RESUMO
One of the capabilities developed by bacteria is the ability to gain large fragments of DNA from other bacteria or to lose portions of their own genomes. Among these exchangeable fragments are the genomic islands (GIs). Nine GIs have been identified in Brucella, and genomic island 3 (GI-3) is shared by two pathogenic species, B. melitensis and B. abortus. GI-3 encodes mostly unknown proteins. One of the aims of this study was to perform pulsed-field gel electrophoresis (PFGE) on field isolates of B. abortus from Chile to determine whether these isolates are clonally related. Furthermore, we focused on the characterization of GI-3, studying its organization and the genetic conservation of the GI-3 sequence using techniques such as tiling-path PCR (TP-PCR) and restriction fragment length polymorphism-PCR (RFLP-PCR). Our results, after PFGE was performed on 69 field isolates of B. abortus from Chile, showed that the strains were genetically homogeneous. To increase the power of genetic discrimination among these strains, we used multiple locus variable-number tandem-repeat (VNTR) analysis with 16 loci (MLVA-16). The results obtained by MLVA-16 showed that the strains of B. abortus were genetically heterogeneous and that most of them clustered according to their geographic origin. Of the genetic loci studied, panel 2B was the one describing the highest diversity in the analysis, as well as locus Bruce19 in panel 2A. In relation to the study of GI-3, our experimental analysis by TP-PCR identified and confirmed that GI-3 is present in all wild strains of B. abortus, demonstrating the high stability of gene cluster GI-3 in Chilean field strains.
Assuntos
Brucella abortus/classificação , Brucella abortus/genética , Brucelose/microbiologia , Variação Genética , Ilhas Genômicas , Animais , Chile , Análise por Conglomerados , DNA Bacteriano/genética , Eletroforese em Gel de Campo Pulsado , Genótipo , Humanos , Tipagem Molecular , Reação em Cadeia da Polimerase , Polimorfismo de Fragmento de RestriçãoRESUMO
The genomic island 3 (GI-3) shared by Brucella melitensis and Brucella abortus contains 29 genes encoding mostly unknown proteins. Within this island, the open reading frames (ORFs) BAB1_0278 and BAB1_0263 are present, BAB1_0278 encodes a hypothetical protein of 64 amino acids sharing a domain with the GcrA superfamily, whereas the amino acid sequence of BAB1_0263 showed 42% identity with an iron regulated Lsr2 protein. We obtained one deletion mutant for each one of these ORFs present within the B. abortus GI-3 named BA-278 and BA-263, respectively. Both mutants were evaluated with respect to their ability to invade and replicate in nonprofessional and professional phagocytes (HeLa and J774.A1 cells) and their virulence in mice. Both mutants invaded efficiently HeLa and J774. A1 cells, however, 48-h post-infection the BA-278 mutant showed a lower intracellular persistence. The deletion of the ORF BAB1_0278, also affected the persistence of B. abortus in the spleens of mice, unlike to the deletion of the ORF BAB1_0263. These results allow us to conclude that BAB1_0278 ORF contributes to virulence of Brucella, since it is necessary to establish an optimal infectious process.
Assuntos
Brucella abortus/genética , Brucella abortus/patogenicidade , Brucelose/veterinária , Ilhas Genômicas , Animais , Brucella melitensis/genética , Células HeLa , Humanos , Camundongos , Camundongos Endogâmicos BALB C , Fases de Leitura Aberta , Deleção de Sequência , Virulência , Fatores de Virulência/genéticaRESUMO
The infC gene of Brucella abortus encoding the translation initiation factor 3 (IF3) was cloned, sequenced and expressed in Escherichia coli. The amino acid sequence analysis predicted a product with 74-80% identity with the IF3 proteins from Mesorhizobium loti, Sinorhizobium meliloti, Aurantimona sp. and Mesorhizobium sp. This protein also show 54% amino acid sequence identity with the E. coli IF3, sharing most of the residues which were described as responsible for the biological activity of this protein. Since we have previously reported the immunoprotective capacity of this Brucella protein, we stimulated lymphoid cells from animals immunized with purified recombinant Brucella IF3 protein "in vitro" with this antigen. The lymphocytes were able to mount a strong proliferative response with concomitant production of gamma interferon, but without the secretion of either IL-4 or antibodies. Thus, immunization with the Brucella recombinant IF3 protein promotes a TH-1 polarized response, allowing us to propose it as a promising candidate antigen for the development of subunit vaccines against Brucella.
Assuntos
Vacina contra Brucelose/imunologia , Brucella abortus/imunologia , Fator de Iniciação 3 em Procariotos/imunologia , Proteínas Recombinantes de Fusão/imunologia , Sequência de Aminoácidos , Animais , Sequência de Bases , Vacina contra Brucelose/genética , Células Cultivadas , Clonagem Molecular , Feminino , Expressão Gênica , Interferon gama/imunologia , Interferon gama/metabolismo , Interleucina-4/imunologia , Interleucina-4/metabolismo , Linfócitos/imunologia , Linfócitos/metabolismo , Camundongos , Camundongos Endogâmicos BALB C , Dados de Sequência Molecular , Fator de Iniciação 3 em Procariotos/genética , Proteínas Recombinantes de Fusão/genética , Alinhamento de Sequência , Vacinas de Subunidades Antigênicas/genética , Vacinas de Subunidades Antigênicas/imunologiaRESUMO
Previously we reported that immunization with Escherichia coli DH5alpha-expressing Brucella abortus Cu/Zn superoxide dismutase [E. coli (pBSSOD)] induces a protective immune response in BALB/c mice. Here we studied the type of immune defense that the recombinant E. coli induces in mice using as our experimental model Brucella superoxide dismutase Cu/Zn presented by J744.A1 to sensitized lymphocytes as the target of specific lysis or as cytokine inductors. The results indicate that E. coli carrying the Cu/Zn gene was able to induce specific cytotoxic T cells, mainly from CD8(+) subpopulation and IFN-gamma-producing cells belonging in their vast majority to the CD4(+) subpopulation.