RESUMO
Defining the mechanisms of Mycobacterium tuberculosis (Mtb) persistence in the host macrophage and identifying mycobacterial factors responsible for it are keys to better understand tuberculosis pathogenesis. The emerging picture from ongoing studies of macrophage deactivation by Mtb suggests that ingested bacilli secrete various virulence determinants that alter phagosome biogenesis, leading to arrest of Mtb vacuole interaction with late endosomes and lysosomes. While most studies focused on Mtb interference with various regulators of the endosomal compartment, little attention was paid to mechanisms by which Mtb neutralizes early macrophage responses such as the NADPH oxidase (NOX2) dependent oxidative burst. Here we applied an antisense strategy to knock down Mtb nucleoside diphosphate kinase (Ndk) and obtained a stable mutant (Mtb Ndk-AS) that displayed attenuated intracellular survival along with reduced persistence in the lungs of infected mice. At the molecular level, pull-down experiments showed that Ndk binds to and inactivates the small GTPase Rac1 in the macrophage. This resulted in the exclusion of the Rac1 binding partner p67(phox) from phagosomes containing Mtb or Ndk-coated latex beads. Exclusion of p67(phox) was associated with a defect of both NOX2 assembly and production of reactive oxygen species (ROS) in response to wild type Mtb. In contrast, Mtb Ndk-AS, which lost the capacity to disrupt Rac1-p67(phox) interaction, induced a strong ROS production. Given the established link between NOX2 activation and apoptosis, the proportion of Annexin V positive cells and levels of intracellular active caspase 3 were significantly higher in cells infected with Mtb Ndk-AS compared to wild type Mtb. Thus, knock down of Ndk converted Mtb into a pro-apoptotic mutant strain that has a phenotype of increased susceptibility to intracellular killing and reduced virulence in vivo. Taken together, our in vitro and in vivo data revealed that Ndk contributes significantly to Mtb virulence via attenuation of NADPH oxidase-mediated host innate immunity.
Assuntos
Proteínas de Bactérias/metabolismo , Imunidade Inata , Macrófagos/imunologia , Mycobacterium tuberculosis/imunologia , Neuropeptídeos/metabolismo , Núcleosídeo-Difosfato Quinase/metabolismo , Tuberculose Pulmonar/imunologia , Proteínas rac1 de Ligação ao GTP/metabolismo , Animais , Proteínas de Bactérias/antagonistas & inibidores , Proteínas de Bactérias/genética , Células da Medula Óssea/citologia , Células da Medula Óssea/enzimologia , Células da Medula Óssea/imunologia , Células da Medula Óssea/microbiologia , Linhagem Celular Transformada , Células Cultivadas , Feminino , Humanos , Macrófagos/enzimologia , Macrófagos/microbiologia , Macrófagos/ultraestrutura , Camundongos , Camundongos Endogâmicos C57BL , Camundongos SCID , Proteínas Mutantes/antagonistas & inibidores , Proteínas Mutantes/genética , Proteínas Mutantes/metabolismo , Mycobacterium tuberculosis/enzimologia , Mycobacterium tuberculosis/patogenicidade , Mycobacterium tuberculosis/fisiologia , NADPH Oxidases/antagonistas & inibidores , NADPH Oxidases/metabolismo , Neuropeptídeos/antagonistas & inibidores , Neuropeptídeos/genética , Núcleosídeo-Difosfato Quinase/antagonistas & inibidores , Núcleosídeo-Difosfato Quinase/genética , Oligorribonucleotídeos Antissenso , Fagossomos/enzimologia , Fagossomos/ultraestrutura , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Tuberculose Pulmonar/enzimologia , Tuberculose Pulmonar/microbiologia , Tuberculose Pulmonar/patologia , Virulência , Proteínas rac1 de Ligação ao GTP/antagonistas & inibidores , Proteínas rac1 de Ligação ao GTP/genéticaRESUMO
CD43 is a large sialylated glycoprotein found on the surface of haematopoietic cells and has been previously shown to be necessary for efficient macrophage binding and immunological responsiveness to Mycobacterium tuberculosis. Using capsular material from M. tuberculosis and recombinant CD43-Fc, we have employed affinity chromatography to show that Cpn60.2 (Hsp65, GroEL), and to a lesser extent DnaK (Hsp70), bind to CD43. Competitive inhibition using recombinant protein and polyclonal F(ab')(2) antibody-mediated epitope masking studies were used to evaluate M. tuberculosis binding to CD43(+/+) versus CD43(-/-) macrophages. Results showed that Cpn60.2, but not DnaK, acts as a CD43-dependent mycobacterial adhesin for macrophage binding. Assessment of the specific binding between Cpn60.2 and CD43 showed it to be saturable, with a comparatively weak affinity in the low micromolar range. We have also shown that the ability of Cpn60.2 to competitively inhibit M. tuberculosis binding to macrophages is shared by the Escherichia coli homologue, GroEL, but not by the mouse and human Hsp60 homologues. These findings add to a growing field of research that implicates molecular chaperones as having extracellular functions, including bacterial adherence to host cells. Thus, CD43 may act as a Pattern Recognition Receptor (PRR) for bacterial homologues of the 60 kDa molecular chaperone.
