RESUMO
Tuberculosis is the most widespread and lethal infectious disease affecting humans. Immunization of mice with plasmid DNA constructs encoding one of the secreted components of Mycobacterium tuberculosis, antigen 85 (Ag85), induced substantial humoral and cell-mediated immune responses and conferred significant protection against challenge with live M. tuberculosis and M. bovis bacille Calmette-Guérin (BCG). These results indicate that immunization with DNA encoding a mycobacterial antigen provides an efficient and simple method for generating protective immunity and that this technique may be useful for defining the protective antigens of M. tuberculosis, leading to the development of a more effective vaccine.
Assuntos
Antígenos de Bactérias/genética , Vacina BCG/imunologia , DNA Bacteriano/imunologia , Mycobacterium tuberculosis/imunologia , Tuberculose/prevenção & controle , Animais , Anticorpos Antibacterianos/sangue , Formação de Anticorpos , Antígenos de Bactérias/imunologia , Vacina BCG/administração & dosagem , Citocinas/imunologia , DNA Bacteriano/administração & dosagem , Modelos Animais de Doenças , Imunidade Celular , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Mycobacterium tuberculosis/genética , Linfócitos T/imunologia , Células Tumorais CultivadasRESUMO
Since the discovery that interferons responses constitute an efficient transition between innate and adaptive immunity to infectious microorganisms, the function and role of these cytokines are better understood. Interferons act on specific cell receptors, which activate well-defined transduction pathways to enhance the expression of hundreds of different Interferon-Stimulated Genes (ISGs) leading to the so-called antiviral state. Several of these genes including those encoding proteins (such as PKR, OAS, and ADAR1) depending on the presence of intracellular double stranded RNA for the activation of their enzymatic function, were studied in more detail. Considerable progress has been made recently in understanding how type I interferons expression is induced in response to virus infection. These imply several Toll-like Receptors (TLRs) and several newly described cytoplasmic protein sensors (RIG-I, MDA5, DAI) that detect the presence of viral nucleic acids and transduce intracellular signals leading to the activation of several members of the family of interferon regulatory factors (IRFs) required for the expression of type I interferons or directly some of the ISGs themselves. The discovery of these transduction pathways derives largely from the study of gene deficient cells and animals. The importance of these proteins is further attested by the discovery of several virally encoded antagonists that can interrupt specifically various steps of either the induction or the action of type I interferons.
Assuntos
Regulação da Expressão Gênica , Interferons/fisiologia , Transdução de Sinais , Receptores Toll-Like/metabolismo , Viroses/imunologia , Humanos , Interferons/genética , Interferons/imunologia , Interferons/metabolismo , Transdução de Sinais/imunologia , Transcrição Gênica/genética , Viroses/genética , Viroses/virologiaRESUMO
We have undertaken the inventory and assembly of the typical subunits of the ABC transporters encoded by the complete genome of Mycobacterium tuberculosis. These subunits, i.e. the nucleotide binding domains (NBDs), the membrane-spanning domains (MSDs) and the substrate binding proteins (SBPs), were identified on the basis of their characteristic stretches of amino acids and/or conserved structure. A total of 45 NBDs present in 38 proteins, of 47 MSDs present in 44 proteins and of 15 SBPs were found to be encoded by M. tuberculosis. Analysis of transcriptional clusters and searches of homology between the identified subunits of the transporters and proteins characterized in other organisms allowed the reconstitution of at least 26 complete (including at least one NBD and one MSD) and 11 incomplete ABC transporters. Sixteen of them were unambiguously classified as importers whereas 21 were presumed to be exporters. By searches of homology with already known transporters from other organisms, potential substrates (peptides, macrolides, carbohydrates, multidrugs, antibiotics, iron, anions) could be attributed to 30 of the ABC transporters identified in M. tuberculosis. The ABC transporters have been further classified in nine different sub-families according to a tree obtained from the clustering of their NBDs. Contrary to Escherichia coli and similarly to Bacillus subtilis, there is an equal representation of extruders and importers. Many exporters were found to be potentially implicated in the transport of drugs, probably contributing to the resistance of M. tuberculosis to many antibiotics. Interestingly, a transporter (absent in E. coli and in B. subtilis) potentially implicated in the export of a factor required for the bacterial attachment to the eukaryotic host cells was also identified. In comparison to E. coli and B. subtilis, there is an under-representation of the importers (with the exception of the phosphate importers) in M. tuberculosis. This may reflect the capacity of this bacterium to synthesize many essential compounds and to grow in the presence of few external nutrients. The genes encoding the ABC transporters occupy about 2.5% of the genome of M. tuberculosis.
