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1.
Methods Mol Biol ; 2314: 77-107, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34235649

RESUMO

The extraction and separation of native mycobacterial proteins remain necessary for antigen discovery, elucidation of enzymes to improve rational drug design, identification of physiologic mechanisms, use as reagents for diagnostics, and defining host immune responses. In this chapter, methods for the manipulation of whole mycobacterial cells and culture exudates are described in detail as these methods are the requisite first steps towards native protein isolation. Specifically, several methods for the inactivation of viable Mycobacterium tuberculosis along with qualification assays are provided, as this is key to safe manipulation of cell pastes for downstream processes. Next, the concentration of spent culture filtrate media in order to permit separation of soluble, secreted proteins is described followed by the separation of mycobacteria extracellular vesicles (MEV) from the remaining soluble proteins in spent media. We then describe the generation of whole-cell lysate and facile separation of lysate into subcellular fractions to afford cell wall, cell membrane, and cytosol-enriched proteins. Due to the hydrophobic nature of cell wall and cell membrane proteins, several extraction protocols to resolve protein subsets (such as extraction with urea and SDS) are also provided. Finally, methods for separation of hydrophobic and hydrophilic proteins from both whole-cell lysate and spent culture media are included. While these methods were optimized for the manipulation of Mycobacterium tuberculosis cells, they have been successfully applied to extract and isolate Mycobacterium leprae, Mycobacterium ulcerans, and Mycobacterium avium proteins.


Assuntos
Proteínas de Bactérias/isolamento & purificação , Proteínas de Bactérias/metabolismo , Membrana Celular/metabolismo , Proteínas de Membrana/isolamento & purificação , Proteínas de Membrana/metabolismo , Mycobacterium tuberculosis/metabolismo , Frações Subcelulares/metabolismo , Proteínas de Bactérias/química , Membrana Celular/química , Proteínas de Membrana/química
2.
J Infect Public Health ; 13(9): 1255-1264, 2020 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-32674978

RESUMO

An alternate host for mycobacteria is Mycobacterium smegmatis which is used frequently. It is a directly budding eco-friendly organism not emulated as human infection. It is mainly useful for the investigation of various microorganisms in the sort of Mycobacteria in cell culture laboratories. Some Mycobacterium species groups that is normal, unsafe ailments, likely to Mycobacterium leprae, Mycobacterium tuberculosis and Mycobacterium bovis. At present, various laboratories are clean and culture this type of species to make an opinion that fascinating route of harmful Mycobacteria. This publication provides aggregate data on cell shape, genome studies, ecology, pathology and utilization of M. smegmatis.


Assuntos
Infecções por Mycobacterium não Tuberculosas/patologia , Mycobacterium smegmatis/metabolismo , Mycobacterium tuberculosis/metabolismo , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Humanos , Lipossomos/metabolismo , Modelos Biológicos , Mycobacterium smegmatis/citologia , Mycobacterium smegmatis/genética , Mycobacterium smegmatis/ultraestrutura
3.
Microbiol Spectr ; 7(3)2019 05.
Artigo em Inglês | MEDLINE | ID: mdl-31172908

RESUMO

Mycobacterium tuberculosis is an ancient master of the art of causing human disease. One important weapon within its fully loaded arsenal is the type VII secretion system. M. tuberculosis has five of them: ESAT-6 secretion systems (ESX) 1 to 5. ESX-1 has long been recognized as a major cause of attenuation of the FDA-licensed vaccine Mycobacterium bovis BCG, but its importance in disease progression and transmission has recently been elucidated in more detail. This review summarizes the recent advances in (i) the understanding of the ESX-1 structure and components, (ii) our knowledge of ESX-1's role in hijacking macrophage function to set a path for infection and dissemination, and (iii) the development of interventions that utilize ESX-1 for diagnosis, drug interventions, host-directed therapies, and vaccines.


