RESUMO
We herein report for the first time the inter- and intramolecular orthogonal cleavage of two ortho-nitrobenzyl (NB) analogues. It is shown that the nitroveratryl (NV) group can be photolyzed with high priority when NV and ortho-nitrobenzyl carbonate (oNBC) are used together as the protecting groups of glycans. Notably, the photolytic products could be used directly in the subsequent glycosylation without further purification. With the above-mentioned orthogonal photolabile protecting group strategy in hand, a Mycobacterium tuberculosis tetrasaccharide and a derivative of glucosyl glycerol were rapidly prepared.
Assuntos
Mycobacterium tuberculosis , Oligossacarídeos , Glicosilação , Estrutura Molecular , Mycobacterium tuberculosis/química , Nitrobenzenos/química , Oligossacarídeos/química , Oligossacarídeos/síntese químicaRESUMO
Mycobacterium tuberculosis can reside and persist in deep tissues; latent tuberculosis can evade immune detection and has a unique mechanism to convert it into active disease through reactivation. M. tuberculosis Rv1421 (MtRv1421) is a hypothetical protein that has been proposed to be involved in nucleotide binding-related metabolism in cell-growth and cell-division processes. However, due to a lack of structural information, the detailed function of MtRv1421 remains unclear. In this study, a truncated N-terminal domain (NTD) of MtRv1421, which contains a Walker A/B-like motif, was purified and crystallized using PEG 400 as a precipitant. The crystal of MtRv1421-NTD diffracted to a resolution of 1.7â Å and was considered to belong to either the C-centered monoclinic space group C2 or the I-centered orthorhombic space group I222, with unit-cell parameters a = 124.01, b = 58.55, c = 84.87â Å, ß = 133.12° or a = 58.53, b = 84.86, c = 90.52â Å, respectively. The asymmetric units of the C2 or I222 crystals contained two or one monomers, respectively. In terms of the binding ability of MtRv1421-NTD to various ligands, uridine diphosphate (UDP) and UDP-N-acetylglucosamine significantly increased the melting temperature of MtRv1421-NTD, which indicates structural stabilization through the binding of these ligands. Altogether, the results reveal that a UDP moiety may be required for the interaction of MtRv1421-NTD as a nucleotide-binding protein with its ligand.
Assuntos
Proteínas de Bactérias , Mycobacterium tuberculosis , Mycobacterium tuberculosis/metabolismo , Mycobacterium tuberculosis/química , Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Proteínas de Bactérias/genética , Cristalografia por Raios X , Ligantes , Ligação Proteica , Domínios Proteicos , Cristalização , Difração de Raios X , Escherichia coli/metabolismo , Escherichia coli/genética , Clonagem Molecular , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Proteínas Recombinantes/genética , Sequência de AminoácidosRESUMO
Tuberculosis necrotizing toxin (TNT) is a protein domain discovered on the outer membrane of Mycobacterium tuberculosis (Mtb), and the fungal pathogen Aspergillus fumigatus. TNT domains have pure NAD(P) hydrolytic activity, setting them apart from other NAD-cleaving domains such as ADP-ribosyl cyclase and Toll/interleukin-1 receptor homology (TIR) domains which form a wider set of products. Importantly, the Mtb TNT domain has been shown to be involved in immune evasion via depletion of the intracellular NAD pool of macrophages. Therefore, an intriguing hypothesis is that TNT domains act as "NAD killers" in host cells facilitating pathogenesis. Here, we explore the phylogenetic distribution of TNT domains and detect their presence solely in bacteria and fungi. Within fungi, we discerned six TNT clades. In addition, X-ray crystallography and AlphaFold2 modeling unveiled clade-specific strategies to promote homodimer stabilization of the fungal enzymes, namely, Ca2+ binding, disulfide bonds, or hydrogen bonds. We show that dimer stabilization is a requirement for NADase activity and that the group-specific strategies affect the active site conformation, thereby modulating enzyme activity. Together, these findings reveal the evolutionary lineage of fungal TNT enzymes, corroborating the hypothesis of them being pure extracellular NAD (eNAD) cleavers, with possible involvement in microbial warfare and host immune evasion.