Assuntos
Transportadores de Cassetes de Ligação de ATP/metabolismo , Proteínas de Bactérias/metabolismo , Mycobacterium tuberculosis/metabolismo , Transportadores de Cassetes de Ligação de ATP/genética , Sequência de Aminoácidos , Aminoácidos/metabolismo , Ânions/metabolismo , Antibacterianos/metabolismo , Fusão Gênica Artificial , Proteínas de Bactérias/genética , Sítios de Ligação , Metabolismo dos Carboidratos , Genoma Bacteriano , Ferro/metabolismo , Macrolídeos/metabolismo , Membranas/metabolismo , Dados de Sequência Molecular , Mycobacterium tuberculosis/genética , Nucleotídeos/metabolismo , Peptídeos/metabolismoRESUMO
The nucleotide sequence of a 1619-bp fragment of Mycobacterium bovis BCG containing the gene that encodes an alcohol dehydrogenase (ADH) has been determined. The M(r) calculated from the deduced amino acid (aa) sequence, as well as the N terminus, are in good accordance with those determined for the ADH purified from M. bovis BCG extracts. The M. bovis BCG cloned adh gene was expressed in Escherichia coli by its own promoter and the synthesized product shows ADH activity in the butane-1-ol-NADP system. Based on comparison of the aa sequence, this enzyme belongs to the zinc-containing, long-chain alcohol/polyol dehydrogenase family, which has been primarily described in eukaryotes. Of the 22 strictly conserved residues in this group, 19 are also conserved in M. bovis BCG ADH (BCGADH).
Assuntos
Álcool Desidrogenase/genética , Mycobacterium bovis/enzimologia , Sequência de Aminoácidos , Sequência de Bases , Southern Blotting , Clonagem Molecular , DNA Bacteriano , Eletroforese em Gel de Ágar , Eletroforese em Gel de Poliacrilamida , Escherichia coli , Genes Bacterianos , Dados de Sequência Molecular , NADP/metabolismo , Mapeamento por Restrição , Homologia de Sequência de AminoácidosRESUMO
By cDNA mutagenesis, we have constructed internal and C-terminal deletions (delta 21-51, delta 52-97, delta 97-104, delta 127-174, delta 97-184 and delta 134-184) in human interleukin-6 (hIL-6). All those deletion-carrying hIL-6 (delta hIL-6) proteins were then produced in Xenopus laevis oocytes and examined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The results show that, at least in frog oocytes, the first potential N-glycosylation site (Asn45) is utilized exclusively. The IL-6 conformation of these deletion-carrying proteins has been studied by immunoprecipitation with two kinds of monoclonal antibodies (mAb's): mAb's that show preference towards denatured hIL-6, or conformation-specific mAb's. The binding pattern of these two series of mAb's indicated that the IL-6 conformation has been largely destroyed for four of our delta-proteins. Proteins delta 21-51 and delta 127-174 have kept a part of the IL-6 tertiary structure since they are still recognized by some conformation-specific mAb's. All of these delta hIL-6 proteins were inactive in the IL-6 hybridoma growth factor (HGF) assay and unable to inhibit the HGF activity of the recombinant human wild-type IL-6 (wt hIL-6). Moreover, the oocyte-synthesized delta hIL-6 (delta 21-51, delta 127-174, delta 97-184, delta 134-184) did not bind to the IL-6 receptor. Finally, we have produced two proteins with aa 29-33 or 97-104 substituted by corresponding murine IL-6 (mIL-6) sequences.(ABSTRACT TRUNCATED AT 250 WORDS)
Assuntos
