A sensor histidine kinase from a plant-endosymbiont bacterium restores the virulence of a mammalian intracellular pathogen.

Alphaproteobacteria include organisms living in close association with plants or animals. This interaction relies partly on orthologous two-component regulatory systems (TCS), with sensor and regulator proteins modulating the expression of conserved genes related to symbiosis/virulence. We assessed the ability of the exoS+Sm gene, encoding a sensor protein from the plant endosymbiont Sinorhizobium meliloti to substitute its orthologous bvrS in the related animal/human pathogen Brucella abortus. ExoS phosphorylated the B. abortus regulator BvrR in vitro and in cultured bacteria, showing conserved biological function. Production of ExoS in a B. abortus bvrS mutant reestablished replication in host cells and the capacity to infect mice. Bacterial outer membrane properties, the production of the type IV secretion system VirB, and its transcriptional regulators VjbR and BvrR were restored as compared to parental B. abortus. These results indicate that conserved traits of orthologous TCS from bacteria living in and sensing different environments are sufficient to achieve phenotypic plasticity and support bacterial survival. The knowledge of bacterial genetic networks regulating host interactions allows for an understanding of the subtle differences between symbiosis and parasitism. Rewiring these networks could provide new alternatives to control and prevent bacterial infection.

A bvrR/bvrS Non-Polar Brucella abortus Mutant Confirms the Role of the Two-Component System BvrR/BvrS in Virulence and Membrane Integrity.

Brucella abortus is a bacterial pathogen causing bovine brucellosis worldwide. This facultative extracellular-intracellular pathogen can be transmitted to humans, leading to a zoonotic disease. The disease remains a public health concern, particularly in regions where livestock farming is present. The two-component regulatory system BvrR/BvrS was described by isolating the attenuated transposition mutants bvrR::Tn5 and bvrS::Tn5, whose characterization led to the understanding of the role of the system in bacterial survival. However, a phenotypic comparison with deletion mutants has not been performed because their construction has been unsuccessful in brucellae and difficult in phylogenetically related Rhizobiales with BvrR/BvrS orthologs. Here, we used an unmarked gene excision strategy to generate a B. abortus mutant strain lacking both genes, called B. abortus ∆bvrRS. The deletion was verified through PCR, Southern blot, Western blot, Sanger sequencing, and whole-genome sequencing, confirming a clean mutation without further alterations at the genome level. B. abortus ∆bvrRS shared attenuated phenotypic traits with both transposition mutants, confirming the role of BvrR/BvrS in pathogenesis and membrane integrity. This B. abortus ∆bvrRS with a non-antimicrobial marker is an excellent tool for continuing studies on the role of BvrR/BvrS in the B. abortus lifestyle.

Virulent Brucella nosferati infecting Desmodus rotundus has emerging potential due to the broad foraging range of its bat host for humans and wild and domestic animals.

Desmodus rotundus, vampire bats, transmit dangerous infections, and brucellosis is a hazardous zoonotic disease, two adversities that coexist in the subtropical and tropical areas of the American continent. Here, we report a 47.89% Brucella infection prevalence in a colony of vampire bats inhabiting the tropical rainforest of Costa Rica. The bacterium induced placentitis and fetal death in bats. Wide-range phenotypic and genotypic characterization placed the Brucella organisms as a new pathogenic species named Brucella nosferati sp. nov., isolated from bat tissues, including the salivary glands, suggesting feeding behavior might favor transmission to their prey. Overall analyses placed B. nosferati as the etiological agent of a reported canine brucellosis case, demonstrating its potential for infecting other hosts. To assess the putative prey hosts, we analyzed the intestinal contents of 14 infected and 23 non-infected bats by proteomics. A total of 54,508 peptides sorted into 7,203 unique peptides corresponding to 1,521 proteins were identified. Twenty-three wildlife and domestic taxa, including humans, were foraged by B. nosferati-infected D. rotundus, suggesting contact of this bacterium with a broad range of hosts. Our approach is appropriate for detecting, in a single study, the prey preferences of vampire bats in a diverse area, demonstrating its suitability for control strategies where vampire bats thrive. IMPORTANCE The discovery that a high proportion of vampire bats in a tropical area is infected with pathogenic Brucella nosferati and that bats forage on humans and many wild and domestic animals is relevant from the perspective of emerging disease prevention. Indeed, bats harboring B. nosferati in their salivary glands may transmit this pathogenic bacterium to other hosts. This potential is not trivial since, besides the demonstrated pathogenicity, this bacterium possesses all the required virulent arsenal of dangerous Brucella organisms, including those that are zoonotic for humans. Our work has settled the basis for future surveillance actions in brucellosis control programs where these infected bats thrive. Moreover, our strategy to identify the foraging range of bats may be adapted for exploring the feeding habits of diverse animals, including arthropod vectors of infectious diseases, and therefore of interest to a broader audience besides experts on Brucella and bats.