Assuntos
Mycobacterium tuberculosis , NAD , Mycobacterium tuberculosis/enzimologia , Mycobacterium tuberculosis/metabolismo , Mycobacterium tuberculosis/genética , Mycobacterium tuberculosis/química , NAD/metabolismo , Domínios Proteicos , Proteínas Fúngicas/química , Proteínas Fúngicas/metabolismo , Proteínas Fúngicas/genética , Cristalografia por Raios X , Aspergillus fumigatus/enzimologia , Aspergillus fumigatus/genética , Aspergillus fumigatus/metabolismo , Aspergillus fumigatus/química , Evolução Molecular , Modelos Moleculares , Filogenia , NAD+ Nucleosidase/metabolismo , NAD+ Nucleosidase/química , NAD+ Nucleosidase/genéticaRESUMO
The unambiguous identification of protein species requires high sequence coverage. In this study, we successfully improved the sequence coverage of early secretory 10 kDa cell filtrate protein (CFP-10) and 6 kDa early secretory antigenic target (ESAT-6) proteins from the Mycobacterium tuberculosis complex (MTC) in broth culture media with the use of the 4-chloro-α-cyanocinnamic acid (Cl-CCA) matrix. Conventional matrices, α-cyano-hydroxy-cinnamic acid (CHCA) and 2,5-dihydroxybenzoic acid (DHB), were also used for comparison. After nanodiamond (ND) extraction, the sequence coverage of the CFP-10 protein was 87% when CHCA and DHB matrices were used, and the ESAT-6 protein was not detected. On the other hand, the sequence coverage for ND-extracted CFP-10 and ESAT-6 could reach 94% and 100%, respectively, when the Cl-CCA matrix was used and with the removal of interference from bovine serum albumin (BSA) protein and α-crystallin (ACR) protein. Matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) was also adopted to analyze the protein mass spectra. A total of 6 prominent ion signals were observed, including ESAT-6 protein peaks at mass-to-charge ratios (m/z) of â¼7931, â¼7974, â¼9768, and â¼9813 and CFP-10 protein peaks at m/z of â¼10 100 and â¼10 660. The ESAT-6 ion signals were always detected concurrently with CFP-10 ion signals, but CFP-10 ion signals could be detected alone without the ESAT-6 ion signals. Furthermore, the newly found ESAT-6 peaks were also confirmed using a Mag-Beads-Protein G kit with an ESAT-6 antibody to capture the ESAT-6 protein, which was also consistent with the sequence coverage analysis.
Assuntos
Antígenos de Bactérias , Proteínas de Bactérias , Mycobacterium tuberculosis , Nanodiamantes , Mycobacterium tuberculosis/química , Proteínas de Bactérias/química , Nanodiamantes/química , Antígenos de Bactérias/química , Antígenos de Bactérias/isolamento & purificação , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz/métodosRESUMO
We discovered an unusual triflic acid-promoted oligomerization of arabinofuranosides during glycosylation of the primary hydroxy group of α-(1 â 5)-linked tetraarabinofuranoside bearing 4-(2-chloroethoxy)phenyl aglycone with α-(1 â 5), ß-(1 â 2)-linked tetraarabinofuranoside containing N-phenyltrifluoroacetimidoyl leaving group, which led to octa-, dodeca- and hexadecaarabinofuranosides. The possible mechanism of triflic acid-promoted oligomerization was proposed. The choice of promoter was found to be a critical factor for the discovered oligomerization of arabinofuranosides. The obtained octa-, dodeca- and hexadecaarabinofuranosides may serve as useful blocks in the synthesis of oligosaccharide fragments of polysaccharides of Mycobacterium tuberculosis.
Assuntos
Arabinose , Mesilatos , Glicosilação , Arabinose/química , Mesilatos/química , Mycobacterium tuberculosis/efeitos dos fármacos , Mycobacterium tuberculosis/química , Configuração de CarboidratosRESUMO
Nitrobindins (Nbs) are all-ß-barrel heme proteins present along the evolutionary ladder. They display a highly solvent-exposed ferric heme group with the iron atom being coordinated by the proximal His residue and a water molecule at the distal position. Ferric nitrobindins (Nb(III)) play a role in the conversion of toxic peroxynitrite (ONOO-) to harmless nitrate, with the value of the second-order rate constant being similar to those of most heme proteins. The value of the second-order rate constant of Nbs increases as the pH decreases; this suggests that Nb(III) preferentially reacts with peroxynitrous acid (ONOOH), although ONOO- is more nucleophilic. In this work, we shed light on the molecular basis of the ONOO- and ONOOH reactivity of ferric Mycobacterium tuberculosis Nb (Mt-Nb(III)) by dissecting the ligand migration toward the active site, the water molecule release, and the ligand binding process by computer simulations. Classical molecular dynamics simulations were performed by employing a steered molecular dynamics approach and the Jarzynski equality to obtain ligand migration free energy profiles for both ONOO- and ONOOH. Our results indicate that ONOO- and ONOOH migration is almost unhindered, consistent with the exposed metal center of Mt-Nb(III). To further analyze the ligand binding process, we computed potential energy profiles for the displacement of the Fe(III)-coordinated water molecule using a hybrid QM/MM scheme at the DFT level and a nudged elastic band approach. These results indicate that ONOO- exhibits a much larger barrier for ligand displacement than ONOOH, suggesting that water displacement is assisted by protonation of the leaving group by the incoming ONOOH.