Deleção Cromossômica , Interleucina-6/genética , Sequência de Aminoácidos , Animais , Anticorpos Monoclonais , Sistema Livre de Células , Clonagem Molecular , Humanos , Interleucina-6/química , Camundongos , Sondas Moleculares , Dados de Sequência Molecular , Coelhos , Reticulócitos , Relação Estrutura-Atividade , Xenopus laevisRESUMO
We report the cloning and sequencing of three M. tuberculosis genes encoding proteins homologous to E. coli PstA, PstC and PstB. They are tentatively called pstA-2, pstC-1 and pstB. They encode proteins of 302, 336 and 275 amino acids, respectively. In E. coli, PstB is the ATP binding component and PstA/PstC are the two hydrophobic subunits of a phosphate permease belonging to the family of ABC (ATP-binding cassette) transporters. In mycobacteria, PstS-1, the phosphate binding subunit (Andersen and Hansen, 1989), is encoded by a gene directly surrounded by pstB, pstC-1 and pstA-2 within a potential operon (pstB, pstS-1, pstC-1, pstA-2). Phosphate uptake by whole, suspension grown, M. bovis BCG cells was measured and could be inhibited by a monoclonal antibody directed against the PstS-1 subunit, suggesting that these genes encode subunits of a functional mycobacterial phosphate permease.
Assuntos
Transportadores de Cassetes de Ligação de ATP/genética , Proteínas de Escherichia coli , Família Multigênica , Mycobacterium bovis/genética , Fosfatos/metabolismo , Sequência de Aminoácidos , Proteínas de Bactérias/genética , Sequência de Bases , Transporte Biológico , DNA Bacteriano , Escherichia coli/metabolismo , Dados de Sequência Molecular , Mycobacterium bovis/metabolismo , ÓperonRESUMO
The IFI-56K and IFI-54K human genes are coordinately regulated by interferon, double-stranded RNA and viruses in a number of cell lines. These genes encode polypeptides of 56 and 54 kDa, respectively, whose function remains to be determined. We analysed the possible structural relatedness between these syntenic and similarly regulated genes. We found that they are very closely related at the protein, mRNA and promoter levels. This suggests that the IFI-56K and IFI-54K genes are members of a gene family, which probably arose from duplication of an ancestor gene.
Assuntos
Cromossomos Humanos Par 10 , Cromossomos Humanos Par 13 , Regulação da Expressão Gênica , Genes , Interferon Tipo I/fisiologia , Sequência de Aminoácidos , Sequência de Bases , Humanos , Dados de Sequência Molecular , Peso MolecularRESUMO
2-5A[ppp(A2'p)n5'A] has been implicated as a mediator in the antiviral action of interferon. Its direct evaluation as an indicator of virus replication is hampered by two limitations: its inability to penetrate intact cells, and its rapid intracellular degradation by (2'-5')phosphodiesterase. These problems could be overcome by using a microinjection technique whereby a phosphodiesterase-resistant analog of 2-A, in which the 2'-terminals adenosine residue is replaced by 2-(9-adenyl)-6-hydroxy-methyl-4-hexylmorpholine, was injected into individual HeLa cells before infection with mengovirus or vesicular stomatitis virus (VSV). This comparative assay with two representatives of different virus classes in a single experimental system pointed to the high sensitivity of VSV to inhibition by 2-5A oligonucleotides, in contrast with the low sensitivity of mengovirus. Microinjection of the hexylmorpholine 2-5A analog led to a much greater reduction in mengovirus yield than did microinjection of 2-5A itself.