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.

Canine brucellosis in Costa Rica reveals widespread Brucella canis infection and the recent introduction of foreign strains.

Brucellosis is a prevalent disease in Costa Rica (CR), with an increasing number of human infections. Close to half of homes in CR have one or more dogs, corresponding to ∼1.4 million canines, most of them in the Central Valley within or near the cities of San José, Heredia, and Alajuela. From 302 dog sera collected from this region, 19 were positive for Brucella canis antigens, and five had antibodies against smooth lipopolysaccharide, suggesting infections by both B. canis and other Brucella species. B. canis strains were isolated in the Central Valley from 26 kennel dogs and three pet dogs, all displaying clinical signs of canine brucellosis. We detected three recent introductions of different B. canis strains in kennels: two traced from Mexico and one from Panama. Multiple locus-variable number tandem repeats (MLVA-16) and whole-genome sequencing (WGSA) analyses showed that B. canis CR strains comprise three main lineages. The tree topologies obtained by WGSA and MLVA-16 just partially agreed, indicating that the latter analysis is not suitable for phylogenetic studies. The fatty acid methyl ester analysis resolved five different B. canis groups, showing less resolution power than the MLVA-16 and WGSA. Lactobacillic acid was absent in linages I and II but present in linage III, supporting the recent introductions of B. canis strains from Mexico. B. canis displaying putative functional cyclopropane synthase for the synthesis of lactobacillic acid are phylogenetically intertwined with B. canis with non-functional protein, indicating that mutations have occurred independently in the various lineages.

Persistence of Brucella abortus lineages revealed by genomic characterization and phylodynamic analysis.

Brucellosis, caused by Brucella abortus, is a major disease of cattle and humans worldwide distributed. Eradication and control of the disease has been difficult in Central and South America, Central Asia, the Mediterranean and the Middle East. Epidemiological strategies combined with phylogenetic methods provide the high-resolution power needed to study relationships between surveillance data and pathogen population dynamics, using genetic diversity and spatiotemporal distributions. This information is crucial for prevention and control of disease spreading at a local and worldwide level. In Costa Rica (CR), the disease was first reported at the beginning of the 20th century and has not been controlled despite many efforts. We characterized 188 B. abortus isolates from CR recovered from cattle, humans and water buffalo, from 2003 to 2018, and whole genome sequencing (WGS) was performed in 95 of them. They were also assessed based on geographic origin, date of introduction, and phylogenetic associations in a worldwide and national context. Our results show circulation of five B. abortus lineages (I to V) in CR, phylogenetically related to isolates from the United States, United Kingdom, and South America. Lineage I was dominant and probably introduced at the end of the 19th century. Lineage II, represented by a single isolate from a water buffalo, clustered with a Colombian sample, and was likely introduced after 1845. Lineages III and IV were likely introduced during the early 2000s. Fourteen isolates from humans were found within the same lineage (lineage I) regardless of their geographic origin within the country. The main CR lineages, introduced more than 100 years ago, are widely spread throughout the country, in contrast to new introductions that seemed to be more geographically restricted. Following the brucellosis prevalence and the farming practices of several middle- and low-income countries, similar scenarios could be found in other regions worldwide.

