Insights into the effect of guanylate-binding protein 1 on the survival of Brucella intracellularly.

Brucellosis is a zoonotic disease that affects wild and domestic animals. It is caused by members of the bacterial genus Brucella. Guanylate-binding protein 1 (GBP1) is associated with microbial infections. However, the role of GBP1 during Brucella infection remains unclear. This investigation aimed to identify the association of GBP1 with brucellosis. Results showed that Brucella infection induced GBP1 upregulation in RAW 264.7 murine macrophages. Small interfering GBP1 targeting RNAs were utilized to explore how GBP1 regulates the survival of Brucella intracellularly. Results revealed that GBP1 knockdown promoted Brucella's survival ability, activated Nod-like receptor (NLR) containing a pyrin domain 3 (NLRP3) and absent in melanoma 2 (AIM2) inflammatory corpuscles, and induced pro-inflammatory cytokines IFN-γ and IL-1ß. Furthermore, Brucella stimulated the expression of GBP1 in bone marrow-derived macrophages (BMDMs) and mice. During the inhibition of GBP1 in BMDMs, the intracellular growth of Brucella increased. In comparison, GBP1 downregulation enhanced the accumulation of Brucella-induced reactive oxygen species (ROS) in macrophages. Overall, the data indicate a significant role of GBP1 in regulating brucellosis and suggest the function underlying its suppressive effect on the survival and growth of Brucella intracellularly.

In silico designed novel multi-epitope mRNA vaccines against Brucella by targeting extracellular protein BtuB and LptD.

Brucella, a gram-negative intracellular bacterium, causing Brucellosis, a zoonotic disease with a range of clinical manifestations, from asymptomatic to fever, fatigue, loss of appetite, joint and muscle pain, and back pain, severe patients have developed serious diseases affecting various organs. The mRNA vaccine is an innovative type of vaccine that is anticipated to supplant traditional vaccines. It is widely utilized for preventing viral infections and for tumor immunotherapy. However, research regarding its effectiveness in preventing bacterial infections is limited. In this study, we analyzed the epitopes of two proteins of brucella, the TonB-dependent outer membrane receptor BtuB and the LPS assembly protein LptD, which is involved in nutrient transport and LPS synthesis in Brucella. In order to effectively stimulate cellular and humoral immunity, we utilize a range of immunoinformatics tools such as VaxiJen, AllergenFPv.1.0 and SignalP 5.0 to design proteins. Finally, five cytotoxic T lymphocyte (CTL) cell epitopes, ten helper T lymphocyte (HTL) cell epitopes, and eight B cell epitopes were selected to construct the vaccine. Computer simulations are also used to verify the immune response of the vaccine. The codon optimization, in silico cloning showed that the vaccine can efficiently transcript and translate in E. coli. The secondary structure of mRNA vaccines and the secondary and tertiary structures of vaccine peptides were predicted and then docked with TLR-4. Finally, the stability of the developed vaccine was confirmed through molecular dynamics simulation. These analyses showed that the design the multi-epitope mRNA vaccine could potentially target extracellular protein of prevalent Brucella, which provided novel strategies for developing the vaccine.

Brucella ceti and Brucella pinnipedialis genome characterization unveils genetic features that highlight their zoonotic potential.

The Gram-negative bacteria Brucella ceti and Brucella pinnipedialis circulate in marine environments primarily infecting marine mammals, where they cause an often-fatal disease named brucellosis. The increase of brucellosis among several species of cetaceans and pinnipeds, together with the report of sporadic human infections, raises concerns about the zoonotic potential of these pathogens on a large scale and may pose a threat to coastal communities worldwide. Therefore, the characterization of the B. ceti and B. pinnipedialis genetic features is a priority to better understand the pathological factors that may impact global health. Moreover, an in-depth functional analysis of the B. ceti and B. pinnipedialis genome in the context of virulence and pathogenesis was not undertaken so far. Within this picture, here we present the comparative whole-genome characterization of all B. ceti and B. pinnipedialis genomes available in public resources, uncovering a collection of genetic tools possessed by these aquatic bacterial species compared to their zoonotic terrestrial relatives. We show that B. ceti and B. pinnipedialis genomes display a wide host-range infection capability and a polyphyletic phylogeny within the genus, showing a genomic structure that fits the canonical definition of closeness. Functional genome annotation led to identifying genes related to several pathways involved in mechanisms of infection, others conferring pan-susceptibility to antimicrobials and a set of virulence genes that highlight the similarity of B. ceti and B. pinnipedialis genotypes to those of Brucella spp. displaying human-infecting phenotypes.