Assuntos
Nucleotídeos de Adenina/farmacologia , Antivirais/farmacologia , Oligorribonucleotídeos/farmacologia , Relação Dose-Resposta a Droga , Células HeLa , Humanos , Interferons/farmacologia , Mengovirus/efeitos dos fármacos , Microinjeções , Vírus da Estomatite Vesicular Indiana/efeitos dos fármacos , Replicação Viral/efeitos dos fármacosRESUMO
Interferon-induced 2-5A synthetases are probably involved in some antiviral actions of interferon. In human cells two different mRNAs (1.6, 1.8 kb long) coding for this protein are transcribed from the same gene and are produced by differential splicing. The relationship between the two mRNAs of different size and the active enzyme is not clear, nor is the cellular localization of the latter known. We have cloned a cDNA corresponding to the 1.6 kb RNA. This cDNA was sequenced and its complete coding region was subcloned into pSP64. The resulting plasmid was used to direct the synthesis of micrograms of capped RNA transcript after linearization in the 3'-non-coding region. A 39 kDa protein was synthesized when this RNA was translated in rabbit reticulocyte lysate. When this capped RNA was introduced by microinjection into Xenopus oocytes, production of 2-5A synthetase was clearly observed in the cytoplasm and 10-30% of the enzyme accumulated with time in the nucleoplasm. Analysis of cytoplasmic homogenates of these oocytes on a glycerol gradient revealed that the enzyme is fully active in the monomeric form.
Assuntos
2',5'-Oligoadenilato Sintetase/genética , Interferon Tipo I/farmacologia , 2',5'-Oligoadenilato Sintetase/biossíntese , Sequência de Aminoácidos , Animais , Sequência de Bases , Núcleo Celular/análise , Sistema Livre de Células , Citoplasma/análise , DNA/análise , DNA Recombinante , Indução Enzimática/efeitos dos fármacos , Feminino , Regulação da Expressão Gênica/efeitos dos fármacos , Humanos , Microinjeções , Oócitos/metabolismo , Splicing de RNA , RNA Mensageiro/biossíntese , RNA Mensageiro/genética , Xenopus laevisRESUMO
Internal deletions of the human interleukin-6 (IL-6) cDNA have been generated in the region encoding residues 29 to 42. Mutant proteins were produced by in vitro transcription-translation or in Escherichia coli and tested for their biological activity using the hybridoma growth factor (HGF) assay or a transcriptional activation assay on human hepatoma cells. The folding of the mutants was also checked by immunoprecipitation with conformation-specific monoclonal antibodies. The results show that only residues 29 to 34 are crucial for IL-6 activity and that the first two amino acids are probably involved in the definition of the IL-6 active site.
Assuntos
Interleucina-6/química , Conformação Proteica , Sequência de Aminoácidos , Anticorpos Monoclonais , Sequência de Bases , Sítios de Ligação/genética , Escherichia coli/metabolismo , Humanos , Interleucina-6/genética , Dados de Sequência Molecular , Mutação/genética , Fragmentos de Peptídeos , Testes de Precipitina , Proteínas Recombinantes/biossíntese , Células Tumorais CultivadasRESUMO
Following the identification of a M. tuberculosis phosphate transporter belonging to the superfamily of ABC transporters, we report on the cloning and sequencing of two additional genes, called pstS-3 and pstC-2, encoding proteins homologous to PstS and PstC of Escherichia coli, respectively. Together with the previously isolated M. tuberculosis gene similar to the E. coli pstA, these are included in a cluster encoding a second putative phosphate transport system. We demonstrate that pstS-3 encodes the previously described Ag 88, a 40 kDa M. bovis BCG culture filtrate antigen (immunodominant in H-2b haplotype type mice). Finally, a signature motif identifying integral transmembrane proteins of prokaryotic phosphate binding-dependent permeases is proposed.
