Type IV secretion systems (T4SS) are multicomponent machineries involved in the translocation of effector molecules across the bacterial cell envelope. The virB operon of Brucella abortus codes for a T4SS that is essential for virulence and intracellular multiplication of the bacterium in the host. Previous studies showed that the virB operon of B. abortus is tightly regulated within the host cells. In order to identify factors implicated in the control of virB expression, we searched for proteins of Brucella that directly bind to the virB promoter (P virB ). Using different procedures, we isolated a 27-kDa protein that binds specifically to P virB . This protein was identified as HutC, the transcriptional repressor of the histidine utilization (hut) genes. Analyses of virB and hut promoter activity revealed that HutC exerts two different roles: it acts as a coactivator of transcription of the virB operon, whereas it represses the hut genes. Such activities were observed both intracellularly and in bacteria incubated under conditions that resemble the intracellular environment. Electrophoresis mobility shift assays (EMSA) and DNase I footprinting experiments revealed the structure, affinity, and localization of the HutC-binding sites and supported the regulatory role of HutC in both hut and virB promoters. Taken together, these results indicate that Brucella coopted the function of HutC to coordinate the Hut pathway with transcriptional regulation of the virB genes, probably as a way to sense its own metabolic state and develop adaptive responses to overcome intracellular host defenses.Type IV secretion systems (T4SS) are multicomponent machineries central to the pathogenesis of many bacterial genera (e.g., Brucella, Agrobacterium, Helicobacter, Legionella, and Bordetella) (4). T4SS function comprises recognition and translocation of specific substrates across the bacterial cell envelope. The nature of the translocated substrates varies from proteins to DNA-protein complexes. In addition to the wellstudied Agrobacterium transferred DNA (T-DNA) and Bordetella pertussis toxin, several translocated effectors have been identified for Helicobacter, Legionella, and Brucella (7). In every case, the translocated molecules alter cellular processes in such a way that allows the pathogen to overcome host defenses.Brucella is a gram-negative bacterium that causes brucellosis, a worldwide zoonosis that affects domestic mammals. Different Brucella species vary in their host preferences. Brucella abortus, Brucella suis, and Brucella melitensis infect cattle, pigs, and goats, respectively, but also infect humans. In animals, the symptoms of the disease are sterility in males and abortion in pregnant females (6). In humans, brucellosis causes undulant fever during the acute phase and, if it reaches chronicity, can lead to endocarditis, osteoarthritis, and neurological damage.Brucella is an intracellular parasite that persists and replicates within host macrophages. After internalization, the bacterium actively controls the matur...

dBrucella 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 ⌬btaE mutant, we showed that BtaE is involved in the binding of B. suis to hyaluronic acid. The B. suis ⌬btaE 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.

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 (P virB ) 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 P virB 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 P virB 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 P virB . 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.Quorum sensing (QS) is a widespread mechanism of gene regulation that mediates bacterial cell-to-cell communication. In Gram-negative bacteria, most of the identified QS circuits consist of components that resemble those of the canonical LuxI/LuxR system of Vibrio fischeri (17). LuxI is the enzyme responsible for the synthesis of an acylated homoserine lactone signaling molecule (AHL) known as an autoinducer whose concentration is proportional to the bacterial population density. As bacterial cell density increases, the extracellular concentration of the autoinducer reaches a threshold value that activates the DNA-binding protein LuxR to control specific gene transcription.In addition to the bioluminescence of Vibrio fischeri, QSrelated systems have been shown to participate in the regulation of many bacterial physiological functions, including biofilm formation and the expression of virulence factors (7,11,16). In Brucella, it was also found that a LuxR-type regulator is directly involved in the control of transcription of important virulence determinants of this facultative intracellular bacterium (9).Brucella is a genus of Gram-negative bacteria that cause brucellosis, a debilitating zoonotic disease that affects different species of domestic mammals. The Brucella species differ in their host specificities. In addition to their animal host, Brucella abortus, Brucella melitensis, and Brucella suis are also able to infect humans. The virulence of Brucella is determined by its ability to survive and replicate wit...