Brucella Outer Membrane Lipoproteins 19 and 16 Differentially Induce Interleukin-18 Response or Pyroptosis in Human Monocytic Cells.

BACKGROUND: Brucella species are Gram-negative intracellular bacteria that causes severe inflammatory diseases in animals and humans. Two major lipoproteins (L19 and L16) of Brucella outer membrane proteins were studied to explore the association with inflammatory response of human monocytes (THP-1). METHODS: Activated THP-1 cells induced with recombinant L19 and L16 were analyzed in comparison with unlipidated forms (U19 and U16) and lipopolysaccharide (LPS) of Brucella melitensis, respectively. RESULTS: Secretion of inflammatory factors tumor necrosis factor-α, interleukin (IL)-6, and IL-1ß was significantly increased from L19, L16, or both stimulated THP-1 cells. High secretion of IL-18 was detected only from L19-induced cells. Signaling of those cytokine responses was identified mainly through the P38-mitogen-activated protein kinase pathway, and signaling of L19-induced IL-1ß response partly occurred via necrosis factor-κB. While exploring different forms of IL-18, we found that L19-induced production of active IL-18 (18 kD) occurred through upregulating NLRP3 and activating caspase-1, whereas L16-induced production of inactive IL-18 fragments (15 kD and 16 kD) occurred through activating caspase-8/3. We also found that L19 upregulated phosphorylation of XIAP for inhibiting caspase-3 activity to cleave IL-18, whereas L16 activated caspase-3 for producing GSDME-N and leading to pyroptosis of THP-1 cells. CONCLUSIONS: Brucella L19 and L16 differentially induce IL-18 response or pyroptosis in THP-1 cells, respectively.

Uncovering the Hidden Credentials of Brucella Virulence.

Bacteria in the genus Brucella are important human and veterinary pathogens. The abortion and infertility they cause in food animals produce economic hardships in areas where the disease has not been controlled, and human brucellosis is one of the world's most common zoonoses. Brucella strains have also been isolated from wildlife, but we know much less about the pathobiology and epidemiology of these infections than we do about brucellosis in domestic animals. The brucellae maintain predominantly an intracellular lifestyle in their mammalian hosts, and their ability to subvert the host immune response and survive and replicate in macrophages and placental trophoblasts underlies their success as pathogens. We are just beginning to understand how these bacteria evolved from a progenitor alphaproteobacterium with an environmental niche and diverged to become highly host-adapted and host-specific pathogens. Two important virulence determinants played critical roles in this evolution: (i) a type IV secretion system that secretes effector molecules into the host cell cytoplasm that direct the intracellular trafficking of the brucellae and modulate host immune responses and (ii) a lipopolysaccharide moiety which poorly stimulates host inflammatory responses. This review highlights what we presently know about how these and other virulence determinants contribute to Brucella pathogenesis. Gaining a better understanding of how the brucellae produce disease will provide us with information that can be used to design better strategies for preventing brucellosis in animals and for preventing and treating this disease in humans.

Brucella suppress STING expression via miR-24 to enhance infection.

Brucellosis, caused by a number of Brucella species, remains the most prevalent zoonotic disease worldwide. Brucella establish chronic infections within host macrophages despite triggering cytosolic innate immune sensors, including Stimulator of Interferon Genes (STING), which potentially limit infection. In this study, STING was required for control of chronic Brucella infection in vivo. However, early during infection, Brucella down-regulated STING mRNA and protein. Down-regulation occurred post-transcriptionally, required live bacteria, the Brucella type IV secretion system, and was independent of host IRE1-RNase activity. STING suppression occurred in MyD88-/- macrophages and was not induced by Toll-like receptor agonists or purified Brucella lipopolysaccharide (LPS). Rather, Brucella induced a STING-targeting microRNA, miR-24-2, in a type IV secretion system-dependent manner. Furthermore, STING downregulation was inhibited by miR-24 anti-miRs and in Mirn23a locus-deficient macrophages. Failure to suppress STING expression in Mirn23a-/- macrophages correlated with diminished Brucella replication, and was rescued by exogenous miR-24. Mirn23a-/- mice were also more resistant to splenic colonization one week post infection. Anti-miR-24 potently suppressed replication in wild type, but much less in STING-/- macrophages, suggesting most of the impact of miR-24 induction on replication occurred via STING suppression. In summary, Brucella sabotages cytosolic surveillance by miR-24-dependent suppression of STING expression; post-STING activation "damage control" via targeted STING destruction may enable establishment of chronic infection.