Assuntos
Transportadores de Cassetes de Ligação de ATP/genética , Proteínas de Bactérias , Proteínas de Transporte/genética , Família Multigênica , Mycobacterium tuberculosis/genética , Transportadores de Cassetes de Ligação de ATP/química , Sequência de Aminoácidos , Sequência de Bases , Sítios de Ligação , Proteínas de Transporte/química , Clonagem Molecular , Sequência Conservada , Brometo de Cianogênio , Eletroforese em Gel de Poliacrilamida , Genes Bacterianos , Immunoblotting , Dados de Sequência Molecular , Mycobacterium tuberculosis/química , Óperon , Peptídeos/química , Proteínas de Ligação a Fosfato , Análise de Sequência de DNARESUMO
There is increasing evidence to implicate a role for CD8(+) T cells in protective immunity against tuberculosis. Recombinant vaccinia (rVV) expressing Mycobacterium tuberculosis (MTB) proteins can be used both as tools to dissect CD8(+) T-cell responses and, in attenuated form, as candidate vaccines capable of inducing a balanced CD4(+)/CD8(+) T-cell response. A panel of rVV was constructed to express four immunodominant secreted proteins of MTB: 85A, 85B and 85C and ESAT-6. A parallel group of rVV was constructed to include the heterologous eukaryotic tissue plasminogen activator (tPA) signal sequence to assess if this would enhance expression and immunogenicity. Clear expression was obtained for 85A, 85B and ESAT-6 and the addition of tPA resulted in N-glycosylation and a 4-10-fold increase in expression. Female C57BL/6 mice were immunised using the rVV-Ag85 constructs, and interleukin-2 and gamma-interferon were assayed using a co-culture of immune splenocytes and recall antigen. There was a marked increase in cytokine production in mice immunised with the tPA-containing constructs. We report the first data demonstrating enhanced immunogenicity of rVV using a tPA signal sequence, which has significant implications for future vaccine design.
Assuntos
Aciltransferases , Antígenos de Bactérias/imunologia , Proteínas de Bactérias/biossíntese , Vacinas Bacterianas/imunologia , Mycobacterium tuberculosis/imunologia , Partícula de Reconhecimento de Sinal , Ativador de Plasminogênio Tecidual/química , Vaccinia virus/metabolismo , Animais , Sequência de Bases , Células Cultivadas , Clonagem Molecular , Técnicas de Cocultura , Feminino , Vetores Genéticos , Glicosilação , Interferon gama/análise , Interleucina-2/análise , Camundongos , Camundongos Endogâmicos C57BL , Dados de Sequência Molecular , Dobramento de ProteínaRESUMO
Interferon (IFN)-inducible human MxA protein mediates resistance against influenza and several other RNA viruses. The MxA gene is under the control of type I IFN and, in certain cell types, is also directly activated by viruses. Here we show that in human macrophages, MxA mRNA levels are upregulated by very low doses of IFN-alpha in a dose-dependent manner. A similar, albeit much weaker, dose-dependent induction was seen with IFN-gamma. The induction was rapid and independent of protein synthesis. Interleukin-6 (IL-6) or tumor necrosis factor-alpha (TNF-alpha) did not influence MxA mRNA levels alone or in combination with IFNs, in spite of the presence of putative response elements of these cytokines in the MxA promoter. We show that the promoter of the MxA gene contains two functional IFN-stimulated response elements (ISRE) near the transcription start site and one homologous ISRE-like element, which is apparently nonfunctional, further upstream. The two proximal ISRE sites are essential for IFN-alpha-induced transcription and appear to be binding sites for IFN-stimulated gene factor 3 complex. In addition, EMSA and DNAse I footprinting analysis demonstrated that Spl binds with high affinity to a region encompassing nucleotides -25 and -50 and, thus, may provide means of interaction with the basal transcriptional machinery.