Assuntos
Simulação de Dinâmica Molecular , Mycobacterium tuberculosis , Ácido Peroxinitroso , Ácido Peroxinitroso/química , Ácido Peroxinitroso/metabolismo , Mycobacterium tuberculosis/química , Hemeproteínas/química , Hemeproteínas/metabolismo , Compostos Férricos/química , Compostos Férricos/metabolismo , TermodinâmicaRESUMO
During the past few decades, a wealth of knowledge has been made available for the transcription machinery in bacteria from the structural, functional and mechanistic point of view. However, comparatively little is known about the homooligomerization of the multisubunit M. tuberculosis RNA polymerase (RNAP) enzyme and its functional relevance. While E. coli RNAP has been extensively studied, many aspects of RNAP of the deadly pathogenic M. tuberculosis are still unclear. We used biophysical and biochemical methods to study the oligomerization states of the core and holoenzymes of M. tuberculosis RNAP. By size exclusion chromatography and negative staining Transmission Electron Microscopy (TEM) studies and quantitative analysis of the TEM images, we demonstrate that the in vivo reconstituted RNAP core enzyme (α2ßß'ω) can also exist as dimers in vitro. Using similar methods, we also show that the holoenzyme (core + σA) does not dimerize in vitro and exist mostly as monomers. It is tempting to suggest that the oligomeric changes that we see in presence of σA factor might have functional relevance in the cellular process. Although reported previously in E. coli, to our knowledge we report here for the first time the study of oligomeric nature of M. tuberculosis RNAP in presence and absence of σA factor.
Assuntos
Proteínas de Bactérias , RNA Polimerases Dirigidas por DNA , Mycobacterium tuberculosis , Multimerização Proteica , Mycobacterium tuberculosis/enzimologia , Mycobacterium tuberculosis/genética , Mycobacterium tuberculosis/química , RNA Polimerases Dirigidas por DNA/metabolismo , RNA Polimerases Dirigidas por DNA/química , RNA Polimerases Dirigidas por DNA/genética , Holoenzimas/química , Holoenzimas/metabolismo , Proteínas de Bactérias/metabolismo , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Microscopia Eletrônica de Transmissão , Fator sigma/metabolismo , Fator sigma/química , Fator sigma/genética , Cromatografia em GelRESUMO
DAT2 is a member of the diacyl trehalose family (DAT) of antigenic glycolipids located in the mycomembrane of Mycobacterium tuberculosis (Mtb). Recently it was shown that the molecular structure of DAT2 had been incorrectly assigned, but the correct structure remained elusive. Herein, the correct molecular structure of DAT2 and its methyl-branched acyl substituent mycolipanolic acid is determined. For this, four different stereoisomers of mycolipanolic acid were prepared in a stereoselective and unified manner, and incorporated into DAT2. A rigorous comparison of the four isomers to the DAT isolated from Mtb H37Rv by NMR, HPLC, GC, and mass spectrometry allowed a structural revision of mycolipanolic acid and DAT2. Activation of the macrophage inducible Ca2+-dependent lectin receptor (Mincle) with all four stereoisomers shows that the natural stereochemistry of mycolipanolic acid / DAT2 provides the strongest activation, which indicates its high antigenicity and potential application in serodiagnostics and vaccine adjuvants.