Type IV secretion systems (T4SS) are multiprotein structures that direct the translocation of specific molecules across the bacterial cell envelope. As in other bacteria, pathogenicity of the genus Brucella essentially depends on the integrity of the T4SS-encoding virB operon, whose expression is regulated by multiple transcription factors belonging to different families. Previously, we identified IHF and HutC, two direct regulators of the virB genes that were isolated from total protein extracts of Brucella. Here, we report the identification of MdrA, a third regulatory element that was isolated using the same screening procedure. This transcription factor, which belongs to the MarR-family of transcriptional regulators, binds at two different sites of the virB promoter and regulates expression in a growth phase-dependent manner. Like other members of the MarR family, specific ligands were able to dissociate MdrA from DNA in vitro. Determination of the MdrA-binding sites by DNase I footprinting and analyses of protein-DNA complexes by electrophoresis mobility shift assays (EMSAs) showed that MdrA competes with IHF and HutC for the binding to the promoter because their target DNA sequences overlap. Unlike IHF, both MdrA and HutC bound to the promoter without inducing bending of DNA. Moreover, the two latter transcription factors activated virB expression to similar extents, and in doing so, they are functionally redundant. Taken together, our results show that MdrA is a regulatory element that directly modulates the activity of the virB promoter and is probably involved in coordinating gene expression in response to specific environmental signals.

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.

This paper describes the results of two experiments regarding porcine reproductive and respiratory syndrome virus (PRRSV1): the first one studied the existence of bottlenecks in an experimental one-to-one model of transmission in pigs; while the second analysed the differences between viral quasi-species in vaccinated pigs that developed shorter or longer viraemias after natural challenge. Serum samples, as well as the initial inoculum, were deep-sequenced and a viral quasi-species was constructed per sample. For the first experiment, the results consistently reported a reduction in the quasi-species diversity after a transmission event, pointing to the existence of bottlenecks during PRRSV1 transmission. However, despite the identified preferred and un-preferred transmitted variants not being randomly distributed along the virus genome, it was not possible to identify any variant producing a structural change in any viral protein. In contrast, the mutations identified in GP2, nsp9 and M of the second experiment pointed to changes in the amino acid charges and the viral RNA-dependent RNA polymerase structure. The fact that the affected proteins are known targets of the immunity against PRRSV, plus the differential level of neutralizing antibodies present in pigs developing short or long viraemias, suggests that the immune response selected those changes.

Regulatory network plasticity is a key attribute underlying changes in bacterial gene expression and a source of phenotypic diversity to interact with the surrounding environment. Here, we sought to study the transcriptional circuit of HutC, a regulator of both metabolic and virulence genes of the facultative intracellular pathogen Brucella. Using in silico and biochemical approaches, we identified a novel functional HutC-binding site upstream of btaE, a trimeric-autotransporter adhesin involved in the attachment of Brucella to host extracellular matrix components. Moreover, we identified two additional regulators, one of which, MdrA, acts in concert with HutC to exert a combinatorial control of both btaE promoter activity and attachment of Brucella to HeLa cells. Analysis of btaE promoter sequences of different species indicated that this HutC-binding site was generated de novo by a single point mutation in a virulent Brucella strain, indicative of a transcriptional rewiring event. In addition to major domain organization differences existing between BtaE proteins within the genus Brucella, our analyses revealed that sequences upstream of btaE display high variability probably associated to intrinsic promoter structural features, which may serve as a substrate for reciprocal selection during co-evolution between this pathogen and its mammalian host.

The existence of bottlenecks during infection of Porcine reproductive and respiratory syndrome virus (PRRSV) was studied in an experimental one-to-one model of transmission in pigs. Besides, the differences between viral quasi-species in vaccinated pigs that developed shorter or longer viremias after natural challenge were analysed. The results consistently reported the existence of bottlenecks during transmission. Several positions along the PRRSV genome were identified as being selected in partially immune animals that developed short viremias. Those positions accumulated in GP2, nsp9 and M proteins and resulted in changes in the protein structure and in the interactions of those proteins with their targets. The fact that the affected proteins are known targets of the immunity against PRRSV suggested that the immune response selected those changes. This pig model can be useful for the study of other pathogens of interest in animals and humans.Author summaryPorcine reproductive and respiratory syndrome (PRRS) is one of the most economically important disease of pigs. It is caused by PRRS virus (PRRSV), a positive-sense, single-stranded RNA virus in the Arteriviridae family within the order Nidovirales. Here, we study the existence of bottlenecks during disease transmission and the differences between viral quasi-species in vaccinated pigs that developed shorter or longer viremias after natural challenge. Our results consistently report the existence of bottlenecks during PRRSV1 transmission and identify several mutations along the viral genome selected by the host immune response that can be clear targets for new vaccine development.