Factores ecoambientales relacionados con potenciales reservorios de Leptospira spp. en la Reserva Ecológica Costanera Sur, Buenos Aires, Argentina / Eco-environmental Factors Related to Potential Reservoirs of Leptospira spp. in the Costanera Sur Ecological Reserve, Buenos Aires, Argentina

Resumen Las modificaciones ambientales generan entornos de interfaz en los que la transmisión de Leptospira spp. puede verse afectada. Con el objetivo de identificar y analizar variables ecoambientales asociadas a potenciales reservorios de Leptospira spp. en la Reserva Ecológica Costanera Sur (RECS) (Buenos Aires, Argentina) y áreas urbanizadas contiguas, se realizó un muestreo por conveniencia en líneas transectas, estratificado y proporcionado. Fueron capturados 170 mamíferos de 11 especies y se analizó la presencia de Leptospira spp. por serología, cultivo y PCR. En áreas conservadas, se registraron mayormente Didelphis albiventris y Deltamys kempi, mientras que Oligoryzomys flavescens también fue hallado en áreas degradadas, y Rattus norvegicus solo fue encontrada en áreas degradadas. No se detectó Leptospira spp. en los mamíferos silvestres. Un perro sin tenedor responsable resultó positivo por serología. Las condiciones ambientales de la RECS sugieren un ecosistema poco propicio para Rattus, evidenciando la importancia de la conservación de áreas naturales en entornos urbanos.

Abstract Environmental modifications generate interface environments in which Leptospira spp. transmission may be affected. In order to identify and analyze eco-environmental variables associated with potential reservoirs of Leptospira spp. in the Reserva Ecológica Costanera Sur (RECS) (Buenos Aires, Argentina) and adjacent urbanized areas, a stratified and proportionate transect line convenience sampling was carried out. A total of 170 mammals of 11 species were captured and analyzed for the presence of Leptospira spp. by serology, culture and PCR. In conserved areas, Didelphis albiventris and Deltamys kempi were mostly recorded, while Oligoryzomys flavescens was also found in degraded areas, and Rattus norvegicus was only found in degraded areas. Leptospira spp. were not detected in wild mammals. One dog without a responsible keeper tested positive by serology. The environmental conditions of the RECS suggest a poor ecosystem for Rattus, demonstrating the importance of the conservation of natural areas in urban environments.

Bottlenecks in the transmission of porcine reproductive and respiratory syndrome virus (PRRSV1) to naïve pigs and the quasi-species variation of the virus during infection in vaccinated pigs.

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.

Analysis of the genetic diversity and mRNA expression level in porcine reproductive and respiratory syndrome virus vaccinated pigs that developed short or long viremias after challenge.

Porcine reproductive and respiratory syndrome virus (PRRSv) infection alters the host's cellular and humoral immune response. Immunity against PRRSv is multigenic and vary between individuals. The aim of the present study was to compare several genes that encode for molecules involved in the immune response between two groups of vaccinated pigs that experienced short or long viremic periods after PRRSv challenge. These analyses include the sequencing of four SLA Class I, two Class II allele groups, and CD163, plus the analysis by quantitative realtime qRT-PCR of the constitutive expression of TLR2, TLR3, TLR4, TLR7, TLR8 and TLR9 mRNA and other molecules in peripheral blood mononuclear cells.

Combinatorial control of adhesion of Brucella abortus 2308 to host cells by transcriptional rewiring of the trimeric autotransporter btaE gene.

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.

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.

A MarR-Type regulator directly activates transcription from the Brucella abortus virB promoter by sharing a redundant role with HutC.

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.

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.

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 (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.

Metabolic control of virulence genes in Brucella abortus: HutC coordinates virB expression and the histidine utilization pathway by direct binding to both promoters.

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.