Assuntos
Antígenos de Bactérias/imunologia , Proteínas de Bactérias/imunologia , Mycobacterium tuberculosis/imunologia , Mycobacterium tuberculosis/metabolismo , Tuberculose/imunologia , Sistemas de Secreção Tipo VII/imunologia , Sistemas de Secreção Tipo VII/metabolismo , Vacina BCG/imunologia , Sistemas de Secreção Bacterianos/metabolismo , Quimiocinas , Interações Hospedeiro-Patógeno , Humanos , Macrófagos/imunologia , Mycobacterium tuberculosis/patogenicidade , Necrose , Fagossomos , Tuberculose/diagnóstico , Tuberculose/tratamento farmacológico , Tuberculose/prevenção & controle , Vacinas , Virulência
4.
Microbiol Spectr ; 7(2)2019 03.
Artigo em Inglês | MEDLINE | ID: mdl-31025625

RESUMO

How do mycobacteria divide? Cell division has been studied extensively in the model rod-shaped bacteria Escherichia coli and Bacillus subtilis, but much less is understood about cell division in mycobacteria, a genus that includes the major human pathogens M. tuberculosis and M. leprae. In general, bacterial cell division requires the concerted effort of many proteins in both space and time to elongate the cell, replicate and segregate the chromosome, and construct and destruct the septum - processes which result in the creation of two new daughter cells. Here, we describe these distinct stages of cell division in B. subtilis and follow with the current knowledge in mycobacteria. As will become apparent, there are many differences between mycobacteria and B. subtilis in terms of both the broad outline of cell division and the molecular details. So, while the fundamental challenge of spatially and temporally organizing cell division is shared between these rod-shaped bacteria, they have solved these challenges in often vastly different ways.


Assuntos
Divisão Celular/fisiologia , Mycobacterium/crescimento & desenvolvimento , Mycobacterium/metabolismo , Bacillus subtilis/genética , Bacillus subtilis/crescimento & desenvolvimento , Bacillus subtilis/metabolismo , Proteínas de Bactérias/metabolismo , Proteínas de Ciclo Celular/metabolismo , Divisão Celular/genética , Parede Celular , Replicação do DNA , Escherichia coli/genética , Escherichia coli/crescimento & desenvolvimento , Escherichia coli/metabolismo , Mycobacterium/genética , Mycobacterium tuberculosis/genética , Mycobacterium tuberculosis/crescimento & desenvolvimento , Mycobacterium tuberculosis/metabolismo
5.
Emerg Microbes Infect ; 8(1): 109-118, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30866765

RESUMO

Of the more than 190 distinct species of Mycobacterium genus, many are economically and clinically important pathogens of humans or animals. Among those mycobacteria that infect humans, three species namely Mycobacterium tuberculosis (causative agent of tuberculosis), Mycobacterium leprae (causative agent of leprosy) and Mycobacterium abscessus (causative agent of chronic pulmonary infections) pose concern to global public health. Although antibiotics have been successfully developed to combat each of these, the emergence of drug-resistant strains is an increasing challenge for treatment and drug discovery. Here we describe the impact of the rapid expansion of genome sequencing and genome/pathway annotations that have greatly improved the progress of structure-guided drug discovery. We focus on the applications of comparative genomics, metabolomics, evolutionary bioinformatics and structural proteomics to identify potential drug targets. The opportunities and challenges for the design of drugs for M. tuberculosis, M. leprae and M. abscessus to combat resistance are discussed.


Assuntos
Proteínas de Bactérias/química , Biologia Computacional/métodos , Mycobacterium/genética , Análise de Sequência de DNA/métodos , Animais , Proteínas de Bactérias/metabolismo , Descoberta de Drogas , Farmacorresistência Bacteriana , Genoma Bacteriano , Humanos , Anotação de Sequência Molecular , Mycobacterium/metabolismo , Mycobacterium abscessus/genética , Mycobacterium abscessus/metabolismo , Mycobacterium leprae/genética , Mycobacterium leprae/metabolismo , Mycobacterium tuberculosis/genética , Mycobacterium tuberculosis/metabolismo , Conformação Proteica , Proteômica
6.
J Biol Chem ; 293(14): 5172-5184, 2018 04 06.
Artigo em Inglês | MEDLINE | ID: mdl-29472294