Assuntos
Aderência Bacteriana , Proteínas de Bactérias/metabolismo , Chaperonina 60/metabolismo , Leucossialina/metabolismo , Macrófagos/imunologia , Macrófagos/microbiologia , Mycobacterium tuberculosis/metabolismo , Adesinas Bacterianas/imunologia , Adesinas Bacterianas/metabolismo , Animais , Membrana Celular/imunologia , Membrana Celular/metabolismo , Membrana Celular/microbiologia , Ensaio de Imunoadsorção Enzimática , Proteínas de Choque Térmico HSP70/metabolismo , Macrófagos/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Mycobacterium tuberculosis/imunologia , Mycobacterium tuberculosis/patogenicidade , Receptores de Reconhecimento de Padrão , Proteínas Recombinantes de Fusão/metabolismoRESUMO
BACKGROUND: H37Rv and H37Ra are well-described laboratory strains of Mycobacterium tuberculosis derived from the same parental strain, H37, that show dramatically different pathogenic phenotypes. METHODOLOGY/PRINCIPAL FINDINGS: In this study, the transcriptomes of the two strains during axenic growth in broth and during intracellular growth within murine bone-marrow macrophages were compared by whole genome expression profiling. We identified and compared adaptations of either strain upon encountering an intracellular environment, and also contrasted the transcriptomes of the two strains while inside macrophages. In the former comparison, both strains induced genes that would facilitate intracellular survival including those involved in mycobactin synthesis and fatty acid metabolism. However, this response was stronger and more extensive for H37Rv than for H37Ra. This was manifested as the differential expression of a greater number of genes and an increased magnitude of expression for these genes in H37Rv. In comparing intracellular transcriptional signatures, fifty genes were found to be differentially expressed between the strains. Of these fifty, twelve were under control of the PhoPR regulon. Further differences between strains included genes whose products were members of the ESAT-6 family of proteins, or were associated with their secretion. CONCLUSIONS/SIGNIFICANCE: Along with the recent identification of single nucleotide polymorphisms in H37Ra when compared to H37Rv, our demonstration of differential expression of PhoP-regulated and ESX-1 region-related genes during macrophage infection further highlights the significance of these genes in the attenuation of H37Ra.
Assuntos
Macrófagos/microbiologia , Mycobacterium tuberculosis/patogenicidade , Transcrição Gênica , Animais , Células Cultivadas , Camundongos , Reação em Cadeia da Polimerase Via Transcriptase Reversa , VirulênciaRESUMO
Mycobacterium tuberculosis remains the most significant single species of bacteria causing disease in mankind. The ability of M. tuberculosis to survive and replicate within host macrophages is a pivotal step in its pathogenesis. Understanding the microenvironments that M. tuberculosis encounters within the macrophage and the adaptations that the bacterium undergoes to facilitate its survival will lead to insights into possible therapeutic targets for improved treatment of tuberculosis. This is urgently needed with the emergence of multi- and extensively drug resistant strains of M. tuberculosis. Significant advances have been made in understanding the macrophage response on encountering M. tuberculosis. Complementary information is also accumulating regarding the counter responses of M. tuberculosis during the various stages of its interactions with the host. As such, a picture is emerging delineating the gene expression of intracellular M. tuberculosis at different stages of the interaction with macrophages.
