P</i><0.01.</p

Comparison between glutamic acid and urocanic acid as inducer signals.

<p>Strain <i>B. abortus</i> 2308 grown in TSB and shifted to MM1 at pH 5.5, in the presence of 5 mM glutamic acid, 5 mM urocanic acid, or without the addition of any carbon source, as indicated. Subsequently, OD₆₀₀ was determined, and samples were analyzed by western blot using anti-VjbR, anti-RibH2, anti-VirB7, anti-HutC or anti-RibH1 polyclonal antibodies.</p

Urocanic acid also induced expression of other <i>Brucella</i> virulence-associated proteins.

<p>(A) Samples corresponding to <i>B. abortus</i> 2308 grown in TSB and shifted to MM1 at pH 5.5, or 7.0 in the absence or presence of 5 mM urocanic acid; were analyzed by western blot using anti-RibH2, anti-VirB7, anti-HutC or anti-RibH1 antibodies. (B) Analysis of <i>ribH2</i> (P<i>_ribH2</i>), <i>virB</i> (P<i>_virB</i>), <i>hut</i> (P<i>_hut</i>), or <i>ribH1</i> (P<i>_ribH1</i>) promoter activity. Strains <i>B. abortus</i> P<i>_ribH2</i>-<i>lacZ</i>, <i>B. abortus</i> P<i>_virB</i>-<i>lacZ</i>, <i>B. abortus</i> P<i>_hut</i>-<i>lacZ</i>, or <i>B. abortus</i> P<i>_ribH1</i>-<i>lacZ</i> were grown in TSB and shifted to MM1 at pH 5.5 in the absence (gray bars) or presence (black bars) of 5 mM urocanic acid. Subsequently, β-galactosidase activities were determined. Values are means ± standard deviations of duplicate samples from a representative of three independent experiments. **, <i>P</i><0.01.</p