Prediction of Structure and Molecular Interaction with DNA of BvrR, a Virulence-Associated Regulatory Protein of Brucella.

Brucellosis, also known as "undulant fever" is a zoonotic disease caused by Brucella, which is a facultative intracellular bacterium. Despite efforts to eradicate this disease, infection in uncontrolled domestic animals persists in several countries and therefore transmission to humans is common. Brucella evasion of the innate immune system depends on its ability to evade the mechanisms of intracellular death in phagocytic cells. The BvrR-BvrS two-component system allows the bacterium to detect adverse conditions in the environment. The BvrS protein has been associated with genes of virulence factors, metabolism, and membrane transport. In this study, we predicted the DNA sequence recognized by BvrR with Gibbs Recursive Sampling and identified the three-dimensional structure of BvrR using I-TASSER suite, and the interaction mechanism between BvrR and DNA with Protein-DNA docking and molecular dynamics (MD) simulation. Based on the Gibbs recursive Sampling analysis, we found the motif AAHTGC (H represents A, C, and T nucleotides) as a possible sequence recognized by BvrR. The docking and EMD simulation results showed that C-terminal effector domain of BvrR protein is likely to interact with AAHTGC sequence. In conclusion, we predicted the structure, recognition motif, and interaction of BvrR with DNA.

A bacterial protease inhibitor protects antigens delivered in oral vaccines from digestion while triggering specific mucosal immune responses.

We report here that a bacterial protease inhibitor from Brucella spp. called U-Omp19 behaves as an ideal constituent for a vaccine formulation against infectious diseases. When co-administered orally with an antigen (Ag), U-Omp19: i) can bypass the harsh environment of the gastrointestinal tract by inhibiting stomach and intestine proteases and consequently increases the half-life of the co-administered Ag at immune inductive sites: Peyer's patches and mesenteric lymph nodes while ii) it induces the recruitment and activation of antigen presenting cells (APCs) and increases the amount of intracellular Ag inside APCs. Therefore, mucosal as well as systemic Ag-specific immune responses, antibodies, Th1, Th17 and CD8(+) T cells are enhanced when U-Omp19 is co-administered with the Ag orally. Finally, this bacterial protease inhibitor in an oral vaccine formulation confers mucosal protection and reduces parasite loads after oral challenge with virulent Toxoplasma gondii.

Newly identified variability in Brucella canis fatty-acid content is associated with geographical origin.

This study compared the fatty-acid profiles of Brucella canis blood culture isolates obtained from infected dogs in the UK, Germany, Japan, South Africa, Peru, Mexico, Colombia, and Argentina, and from a human clinical case in Argentina, to a bank of isolates obtained from canine outbreaks in the USA. Analysis of a total of 42 B. canis isolates and one reference strain found a marked variation within the species. Fatty-acid analysis showed that only the isolates from Argentina, Colombia, and Mexico, which included the human B. canis isolate, contained a specific fatty acid, 19:0 cyclopropane (lactobacillic acid), w8c (cis-11,12-methylene octadecanoic acid), and that this fatty acid, when present, made up a large percentage of overall fatty-acid content. Prior to this study, the cellular fatty-acid 19:0 cyclopropane had been identified in all of the species of Brucella considered to be pathogenic to humans (B. abortus, B. melitensis, B. suis) except for B. canis. Discovering that this fatty acid not only occurs in B. canis, but also that it is only present in some strains of the species provides a new focus for investigations aimed at identifying the cause of reported geographical variability in human B. canis infection, and at finding predictors of biological behaviour and human pathogenicity within this Brucella species.

Brucella sp. como bioagente da bursite cervical em bovinos abatidos no Município de São Luís, Maranhão: uma abordagem sorológica, imunohistoquímica, microbiológica e molecular