Assuntos
Adaptação Fisiológica , Regulação Bacteriana da Expressão Gênica , Macrófagos/microbiologia , Mycobacterium tuberculosis/fisiologia , Estresse Fisiológico , Interações Hospedeiro-Patógeno , HumanosRESUMO
Mycobacterium tuberculosis, the causative agent of tuberculosis, initially contacts host cells with elements of its outer cell wall, or capsule. We have shown that capsular material from the surface of M. tuberculosis competitively inhibits the nonopsonic binding of whole M. tuberculosis bacilli to macrophages in a dose-dependent manner that is not acting through a global inhibition of macrophage binding. We have further demonstrated that isolated M. tuberculosis capsular proteins mediate a major part of this inhibition. Two-dimensional polyacrylamide gel electrophoresis analysis of the capsular proteins showed the presence of a wide variety of protein species, including proportionately high levels of the Cpn60.2 (Hsp65, GroEL2) and DnaK (Hsp70) molecular chaperones. Both of these proteins were subsequently detected on the bacterial surface. To determine whether these molecular chaperones play a role in bacterial binding, recombinant Cpn60.2 and DnaK were tested for their ability to inhibit the association of M. tuberculosis bacilli with macrophages. We found that recombinant Cpn60.2 can inhibit approximately 57% of bacterial association with macrophages, while DnaK was not inhibitory at comparable concentrations. Additionally, when polyclonal F(ab')(2) fragments of anti-Cpn60.2 and anti-DnaK were used to mask the surface presentation of these molecular chaperones, a binding reduction of approximately 34% was seen for anti-Cpn60.2 F(ab')(2), while anti-DnaK F(ab')(2) did not significantly reduce bacterial association with macrophages. Thus, our findings suggest that while M. tuberculosis displays both surface-associated Cpn60.2 and DnaK, only Cpn60.2 demonstrates adhesin functionality with regard to macrophage interaction.
Assuntos
Adesinas Bacterianas/fisiologia , Aderência Bacteriana , Proteínas de Bactérias/fisiologia , Chaperonina 60/fisiologia , Proteínas de Choque Térmico HSP70/fisiologia , Macrófagos/microbiologia , Chaperonas Moleculares/fisiologia , Mycobacterium tuberculosis/patogenicidade , Adesinas Bacterianas/análise , Animais , Cápsulas Bacterianas/química , Proteínas de Bactérias/análise , Células Cultivadas , Chaperonina 60/análise , Eletroforese em Gel Bidimensional , Proteínas de Choque Térmico HSP70/análise , Humanos , Camundongos , Camundongos Endogâmicos BALB C , Chaperonas Moleculares/análise , Mycobacterium tuberculosis/químicaRESUMO
To identify genes involved in the intracellular survival of Mycobacterium tuberculosis we compared the transcriptomes of virulent (H37Rv) and attenuated (H37Ra) strains during their interaction with murine bone-marrow-derived macrophages. Expression profiling was accomplished via the bacterial artificial chromosome fingerprint array (BACFA) technique. Genes identified with BACFA, and confirmed via qPCR to be upregulated in the attenuated H37Ra at 168 h post-infection, were frdB, frdC and frdD. Genes upregulated in the virulent H37Rv were pks2, aceE and Rv1571. Further qPCR analysis of these genes at 4 and 96 h post-infection revealed that the frd operon (encoding the fumarate reductase enzyme complex) is expressed at higher levels in the virulent H37Rv at earlier time points while the expression of aceE and pks2 is higher in the virulent strain throughout the course of infection. Assessment of frd transcripts in oxygen-limited cultures of M. tuberculosis H37Ra and H37Rv showed that the attenuated strain displayed a lag in frdA and frdB expression at the onset of microaerophilic culture, when compared to microaerophilic cultures of H37Rv and aerated cultures of H37Ra. Lastly, treatment of intracellular bacteria with a putative inhibitor of fumarate reductase resulted in a significant reduction of bacterial growth.
