RESUMO
Mycobacterium tuberculosis and M. smegmatis form drug-tolerant biofilms through dedicated genetic programs. In support of a stepwise process regulating biofilm production in mycobacteria, it was shown elsewhere that lsr2 participates in intercellular aggregation, while groEL1 was required for biofilm maturation in M. smegmatis. Here, by means of RNA-Seq, we monitored the early steps of biofilm production in M. bovis BCG, to distinguish intercellular aggregation from attachment to a surface. Genes encoding for the transcriptional regulators dosR and BCG0114 (Rv0081) were significantly regulated and responded differently to intercellular aggregation and surface attachment. Moreover, a M. tuberculosis H37Rv deletion mutant in the Rv3134c-dosS-dosR regulon, formed less biofilm than wild type M. tuberculosis, a phenotype reverted upon reintroduction of this operon into the mutant. Combining RT-qPCR with microbiological assays (colony and surface pellicle morphologies, biofilm quantification, Ziehl-Neelsen staining, growth curve and replication of planktonic cells), we found that BCG0642c affected biofilm production and replication of planktonic BCG, whereas ethR affected only phenotypes linked to planktonic cells despite its downregulation at the intercellular aggregation step. Our results provide evidence for a stage-dependent expression of genes that contribute to biofilm production in slow-growing mycobacteria.
Assuntos
Proteínas de Bactérias/genética , Biofilmes , Mycobacterium bovis/genética , Mycobacterium tuberculosis/genética , Vacina BCG/genética , Vacina BCG/metabolismo , Aderência Bacteriana , Proteínas de Bactérias/metabolismo , Regulação Bacteriana da Expressão Gênica , Mycobacterium bovis/metabolismo , Mycobacterium tuberculosis/fisiologia , Óperon , Regulon , Transcrição GênicaRESUMO
Bovine tuberculosis is a chronic inflammatory disease that causes granuloma formation. Characterization of granulomatous lesions of Mycobacterium bovis (M. bovis) experimentally infected cattle has helped to better understand the pathogenesis of this disease. However, few studies have described granulomas found in M. bovis naturally infected cattle. The aim of this work was to examine granulomas from Holstein-Friesian cattle naturally infected with M. bovis from a dairy basin located in the central region of Mexico. Tissue samples from thirty-two cattle with lesions suggestive of tuberculosis were collected post-mortem. Fifteen of the 32 sampled animals (46.8%) were 4 months of age or younger (calves), whereas the rest (53.2%, 17/32) were over one year old (adults). Macroscopic lesions suggestive of tuberculosis were found in the mediastinal lymph node chain of all animals (32/32). From the 1,143 granulomatous lesions that were microscopically analyzed, 34.6% (396/1143) were collected from adult animals and subsequently classified according to the nomenclature suggested by Wangoo et al., 2005. Surprisingly, lesions from calf tissues showed an atypical pattern which could not be fitted into the established developmental stages of this classification. Granulomatous lesions found in calves covered most of the affected organ, histologically showed large necrotic areas with central calcification, absence of a connective tissue capsule, and few giant cells. Also, there was a higher percentage of lesions with acid-fast bacilli (AFB) when compared to studied granulomas in adults. Growth of Mycobacterium spp was detected in 11 bacteriological tissue cultures. Genotypic identification of M. bovis was performed by DNA extraction from bacterial isolates, formalin-fixed and paraffin-embedded (FFPE) tissues and samples without bacterial isolation. M. bovis was detected by PCR in 84.3% (27/32) of the studied cases; whereas other AFB were observed in tissues of the remaining sampled animals (5/32). Our results describe atypical granuloma formation in calves 4 months of age or younger, naturally infected with M. bovis. These findings contribute to better understanding the physiopathology of M. bovis infection in cattle.