RESUMO

Mycolic acids are the hallmark of the cell envelope in mycobacteria, which include the important human pathogens Mycobacterium tuberculosis and Mycobacterium leprae Mycolic acids are very long C60-C90 α-alkyl ß-hydroxy fatty acids having a variety of functional groups on their hydrocarbon chain that define several mycolate types. Mycobacteria also produce an unusually large number of putative epoxide hydrolases, but the physiological functions of these enzymes are still unclear. Here, we report that the mycobacterial epoxide hydrolase EphD is involved in mycolic acid metabolism. We found that orthologs of EphD from M. tuberculosis and M. smegmatis are functional epoxide hydrolases, cleaving a lipophilic substrate, 9,10-cis-epoxystearic acid, in vitro and forming a vicinal diol. The results of EphD overproduction in M. smegmatis and M. bovis BCG Δhma strains producing epoxymycolic acids indicated that EphD is involved in the metabolism of these forms of mycolates in both fast- and slow-growing mycobacteria. Moreover, using MALDI-TOF-MS and 1H NMR spectroscopy of mycolic acids and lipids isolated from EphD-overproducing M. smegmatis, we identified new oxygenated mycolic acid species that accumulated during epoxymycolate depletion. Disruption of the ephD gene in M. tuberculosis specifically impaired the synthesis of ketomycolates and caused accumulation of their precursor, hydroxymycolate, indicating either direct or indirect involvement of EphD in ketomycolate biosynthesis. Our results clearly indicate that EphD plays a role in metabolism of oxygenated mycolic acids in mycobacteria.


Assuntos
Epóxido Hidrolases/metabolismo , Ácidos Micólicos/metabolismo , Parede Celular/metabolismo , Ácidos Graxos/metabolismo , Metabolismo dos Lipídeos/fisiologia , Lipídeos/fisiologia , Espectrometria de Massas/métodos , Mycobacterium/metabolismo , Mycobacterium smegmatis/metabolismo , Mycobacterium tuberculosis/metabolismo
8.
Curr Drug Targets ; 18(16): 1904-1918, 2017 Nov 30.
Artigo em Inglês | MEDLINE | ID: mdl-28699515

RESUMO

BACKGROUND: Mycobacteria genus is responsible for deadly diseases like tuberculosis and leprosy. Cell wall of bacteria belonging to this genus is unique in many ways. It plays a major role in the pathogenesis and intracellular survival inside the host. In intracellular pathogens, their cell wall acts as molecular shield and interacts with host cell milieu to modulate host defense responses. OBJECTIVES: In this review, we summarize the factors that participate in the biosynthesis of unique mycobacterial cell wall, understand their potential as drug targets and the recent developments where they have been evaluated as possible drug targets. RESULTS: Several cell wall associated factors that play crucial roles in the synthesis of cell wall components like Antigen 85 complex, Glycosyltransferases (GTs), LM (lipomannan) and LAM (lipoarabinomannan), mAGP Complex, lipolytic enzyme have been categorically documented. Most of the presently used anti TB regimens interrupted cell wall synthesis, but the emergence of drug resistant strains made it mandatory to identify new drug targets. Novel drug candidates which could inhibit the synthesis of cell wall components have been thoroughly studied worldwide. CONCLUSION: Studies demonstrated that the cell wall components are unique in terms of their contribution in mycobacterium pathogenesis. Targeting these can hamper the growth of M. tuberculosis. In this study, we scrutinize the drugs under trials and the potential candidates screened through in silico findings.


Assuntos
Antituberculosos/farmacologia , Parede Celular/efeitos dos fármacos , Mycobacterium tuberculosis/patogenicidade , Tuberculose/tratamento farmacológico , Fatores de Virulência/metabolismo , Antituberculosos/química , Antituberculosos/uso terapêutico , Proteínas de Bactérias/metabolismo , Vias Biossintéticas/efeitos dos fármacos , Parede Celular/metabolismo , Ensaios Clínicos como Assunto , Simulação por Computador , Desenho de Fármacos , Regulação Bacteriana da Expressão Gênica/efeitos dos fármacos , Humanos , Mycobacterium tuberculosis/efeitos dos fármacos , Mycobacterium tuberculosis/metabolismo
9.
Am J Phys Anthropol ; 162(1): 143-156, 2017 01.
Artigo em Inglês | MEDLINE | ID: mdl-27704524