Assuntos
Proteínas de Bactérias/metabolismo , Expressão Gênica , Macrófagos/microbiologia , Mycobacterium tuberculosis/crescimento & desenvolvimento , Mycobacterium tuberculosis/patogenicidade , Succinato Desidrogenase/metabolismo , Animais , Proteínas de Bactérias/genética , Cromossomos Artificiais Bacterianos/genética , Humanos , Camundongos , Mycobacterium tuberculosis/genética , Mycobacterium tuberculosis/metabolismo , Oxigênio/metabolismo , Succinato Desidrogenase/genética , VirulênciaRESUMO
Although microarray technology has become more widespread as a discovery tool for bacterial pathogenesis, it remains a method available only to laboratories with access to expensive equipment and costly analysis software. Mycobacterium tuberculosis, the causative agent for tuberculosis (TB), afflicts one-third of the global population, and kills between 2 and 3 million people per year. While the majority of cases of TB occur in developing areas of the world, facilities in these regions may not be able to support microarray analysis. Additionally, a major limitation of microarrays is that only genes on the array are being assayed. With acquired virulence and drug resistance in microbes, a method less dependent on a predetermined list of gene targets is advantageous. We present a method of expression analysis based on bacterial artificial chromosomes (BACs) that can be applied with standard laboratory equipment and free analysis software. This technique, bacterial artificial chromosome fingerprint arrays (BACFA), was developed and utilised to identify expression differences between intracellular strains of M. tuberculosis, one virulent (H37Rv) and one attenuated (H37Ra). Southern blots of restriction-enzyme digested BAC fragments were sequentially hybridised with strain-specific cDNA probes to generate expression profiles that were used to isolate expression differences in broth grown and intracellular bacteria. Repeat comparisons of intracellular profiles via BACFA identified genomic regions differentially expressed by the two strains. Quantitative real-time PCR was used to assess the genes located in these fragments in order to confirm or deny the differential regulation of genes. In total, we identified six genes that were differentially regulated between strains inside the host cell (pks2, aceE, Rv1571, and frdBCD). We report that BACFA is an effective technique in the expression analysis of bacteria and can be considered complementary to the high-throughput analysis offered by microarrays.
Assuntos
Cromossomos Artificiais Bacterianos/genética , Impressões Digitais de DNA/métodos , Mycobacterium tuberculosis/genética , Análise de Sequência com Séries de Oligonucleotídeos/métodos , Transcrição Gênica , Tuberculose Pulmonar/microbiologia , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Sequência de Bases , Regulação Bacteriana da Expressão Gênica , Humanos , Dados de Sequência Molecular , Mycobacterium tuberculosis/isolamento & purificação , Tuberculose Pulmonar/diagnósticoRESUMO
Establishment of Tuberculosis infection begins with the successful entry and survival of the pathogen within macrophages. We previously showed that macrophage CD43 is required for optimal uptake and growth inhibition of Mycobacterium tuberculosis both in vitro and in vivo. Here, we explore the mechanisms by which CD43 restricts mycobacterial growth in murine macrophages. We found that although M. tuberculosis grows more readily in resting CD43-/- macrophages, priming of cells with IFN-gamma returns the bacterial growth rate to that seen in CD43+/+ cells. To discern the mechanisms by which M. tuberculosis exhibits enhanced growth within resting CD43-/- macrophages, we assessed the induction of inflammatory mediators in response to infection. We found that absence of CD43 resulted in reduced production of TNF-alpha, IL-12 and IL-6 by M. tuberculosis-infected macrophages. We also found that infected resting, but not activated CD43-/- macrophages, showed decreased apoptosis and increased necrosis. Exogenous addition of the pro-inflammatory cytokine TNF-alpha restored control of M. tuberculosis growth and induction of apoptosis to CD43+/+ levels. We propose that CD43 is involved in the inflammatory response to M. tuberculosis and, through the induction of pro-inflammatory mediators, can regulate apoptosis to control intracellular growth of the bacterium.