Occupational exposure to Brucella spp.: A systematic review and meta-analysis.

Brucellosis is a neglected zoonotic disease of remarkable importance worldwide. The focus of this systematic review was to investigate occupational brucellosis and to identify the main infection risks for each group exposed to the pathogen. Seven databases were used to identify papers related to occupational brucellosis: CABI, Cochrane, Pubmed, Scielo, Science Direct, Scopus and Web of Science. The search resulted in 6123 studies, of which 63 were selected using the quality assessment tools guided from National Institutes of Health (NIH) and Case Report Guidelines (CARE). Five different job-related groups were considered greatly exposed to the disease: rural workers, abattoir workers, veterinarians and veterinary assistants, laboratory workers and hunters. The main risk factors and exposure sources involved in the occupational infection observed from the analysis of the articles were direct contact with animal fluids, failure to comply with the use of personal protective equipment, accidental exposure to live attenuated anti-brucellosis vaccines and non-compliance with biosafety standards. Brucella species frequently isolated from job-related infection were Brucella melitensis, Brucella abortus, Brucella suis and Brucella canis. In addition, a meta-analysis was performed using the case-control studies and demonstrated that animal breeders, laboratory workers and abattoir workers have 3.47 [95% confidence interval (CI); 1.47-8.19] times more chance to become infected with Brucella spp. than others individuals that have no contact with the possible sources of infection. This systematic review improved the understanding of the epidemiology of brucellosis as an occupational disease. Rural workers, abattoir workers, veterinarians, laboratory workers and hunters were the groups more exposed to occupational Brucella spp. infection. Moreover, it was observed that the lack of knowledge about brucellosis among frequently exposed professionals, in addition to some behaviors, such as negligence in the use of individual and collective protective measures, increases the probability of infection.

Functional analysis of Bucella reveals transcriptional regulation of MarR.

Brucellosis is a zoonotic infectious disease caused by Brucella infection. MarR-family transcription factors are closely related to diverse physiological functions necessary for many pathogens adaptation to environmental changes. However, whether the MarR-family transcription factors are involved in virulence, mediated inflammatory responses and regulated virulence gene expression in the intracellular pathogen Brucella are still unknown. Therefore, we created a 2308ΔMarR6 mutant of B. abortus 2308 (S2308). Virulence and inflammatory cytokines assays were performed using a murine macrophage cell line (RAW 264.7). We also performed chromatin immunoprecipitation of MarR6 followed by next-generation sequencing (ChIP-seq). The results showed that 2308ΔMarR6 was significantly reduced survival capability in RAW 264.7. After the macrophages were infected with 2308ΔMarR6, the levels of tumor necrosis factor-α (TNF-α), interleukin-1ß (IL-1ß), interleukin-6 (IL-6), interleukin-12 (IL-12), interferon-gamma (IFN-γ) and macrophage chemoattractant protein-1 (MCP-1) were decreased and were significantly lower than that for the S2308-infected group, indicating that the 2308ΔMarR6 mutant could reduce the secretion of inflammatory cytokines. Furthermore, we detected 122 intergenic ChIP-seq peaks of MarR6 binding distributed across the Brucella genome. Taken together, the research has recorded valuable data about MarR6. Our findings are of great significance in elucidating the function of MarR6.

Lethality of Brucella microti in a murine model of infection depends on the wbkE gene involved in O-polysaccharide synthesis.

Brucella microti was isolated a decade ago from wildlife and soil in Europe. Compared to the classical Brucella species, it exhibits atypical virulence properties such as increased growth in human and murine macrophages and lethality in experimentally infected mice. A spontaneous rough (R) mutant strain, derived from the smooth reference strain CCM4915T, showed increased macrophage colonization and was non-lethal in murine infections. Whole-genome sequencing and construction of an isogenic mutant of B. microti and Brucella suis 1330 revealed that the R-phenotype was due to a deletion in a single gene, namely wbkE (BMI_I539), encoding a putative glycosyltransferase involved in lipopolysaccharide (LPS) O-polysaccharide biosynthesis. Complementation of the R-strains with the wbkE gene restored the smooth phenotype and the ability of B. microti to kill infected mice. LPS with an intact O-polysaccharide is therefore essential for lethal B. microti infections in the murine model, demonstrating its importance in pathogenesis.

