The Brucella abortus two-component system response regulator BvrR binds to three DNA regulatory boxes in the upstream region of omp25.

Brucella abortus is a facultative extracellular-intracellular bacterial zoonotic pathogen worldwide. It is also a major cause of abortion in bovines, generating economic losses. The two-component regulatory system BvrR/BvrS modulates the expression of genes required to transition from extracellular to intracellular lifestyles. However, few regulatory regions of BvrR direct target genes have been studied. In this study, we characterized the regulatory region of omp25, a gene encoding an outer membrane protein that is positively regulated by TCS BvrR/BvrS. By omp25-lacZ reporter fusions and ß-galactosidase activity assays, we found that the region between-262 and + 127 is necessary for transcriptional activity, particularly a 111-bp long fragment located from-262 to -152. In addition, we demonstrated the binding of P-BvrR to three sites within the -140 to +1 region. Two of these sites were delimited between -18 to +1 and - 99 to -76 by DNase I footprinting and called DNA regulatory boxes 1 and 2, respectively. The third binding site (box 3) was delimited from -140 to -122 by combining EMSA and fluorescence anisotropy results. A molecular docking analysis with HDOCK predicted BvrR-DNA interactions between 11, 13, and 12 amino acid residue-nucleotide pairs in boxes 1, 2, and 3, respectively. A manual sequence alignment of the three regulatory boxes revealed the presence of inverted and non-inverted repeats of five to eight nucleotides, partially matching DNA binding motifs previously described for BvrR. We propose that P-BvrR binds directly to up to three regulatory boxes and probably interacts with other transcription factors to regulate omp25 expression. This gene regulation model could apply to other BvrR target genes and to orthologs of the TCS BvrR/BvrS and Omp25 in phylogenetically closed Rhizobiales.

The regulon of Brucella abortus two-component system BvrR/BvrS reveals the coordination of metabolic pathways required for intracellular life.

Brucella abortus is a facultative intracellular pathogen causing a severe zoonotic disease worldwide. The two-component regulatory system (TCS) BvrR/BvrS of B. abortus is conserved in members of the Alphaproteobacteria class. It is related to the expression of genes required for host interaction and intracellular survival. Here we report that bvrR and bvrS are part of an operon composed of 16 genes encoding functions related to nitrogen metabolism, DNA repair and recombination, cell cycle arrest, and stress response. Synteny of this genomic region within close Alphaproteobacteria members suggests a conserved role in coordinating the expression of carbon and nitrogen metabolic pathways. In addition, we performed a ChIP-Seq analysis after exposure of bacteria to conditions that mimic the intracellular environment. Genes encoding enzymes at metabolic crossroads of the pentose phosphate shunt, gluconeogenesis, cell envelope homeostasis, nucleotide synthesis, cell division, and virulence are BvrR/BvrS direct targets. A 14 bp DNA BvrR binding motif was found and investigated in selected gene targets such as virB1, bvrR, pckA, omp25, and tamA. Understanding gene expression regulation is essential to elucidate how Brucella orchestrates a physiological response leading to a furtive pathogenic strategy.

A Sinorhizobium meliloti and Agrobacterium tumefaciens ExoR ortholog is not crucial for Brucella abortus virulence.

Brucella is a facultative extracellular-intracellular pathogen that belongs to the Alphaproteobacteria class. Precise sensing of environmental changes and a proper response mediated by a gene expression regulatory network are essential for this pathogen to survive. The plant-related Alphaproteobacteria Sinorhizobium meliloti and Agrobacterium tumefaciens also alternate from a free to a host-associated life, where a regulatory invasion switch is needed for this transition. This switch is composed of a two-component regulatory system (TCS) and a global inhibitor, ExoR. In B. abortus, the BvrR/BvrS TCS is essential for intracellular survival. However, the presence of a TCS inhibitor, such as ExoR, in Brucella is still unknown. In this work, we identified a genomic sequence similar to S. meliloti exoR in the B. abortus 2308W genome, constructed an exoR mutant strain, and performed its characterization through ex vivo and in vivo assays. Our findings indicate that ExoR is related to the BvrR phosphorylation state, and is related to the expression of known BvrR/BrvS gene targets, such as virB8, vjbR, and omp25 when grown in rich medium or starving conditions. Despite this, the exoR mutant strain showed no significant differences as compared to the wild-type strain, related to resistance to polymyxin B or human non-immune serum, intracellular replication, or infectivity in a mice model. ExoR in B. abortus is related to BvrR/BvrS as observed in other Rhizobiales; however, its function seems different from that observed for its orthologs described in A. tumefaciens and S. meliloti.