Assuntos
Apoptose , Citosol/microbiologia , Leucossialina/imunologia , Macrófagos/microbiologia , Mycobacterium tuberculosis/imunologia , Fator de Necrose Tumoral alfa/imunologia , Animais , Células Cultivadas , Contagem de Colônia Microbiana , Interleucina-12/biossíntese , Interleucina-6/biossíntese , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Fator de Necrose Tumoral alfa/biossínteseRESUMO
Alterations to the composition or architecture of the mycobacterial cell envelope can affect the macrophage infectivity of the bacillus. To further characterize the mycobacterial gene products that modulate the interaction with host cells, we employed transposon mutagenesis and screened for mutants that demonstrated an enhanced binding affinity toward macrophages. After successive rounds of mutant selection and enrichment, a total of five mutants were isolated that harboured gene disruptions within loci involved in lipid synthetic pathways as well as genes coding for putative hypothetical proteins. One mutant in particular, with a disruption in the Rv3826 gene (fadD23), was repeatedly isolated during library screening. Analysis of the cell envelope constituents of the Tn : : fadD23 strain revealed a lack of sulfolipid production which was restored following complementation with the wild-type gene.
Assuntos
Elementos de DNA Transponíveis , Lipídeos/biossíntese , Macrófagos/microbiologia , Mycobacterium tuberculosis/patogenicidade , Linhagem Celular , Humanos , Mutagênese Insercional , Mutação , Mycobacterium tuberculosis/genética , Mycobacterium tuberculosis/metabolismoRESUMO
The interaction of host macrophage (Mphi) and Mycobacterium tuberculosis (Mtb) is mediated by cell surface receptors and is important in establishing intracellular infection. Mphis can kill invading organisms via reactive oxygen intermediates (ROI) and reactive nitrogen intermediates (RNI). Using a Complement Receptor 3 (CR3) knockout mouse model we have examined whether the presence of CR3 affects the binding and uptake of viable Mtb by Mphis, the survival of the ingested bacteria and the induction of ROI and RNI during this interaction. We show that, although CR3 plays a role in the uptake of viable Mtb, the receptor plays no role in the subsequent survival of the bacteria. The finding holds true for resident Mphis and for interferon-gamma (IFN-gamma) activated Mphis, both in the absence and presence of serum opsonins. Activation of Mphi populations with IFN-gamma significantly inhibits the growth of Mtb in host Mphis and enhances the production of ROI and RNI. However, the presence of CR3 was not critical in any of these mechanisms. Furthermore, we demonstrate that the control of intracellular growth of Mtb in IFN-gamma activated Mphis is not mediated by a direct effect of RNI.
Assuntos
Antígeno de Macrófago 1/fisiologia , Macrófagos/microbiologia , Mycobacterium tuberculosis/fisiologia , Óxido Nítrico/fisiologia , Superóxidos/metabolismo , Tuberculose/metabolismo , Tuberculose/microbiologia , Animais , Inibidores Enzimáticos/farmacologia , Feminino , Interferon gama/imunologia , Ativação de Macrófagos/imunologia , Antígeno de Macrófago 1/imunologia , Antígeno de Macrófago 1/metabolismo , Macrófagos/imunologia , Macrófagos/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Mycobacterium tuberculosis/crescimento & desenvolvimento , Mycobacterium tuberculosis/imunologia , Mycobacterium tuberculosis/metabolismo , Óxido Nítrico/antagonistas & inibidores , Óxido Nítrico/biossíntese , Óxido Nítrico/imunologia , Organismos Livres de Patógenos Específicos , Superóxido Dismutase/farmacologia , Superóxidos/antagonistas & inibidores , Superóxidos/imunologia , ômega-N-Metilarginina/farmacologiaRESUMO
We explored the role of macrophage (Mphi) CD43, a transmembrane glycoprotein, in the pathogenesis of Mycobacterium tuberculosis. Using gene-deleted mice (CD43-/-), we assessed the association of the bacterium with distinct populations of Mphi and found that CD43-/- Mphi bound less M. tuberculosis than CD43+/+ Mphi. Increased infective doses did not abrogate this difference. However, reduced association due to the absence of CD43 could be overcome by serum components. Mphi from heterozygote mice, which express 50% of wild-type CD43, bound more bacteria than CD43-/- but less than CD43+/+, proving that the gene dose of CD43 correlates with binding of M. tuberculosis. Furthermore, the reduced ability of CD43-/- Mphi to bind bacteria was restricted to mycobacterial species. We also found that the survival and replication of M. tuberculosis within Mphi was enhanced significantly in the absence of CD43, making this the first demonstration that the mechanism of mycobacterial entry influences its subsequent growth. Most importantly, we show here that the absence of CD43 in mice aerogenically infected with M. tuberculosis results in an increased bacterial load during both the acute and chronic stages of infection and more rapid development of granulomas, with greater lung involvement and distinctive cellularity.
