RESUMO
Mycobacterium bovis causes tuberculosis in a wide variety of mammals, with strong tropism for cattle and eventually humans. P27, also called LprG, is among the proteins involved in the mechanisms of the virulence and persistence of M. bovis and Mycobacterium tuberculosis Here, we describe a novel function of P27 in the interaction of M. bovis with its natural host cell, the bovine macrophage. We found that a deletion in the p27-p55 operon impairs the replication of M. bovis in bovine macrophages. Importantly, we show for the first time that M. bovis arrests phagosome maturation in a process that depends on P27. This effect is P27 specific since complementation with wild-type p27 but not p55 fully restored the wild-type phenotype of the mutant strain; this indicates that P55 plays no important role during the early events of M. bovis infection. In addition, we also showed that the presence of P27 from M. smegmatis decreases the association of LAMP-3 with bead phagosomes, indicating that P27 itself blocks phagosome-lysosome fusion by modulating the traffic machinery in the cell host.
Assuntos
Lipoproteínas/metabolismo , Macrófagos/microbiologia , Macrófagos/fisiologia , Mycobacterium bovis/fisiologia , Fagossomos/metabolismo , Fagossomos/microbiologia , Animais , Bovinos , Pontos de Checagem do Ciclo Celular , Expressão Gênica , Células HeLa , Humanos , Lipoproteínas/genética , Viabilidade Microbiana , Mutação , Óxido Nítrico Sintase Tipo II/genética , Óxido Nítrico Sintase Tipo II/metabolismo , ÓperonRESUMO
BACKGROUND: Tuberculosis is one of the leading causes of mortality throughout the world. Mycobacterium tuberculosis, the agent of human tuberculosis, has developed strategies involving proteins and other compounds called virulence factors to subvert human host defences and damage and invade the human host. Among these virulence-related proteins are the Mce proteins, which are encoded in the mce1, mce2, mce3 and mce4 operons of M. tuberculosis. The expression of the mce2 operon is negatively regulated by the Mce2R transcriptional repressor. Here we evaluated the role of Mce2R during the infection of M. tuberculosis in mice and macrophages and defined the genes whose expression is in vitro regulated by this transcriptional repressor. RESULTS: We used a specialized transduction method for generating a mce2R mutant of M. tuberculosis H37Rv. Although we found equivalent replication of the MtΔmce2R mutant and the wild type strains in mouse lungs, overexpression of Mce2R in the complemented strain (MtΔmce2RComp) significantly impaired its replication. During in vitro infection of macrophages, we observed a significantly increased association of the late endosomal marker LAMP-2 to MtΔmce2RComp-containing phagosomes as compared to MtΔmce2R and the wild type strains. Whole transcriptional analysis showed that Mce2R regulates mainly the expression of the mce2 operon, in the in vitro conditions studied. CONCLUSIONS: The findings of the current study indicate that Mce2R weakly represses the in vivo expression of the mce2 operon in the studied conditions and argue for a role of the proteins encoded in Mce2R regulon in the arrest of phagosome maturation induced by M. tuberculosis.
Assuntos
Antígenos de Bactérias/biossíntese , Proteínas de Bactérias/biossíntese , Regulação Bacteriana da Expressão Gênica , Mycobacterium tuberculosis/fisiologia , Proteínas Repressoras/metabolismo , Fatores de Virulência/biossíntese , Animais , Modelos Animais de Doenças , Deleção de Genes , Perfilação da Expressão Gênica , Pulmão/microbiologia , Macrófagos/microbiologia , Camundongos , Camundongos Endogâmicos BALB C , Mycobacterium tuberculosis/genética , Mycobacterium tuberculosis/crescimento & desenvolvimento , Mycobacterium tuberculosis/patogenicidade , Óperon , Transdução Genética , Tuberculose/microbiologia , Tuberculose/patologiaRESUMO
A Mycobacterium bovis strain deleted in mce2A and mce2B genes (M. bovis Δmce2) was tested as an experimental vaccine in cattle challenged with a virulent M. bovis strain. Three-and-a-half-month old calves (n = 5 to 6 per group) were vaccinated and challenged with a virulent strain of M. bovis by the intratracheal route 9 weeks after vaccination. A non-vaccinated group and a group vaccinated with BCG were included as controls. Blood samples were collected to measure IFN-γ by an interferon-gamma release assay (IGRA), cytometry and cytokine responses of bovine purified protein derivative (PPD) restimulated peripheral blood mononuclear cells (PBMCs). The IGRA test showed IFN-γ values similar to pre-vaccination except for the animals vaccinated with M. bovis Δmce2, where a significant increase was observed at 30 days post-vaccination. The expression of IL-2R on CD4(+) cells in response to PPD from the animals vaccinated with Δmce2 increased at 15 days post-vaccination compared to cells from non-vaccinated