Assuntos
Granuloma , Mycobacterium bovis , Tuberculose Bovina , Animais , Bovinos , Granuloma/genética , Granuloma/metabolismo , Granuloma/microbiologia , Granuloma/veterinária , Linfonodos/microbiologia , Linfonodos/patologia , México , Mycobacterium bovis/genética , Mycobacterium bovis/metabolismo , Especificidade de Órgãos , Tuberculose Bovina/genética , Tuberculose Bovina/metabolismo , Tuberculose Bovina/patologiaRESUMO
Mycobacterium bovis is the causative agent of tuberculosis in farms, wildlife and causes sporadic disease in humans. Despite the high similitude in genome sequence between M. bovis strains, some strains like the wild boar 04-303 isolate show a highly virulent phenotype in animal models. Comparative studies will contribute to link protein expression with the virulence phenotype. In vitro, the 04-303 strain was more phagocytized by J774A.1 macrophages in comparison with 444 strain (a cow isolate with the same genotype) and BCG. The secretome of these strains showed a significant proportion of shared proteins (368 spots). Among the proteins only visualized in the secretome of the 04-303 strain, we identify the nine most abundant proteins by LC-MS/MS. The most relevant were EsxA and EsxB proteins, which are encoded in the RD1 region, deleted in BCG strains. These proteins are the major virulence factor of M. tuberculosis. The other proteins identified belong to functional categories of virulence, detoxification, and adaptation; lipid metabolism; and cell wall and cell processes. The relatively high proportion of proteins involved in the cell wall and cell process is consistent with the previously described variation among M. bovis genomes.
Assuntos
Proteínas de Bactérias/análise , Proteínas de Bactérias/metabolismo , Mycobacterium bovis/química , Mycobacterium bovis/metabolismo , Proteoma/análise , Fatores de Virulência/análise , Animais , Cromatografia Líquida , Macrófagos/imunologia , Macrófagos/microbiologia , Mycobacterium bovis/imunologia , Mycobacterium bovis/isolamento & purificação , Mycobacterium tuberculosis , Fagocitose , Sus scrofa , Espectrometria de Massas em TandemRESUMO
We constructed recombinant Bacille Calmette-Guérin (rBCG) that secreted human interferon alpha 2b (hIFNα-2b), and investigated its antitumor effects on bladder cancer cells in vitro. The recombinant plasmid phIFN-α-2b was constructed using pMAO-4 and transformed into BCG. The supernatant was collected at various times and IFN-γ, interleukin (IL)-12, and tumor necrosis factor (TNF)-α were detected using an enzyme-linked immunosorbent assay. EJ cells were cultivated for 24, 48, and 72 h, together with rBCG, wild-type BCG (wBCG), or wBCG+IFN-α-2b. rBCG capable of secreting cytokine IFNα-2b was constructed. On the 4th day of culture, the IFNα-2b secreted by rBCG reached a maximum. wBCG and rBCG showed no significant difference on cell growth rate over 7 days of incubation in 7H9 medium. wBCG and rBCG were both positive for acid-fast staining, and showed mycobacterial characteristics of intercellular connection in clusters with no clear abnormalities. Higher levels of IFN-γ, TNF-α, and IL-12 were induced by rBCG compared with wBCG or MAO4-rBCG (P < 0.05). rBCG may induce lymphocyte proliferation; the proliferation ratio was higher than those induced by wBCG and wBCG+IFN. rBCG had direct anti-proliferative effects on EJ cells. An MTT assay showed that rBCG inhibited the proliferation of bladder cancer cells and had more activity compared with wBCG (P < 0.05). The highest anti-tumor activity of lymphocytes was stimulated by rBCG (20.31-51.22%). rBCG-IFNα-2b induces enhanced cytotoxicity against bladder cancer cells in vitro and may be used as an alternative to BCG for bladder cancer patients.