Assuntos
Antígenos CD/fisiologia , Inibidores do Crescimento/fisiologia , Macrófagos Alveolares/imunologia , Macrófagos Alveolares/microbiologia , Macrófagos Peritoneais/imunologia , Macrófagos Peritoneais/microbiologia , Mycobacterium tuberculosis/crescimento & desenvolvimento , Mycobacterium tuberculosis/imunologia , Sialoglicoproteínas/fisiologia , Doença Aguda , Administração por Inalação , Animais , Antígenos CD/biossíntese , Antígenos CD/genética , Aderência Bacteriana/genética , Aderência Bacteriana/imunologia , Células da Medula Óssea/imunologia , Células da Medula Óssea/microbiologia , Adesão Celular/genética , Adesão Celular/imunologia , Membrana Celular/imunologia , Membrana Celular/metabolismo , Membrana Celular/microbiologia , Doença Crônica , Dosagem de Genes , Inibidores do Crescimento/biossíntese , Inibidores do Crescimento/deficiência , Inibidores do Crescimento/genética , Imunofenotipagem , Leucossialina , Listeria monocytogenes/genética , Listeria monocytogenes/imunologia , Macrófagos Alveolares/patologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Mycobacterium tuberculosis/patogenicidade , Proteínas Opsonizantes/metabolismo , Salmonella typhimurium/genética , Salmonella typhimurium/imunologia , Sialoglicoproteínas/biossíntese , Sialoglicoproteínas/deficiência , Sialoglicoproteínas/genética , Tuberculose Pulmonar/genética , Tuberculose Pulmonar/imunologia , Tuberculose Pulmonar/microbiologia , Tuberculose Pulmonar/patologiaRESUMO
Bacillus Calmette-Guerin (BCG) vaccine has failed to control the global tuberculosis (TB) epidemic, and there is a lack of safe and effective mucosal vaccines capable of potent protection against pulmonary TB. A recombinant replication-deficient adenoviral-based vaccine expressing an immunogenic Mycobacterium tuberculosis Ag Ag85A (AdAg85A) was engineered and evaluated for its potential to be used as a respiratory mucosal TB vaccine in a murine model of pulmonary TB. A single intranasal, but not i.m., immunization with AdAg85A provided potent protection against airway Mycobacterium tuberculosis challenge at an improved level over that by cutaneous BCG vaccination. Systemic priming with an Ag85A DNA vaccine and mucosal boosting with AdAg85A conferred a further enhanced immune protection which was remarkably better than BCG vaccination. Such superior protection triggered by AdAg85 mucosal immunization was correlated with much greater retention of Ag-specific T cells, particularly CD4 T cells, in the lung and was shown to be mediated by both CD4 and CD8 T cells. Thus, adenoviral TB vaccine represents a promising novel vaccine platform capable of potent mucosal immune protection against TB. Our study also lends strong evidence that respiratory mucosal vaccination is critically advantageous over systemic routes of vaccination against TB.