Brucella sp. como bioagente da bursite cervical em bovinos abatidos no Município de São Luís, Maranhão: uma abordagem sorológica, imunohistoquímica, microbiológica e molecular. [Brucella sp. like a bioagent of cervical bursitis in cattle slaughtered in São Luís, Maranhão: a serological approach, immunohistochemistry, and molecular microbiological]. 2012. 82f. Dissertação (Mestrado em Ciência Animal) – Universidade Estadual do Maranhão, São Luís, 2012. RESUMO A brucelose é uma antropozoonose de evolução preferencialmente crônica, caracterizada pela infecção do sistema fagocítico mononuclear, por bactérias do gênero Brucella. Considerando a hipótese do abate de animais infectados e o seu risco potencial ao homem, a presente pesquisa buscou avaliar a Brucella sp. como bioagente da bursite cervical em bovinos abatidos no Município de São Luís, Maranhão. Foram coletadas amostras de sangue, com e sem E.D.T.A (ácido etilenodiamino tetra-acético), fragmentos de bolsa cervical e linfonodos correspondentes de 47 bovinos abatidos em frigoríficos sob Inspeção Municipal, que apresentaram bursite cervical postmortem. A partir deste material coletado estimou-se a freqüência do bioagente Brucella sp. nas diferentes abordagens diagnósticas: sorologia, histopatologia, imunohistoquímica e PCR. Na sorologia de animais acima de 24 meses, 85,1% das amostras foram reagentes ao teste do AAT, confirmando-se a positividade em 78,7% através dos testes de soroaglutinação lenta e 2-mercaptoetanol. Na histopatologia foi possível observar que a maioria das amostras de bolsa cervical e linfonodos apresentaram infiltrado inflamatório linfohistioplasmocitário, mais comumente associado ao quadro crônico da doença. Todas as amostras de sangue total coletadas foram submetidas a PCR convencional, não sendo verificada a amplificação de produto de DNA compatível com Brucella sp. As 47 amostras coletadas foram submetidas ao isolamento bacteriano, sendo, posteriormente, efetuada a PCR a partir de tecidos (bolsa cervical e linfonodos correspondentes). Nesta PCR, evidenciou-se que 63,8% das amostras apresentaram um amplificado de 223pb, condizentes com o gênero Brucella sp. As amostras nas quais foram confirmadas o gênero Brucella, foram submetidas ao PCR multiplex AMOS – ENHANCED, objetivando verificar quais espécies desta bactéria estavam presentes nas amostras analisadas, constatou-se que em 28,0% foram identificadas duas bandas de 498 e 178pb, correspondentes ao biovar 1, 2, 4; em 30,0% das amostras foi identificada somente uma banda de 178pb, correspondente ao biovar 3, 5, 6, 9; 6,0% das amostras foram negativas na PCR multiplex e em 36,0% não houve isolamento de colônias, demonstrando padrões diferenciados entre os amplificados. Os resultados obtidos com a conclusão deste estudo puderam fornecer subsídios para futuras publicações científicas, além de contribuir com dados para o entendimento epidemiológico, isolamento e caracterização das espécies de Brucella sp., que acometem o rebanho bovino maranhense.

The ß-scaffold of the LOV domain of the Brucella light-activated histidine kinase is a key element for signal transduction.

Light-oxygen-voltage (LOV) domains are blue-light-activated signaling modules present in a wide range of sensory proteins. Among them, the histidine kinases are the largest group in prokaryotes (LOV-HK). Light modulates the virulence of the pathogenic bacteria Brucella abortus through LOV-HK. One of the striking characteristic of Brucella LOV-HK is the fact that the protein remains activated upon light sensing, without recovering the basal state in the darkness. In contrast, the light state of the isolated LOV domain slowly returns to the dark state. To gain insight into the light activation mechanism, we have characterized by X-ray crystallography and solution NMR spectroscopy the structure of the LOV domain of LOV-HK in the dark state and explored its light-induced conformational changes. The LOV domain adopts the α/ß PAS (PER-ARNT-SIM) domain fold and binds the FMN cofactor within a conserved pocket. The domain dimerizes through the hydrophobic ß-scaffold in an antiparallel way. Our results point to the ß-scaffold as a key element in the light activation, validating a conserved structural basis for light-to-signal propagation in LOV proteins.

A recombinant subunit vaccine based on the insertion of 27 amino acids from Omp31 to the N-terminus of BLS induced a similar degree of protection against B. ovis than Rev.1 vaccination.

The development of an effective subunit vaccine against brucellosis is a research area of intense interest. The enzyme lumazine synthase from Brucella spp. (BLS) is highly immunogenic, presumably due to its decameric arrangement and remarkable stability. In this work we decided to develop a chimera with the scaffold protein BLS decorated with 10 copies of a known protective epitope derived from an outer membrane protein of 31kDa (Omp31) from Brucella spp. Vaccination of BALB/c mice with the chimera as a recombinant protein (rBLSOmp31) provided the best protection level against Brucella ovis, which was higher than the given by the co-delivery of both recombinant proteins (rBLS + rOmp31) and similar than the control vaccine Brucella melitensis strain Rev.1. Moreover rBLSOmp31 induced protection against Brucella melitensis but to a lesser degree than Rev.1. The chimera induced a strong humoral response against the inserted peptide. It also induced peptide- and BLS-specific T helper 1 and cytotoxic T responses. In conclusion, our results indicate that BLSOmp31 could be a useful candidate for the development of subunit vaccines against brucellosis since it elicits humoral, T helper and cytotoxic immune responses and protection against smooth and rough species of Brucella.

Intracellular lifestyle of Brucella spp. Common genes with other animal pathogens, plant pathogens, and endosymbionts.

Brucella spp. are intracellular pathogens that belong, like Agrobacterium, Rhizobium and Rickettsia, to the alpha-2-subgroup of proteobacteria. The genome organization of most Brucella spp. is characterized by the presence of two chromosomes. The intracellular lifestyle of Brucella, as well as the possible genes involved in pathogenesis and host cell signaling, are discussed, including the presence of genes with high similarity to those from other animal pathogens, plant pathogens and endosymbionts.