Assuntos
Antineoplásicos/farmacologia , Carcinoma de Células de Transição/terapia , Interferon-alfa/farmacologia , Mycobacterium bovis/imunologia , Neoplasias da Bexiga Urinária/terapia , Apoptose , Carcinoma de Células de Transição/imunologia , Linhagem Celular Tumoral/efeitos dos fármacos , Proliferação de Células , Sobrevivência Celular/efeitos dos fármacos , Citocinas/metabolismo , Ensaios de Seleção de Medicamentos Antitumorais , Humanos , Fatores Imunológicos/farmacologia , Interferon alfa-2 , Mycobacterium bovis/metabolismo , Proteínas Recombinantes/farmacologia , Células Th1/metabolismo , Células Th1/fisiologia , Neoplasias da Bexiga Urinária/imunologiaRESUMO
Upon oxygen shift-down, Mycobacterium tuberculosis complex bacteria can induce a genetic program characterized by halted duplication, which is called Non-replicating persistence (NRP). During this phase, at least 48 genes, collectively named Dormancy survival regulator (DosR) regulon, are important for the long-term survival of bacilli under a non-respiring state, a condition that bacilli encounter inside granulomatous lesions. It remains unclear whether expression of NRP genes occurs within the tissue of Mycobacterium bovis naturally infected cattle. In order to start dissecting this question, total RNA from bovine lymph node tissues of sacrificed tuberculin reacting animals was isolated and transcription of genes required for in vivo duplication (esxB and fbpB) and in vitro NRP (hspX, pfkB, and mb2660c) were analyzed by RT-PCR approaches. Detection of transcripts was positive in bovine tissue samples for genes hspX, pfkB, and mb2660c in 84, 32, and 21%, respectively. NRP genes were upregulated even in animals with a negative IFN-γ in vitro test, and the expression of NRP genes occurred more often than expression of the esxB gene.
Assuntos
Proteínas de Bactérias/metabolismo , Regulação Bacteriana da Expressão Gênica , Linfonodos/microbiologia , Mycobacterium bovis/crescimento & desenvolvimento , Regulação para Cima , Animais , Proteínas de Bactérias/genética , Bovinos , Interferon gama/biossíntese , Testes de Liberação de Interferon-gama , Mycobacterium bovis/genética , Mycobacterium bovis/metabolismo , Mycobacterium bovis/patogenicidade , Regulon , Testes Cutâneos , Teste Tuberculínico , Tuberculose Bovina/microbiologiaRESUMO
Infection of bovines with Mycobacterium bovis causes important financial hardship in many countries presenting also a risk for humans. M. bovis is known to be adapted to survive and thrive within the intramacrophage environment. In spite of its relevance, at present the information about macrophage expression patterns is scarce, particularly regarding the bovine host. In this study, transcriptomic analysis was used to detect genes differentially expressed in macrophages derived from peripheral blood mononuclear cells at early stages of infection with two Argentinean strains of M. bovis, a virulent and an attenuated strains. The results showed that the number of differentially expressed genes in the cells infected with the virulent strain (5) was significantly lower than those in the cells infected with the attenuated strain (172). Several genes were more strongly expressed in infected macrophages. Among them, we detected encoding transcription factors, anthrax toxin receptor, cell division and apoptosis regulator, ankyrin proteins, cytoskeleton proteins, protein of cell differentiation, and regulators of endocytic traffic of membrane. Quantitative real-time PCR of a selected group of differentially expressed genes confirmed the microarrays results. Altogether, the present results contribute to understanding the mechanisms involved in the early interaction of M. bovis with the bovine macrophage.
Assuntos
Regulação da Expressão Gênica , Macrófagos/metabolismo , Monócitos/metabolismo , Mycobacterium bovis/metabolismo , Transcrição Gênica , Tuberculose Bovina/metabolismo , Animais , Argentina , Bovinos , Macrófagos/imunologia , Macrófagos/microbiologia , Monócitos/imunologia , Monócitos/microbiologia , Mycobacterium bovis/imunologia , Mycobacterium bovis/isolamento & purificação , Tuberculose Bovina/imunologia , Tuberculose Bovina/microbiologiaRESUMO
When grown in the presence of exogenous collagen I, Mycobacterium bovis BCG was shown to form clumps. Scanning electron microscopy examination of these clumps revealed the presence of collagen fibres cross-linking the bacilli. Since collagen is a major constituent of the eukaryotic extracellular matrices, we assayed BCG cytoadherence in the presence of exogenous collagen I. Collagen increased the interaction of the bacilli with A549 type II pneumocytes or U937 macrophages, suggesting that BCG is able to recruit collagen to facilitate its attachment to host cells. Using an affinity chromatography approach, we have isolated a BCG collagen-binding protein corresponding to the previously described mycobacterial laminin-binding histone-like protein (LBP/Hlp), a highly conserved protein associated with the mycobacterial cell wall. Moreover, Mycobacterium leprae LBP/Hlp, a well-characterized adhesin, was also able to bind collagen I. Finally, using recombinant fragments of M. leprae LBP/Hlp, we mapped the collagen-binding activity within the C-terminal domain of the adhesin. Since this protein was already shown to be involved in the recognition of laminin and heparan sulphate-containing proteoglycans, the present observations reinforce the adhesive activities of LBP/Hlp, which can be therefore considered as a multifaceted mycobacterial adhesin, playing an important role in both leprosy and tuberculosis pathogenesis.