Assuntos
Glicolipídeos , Mycobacterium tuberculosis , Mycobacterium tuberculosis/imunologia , Mycobacterium tuberculosis/química , Glicolipídeos/química , Glicolipídeos/síntese química , Glicolipídeos/imunologia , Estereoisomerismo , Estrutura MolecularRESUMO
BACKGROUND: The intracellular concentration of heavy-metal cations, such as copper, nickel, and zinc is pivotal for the mycobacterial response to the hostile environment inside macrophages. To date, copper transport mediated by P-type ATPases across the mycobacterial plasma membrane has not been sufficiently explored. RESULTS: In this work, the ATPase activity of the putative Mycobacterium tuberculosis P1B-type ATPase CtpB was associated with copper (I) transport from mycobacterial cells. Although CtpB heterologously expressed in M. smegmatis induced tolerance to toxic concentrations of Cu2+ and a metal preference for Cu+, the disruption of ctpB in M. tuberculosis cells did not promote impaired cell growth or heavy-metal accumulation in whole mutant cells in cultures under high doses of copper. In addition, the Cu+ ATPase activity of CtpB embedded in the plasma mem-brane showed features of high affinity/slow turnover ATPases, with enzymatic parametersKM 0.19 ± 0.04 µM and Vmax 2.29 ± 0.10 nmol/mg min. In contrast, the ctpB gene transcription was activated in cells under culture conditions that mimicked the hostile intraphagosomal environment, such as hypoxia, nitrosative and oxidative stress, but not under high doses of copper. CONCLUSIONS: The overall results suggest that M. tuberculosis CtpB is associated with Cu+ transport from mycobacterial cells possibly playing a role different from copper detoxification.
Assuntos
Membrana Celular/metabolismo , ATPases Transportadoras de Cobre/metabolismo , Mycobacterium tuberculosis/metabolismo , Mycobacterium tuberculosis/químicaRESUMO
ABSTRACT Introducción. Los aislamientos de Mycobacterium tuberculosis pertenecientes al linaje Beijing se consideran especialmente virulentos y transmisibles, y con mayor tendencia a la adquisición de resistencia. El linaje Beijing se ha reportado en todo el mundo; sin embargo, en Latinoamérica los estudios al respecto son más escasos. En el único estudio multinacional llevado a cabo en la región, se detectó una distribución heterogénea del linaje, y no se le encontró en Chile, Colombia y Ecuador, aunque en estudios nacionales posteriores se identificaron aislamientos en Chile y Colombia. Objetivo. Rastrear la presencia del linaje Beijing de M. tuberculosis en Ecuador, único país en la región en el que aún no se reporta. Materiales y métodos. Se analizó una muestra de conveniencia (2006-2012) en dos hospitales que atendían poblaciones diferentes. La genotipificación de los aislamientos de M. tuberculosis se hizo mediante la plataforma 24-MIRU-VNTR. La asignación de linajes se hizo mediante la comparación de los patrones genotípicos con los incluidos en la plataforma MIRU-VNTRplus, y aquellos pertenecientes al linaje Beijing fueron confirmados mediante reacción en cadena de la polimerasa específica de alelo. Resultados. Se detectó el primer aislamiento Beijing en Ecuador, en una circunstancia epidemiológica inesperada: un paciente de la región andina, proveniente de una comunidad con escasa movilidad y alejada de las fronteras con los países limítrofes, Perú y Colombia, en los que ya se han identificado aislamientos de M. tuberculosis pertenecientes al linaje Beijing. Conclusiones. En este trabajo se reporta por primera vez la presencia del linaje Beijing de M. tuberculosis en Ecuador en un contexto epidemiológico inusual que merece especial atención.
RESUMEN Introduction: Mycobacterium tuberculosis Beijing lineage isolates are considered to be especially virulent, transmissible and prone to acquire resistances. Beijing strains have been reported worldwide, but studies in Latin America are still scarce. The only multinational study performed in the region indicated a heterogeneous distribution for this lineage, which was absent in Chile, Colombia and Ecuador, although further studies found the lineage in Chile and Colombia. Objective: To search for the presence of the Beijing lineage in Ecuador, the only country in the region where it remains unreported. Materials and methods: We obtained a convenience sample (2006-2012) from two hospitals covering different populations. The isolates were genotyped using 24-MIRU-VNTR. Lineages were assigned by comparing their patterns to those in the MIRU-VNTRplus platform. Isolates belonging to the Beijing lineage were confirmed by allele-specific PCR. Results: We identified the first Beijing isolate in Ecuador in an unexpected epidemiological scenario: A patient was infected in the Andean region, in a population with low mobility and far from the borders of the neighboring countries where Beijing strains had been previously reported. Conclusion: This is the first report of the presence of the Beijing lineage in Ecuador in an unusual epidemiological context that deserves special attention.