Assuntos
Antivirais/genética , Antivirais/farmacologia , GTP Fosfo-Hidrolases/genética , Proteínas de Ligação ao GTP , Interferon-alfa/farmacologia , Regiões Promotoras Genéticas , Proteínas/genética , Sequência de Bases , Proteínas de Ligação a DNA/metabolismo , Humanos , Fator Gênico 3 Estimulado por Interferon , Fator Gênico 3 Estimulado por Interferon, Subunidade gama , Macrófagos/efeitos dos fármacos , Macrófagos/metabolismo , Dados de Sequência Molecular , Proteínas de Resistência a Myxovirus , RNA Mensageiro/biossíntese , Elementos de Resposta , Estimulação Química , Fatores de Transcrição/metabolismo , Regulação para CimaRESUMO
Human interleukin 1 (IL 1) exerts an interferon-like antiviral activity in certain strains of human diploid fibroblasts. It is shown that this antiviral effect is indirect, in that it is mediated by production of interferon-beta. IL 1 also induces synthesis of the mRNA of another secreted protein, 26K, whose biological function is still controversial. Finally, IL 1 has bone marrow colony stimulating activity which is shown to be due to induction of colony stimulating factor in adherent phagocytic cells present in the bone marrow cultures. These findings emphasize the concept that several of the varied biological effects of IL 1 may be indirect.
Assuntos
Indutores de Interferon , Interferon Tipo I/biossíntese , Interleucina-1/farmacologia , Células da Medula Óssea , Células Cultivadas , Ensaio de Unidades Formadoras de Colônias , Fatores Estimuladores de Colônias/metabolismo , Cicloeximida/farmacologia , Fibroblastos , Regulação da Expressão Gênica , Hematopoese/efeitos dos fármacos , Humanos , RNA Mensageiro/genética , Proteínas Recombinantes/farmacologiaRESUMO
DNA plasmids encoding Mycobacterium tuberculosis antigen 85 (Ag85) were tested as vaccines in animal models. Ag85 DNA induced relevant immune responses (i.e. T helper (Th) cells, Th1 cytokines and cytotoxic T lymphocytes) and was protective in mouse and guinea pig models of mycobacterial disease. Therefore, DNA vaccination holds promise as an effective means of preventing tuberculosis in humans. Furthermore, this technique is amenable to identifying the protective antigens of M. tuberculosis.
Assuntos
Aciltransferases , Antígenos de Bactérias/imunologia , Vacina BCG/uso terapêutico , Tuberculose Pulmonar/prevenção & controle , Vacinas de DNA/uso terapêutico , Animais , Anticorpos Antibacterianos/sangue , Antígenos de Bactérias/genética , Vacina BCG/imunologia , Feminino , Cobaias , Imunidade Celular , Camundongos , Camundongos Endogâmicos BALB C , Vacinação , Vacinas de DNA/imunologiaRESUMO
OBJECTIVE: To determine the feasibility of prenatal diagnosis of fetal cytomegalovirus (CMV) infection. METHODS: Fifty-two pregnant women were investigated in our unit between October 1985 and July 1992. The diagnostic procedures included ultrasound examination, amniocentesis, and fetal blood sampling. Specific tests for CMV infection included specific immunoglobulin (Ig) M antibodies, viral culture, and amplification of CMV DNA by polymerase chain reaction. Nonspecific tests included white blood cell count, hemoglobin, hematocrit, platelets, and gamma-glutamyl transferase determination. RESULTS: The combination of tests allowed an antenatal diagnosis of CMV in 13 of the 16 infected fetuses (sensitivity 81%). Amniocentesis allowed the diagnosis in 12 of the 13 antenatally diagnosed cases. The sensitivity of CMV IgM antibody detection in fetal blood was 69%. The culture of fetal blood was never positive. Thrombocytopenia was present in six cases, and ultrasound was abnormal in five. CONCLUSIONS: Amniotic fluid is the best sample to diagnose CMV infection, and fetal blood sampling and sonography are important to assess the fetal condition. Our experience underscores the importance of repetitive sampling.