Monitoring the course of Brucella infection with qPCR-based detection.

OBJECTIVES: To determine blood Brucella DNA loads between brucellosis patients and those without brucellosis. METHODS: The patient group included 350 brucellosis patients. The control was composed of 200 subjects without brucellosis. The extracted DNA from blood was tested by quantitative polymerase chain reaction (qPCR). The cutoff value was determined by receiver operating characteristic curve analysis. A portion of the brucellosis patients were monitored by qPCR during therapy. RESULTS: The detection limit of qPCR was between 1E+01cfu/µL and 1E+08cfu/µL. The standard curve R2 reached 0.998. The cutoff value was 4E+01cfu/µL, which was determined by comparison of the patient group and the control. The qPCR assay had a specificity of 100% and a sensitivity of 93.14%. The monitoring results showed that the Brucella DNA load decreased in most patients during the first 4 weeks of treatment. One patient with bad treatment compliance showed a rebound. CONCLUSIONS: The qPCR results were in accordance with the course of brucellosis in the clinic. The DNA load often reflects the situation of the Brucella-infected patient. The cutoff value provides an important reference of infection. This qPCR-based method can be used to assist in the diagnosis of brucellosis and to adjust the therapy.

RNA-seq reveals the critical role of Lon protease in stress response and Brucella virulence.

The Brucella spp encounter stressful environment inside their host cells. The Lon protein is an important protease related to cellular protein degradation and resistance to stress in Brucella. However, the molecular mechanism between Lon protein and stress response was still unknown. In this study, it was found that the lon mutant exhibited obvious growth defect in TSB medium, compared with its parent strain. In addition, our results indicated that Lon protein was involved in resistance to various stress conditions and all the ß-lactam antibiotics tested. Although deletion of this protease did not affect Brucella virulence in macrophage, the mutant strain was significantly attenuated in mice infection model at 1 week post infection, and the expression level of several cytokine genes was significantly changed in vivo. To gain insight into the genetic basis for the distinctive phenotypic properties exhibited by the lon mutant strain, RNA-seq was performed, and the result showed that various genes involved in stress response, quorum sensing and transcriptional regulation were significantly altered in Δlon strain. Overall, these studies have preliminary uncovered the molecular mechanism between Lon protease, stress response and bacterial virulence.

Lable-free based comparative proteomic analysis of secretory proteins of rough Brucella mutants.

Brucella rough mutants are reported to induce infected macrophage death, which is type IV secretion system (T4SS) dependent. T4SS and its secretory proteins play a major role in host-bacteria interactions, but the crucial secretory proteins to promote macrophage death during Brucella rough mutant infection have not been characterized. In this study, we found that T4SS components played no role for macrophage death induced by Brucella rough mutant infection, but some T4SS effectors did. Proteomics of secretory proteins from Brucella rough mutants ΔrfbE and ΔrfbEΔvirB123 was analyzed by liquid chromatography/tandem mass spectrometry and 861 unique proteins were identified, among which 37 were differential secretory proteins. Gene ontology and pathway analysis showed that differential secretory proteins involved in cellular process and metabolic process, distributed in the cell and membrane, possessed molecular function of catalytic activity and binding, and were associated with ribosome, NOD-like receptor signaling pathway, two-component system and bacterial secretion system. Cell death analysis showed that T4SS effector VceC, and two differential secretory proteins OmpW family protein (BAB1_1579) and protein BAB1_1185 were associated with Brucella cytotoxicity. This study provides new insights into the molecular mechanisms associated with Brucella cytotoxicity and valuable information for screening vaccine candidates for Brucella. SIGNIFICANCE: Brucella rough mutants induce infected macrophage death, which is T4SS dependent. In the present report, a comparative proteomics analysis revealed 37 differential secretory proteins between Brucella rough mutants ΔrfbE and ΔrfbEΔvirB123. Further study demonstrated OmpW family protein (BAB1_1579) and uncharacterized protein BAB1_1185, two differential secretory proteins, were associated with Brucella cytotoxicity. This study provides novel information of the secretory proteins from the Brucella rough mutants and their effects on the Brucella cytotoxicity.