group. Vaccination of cattle with M. bovis Δmce2 induced the highest (P < 0.05) expression of IFN-γ and IL-17 mRNA upon PPD stimulation of PBMCs compared to vaccination with BCG or that for the non-vaccinated group. There was a weak positive correlation between the production of these proinflammatory cytokines post-vaccination and reduced pathology scores post-challenge. The animals were euthanized and necropsied 100 days after challenge. The group vaccinated with M. bovis Δmce2 displayed a significantly lower histopathological score for lesions in lungs and pulmonary lymph nodes than for the other groups (P < 0.05). A marked positive reaction to tuberculin intradermal test was observed post-vaccination in animals vaccinated with M. bovis Δmce2 compared to those vaccinated with BCG or the non-vaccinated group. In contrast, after challenge, non-vaccinated animals had greater skin test responses than the vaccinated animals. In summary, M. bovis Δmce2 is a promising vaccine candidate to control M. bovis pathogenesis in cattle.
Assuntos
Antígenos de Bactérias/genética , Proteínas de Bactérias/genética , Mycobacterium bovis/genética , Vacinas contra a Tuberculose/imunologia , Tuberculose Bovina/prevenção & controle , Animais , Antígenos de Bactérias/imunologia , Vacina BCG , Carga Bacteriana , Proteínas de Bactérias/imunologia , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD8-Positivos/imunologia , Bovinos , Citocinas/biossíntese , Citocinas/sangue , Citocinas/genética , Deleção de Genes , Interferon gama/biossíntese , Testes de Liberação de Interferon-gama/métodos , Ativação Linfocitária/imunologia , Subpopulações de Linfócitos/imunologia , Mycobacterium bovis/patogenicidade , Tuberculina/imunologia , Teste Tuberculínico , Tuberculose Bovina/imunologia , Tuberculose Bovina/microbiologia , Tuberculose Bovina/patologia , Vacinas Atenuadas/imunologia , VirulênciaRESUMO
Mycobacterium bovis is the causative agent of tuberculosis in cattle but also infects other animals, including humans. Previous studies in cattle have demonstrated that the protection induced by BCG is not complete. In order to improve the protection efficacy of BCG, in this study we overexpressed Ag85B in a BCG Pasteur strain, by using an expression system based on the use of an auxotrophic strain for the leucine amino acid, and complementation with leuD. We found that vaccination of cattle with BCG overexpressing Ag85B induced higher production of IL-17 and IL-4 mRNA upon purified protein derivative (PPDB) stimulation of peripheral blood mononuclear cells (PBMCs) than vaccination with BCG. Moreover, the IL-17 mRNA expression after vaccination negatively correlated with disease severity resulting from a subsequent challenge with M. bovis, suggesting that this cytokine is a potential biomarker of cattle protection against bovine tuberculosis. Importantly, vaccination with the recombinant BCG vaccine protected cattle better than the wild-type BCG Pasteur.
Assuntos
Vacina BCG/administração & dosagem , Doenças dos Bovinos/prevenção & controle , Mycobacterium bovis/imunologia , Tuberculose Bovina/prevenção & controle , Animais , Bovinos , Doenças dos Bovinos/imunologia , Primers do DNA , Citometria de Fluxo , Interferon gama/biossíntese , Reação em Cadeia da Polimerase , Teste Tuberculínico , Tuberculose Bovina/imunologiaRESUMO
Mycobacterium bovis is the causative agent of most cases of bovine tuberculosis. The identification of bTB biomarkers in specific stages of the disease will contribute to a better understanding of the immunopathology associated with tuberculosis and will enable their use in disease diagnosis and prognosis. The aim of this study was to evaluate the gene expression profile induced after specific stimulation of bovine peripheral blood mononuclear cells from cattle infected with M. bovis using the Affymetrix® GeneChip® Bovine Genome Array. A total of 172 genes showed differential expression profile that was statistically significant with log2-fold change >2.5 and <-2.5. Twenty-four out of these genes were upregulated and 148 were downregulated in bovine peripheral blood mononuclear cells of M. bovis-infected cattle. The highest differentially-expressed genes were related to immune and inflammatory responses, apoptosis, endocytosis, cellular trafficking and genes encoding proteins involved in cellular matrix degradation. Microarray results were confirmed in another group of infected cattle by RT-qPCR for the CD14, IL-1R, THBS1, MMP9 and FYVE genes. This study confirms previous findings that have shown that M. bovis infection in cattle results in the downregulation of immune response-related genes. Moreover, it validates the use of microarray platforms in combination with RT-qPCR to identify biomarkers of bovine tuberculosis. In addition, we propose CD14, IL-1R, THBS1, MMP9 and FYVE as potential biomarkers of bovine tuberculosis.
