RESUMO
The purinergic P2X7 receptor (P2X7R) is a sensor of extracellular ATP, a damage-associated molecule that is released from necrotic cells and that induces pro-inflammatory cytokine production and cell death. To investigate whether the innate immune response to damage signals could contribute to the development of pulmonary necrotic lesions in severe forms of tuberculosis, disease progression was examined in C57BL/6 and P2X7R-/- mice that were intratracheally infected with highly virulent mycobacterial strains (Mycobacterium tuberculosis strain 1471 of the Beijing genotype family and Mycobacterium bovis strain MP287/03). The low-dose infection of C57BL/6 mice with bacteria of these strains caused the rapid development of extensive granulomatous pneumonia with necrotic areas, intense bacillus dissemination and anticipated animal death. In contrast, in P2X7R-/- mice, the lung pathology presented with moderate infiltrates of mononuclear leukocytes without visible signs of necrosis; the disease attenuation was accompanied by a delay in mortality. In vitro, the hypervirulent mycobacteria grew rapidly inside macrophages and induced death by a P2X7R-dependent mechanism that facilitated the release of bacilli. Furthermore, these bacteria were resistant to the protective mechanisms elicited in macrophages following extracellular ATP stimulation. Based on this study, we propose that the rapid intracellular growth of hypervirulent mycobacteria results in massive macrophage damage. The ATP released by damaged cells engages P2X7R and accelerates the necrotic death of infected macrophages and the release of bacilli. This vicious cycle exacerbates pneumonia and lung necrosis by promoting widespread cell destruction and bacillus dissemination. These findings suggest the use of drugs that have been designed to inhibit the P2X7R as a new therapeutic approach to treat the aggressive forms of tuberculosis.
Assuntos
Macrófagos , Mycobacterium tuberculosis/imunologia , Mycobacterium tuberculosis/patogenicidade , Receptores Purinérgicos P2X7 , Tuberculose Pulmonar , Trifosfato de Adenosina/imunologia , Animais , Humanos , Macrófagos/imunologia , Macrófagos/microbiologia , Macrófagos/patologia , Camundongos , Camundongos Knockout , Mycobacterium bovis/imunologia , Mycobacterium bovis/patogenicidade , Receptores Purinérgicos P2X7/genética , Receptores Purinérgicos P2X7/imunologia , Tuberculose Pulmonar/genética , Tuberculose Pulmonar/imunologia , Tuberculose Pulmonar/patologiaRESUMO
Strains of the Beijing genotype family of Mycobacterium tuberculosis are a cause of particular concern because of their increasing dissemination in the world and their association with drug resistance. Phylogenetically, this family includes distinct ancient and modern sublineages. The modern strains, contrary to the ancestral counterparts, demonstrated increasing prevalence in many world regions that suggest an enhanced bacterial pathogenicity. We therefore evaluated virulence of modern versus ancient Beijing strains with similar epidemiological and genotype characteristics. For this, we selected six strains that had very similar 24-locus mycobacterial interspersed repetitive-unit-variable-number tandem-repeat (MIRU-VNTR) typing profiles and belonged to the region of difference 181 (RD181) subgroup but differed using markers (mutT2 and mutT4 genes and NTF locus) that discriminate between modern and ancient Beijing sublineages. The strains were isolated from native patients in Brazil and Mozambique, countries with a low prevalence of Beijing strains. The virulence levels of these strains were determined in models of pulmonary infection in mice and in vitro macrophage infection and compared with that of a strain from Russia, part of the epidemic and hypervirulent Beijing clone B0/W148, and of the laboratory strain H37Rv. The results showed that two of the three modern Beijing strains were highly pathogenic, exhibiting levels of virulence comparable with that of the epidemic Russian strain. In contrast, all isolates of the ancient sublineage displayed intermediate or low virulence. The data obtained demonstrate that the strains of the modern Beijing sublineage are more likely to exhibit highly virulent phenotypes than ancient strains and suggest that genetic alterations characteristic of the modern Beijing sublineage favor selection of highly virulent bacteria.
Assuntos
Mycobacterium tuberculosis/genética , Mycobacterium tuberculosis/patogenicidade , Tuberculose Resistente a Múltiplos Medicamentos/microbiologia , Tuberculose Resistente a Múltiplos Medicamentos/patologia , Animais , Brasil , Células Cultivadas , Modelos Animais de Doenças , Genótipo , Humanos , Macrófagos/microbiologia , Camundongos Endogâmicos C57BL , Tipagem Molecular , Moçambique , Mycobacterium tuberculosis/classificação , Mycobacterium tuberculosis/isolamento & purificação , Federação Russa , Tuberculose Pulmonar/microbiologia , Tuberculose Pulmonar/patologiaRESUMO
The epidemiologically important Mycobacterium tuberculosis Beijing genotype strains, highly endemic in East Asia, have become an emerging infection in certain geographic areas, including Russia, because of its increasing prevalence and association with multidrug resistance (MDR). The aim was to verify whether MDR Beijing strains circulating in the emerging regions present some biological particularities that could contribute to their success in causing disease in comparison with the sporadic strains from locations with low prevalence of the Beijing genotype. We evaluated virulence-associated characteristics of the MDR Beijing strains isolated in Russia and compared them with those of the drug-resistant and susceptible Beijing strains from Brazil and reference H37Rv strain. We found that Russian MDR strains demonstrated an increased bacterial fitness and growth in THP-1 macrophage-like cells, as well as a higher capacity to induce non-protective cytokine synthesis and necrotic macrophage death. By contrast, the biological properties of the strains isolated in Brazil largely resembled those of the H37Rv strain, with the exception of the drug-resistant isolates that presented significantly reduced fitness. The data demonstrate that the emerging MDR strains of the Beijing genotype circulating in Russia do express a pattern of properties associated with the enhanced virulence favouring its clonal dissemination in this region.