Brucella - Virulence Factors, Pathogenesis and Treatment.

Brucellae are Gram-negative, small rods infecting mammals and capable of causing disease called brucellosis. The infection results in abortion and sterility in domestic animals (sheeps, pigs, rams etc). Especially dangerous for humans are: Brucella melitensis, Brucella suis, Brucella abortus, and Brucella canis that trigger unspecific symptoms (flu-like manifestation). Brucella rods are introduced via host cells, by inhalation, skin abrasions, ingestion or mucosal membranes. The most important feature of Brucella is the ability to survive and multiply within both phagocytic and non-phagocytic cells. Brucella does not produce classical virulence factors: exotoxin, cytolisins, exoenzymes, plasmids, fimbria, and drug resistant forms. Major virulence factors are: lipopolysaccharide (LPS), T4SS secretion system and BvrR/BvrS system, which allow interaction with host cell surface, formation of an early, late BCV (Brucella Containing Vacuole) and interaction with endoplasmic reticulum (ER) when the bacteria multiply. The treatment of brucellosis is based on two-drug therapy, the most common combinations of antibiotics are: doxycycline with rifampicin or fluoroquinolones with rifampicin. Currently, also other methods are used to disrupt Brucella intracellular replication (tauroursodeoxycholic acid or ginseng saponin fraction A).

Application of real-time quantitative PCR assays for detecting marine Brucella spp. in fish.

Brucella ceti and Brucella pinnipedialis have been documented as occurring in marine mammals, and B. ceti has been identified in 3 naturally acquired human cases. Seroconversion and infection patterns in Pacific Northwest harbor seals ( Phoca vitulina richardii) and North Atlantic hooded seals ( Cystophora cristata) indicate post-weaning exposure through prey consumption or lungworm infection, suggesting fish and possibly invertebrates play an epizootiologic role in marine Brucella transmission and possible foodborne risk to humans. We determined if real-time quantitative PCR (qPCR) assays can detect marine Brucella DNA in fish DNA. Insertion sequence (IS) 711 gene and sequence type (ST)27 primer-probe sets were used to detect Brucella associated with marine mammals and human zoonotic infections, respectively. First, DNA extracts from paired-species fish (containing 2 species) samples were tested and determined to be Brucella DNA negative using both IS 711 and ST27 primer-probe sets. A representative paired-species fish DNA sample was spiked with decreasing concentrations of B. pinnipedialis DNA to verify Brucella detection by the IS 711 primer-probe within fish DNA. A standard curve, developed using isolated DNA from B. pinnipedialis, determined the limit of detection. Finally, the IS 711 primer-probe was used to test Atlantic cod ( Gadus morhua) DNA extracts experimentally infected with the B. pinnipedialis hooded seal strain. In culture-positive cod tissue, the IS 711 limit of detection was ~1 genome copy of Brucella. Agreement between culture and PCR results for the 9 positive and 9 negative cod tissues was 100%. Although a larger sample set is required for validation, our study shows that qPCR can detect marine Brucella in fish.

Brucella Rough Mutant Induce Macrophage Death via Activating IRE1α Pathway of Endoplasmic Reticulum Stress by Enhanced T4SS Secretion.

Brucella is a Gram-negative facultative intracellular pathogen that causes the worldwide zoonosis, known as brucellosis. Brucella virulence relies mostly on its ability to invade and replicate within phagocytic cells. The type IV secretion system (T4SS) and lipopolysaccharide are two major Brucella virulence factors. Brucella rough mutants reportedly induce the death of infected macrophages, which is T4SS dependent. However, the underlying molecular mechanism remains unclear. In this study, the T4SS secretion capacities of Brucella rough mutant and its smooth wild-type strain were comparatively investigated, by constructing the firefly luciferase fused T4SS effector, BPE123 and VceC. In addition, quantitative real-time PCR and western blotting were used to analyze the T4SS expression. The results showed that T4SS expression and secretion were enhanced significantly in the Brucella rough mutant. We also found that the activity of the T4SS virB operon promoter was notably increased in the Brucella rough mutant, which depends on quorum sensing-related regulators of VjbR upregulation. Cell infection and cell death assays revealed that deletion of vjbR in the Brucella rough mutant absolutely abolished cytotoxicity within macrophages by downregulating T4SS expression. This suggests that up-regulation of T4SS promoted by VjbR in rough mutant ΔrfbE contribute to macrophage death. In addition, we found that the Brucella rough mutant induce macrophage death via activating IRE1α pathway of endoplasmic reticulum stress. Taken together, our study provide evidence that in comparison to the Brucella smooth wild-type strain, VjbR upregulation in the Brucella rough mutant increases transcription of the virB operon, resulting in overexpression of the T4SS gene, accompanied by the over-secretion of effecter proteins, thereby causing the death of infected macrophages via activating IRE1α pathway of endoplasmic reticulum stress, suggesting novel insights into the molecular mechanisms associated with Brucella rough mutant-induced macrophage cytotoxicity.