Assuntos
Regulação Bacteriana da Expressão Gênica , Leucócitos Mononucleares/metabolismo , Leucócitos Mononucleares/microbiologia , Mycobacterium bovis/metabolismo , Tuberculose Bovina/microbiologia , Animais , Apoptose , Biomarcadores/metabolismo , Bovinos , Endocitose , Perfilação da Expressão Gênica , Inflamação , Modelos Genéticos , Análise de Sequência com Séries de Oligonucleotídeos , Transcrição GênicaRESUMO
The generation of efficient candidate vaccines against bovine tuberculosis will contribute to the control of this zoonotic disease. Rationally attenuated Mycobacterium bovis strains generated by knockout of virulence genes are promising candidate vaccines. However, to be effective, these candidate vaccines should at least maintain the immunological properties of their virulent parental M. bovis strains. Therefore, the aim of this study was to obtain an M. bovis strain deleted in the mce2 genes and evaluate the effect of the mutation on the immunological profile elicited by the bacteria in cattle. We showed that the activation of CD4+ T cells in cattle inoculated with the mutant strain was equivalent to that in animals inoculated with the parental strain. Moreover, after in vitro stimulation, peripheral blood mononuclear cells from animals inoculated with the mutant produced higher levels of mRNA Th-1 cytokines than the parental strain. Therefore, these results indicate that the mce2 mutant is a promising candidate vaccine against bovine tuberculosis.
Assuntos
Bovinos/imunologia , Mycobacterium bovis/genética , Mycobacterium bovis/imunologia , Vacinas contra a Tuberculose/genética , Vacinas contra a Tuberculose/imunologia , Tuberculose Bovina/imunologia , Animais , Antígenos de Bactérias/genética , Proteínas de Bactérias/genética , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD8-Positivos/imunologia , Células Cultivadas , Citocinas/genética , Citocinas/imunologia , Citocinas/metabolismo , Técnicas de Inativação de Genes , Interações Hospedeiro-Patógeno , Leucócitos Mononucleares/imunologia , Leucócitos Mononucleares/metabolismo , Reação em Cadeia da Polimerase , RNA Mensageiro/análise , Vacinas contra a Tuberculose/administração & dosagem , Tuberculose Bovina/prevenção & controleRESUMO
The Mycobacterium smegmatis genome contains six operons designated mce (mammalian cell entry). These operons, which encode membrane and exported proteins, are highly conserved in pathogenic and non-pathogenic mycobacteria. Although the function of the Mce protein family has not yet been established in Mycobacterium smegmatis, the requirement of the mce4 operon for cholesterol utilization and uptake by Mycobacterium tuberculosis has recently been demonstrated. In this study, we report the construction of an M. smegmatis knock-out mutant deficient in the expression of all six mce operons. The consequences of these mutations were studied by analyzing physiological parameters and phenotypic traits. Differences in colony morphology, biofilm formation and aggregation in liquid cultures were observed, indicating that mce operons of M. smegmatis are implicated in the maintenance of the surface properties of the cell. Importantly, the mutant strain showed reduced cholesterol uptake when compared to the parental strain. Further cholesterol uptake studies using single mce mutant strains showed that the mutation of operon mce4 was reponsible for the cholesterol uptake failure detected in the sextuple mce mutant. This finding demonstrates that mce4operon is involved in cholesterol transport in M. smegmatis.