RESUMO

It is possible that during long lasting chronic infections such as tuberculosis (TB) and leprosy individuals who generate a stronger immune response will produce a chronic shift in the systemic levels of inflammatory proteins. Consequently, the systemic immunological shift could affect inflammatory responses against other persistent pathogens such as Porphyromonas gingivalis associated with periodontal disease (PD). OBJECTIVE: To determine if in vitro exposure to Mycobacterium tuberculosis or M. leprae lysates impacts subsequent immune responses to P. gingivalis; and to propose a new dialogue between experimental immunology and paleopathology. MATERIAL AND METHODS: We sequentially (2 days protocol) exposed peripheral blood mononuclear cells (PBMCs) from healthy donors to bacterial lysates either from M. tuberculosis, or M. leprae, or P. gingivalis. After collecting all supernatants, we measured the expression of immune proteins TNFα and IFNγ using an enzyme-linked immunosorbent assay. RESULTS: Early exposure (day 1) of PBMCs to M. leprae or M. tuberculosis lysates induces an inflammatory shift detected by the increase of TNFα and IFNγ when the same cells are subsequently (day 2) exposed to oral pathogen P. gingivalis. DISCUSSION: By extrapolating these results, we suggest that chronic infections, such as TB and leprosy, could generate a systemic immunological shift that can affect other inflammatory processes such the one present in PD. We propose that the presence and severity of PD should be explored as a proxy for inflammatory status or competence when reconstructing the health profile in past populations.


Assuntos
Inflamação/imunologia , Inflamação/microbiologia , Hanseníase/microbiologia , Mycobacterium leprae/imunologia , Mycobacterium tuberculosis/imunologia , Tuberculose/microbiologia , Arqueologia , Citocinas/imunologia , Citocinas/metabolismo , Humanos , Leucócitos Mononucleares , Mycobacterium leprae/metabolismo , Mycobacterium tuberculosis/metabolismo , Porphyromonas gingivalis/imunologia
10.
J Bacteriol ; 198(15): 2020-8, 2016 08 01.
Artigo em Inglês | MEDLINE | ID: mdl-27185825

RESUMO

UNLABELLED: Phthiocerol dimycocerosates (PDIM) are a group of cell surface-associated apolar lipids of Mycobacterium tuberculosis and closely related mycobacteria, such as Mycobacterium bovis and Mycobacterium leprae A characteristic methoxy group of these lipids is generated from the methylation of a hydroxyl group of the direct precursors, the phthiotriols. The precursors arise from the reduction of phthiodiolones, the keto intermediates, by a ketoreductase. The putative phthiodiolone ketoreductase (PKR) is encoded by Rv2951c in M. tuberculosis and BCG_2972c in M. bovis BCG, and these open reading frames (ORFs) encode identical amino acid sequences. We investigated the cofactor requirement of the BCG_2972c protein. A comparative analysis based on the crystallographic structures of similar enzymes identified structural elements for binding of coenzyme F420 and hydrophobic phthiodiolones in PKR. Coenzyme F420 is a deazaflavin coenzyme that serves several key functions in pathogenic and nonpathogenic mycobacteria. We found that an M. bovis BCG mutant lacking F420-dependent glucose-6-phosphate dehydrogenase (Fgd), which generates F420H2 (glucose-6-phosphate + F420 → 6-phosphogluconate + F420H2), was devoid of phthiocerols and accumulated phthiodiolones. When the mutant was provided with F420H2, a broken-cell slurry of the mutant converted accumulated phthiodiolones to phthiocerols; F420H2 was generated in situ from F420 and glucose-6-phosphate by the action of Fgd. Thus, the reaction mixture was competent in reducing phthiodiolones to phthiotriols (phthiodiolones + F420H2 → phthiotriols + F420), which were then methylated to phthiocerols. These results established the mycobacterial phthiodiolone ketoreductase as an F420H2-dependent enzyme (fPKR). A phylogenetic analysis of close homologs of fPKR revealed potential F420-dependent lipid-modifying enzymes in a broad range of mycobacteria. IMPORTANCE: Mycobacterium tuberculosis is the causative agent of tuberculosis, and phthiocerol dimycocerosates (PDIM) protect this pathogen from the early innate immune response of an infected host. Thus, the PDIM synthesis system is a potential target for the development of effective treatments for tuberculosis. The current study shows that a PDIM synthesis enzyme is dependent on the coenzyme F420 F420 is universally present in mycobacteria and absent in humans. This finding expands the number of experimentally validated F420-dependent enzymes in M. tuberculosis to six, each of which helps the pathogen to evade killing by the host immune system, and one of which activates an antituberculosis drug, PA-824. This work also has relevance to leprosy, since similar waxy lipids are found in Mycobacterium leprae.