Assuntos
Infecções por Citomegalovirus/diagnóstico , Doenças Fetais/diagnóstico , Diagnóstico Pré-Natal , Amniocentese , Anticorpos Antivirais/sangue , Citomegalovirus/imunologia , Citomegalovirus/isolamento & purificação , Infecções por Citomegalovirus/sangue , Infecções por Citomegalovirus/congênito , Infecções por Citomegalovirus/epidemiologia , Feminino , Sangue Fetal , Doenças Fetais/sangue , Doenças Fetais/epidemiologia , Humanos , Imunoglobulina M/sangue , Gravidez , Estudos Prospectivos , Fatores de Risco , Sensibilidade e Especificidade , Ultrassonografia Pré-NatalRESUMO
We report a case of recurrent granulomatous tenosynovitis with M. intracellulare in a 55-year-old HIV negative diabetic woman. Identification of the causative agent further than belonging to the M. avium-intracellulare complex is provided by specific PCR-amplification of genomic DNA and sequencing of an hypervariable region within its 16S RNA gene. Sixteen months antibiotic regimen of rifabutin and clarithromycin led to a complete resolution of the tenosynovitis.
Assuntos
Mãos , Complexo Mycobacterium avium , Tenossinovite/microbiologia , Feminino , Granuloma , Humanos , Pessoa de Meia-Idade , Complexo Mycobacterium avium/classificação , Complexo Mycobacterium avium/genética , Recidiva , Tenossinovite/tratamento farmacológico , Resultado do TratamentoRESUMO
Non-specific phosphomonoesterase activities (alkaline phosphatase (EC 3.1.3.1) and acid phosphatase (EC 3.1.3.2)) were examined at the cell surface of Mycobacterium bovis BCG. Using p-nitrophenylphosphate as the substrate, peaks of phosphatase activity were detected at pH 6.0, pH 10.0 and pH 12.0, suggesting the presence of one acid phosphatase and two alkaline phosphatases with distinct optimum pH values. Contrary to the situation observed in several other microorganisms, the expression of these enzymes is not regulated by the environmental inorganic phosphate concentration.
Assuntos
Fosfatase Ácida/metabolismo , Fosfatase Alcalina/metabolismo , Mycobacterium bovis/enzimologia , Fosfatos/metabolismo , Fosfatase Ácida/biossíntese , Fosfatase Alcalina/biossíntese , Membrana Celular/enzimologia , Meios de Cultura , Indução Enzimática , Concentração de Íons de Hidrogênio , Mycobacterium bovis/crescimento & desenvolvimentoRESUMO
The structure and distribution of a Mycobacterium bovis BCG insertion element of the IS21 family were investigated. Several IS21-like elements found in mycobacterial genomes were separated in four types, following their nucleic acid similarities. The M. bovis BCG IS21 element is highly similar to IS1533 (class I), 70% similar to IS1534 (class II), 52% similar to IS1532 (class III) of Mycobacterium tuberculosis, and 54% similar to both an Mycobacterium avium serovar 2 and an M. avium silvaticum IS (class IV). The M. bovis BCG IS21 element of the class I appears to be present in a single copy in the genome of M. bovis BCG, M. bovis, M. tuberculosis and Mycobacterium africanum and to be absent from all other tested species of the Corynebacteria-Mycobacteria-Nocardia group.
Assuntos
Proteínas de Bactérias/genética , Elementos de DNA Transponíveis , Mycobacterium bovis/genética , Sequência de Aminoácidos , Proteínas de Bactérias/química , Sequência de Bases , DNA Bacteriano/genética , Dados de Sequência Molecular , Alinhamento de Sequência , Análise de Sequência de DNARESUMO
We previously identified a 70-kDa serine/threonine protein kinase (MbK or PknD) from Mycobacterium tuberculosis Erdman containing a transmembrane domain and bearing a 270-amino acid N-terminal kinase domain. With the use of a polyclonal serum, Mbk has now been identified by Western blotting in protein extracts from M. tuberculosis and confirmed to be localised in the envelope. An identical mbk gene has been found by sequencing different M. tuberculosis and M. africanum strains. Surprisingly, in two virulent M. bovis strains and four different strains of M. bovis BCG, an additional adenine after position 829 of the open reading frame was found that produces a frame shift resulting in a predicted truncated, presumably free cytoplasmic protein, encoding only the N-terminal 30-kDa Mbk kinase domain. This sequence polymorphism has been confirmed by Western blot analysis of M. bovis BCG protein extracts.