Assuntos
Doenças Transmissíveis Emergentes/microbiologia , Mycobacterium tuberculosis/fisiologia , Tuberculose Resistente a Múltiplos Medicamentos/microbiologia , Adolescente , Adulto , Análise de Variância , Brasil/epidemiologia , Morte Celular , Linhagem Celular , Doenças Transmissíveis Emergentes/epidemiologia , Citocinas/metabolismo , Feminino , Genótipo , Humanos , Macrófagos/metabolismo , Macrófagos/microbiologia , Masculino , Pessoa de Meia-Idade , Mycobacterium tuberculosis/genética , Mycobacterium tuberculosis/crescimento & desenvolvimento , Mycobacterium tuberculosis/patogenicidade , Federação Russa/epidemiologia , Tuberculose Resistente a Múltiplos Medicamentos/epidemiologia , Virulência/genéticaRESUMO
The epidemiologically important Mycobacterium tuberculosis Beijing genotype strains, highly endemic in East Asia, have become an emerging infection in certain geographic areas, including Russia, because of its increasing prevalence and association with multidrug resistance (MDR). The aim was to verify whether MDR Beijing strains circulating in the emerging regions present some biological particularities that could contribute to their success in causing disease in comparison with the sporadic strains from locations with low prevalence of the Beijing genotype. We evaluated virulence-associated characteristics of the MDR Beijing strains isolated in Russia and compared them with those of the drug-resistant and susceptible Beijing strains from Brazil and reference H37Rv strain. We found that Russian MDR strains demonstrated an increased bacterial fitness and growth in THP-1 macrophage-like cells, as well as a higher capacity to induce non-protective cytokine synthesis and necrotic macrophage death. By contrast, the biological properties of the strains isolated in Brazil largely resembled those of the H37Rv strain, with the exception of the drug-resistant isolates that presented significantly reduced fitness. The data demonstrate that the emerging MDR strains of the Beijing genotype circulating in Russia do express a pattern of properties associated with the enhanced virulence favouring its clonal dissemination in this region.
Assuntos
Humanos , Masculino , Feminino , Adolescente , Adulto , Pessoa de Meia-Idade , Virulência/genética , Brasil/epidemiologia , Federação Russa/epidemiologia , Tuberculose Resistente a Múltiplos Medicamentos/epidemiologia , Doenças Transmissíveis Emergentes , Macrófagos , Mycobacterium tuberculosis/crescimento & desenvolvimento , Mycobacterium tuberculosis/fisiologia , Mycobacterium tuberculosis/genética , Mycobacterium tuberculosis/patogenicidadeRESUMO
Macrophage migration and adhesion are important for the control of mycobacterial infection and are critically dependent on the reorganization of the cytoskeleton. Mycobacteria elicit rapid morphological changes, such as cell spreading, a process relevant to in vivo changes of macrophage shape during extravasation and migration. In this study, we investigated the BCG mycobacteria-induced signaling events leading to macrophage cytoskeletal rearrangements employing specific pharmacological inhibitors to suppress distinct kinase pathways known to be elicited by infection. Viable or lysed mycobacteria, as well as purified cell wall lipoprotein p19, TLR2 agonist, induced RAW264.7 cells to extend actin-rich pseudopods, which impart radial spreading within 3 h, leading later to persistent cell polarization. BCG induced rapid activation of phosphatidylinositol 3-kinase, PI3K, activation that was recruited to the activated TLR2 receptor. TLR2- neutralizing antibody inhibited macrophage spreading and PI3K activation induced by p19. Additionally, BCG induced spreading and polarization of bone marrow-derived macrophages from TLR2- expressing mice in contrast to their TLR2-knockout counterparts. Neither MEK1/ERK, p38 MAPK, nor NF-kappaB activation were important for the early cytoskeletal rearrangements observed, although suppression of these pathways is known to inhibit chemokine secretion by activated macrophages. Beta2-integrins blockade with a corresponding antibody inhibited macrophage spreading and polarization but had no effect on pseudopodia protrusions demonstrating the downstream position of integrin-mediated adhesion in PI3K- dependent signaling pathway leading to the motility phenotype. The obtained data demonstrate that the direct effect of mycobacteria on macrophage shape might be mediated through TLR2-dependent PI3K activation.