A Brucella spp. Isolate from a Pac-Man Frog (Ceratophrys ornata) Reveals Characteristics Departing from Classical Brucellae.

Brucella are highly infectious bacterial pathogens responsible for brucellosis, a frequent worldwide zoonosis. The Brucella genus has recently expanded from 6 to 11 species, all of which were associated with mammals; The natural host range recently expanded to amphibians after some reports of atypical strains from frogs. Here we describe the first in depth phenotypic and genetic characterization of a Brucella strains isolated from a frog. Strain B13-0095 was isolated from a Pac-Man frog (Ceratophyrus ornate) at a veterinary hospital in Texas and was initially misidentified as Ochrobactrum anthropi. We found that B13-0095 belongs to a group of early-diverging brucellae that includes Brucella inopinata strain BO1 and the B. inopinata-like strain BO2, with traits that depart significantly from those of the "classical" Brucella spp. Analysis of B13-0095 genome sequence revealed several specific features that suggest that this isolate represents an intermediate between a soil associated ancestor and the host adapted "classical" species. Like strain BO2, B13-0095 does not possess the genes required to produce the perosamine based LPS found in classical Brucella, but has a set of genes that could encode a rhamnose based O-antigen. Despite this, B13-0095 has a very fast intracellular replication rate in both epithelial cells and macrophages. Finally, another major finding in this study is the bacterial motility observed for strains B13-0095, BO1, and BO2, which is remarkable for this bacterial genus. This study thus highlights several novel characteristics in strains belonging to an emerging group within the Brucella genus. Accurate identification tools for such atypical Brucella isolates and careful evaluation of their zoonotic potential, are urgently required.

Long-term and large-scale epidemiology of Brucella infection in baleen whales and sperm whales in the western North Pacific and Antarctic Oceans.

In a long-term, large-scale serologic study in the western North Pacific Ocean, anti-Brucella antibodies were detected in common minke whales (Balaenoptera acutorostrata) in the 1994-2010 offshore surveys (21%, 285/1353) and in the 2006-2010 Japanese coastal surveys (20%, 86/436), in Bryde's whales (B. edeni brydei) in the 2000-2010 offshore surveys (9%, 49/542), in sei whales (B. borealis) in the 2002-2010 offshore surveys (5%, 40/788) and in sperm whales (Physeter macrocephalus) in the 2000-2010 offshore surveys (8%, 4/50). Anti-Brucella antibodies were not detected in 739 Antarctic minke whales (B. bonaerensis) in the 2000-2010 Antarctic surveys. This suggests that Brucella was present in the four large whale populations inhabiting the western North Pacific, but not in the Antarctic minke whale population. By PCR targeting for genes of outer membrane protein 2, the Brucella infection was confirmed in tissue DNA samples from Bryde's whales (14%, 2/14), sei whales (11%, 1/9) and sperm whales (50%, 2/4). A placental tissue and an apparently healthy fetus from a sperm whale were found to be PCR-positive, indicating that placental transmission might have occurred and the newborn could act as a bacterial reservoir. Marked granulomatous testes were observed only in mature animals of the three species of baleen whales in the western North Pacific offshore surveys, especially in common minke whales, and 29% (307/1064) of total mature males had abnormal testes. This study provides an insight into the status of marine Brucella infection at a global level.

Brucella vulpis sp. nov., isolated from mandibular lymph nodes of red foxes (Vulpes vulpes).