Brucella abortus Senses the Intracellular Environment through the BvrR/BvrS Two-Component System, Which Allows B. abortus To Adapt to Its Replicative Niche.

Brucella abortus is a facultative extracellular-intracellular pathogen belonging to a group of Alphaproteobacteria that establishes close interactions with animal cells. This bacterium enters host cells in a membrane-bound compartment, avoiding the lysosomal route and reaching the endoplasmic reticulum through the action of the type IV secretion system, VirB. In this work, we demonstrate that the BvrR/BvrS two-component system senses the intracellular environment to mount the transcriptional response required for intracellular life adaptation. By combining a method to purify intracellularly extracted bacteria with a strategy that allows direct determination of BvrR phosphorylation, we showed that upon entrance to host cells, the regulatory protein BvrR was activated (BvrR-P) by phosphorylation at aspartate 58. This activation takes place in response to intracellular cues found in early compartments, such as low pH and nutrient deprivation. Furthermore, BvrR activation was followed by an increase in the expression of VjbR and VirB. The in vitro activation of this BvrR-P/VjbR/VirB virulence circuit rescued B. abortus from the inhibition of intracellular replication induced by bafilomycin treatment of cells, demonstrating the relevance of this mechanism for intracellular bacterial survival and replication. All together, our results indicate that B. abortus senses the transition from the extracellular to the intracellular milieu through BvrR/BvrS, allowing the bacterium to transit safely to its replicative niche. These results serve as a working model for understanding the role of this family of two-component systems in the adaptation to intracellular life of Alphaproteobacteria.

Brucella Genetic Variability in Wildlife Marine Mammals Populations Relates to Host Preference and Ocean Distribution.

Intracellular bacterial pathogens probably arose when their ancestor adapted from a free-living environment to an intracellular one, leading to clonal bacteria with smaller genomes and less sources of genetic plasticity. Still, this plasticity is needed to respond to the challenges posed by the host. Members of the Brucella genus are facultative-extracellular intracellular bacteria responsible for causing brucellosis in a variety of mammals. The various species keep different host preferences, virulence, and zoonotic potential despite having 97-99% similarity at genome level. Here, we describe elements of genetic variation in Brucella ceti isolated from wildlife dolphins inhabiting the Pacific Ocean and the Mediterranean Sea. Comparison with isolates obtained from marine mammals from the Atlantic Ocean and the broader Brucella genus showed distinctive traits according to oceanic distribution and preferred host. Marine mammal isolates display genetic variability, represented by an important number of IS711 elements as well as specific IS711 and SNPs genomic distribution clustering patterns. Extensive pseudogenization was found among isolates from marine mammals as compared with terrestrial ones, causing degradation in pathways related to energy, transport of metabolites, and regulation/transcription. Brucella ceti isolates infecting particularly dolphin hosts, showed further degradation of metabolite transport pathways as well as pathways related to cell wall/membrane/envelope biogenesis and motility. Thus, gene loss through pseudogenization is a source of genetic variation in Brucella, which in turn, relates to adaptation to different hosts. This is relevant to understand the natural history of bacterial diseases, their zoonotic potential, and the impact of human interventions such as domestication.

Brucella abortus Strain 2308 Wisconsin Genome: Importance of the Definition of Reference Strains.

Brucellosis is a bacterial infectious disease affecting a wide range of mammals and a neglected zoonosis caused by species of the genetically homogenous genus Brucella. As in most studies on bacterial diseases, research in brucellosis is carried out by using reference strains as canonical models to understand the mechanisms underlying host pathogen interactions. We performed whole genome sequencing analysis of the reference strain B. abortus 2308 routinely used in our laboratory, including manual curated annotation accessible as an editable version through a link at https://en.wikipedia.org/wiki/Brucella#Genomics. Comparison of this genome with two publically available 2308 genomes showed significant differences, particularly indels related to insertional elements, suggesting variability related to the transposition of these elements within the same strain. Considering the outcome of high resolution genomic techniques in the bacteriology field, the conventional concept of strain definition needs to be revised.