Corrigendum: Genetic and Phenotypic Characterization of the Etiological Agent of Canine Orchiepididymitis Smooth Brucella sp. BCCN84.3.

[This corrects the article DOI: 10.3389/fvets.2019.00175.].

Genetic and Phenotypic Characterization of the Etiological Agent of Canine Orchiepididymitis Smooth Brucella sp. BCCN84.3.

Members of the genus Brucella cluster in two phylogenetic groups: classical and non-classical species. The former group is composed of Brucella species that cause disease in mammals, including humans. A Brucella species, labeled as Brucella sp. BCCN84.3, was isolated from the testes of a Saint Bernard dog suffering orchiepididymitis, in Costa Rica. Following standard microbiological methods, the bacterium was first defined as "Brucella melitensis biovar 2." Further molecular typing, identified the strain as an atypical "Brucella suis." Distinctive Brucella sp. BCCN84.3 markers, absent in other Brucella species and strains, were revealed by fatty acid methyl ester analysis, high resolution melting PCR and omp25 and omp2a/omp2b gene diversity. Analysis of multiple loci variable number of tandem repeats and whole genome sequencing demonstrated that this isolate was different from the currently described Brucella species. The smooth Brucella sp. BCCN84.3 clusters together with the classical Brucella clade and displays all the genes required for virulence. Brucella sp. BCCN84.3 is a species nova taxonomical entity displaying pathogenicity; therefore, relevant for differential diagnoses in the context of brucellosis. Considering the debate on the Brucella species concept, there is a need to describe the extant taxonomical entities of these pathogens in order to understand the dispersion and evolution.

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.

Epidemiology of bovine brucellosis in Costa Rica: Lessons learned from failures in the control of the disease.

Brucellosis, caused by Brucella abortus is a major disease of cattle and a zoonosis. In order to estimate the bovine brucellosis prevalence in Costa Rica (CR), a total 765 herds (13078 bovines) from six regions of CR were randomly sampled during 2012-2013. A non-random sample of 7907 herds (532199 bovines) of the six regions, arriving for diagnoses during 2014-2016 to the Costa Rican Animal Health Service was also studied. The prevalence estimated by Rose Bengal test (RBT) ranged from 10.5%-11.4%; alternatively, the prevalence estimated by testing the RBT positives in iELISA, ranged from 4.1%-6.0%, respectively. However, cattle in CR are not vaccinated with B. abortus S19 but with RB51 (vaccination coverage close to 11%), and under these conditions the RBT displays 99% specificity and 99% sensitivity. Therefore, the RBT herd depicted in the random analysis stands as a feasible assessment and then, the recommended value in case of planning an eradication program in CR. Studies of three decades reveled that bovine brucellosis prevalence has increased in CR. B. abortus was identified by biochemical and molecular studies as the etiological agent of bovine brucellosis. Multiple locus variable-number tandem repeat analysis-16 revealed four B. abortus clusters. Cluster one and three are intertwined with isolates from other countries, while clusters two and four have only representatives from CR. Cluster one is widely distributed in all regions of the country and may be the primary B. abortus source. The other clusters seem to be restricted to specific areas in CR. The implications of our findings, in relation to the control of the disease in CR, are critically discussed.

Brucella neotomae Infection in Humans, Costa Rica.

Several species of Brucella are known to be zoonotic, but B. neotomae infection has been thought to be limited to wood rats. In 2008 and 2011, however, B. neotomae was isolated from cerebrospinal fluid of 2 men with neurobrucellosis. The nonzoonotic status of B. neotomae should be reassessed.

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.