Assuntos
Adenoviridae/genética , Adenoviridae/imunologia , Mucosa Respiratória/imunologia , Vacinas contra a Tuberculose/imunologia , Tuberculose Pulmonar/prevenção & controle , Vacinas de DNA/imunologia , Vacinas Virais/imunologia , Aciltransferases/biossíntese , Aciltransferases/genética , Administração Intranasal , Animais , Antígenos de Bactérias/biossíntese , Antígenos de Bactérias/genética , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD8-Positivos/imunologia , Feminino , Vetores Genéticos , Imunidade Celular/genética , Imunização Secundária , Injeções Intramusculares , Injeções Subcutâneas , Camundongos , Camundongos Endogâmicos BALB C , Mycobacterium bovis/imunologia , Mycobacterium tuberculosis/genética , Mycobacterium tuberculosis/imunologia , Mucosa Respiratória/microbiologia , Mucosa Respiratória/virologia , Vacinas contra a Tuberculose/administração & dosagem , Vacinas contra a Tuberculose/genética , Tuberculose Pulmonar/imunologia , Tuberculose Pulmonar/virologia , Vacinas de DNA/administração & dosagem , Vacinas de DNA/genética , Vacinas Sintéticas/administração & dosagem , Vacinas Sintéticas/genética , Vacinas Sintéticas/imunologia , Vacinas Virais/administração & dosagem , Vacinas Virais/genéticaRESUMO
Mycobacterium tuberculosis, the causative agent of tuberculosis, is a facultative intracellular pathogen that infects macrophages and other host cells. We show that sonication of M. tuberculosis results in the removal of material from the surface capsule-like layer of the bacteria, resulting in an enhanced propensity of the bacteria to bind to macrophages. This effect is observed with disparate murine and human macrophage populations though, interestingly, not with freshly explanted alveolar macrophages. Enhanced binding to macrophages following sonication is significantly greater within members of the M. tuberculosis family (pathogens) than within the Mycobacterium avium complex (opportunistic pathogens) or for Mycobacterium smegmatis (saprophyte). Sonication does not affect the viability or the surface hydrophobicity of M. tuberculosis but does result in changes in surface charge and in the binding of mannose-specific lectins to the bacterial surface. The increased binding of sonicated M. tuberculosis was not mediated through complement receptor 3. These results provide evidence that the surface capsule on members of the M. tuberculosis family may be an important virulence factor involved in the survival of M. tuberculosis in the mammalian host. They also question the view that M. tuberculosis is readily ingested by any macrophage it encounters and support the contention that M. tuberculosis, like many other microbial pathogens, has an antiphagocytic capsule that limits and controls the interaction of the bacterium with macrophages.
Assuntos
Cápsulas Bacterianas/fisiologia , Parede Celular/metabolismo , Macrófagos Peritoneais/microbiologia , Mycobacterium tuberculosis/citologia , Mycobacterium tuberculosis/fisiologia , Fagocitose , Polissacarídeos/metabolismo , Animais , Aderência Bacteriana/efeitos dos fármacos , Cápsulas Bacterianas/ultraestrutura , Parede Celular/química , Meios de Cultura Livres de Soro/farmacologia , Humanos , Interações Hidrofóbicas e Hidrofílicas , Lectinas/metabolismo , Macrófagos Alveolares , Camundongos , Complexo Mycobacterium avium/citologia , Mycobacterium smegmatis/citologia , Mycobacterium tuberculosis/classificação , Mycobacterium tuberculosis/ultraestrutura , Sonicação , Eletricidade Estática , Propriedades de Superfície , Seringas , Virulência , Fatores de VirulênciaRESUMO
The function of the Mycobacterium tuberculosis eukaryotic-like protein serine/threonine kinase PknG was investigated by gene knock-out and by expression and biochemical analysis. The pknG gene (Rv0410c), when cloned and expressed in Escherichia coli, encodes a functional kinase. An in vitro kinase assay of the recombinant protein demonstrated that PknG can autophosphorylate its kinase domain as well as its 30 kDa C-terminal portion, which contains a tetratricopeptide (TPR) structural signalling motif. Western analysis revealed that PknG is located in the cytosol as well as in mycobacterial membrane. The pknG gene was inactivated by allelic exchange in M. tuberculosis. The resulting mutant strain causes delayed mortality in SCID mice and displays decreased viability both in vitro and upon infection of BALB/c mice. The reduced growth of the mutant was more pronounced in the stationary phase of the mycobacterial growth cycle and when grown in nutrient-depleted media. The PknG-deficient mutant accumulates glutamate and glutamine. The cellular levels of these two amino acids reached approximately threefold of their parental strain levels. Higher cellular levels of the amine sugar-containing molecules, GlcN-Ins and mycothiol, which are derived from glutamate, were detected in the DeltapknG mutant. De novo glutamine synthesis was shown to be reduced by 50%. This is consistent with current knowledge suggesting that glutamine synthesis is regulated by glutamate and glutamine levels. These data support our hypothesis that PknG mediates the transfer of signals sensing nutritional stress in M. tuberculosis and translates them into metabolic adaptation.