Assuntos
Humanos , Tuberculose/genética , Tuberculose/epidemiologia , Reação em Cadeia da Polimerase/métodos , Mycobacterium tuberculosis/isolamento & purificação , Mycobacterium tuberculosis/genética , Variação Genética , Chile , Evolução Molecular , Equador , Pequim , Genótipo , Mycobacterium tuberculosis/químicaRESUMO
Resumen Introducción: La metodología de GenoType(r) MTBDRplus V.2 es una técnica molecular aprobada por la Organización Mundial de la Salud y la Organización Panamericana de la Salud para la detección de las mutaciones en el gen rpoβ del complejo Mycobacterium tuberculosis, las cuales confieren resistencia a la rifampicina, y las de los genes katG e inhA que la confieren frente a la isoniacida. Debido a la variación genética en las cepas circulantes a nivel mundial, los programas nacionales de control de la tuberculosis deben comprobar el desempeño de los nuevos métodos de diagnóstico para su aplicación como prueba rápida. Objetivo: Describir las mutaciones detectadas mediante la técnica GenoType(r) MTBDRplus V.2 en muestras pulmonares y aislamientos de M. tuberculosis procesados en el Laboratorio Nacional de Referencia del Instituto Nacional de Salud durante el 2014. Materiales y métodos: Se hizo un estudio retrospectivo descriptivo que determinó la expresión de los genes inhA, KatG y rpoβ responsables de la resistencia a isoniacida y rifampicina, utilizando la técnica GenoType(r) MTBDRplus V.2 en 837 muestras y aislamientos de casos de tuberculosis. Resultados. Se obtuvieron 689 resultados de pruebas: 581(84,3 %) sensibles, 58 (8,4 %) resistentes y 50 (7,2 %) multirresistentes. Se detectaron diversas mutaciones en el gen rpoβ, de las cuales la más frecuente fue la Ser531Leu (36,6 %), seguida por la Asp516Val (21,6 %), en tanto que en el gen katG la más frecuente fue la Ser315Thr1 (91,9 %). Conclusiones: Se detectaron varias mutaciones en los casos resistentes reportados en el país, con frecuencias similares a las reportadas en otros países de la región de América del Sur.
Abstract Introduction: The GenoType(r)MTBDRplusV.2 assay is a molecular technique endorsed by the World Health Organization and the Pan American Health Organization that allows for the identification of the Mycobacterium tuberculosis complex and the detection of mutations in the rpoβ gene for rifampicin resistance, and katG and inhA genes for isoniazid resistance. Due to the genetic variability in the circulating strains around the world, the national tuberculosis control programs should assess the performance of these new diagnostic technologies and their use under program conditions as rapid tests. Objective: To describe the mutations identified by the GenoType(r)MTBDRplusV.2 assay in pulmonary samples and Mycobacterium tuberculosis isolates in the Laboratorio Nacional de Referencia of the Instituto Nacional de Salud in 2014. Materials and methods. We conducted a retrospective, descriptive study to detect the expression of inhA, KatG and rpoβ genes, responsible for resistence against isoniazid and rifampicin using the GenoType(r) MTBDRplus V.2 assay in 837 samples and isolates from tuberculosis cases. Results: Several mutations in the rpoβ gene were identified. Ser531Leu was the most frequent (36.6%) followed by Asp516Val (21.6%), while Ser315Thr1 was the most frequent mutation in the katG gene (91.9%). Conclusions: We were able to identify different mutations present in MDR-TB strains in the country, with frequencies similar to those reported in other countries in the South American region.
Assuntos
Humanos , Rifampina/farmacologia , Tuberculose Resistente a Múltiplos Medicamentos/microbiologia , Técnicas de Genotipagem/métodos , Isoniazida/farmacologia , Mycobacterium tuberculosis/efeitos dos fármacos , Antituberculosos/farmacologia , Rifampina/química , Testes de Sensibilidade Microbiana/métodos , Estudos Retrospectivos , Tuberculose Resistente a Múltiplos Medicamentos/tratamento farmacológico , Colômbia , Genótipo , Isoniazida/química , Mutação , Mycobacterium tuberculosis/química , Antituberculosos/químicaRESUMO
Diagnosis of tuberculosis a problem, specially in the regions harboring an abundance of both pathogenic and non-pathogenic mycobacteria. This study was undertaken to assess in such a situation the predictive value of proliferative T cell response to a peptide epitope ('38G') of the 38 kDa membrane protein of Mycobacterium tuberculosis. 3[H]-thymidine incorporation assays were done with peripheral blood mononuclear cells of tuberculoid leprosy and pulmonary tuberculosis patients. The donors were also classified as PPD responders (Stimulation Index, SI> 3) or non-responders (SI 3 in 54 per cent subjects) and non-responders (SI > 3 in 29 per cent subjects). However, it did not differentiate between leprosy and tuberculosis. The study supports use of liposomes as adjuvant vehicles for antigenic peptides designed to activate human T cells.