Two slow-growing, Gram-negative, non-motile, non-spore-forming, coccoid bacteria (strains F60T and F965), isolated in Austria from mandibular lymph nodes of two red foxes (Vulpes vulpes), were subjected to a polyphasic taxonomic analysis. In a recent study, both isolates were assigned to the genus Brucella but could not be attributed to any of the existing species. Hence, we have analysed both strains in further detail to determine their exact taxonomic position and genetic relatedness to other members of the genus Brucella. The genome sizes of F60T and F965 were 3 236 779 and 3 237 765 bp, respectively. Each genome consisted of two chromosomes, with a DNA G+C content of 57.2 %. A genome-to-genome distance of >80 %, an average nucleotide identity (ANI) of 97 % and an average amino acid identity (AAI) of 98 % compared with the type species Brucella melitensis confirmed affiliation to the genus. Remarkably, 5 % of the entire genetic information of both strains was of non-Brucella origin, including as-yet uncharacterized bacteriophages and insertion sequences as well as ABC transporters and other genes of metabolic function from various soil-living bacteria. Core-genome-based phylogenetic reconstructions placed the novel species well separated from all hitherto-described species of the genus Brucella, forming a long-branched sister clade to the classical species of Brucella. In summary, based on phenotypic and molecular data, we conclude that strains F60T and F965 are members of a novel species of the genus Brucella, for which the name Brucella vulpis sp. nov. is proposed, with the type strain F60T ( = BCCN 09-2T = DSM 101715T).

Brucella ovis PA mutants for outer membrane proteins Omp10, Omp19, SP41, and BepC are not altered in their virulence and outer membrane properties.

Mutants in several genes have been obtained on the genetic background of virulent rough (lacking O-polysaccharide) Brucella ovis PA. The target genes encode outer membrane proteins previously associated with the virulence of smooth (bearing O-polysaccharide chains in the lipopolysaccharide) Brucella strains. Multiple attempts to delete omp16, coding for a homologue to peptidoglycan-associated lipoproteins, were unsuccessful, which suggests that Omp16 is probably essential for in vitro survival of B. ovis PA. Single deletion of omp10 or omp19-that encode two other outer membrane lipoproteins--was achieved, but the simultaneous removal of both genes failed, suggesting an essential complementary function between both proteins. Two other deletion mutants, defective in the Tol-C-homologue BepC or in the SP41 adhesin, were also obtained. Surprisingly when compared to previous results obtained with smooth Brucella, none of the B. ovis mutants showed attenuation in the virulence, either in the mouse model or in cellular models of professional and non-professional phagocytes. Additionally, and in contrast to the observations reported with smooth Brucella strains, several properties related to the outer membrane remained almost unaltered. These results evidence new distinctive traits between naturally rough B. ovis and smooth brucellae.

Brucella discriminates between mouse dendritic cell subsets upon in vitro infection.

Brucella is a Gram-negative bacterium responsible for brucellosis, a worldwide re-emerging zoonosis. Brucella has been shown to infect and replicate within Granulocyte macrophage colony-stimulating factor (GMCSF) in vitro grown bone marrow-derived dendritic cells (BMDC). In this cell model, Brucella can efficiently control BMDC maturation. However, it has been shown that Brucella infection in vivo induces spleen dendritic cells (DC) migration and maturation. As DCs form a complex network composed by several subpopulations, differences observed may be due to different interactions between Brucella and DC subsets. Here, we compare Brucella interaction with several in vitro BMDC models. The present study shows that Brucella is capable of replicating in all the BMDC models tested with a high infection rate at early time points in GMCSF-IL15 DCs and Flt3l DCs. GMCSF-IL15 DCs and Flt3l DCs are more activated than the other studied DC models and consequently intracellular bacteria are not efficiently targeted to the ER replicative niche. Interestingly, GMCSF-DC and GMCSF-Flt3l DC response to infection is comparable. However, the key difference between these 2 models concerns IL10 secretion by GMCSF DCs observed at 48 h post-infection. IL10 secretion can explain the weak secretion of IL12p70 and TNFα in the GMCSF-DC model and the low level of maturation observed when compared to GMCSF-IL15 DCs and Flt3l DCs. These models provide good tools to understand how Brucella induce DC maturation in vivo and may lead to new therapeutic design using DCs as cellular vaccines capable of enhancing immune response against pathogens.