Assuntos
Regulação Bacteriana da Expressão Gênica , Ácido Glutâmico/metabolismo , Glutamina/metabolismo , Mycobacterium tuberculosis/crescimento & desenvolvimento , Proteínas Serina-Treonina Quinases/metabolismo , Animais , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Western Blotting , Membrana Celular/química , Clonagem Molecular , Citoplasma/química , Escherichia coli/genética , Escherichia coli/metabolismo , Deleção de Genes , Genes Bacterianos , Camundongos , Camundongos Endogâmicos BALB C , Camundongos SCID , Mutagênese Insercional , Mycobacterium tuberculosis/genética , Mycobacterium tuberculosis/metabolismo , Mycobacterium tuberculosis/patogenicidade , Proteínas Serina-Treonina Quinases/genética , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/isolamento & purificação , Tuberculose/mortalidade , Virulência/genéticaRESUMO
Annually, Mycobacterium tuberculosis is the cause of approximately three million deaths worldwide. It would appear that currently available therapies for this disease are inadequate. The identification of genes involved in mycobacterial virulence will facilitate the design of new prophylactic and therapeutic interventions. A method for high-resolution comparison of bacterial genomes has been developed to facilitate the identification of genes possibly involved in the virulence of clinically relevant mycobacteria. This 'two-dimensional bacterial genome display' (2DBGD) method utilizes two-dimensional DNA electrophoresis to separate, on the basis of size and G+C content, genomic fragments generated with different restriction endonucleases. The use of this method to identify genomic differences between species, strains and, most importantly, isogenic mutants of mycobacteria is reported. That 2DBGD can be used to identify differences resulting from either insertional mutagenesis using a gentamicin-resistance gene or from a frameshift mutation is demonstrated.
Assuntos
Técnicas de Tipagem Bacteriana , DNA Bacteriano/análise , Genoma Bacteriano , Mutação , Mycobacterium/classificação , Proteínas de Bactérias/genética , Composição de Bases , Eletroforese em Gel Bidimensional/métodos , Humanos , Mycobacterium/genética , Infecções por Mycobacterium/microbiologia , Complexo Mycobacterium avium/genética , Mycobacterium tuberculosis/genética , Mycobacterium tuberculosis/patogenicidade , Mapeamento por Restrição , Especificidade da EspécieRESUMO
High-resolution comparison of bacterial genomes facilitates the identification of the genetic changes responsible for clinically relevant phenotypes. For this purpose we have established a method for the display and comparison of high G+C bacterial genomes in two dimensions. Here we describe the application of two-dimensional bacterial genomic display to resolve the genomes of Bordetella pertussis, Mycobacterium avium and Mycobacterium tuberculosis, and its utility in strain comparison and detection of insertion and substitution mutations.
Assuntos
Bactérias/genética , DNA Bacteriano/genética , Eletroforese em Gel Bidimensional/métodos , Genoma Bacteriano , Bactérias/patogenicidade , Composição de Bases , Bordetella pertussis/genética , Análise Mutacional de DNA , DNA Bacteriano/química , Mutação , Mycobacterium avium/genética , Mycobacterium tuberculosis/genética , Reprodutibilidade dos Testes , Especificidade da Espécie , Virulência/genéticaRESUMO
Allelic replacement will be a vital tool for understanding gene function in mycobacteria. Disruption of the chromosomal hisD gene of Mycobacterium smegmatis by standard gene replacement methods was surprisingly difficult, with most products being caused by illegitimate recombination (IR) events. A recombination assay was therefore developed and used to optimize conditions for homologous recombination (HR) in M. smegmatis. Treatment of competent cells with UV, hydrogen peroxide or mitomycin C did not improve the frequency of HR; however, treatment of the DNA with alkali or UV enhanced recombination frequency, while boiling did not. Applying these observations to allele replacement, UV and alkali treatment of transforming DNA increased HR events with pyrF and hisD, while the level of IR was unchanged. The introduction of ss phagemid DNA improved the level of HR and abolished IR. In Mycobacterium intracellulare the use of alkali-denatured DNA increased the numbers of recombinants obtained with an inactivated 19Ag gene, while in Mycobacterium tuberculosis, inactivation of a putative haemolysin gene, tlyA, was achieved using both UV-irradiated DNA and ss phagemid DNA. Significantly, IR, which has been reported to be a problem in this species, was not observed. Thus, four genes in three species were successfully knocked-out using non-replicating DNA pretreated with alkali, UV or in an ss form. The use of these methods to enhance HR will greatly facilitate experiments to inactivate other genes in these important species.