Assuntos
Proteínas de Bactérias/metabolismo , Desidrogenases de Carboidrato/metabolismo , Regulação Bacteriana da Expressão Gênica/fisiologia , Lipídeos/biossíntese , Mycobacterium bovis/metabolismo , Mycobacterium tuberculosis/metabolismo , Sequência de Aminoácidos , Proteínas de Bactérias/genética , Desidrogenases de Carboidrato/genética , Mycobacterium bovis/enzimologia , Mycobacterium tuberculosis/enzimologia , Filogenia
11.
Crit Rev Microbiol ; 42(5): 738-58, 2016 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-26089025

RESUMO

The method of genotyping by variable number tandem repeats (VNTRs) facilitates the epidemiological studies of different Mycobacterium species worldwide. Until now, the VNTR method is not fully understood, for example, its discovery, function and classification. The inconsistent nomenclature and terminology of VNTR is especially confusing. In this review, we first describe in detail the VNTRs in Mycobacterium tuberculosis (M. tuberculosis), as this pathogen resulted in more deaths than any other microbial pathogen as well as for which extensive studies of VNTRs were carried out, and then we outline the recent progress of the VNTR-related epidemiological research in several other Mycobacterium species, such as M. abscessus, M. africanum, M. avium, M. bovis, M. canettii, M. caprae, M. intracellulare, M. leprae, M. marinum, M. microti, M. pinnipedii and M. ulcerans from different countries and regions. This article is aimed mainly at the practical notes of VNTR to help the scientists in better understanding and performing this method.


Assuntos
Técnicas de Tipagem Bacteriana/métodos , Repetições Minissatélites , Mycobacterium tuberculosis/genética , Mycobacterium tuberculosis/isolamento & purificação , Tuberculose/microbiologia , Animais , Genótipo , Humanos , Mycobacterium tuberculosis/classificação , Mycobacterium tuberculosis/metabolismo
12.
Methods Mol Biol ; 1285: 47-75, 2015.
Artigo em Inglês | MEDLINE | ID: mdl-25779310

RESUMO

The extraction and isolation of native bacterial proteins continue to be valuable technical pursuits in order to understand bacterial physiology, screen for virulence determinants, and describe antigens. In this chapter, methods for the manipulation of whole mycobacterial cells are described in detail. Specifically, the concentration of spent culture filtrate media is described in order to permit separation of soluble, secreted proteins; several discrete separation techniques, including precipitation of protein mixtures with ammonium sulfate and separation of proteins by hydrophobic chromatography are also provided. Similarly, the generation of whole cell lysate and facile separation of lysate into subcellular fractions to afford cell wall, cell membrane, and cytosol enriched proteins is described. Due to the hydrophobic nature of cell wall and cell membrane proteins, several extraction protocols to resolve protein subsets (such as extraction with urea and SDS) are also provided, as well as a separation technique (isoelectric focusing) that can be applied to separate hydrophobic proteins. Lastly, two commonly used analytical techniques, in-gel digestion of proteins for LC-MS and analysis of intact proteins by MALDI-ToF MS, are provided for rapid analysis of discrete proteins within subcellular or chromatographic fractions. While these methods were optimized for the manipulation of Mycobacterium tuberculosis cells, they have been successfully applied to extract and isolate Mycobacterium leprae, Mycobacterium ulcerans, and Mycobacterium avium proteins. In addition, a number of these methods may be applied to extract and analyze mycobacterial proteins from cell lines and host derived samples.