Assuntos
Colesterol/farmacocinética , Genes Bacterianos , Mycobacterium smegmatis/genética , Mycobacterium smegmatis/metabolismo , Óperon/genética , Deleção de Sequência , Virulência/genética , Regulação Bacteriana da Expressão Gênica , Técnicas de Inativação de Genes , Mutação , Mycobacterium smegmatis/patogenicidade , Mycobacterium tuberculosis/genética , Mycobacterium tuberculosis/metabolismoRESUMO
Integrity of p27-p55 operon has been demonstrated to be crucial for replication of Mycobacterium tuberculosis, the main agent of human tuberculosis, in the mouse model of infection. However, the individual contribution of each gene of the operon for the virulence of pathogenic Mycobacterium spp. still remains unclear. The operon is formed by two genes, p27 and p55. p27 gene encodes a lipoprotein that binds triacylated glycolipids and modulates the host immune responses by inhibiting the MHC-II Ag processing. Besides, p55 encodes an efflux pump that, together with P27, is involved in resistance to drugs. In this study, we evaluated the individual contribution of P27 and P55 to the virulence of Mycobacterium bovis, the etiological agent for bovine tuberculosis. Knockout mutation of p27-p55 operon in M. bovis severely decreased the virulence of the bacteria when assessed in a progressive model of pulmonary tuberculosis in Balb/c mice. In addition, the mutant strain showed poor replication in a murine macrophagic cell line. Virulence and intracellular replication were only restored when the mutant strain was complemented with a copy of the whole operon. The reintroduction of p55 into the mutant strain partially restored the virulence of the bacteria while no complementation was achieved with p27 individual gene.
Assuntos
Proteínas de Bactérias/genética , Deleção de Genes , Lipoproteínas/genética , Macrófagos/microbiologia , Proteínas de Membrana Transportadoras/genética , Viabilidade Microbiana , Mycobacterium bovis/patogenicidade , Tuberculose Bovina/microbiologia , Animais , Bovinos , Linhagem Celular , Contagem de Colônia Microbiana , Modelos Animais de Doenças , Teste de Complementação Genética , Pulmão/microbiologia , Camundongos , Camundongos Endogâmicos BALB C , Mycobacterium bovis/genética , Óperon , Tuberculose Bovina/patologia , Tuberculose Pulmonar/microbiologia , Tuberculose Pulmonar/patologia , Virulência , Fatores de Virulência/genéticaRESUMO
The identification of bovine tuberculosis (bTB) biomarkers in specific stages of the disease will contribute to a better understanding of the immunopathology associated with tuberculosis and to improve the disease diagnosis and prognosis. The aim of this study was to understand the changing profile of the immune responses during the course of infection and to identify biomarkers associated with pathology. Here we describe the immune response developed in experimentally infected cattle with field Mycobacterium bovis strains. Blood samples were taken from each animal at different time points after M. bovis intratracheal infection and lymphocyte subset activation and cytokine mRNA expression were determined from peripheral blood mononuclear cells in response to purified protein derivative (PPDB). We found that CD4 and CD8 activation during the early stages of infection, together with IL-17 gene expression, were positively associated with pathology. The results of this study provide evidences of the role of IL-17 in the immunopathology of tuberculosis and support the use of IL-17 as a potential biomarker with predictive value of prognosis in bTB.
Assuntos
Linfócitos T CD4-Positivos/imunologia , Regulação Bacteriana da Expressão Gênica/imunologia , Interleucina-17/metabolismo , Mycobacterium bovis/imunologia , Tuberculose Bovina/imunologia , Animais , Biomarcadores/metabolismo , Bovinos , Interleucina-17/genética , Ativação Linfocitária , Mycobacterium bovis/genética , Valor Preditivo dos Testes , Tuberculina/imunologia , Tuberculose Bovina/genéticaRESUMO
A number of studies have determined the contribution of Th1 and Th2 responses to the protective immunity and pathology of Mycobacterium bovis infection. However, much of that information is derived from experimentally infecting cattle with M. bovis and few data from naturally infected animals are available. The aim of this study was to characterize the immunological profile towards M. bovis antigens of naturally infected cattle by measurement of cytokine mRNA expression in PBMC, and to determine which lymphocyte subsets are involved in recall responses of PBMC from M. bovis infected cattle to M. bovis antigens. Consistent with data from cattle experimentally infected with M. bovis, naturally infected animals were found to display a Th1 cytokine profile in response to M. bovis PPDB stimulation. Production of IFN-gamma mRNA by PBMC after PPDB stimulation statistically distinguishes between infected and healthy herds, suggesting that this molecule is usable as an M. bovis-infection marker. As happens in experimentally infected cows, CD4, CD8 and gammadeltaTCR cells from a herd naturally infected with M. bovis are the predominant T cell subsets expanded in response to PPDB.