Assuntos
Humanos , Antígenos de Bactérias/administração & dosagem , Antígenos de Bactérias/genética , Antígenos de Bactérias/química , Ativação Linfocitária , Dados de Sequência Molecular , Diagnóstico Diferencial , Epitopos/administração & dosagem , Epitopos/genética , Epitopos/química , Hanseníase Tuberculoide/diagnóstico , Hanseníase Tuberculoide/imunologia , Linfócitos T/imunologia , Mycobacterium tuberculosis/genética , Mycobacterium tuberculosis/imunologia , Mycobacterium tuberculosis/química , Sequência de Aminoácidos , Teste Tuberculínico , Tuberculose Pulmonar/diagnóstico , Tuberculose Pulmonar/imunologia , Lipossomos , Peso MolecularRESUMO
Se realiza el estudio mediante técnicas cromatográficas, de un grupo de cepas de Mycobacterium tuberculosis aisladas de un brote en pacientes infectados con el virus VIH. Se utilizaron como referencia un grupo de cepas de M. tuberculosis de pacientes sintomáticos respiratorios (SR+14) y cepas patrones de la colección del laboratorio, con el objetivo de comparar cualitativamente los patrones cromatográficos descritos por las cepas aisladas de pacientes. Se obtuvieron y compararon los perfiles cromatográficos de las cepas aisladas de pacientes (ST+) y de VIH+ por la técnica de cromatografía en capa delgada. Se identificó cada uno de los ácidos grasos presentes por la técnica de cromatografía de gases acoplada a espectrometría de masas. Todas las cepas estudiadas fueron clasificadas como Mycobacterium tuberculosis. Por resultados obtenidos se demuestra la utilidad de las técnicas cromatográficas como técnicas alternativas para el diagnóstico micobacteriano.
A group of strains of Mycobacterium tuberculosis isolated from an outbreak in HIV-infected patients was studied by chromatographic techniques.A group of strains of M. Tuberculosis from symptomatic respiratory patients (SR + 14) and patterns strains from the laboratory collection were used as a reference aimed at making a qualitative comparison of the chromatographic patterns described by the strains isolated from patients. The chromatographic profiles of the strains isolated from patients (SR +) and fro HIV + were obtained and comparede by thin layer chromatography (TLC). Each of the present fatty acids was identified by using the gas chromatography technique (GC) coupled to mass spectrum analysis. All the studied strains were classified as Mycobacterium tuberculosis. According to the results attained, the usefulness of the chromatographic techniques as alternative techniques for the mycobacterial diagnosis is demonstrated.
Assuntos
Humanos , Infecções Oportunistas Relacionadas com a AIDS/microbiologia , Surtos de Doenças , HIV-1 , Mycobacterium tuberculosis/química , Tuberculose Pulmonar/microbiologia , Cuba , Cromatografia Gasosa-Espectrometria de Massas/métodos , Cromatografia Gasosa-Espectrometria de Massas/estatística & dados numéricos , Mycobacterium tuberculosis/isolamento & purificação , Padrões de ReferênciaRESUMO
New diagnostic tests and vaccines for tuberculosis are being developed by means of a strategy based on the study of antigens exclusive to Mycobacterium tuberculosis. These antigens were initially identified by Western blots using sera from active pulmonary tuberculosis patients against sonic extracts from M. tuberculosis and M. bovis BCG. Several proteins present in the M. tuberculosis but absent in the Mycobacterium bovis BCG sonic extracts were selected and are currently under investigation. One of these, denoted MTP40, has been extensively studied. The nucleotide sequence of the mtp40 gene has been obtained; hybridization studies have shown that this DNA fragment is exclusive to M. tuberculosis. Using this genomic fragment, a polymerase chain reaction (PCR)-based diagnostic test which allows the specific identification of a minimum of 10 fg of M. tuberculosis DNA was developed. The diagnostic assay is now being tested on uncultured clinical samples in order to determine its usefulness in routine diagnosis. Peptides synthesized from the derived sequence for the MTP40 protein and also from other M. tuberculosis proteins are now being studied as possible candidates for a new generation of synthetic vaccines against tuberculosis