Assuntos
Animais , Humanos , Aderência Bacteriana , Colágeno Tipo I/farmacologia , Mycobacterium bovis/metabolismo , Mycobacterium leprae/metabolismo , Aderência Bacteriana/imunologia , Proteínas de Transporte/imunologia , Proteínas de Transporte/metabolismo , Colágeno Tipo I/metabolismo , Histonas/metabolismo , Mycobacterium bovis/imunologia , Mycobacterium leprae/imunologiaRESUMO
BACKGROUND: Takayasu's arteritis (TA) is a chronic inflammatory disease affecting the large arteries and their branches; its etiology is still unknown. In individuals suffering from TA, arterial inflammation progresses to stenosis and/or occlusion, leading to organ damage and affecting survival. Relation of TA with Mycobacterium tuberculosis has been known, but there have been only a few systematic studies focusing on this association. The IS6110 sequence identifies the Mycobacterium tuberculosis complex and the HupB establishes the differences between M. tuberculosis and M. bovis. Our objective was to search the presence of IS6110 and HupB genes in aorta of patients with TA. METHODS: We analyzed aorta tissues embedded in paraffin from 5760 autopsies obtained from our institution, we divided the selected samples as cases and controls; CASES: aortic tissues of individuals with Takayasu's arteritis. Control positive: aortic tissues (with tuberculosis disease confirmed) and control negative with other disease aortic (atherosclerosis). RESULTS: Of 181 selected aorta tissues, 119 fulfilled the corresponding criteria for TA, TB or atherosclerosis. Thus 33 corresponded to TA, 33 to tuberculosis (TB) and 53 to atherosclerosis. The mean age was 22 ± 13, 41 ± 19, and 57 ± 10, respectively. IS6110 and HupB sequences were detected in 70% of TA tissues, 82% in tuberculosis, and in 32% with atherosclerosis. Important statistical differences between groups with TA, tuberculosis versus atherosclerosis (p = 0.004 and 0.0001, respectively) were found. CONCLUSION: We identified a higher frequency of IS6110 and HupB genes in aortic tissues of TA patients. This data suggests that arterial damage could occur due to previous infection with M. tuberculosis.
Assuntos
Aorta/metabolismo , Aorta/microbiologia , Proteínas de Bactérias/metabolismo , Histonas/metabolismo , Mycobacterium bovis/metabolismo , Mycobacterium tuberculosis/metabolismo , Arterite de Takayasu/metabolismo , Arterite de Takayasu/microbiologia , Adolescente , Adulto , Idoso , Criança , Feminino , Humanos , Técnicas In Vitro , Masculino , Pessoa de Meia-Idade , Adulto JovemRESUMO
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
When grown in the presence of exogenous collagen I, Mycobacterium bovis BCG was shown to form clumps. Scanning electron microscopy examination of these clumps revealed the presence of collagen fibres cross-linking the bacilli. Since collagen is a major constituent of the eukaryotic extracellular matrices, we assayed BCG cytoadherence in the presence of exogenous collagen I. Collagen increased the interaction of the bacilli with A549 type II pneumocytes or U937 macrophages, suggesting that BCG is able to recruit collagen to facilitate its attachment to host cells. Using an affinity chromatography approach, we have isolated a BCG collagen-binding protein corresponding to the previously described mycobacterial laminin-binding histone-like protein (LBP/Hlp), a highly conserved protein associated with the mycobacterial cell wall. Moreover, Mycobacterium leprae LBP/Hlp, a well-characterized adhesin, was also able to bind collagen I. Finally, using recombinant fragments of M. leprae LBP/Hlp, we mapped the collagen-binding activity within the C-terminal domain of the adhesin. Since this protein was already shown to be involved in the recognition of laminin and heparan sulphate-containing proteoglycans, the present observations reinforce the adhesive activities of LBP/Hlp, which can be therefore considered as a multifaceted mycobacterial adhesin, playing an important role in both leprosy and tuberculosis pathogenesis.