Assuntos
Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Fracionamento Celular , Mycobacterium tuberculosis/metabolismo , Proteínas de Bactérias/isolamento & purificação , Fracionamento Celular/métodos , Interações Hidrofóbicas e Hidrofílicas , Focalização Isoelétrica , Espectrometria de Massas , Solubilidade , Frações Subcelulares
13.
Cell Physiol Biochem ; 35(4): 1276-88, 2015.
Artigo em Inglês | MEDLINE | ID: mdl-25721573

RESUMO

BACKGROUND: The early secreted antigenic target 6-kDa protein (ESAT-6) of Mycobacterium tuberculosis (Mtb) not only acts as a key player for virulence but also exhibits a strong immunotherapeutic potential against Mtb. However, little is known about the molecular basis for its potential in immunotherapy. The present study was designed to unravel the role of miRNA-155 in ESAT-6-mediated enhancement of host immunity and apoptosis in macrophages. METHODS: Lentivirus-mediated miR-155 sponge and miR-155 and SOCS1 overexpression vectors were developed in macrophages. TLR2- or p65-specific siRNA knockdown was employed to silence TLR2 or p65. Quantitative polymerase chain reaction and western blotting analyses were performed to determine mRNA and protein expression levels, respectively. Macrophage apoptosis was analyzed by flow cytometry. RESULTS: ESAT-6 significantly increased miR-155 expression, which was dependent on TLR2/NF-κB activation in macrophages. Induced expression of miRNA-155 was required for the ESAT-6-mediated protective immune response and macrophage apoptosis. ESAT-6 promoted macrophage apoptosis by targeting the miR-155-SOCS1 pathway. The differential expression levels of TLR2, BIC, and SOCS1 were involved in regulating the immune response in human peripheral blood mononuclear cells of patients with active tuberculosis (TB) and latent TB (LTB). CONCLUSION: ESAT-6 promotes apoptosis of macrophages via targeting the miRNA155-SOCS1 interaction.


Assuntos
Antígenos de Bactérias/farmacologia , Apoptose/efeitos dos fármacos , MicroRNAs/metabolismo , Mycobacterium tuberculosis/metabolismo , Proteínas Supressoras da Sinalização de Citocina/metabolismo , Tuberculose/patologia , Animais , Antígenos de Bactérias/genética , Antígenos de Bactérias/metabolismo , Linhagem Celular , Humanos , Interleucina-6/genética , Interleucina-6/metabolismo , Leucócitos Mononucleares/metabolismo , Macrófagos/citologia , Macrófagos/efeitos dos fármacos , Macrófagos/metabolismo , Camundongos , Interferência de RNA , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/genética , Proteínas Recombinantes/farmacologia , Proteínas Supressoras da Sinalização de Citocina/genética , Receptor 2 Toll-Like/antagonistas & inibidores , Receptor 2 Toll-Like/genética , Receptor 2 Toll-Like/metabolismo , Fator de Transcrição RelA/antagonistas & inibidores , Fator de Transcrição RelA/genética , Fator de Transcrição RelA/metabolismo , Tuberculose/metabolismo , Fator de Necrose Tumoral alfa/genética , Fator de Necrose Tumoral alfa/metabolismo
14.
J Struct Biol ; 188(2): 156-64, 2014 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-25260828

RESUMO

Among the few proteins shown to be secreted by the Tat system in Mycobacterium tuberculosis, Rv2525c is of particular interest, since its gene is conserved in the minimal genome of Mycobacterium leprae. Previous evidence linked this protein to cell wall metabolism and sensitivity to ß-lactams. We describe here the crystal structure of Rv2525c that shows a TIM barrel-like fold characteristic of glycoside hydrolases of the GH25 family, which includes prokaryotic and phage-encoded peptidoglycan hydrolases. Structural comparison with other members of this family combined with substrate docking suggest that, although the 'neighbouring group' catalytic mechanism proposed for this family still appears as the most plausible, the identity of residues involved in catalysis in GH25 hydrolases might need to be revised.


Assuntos
Proteínas de Bactérias/metabolismo , Produtos do Gene tat/metabolismo , Mycobacterium tuberculosis/metabolismo , N-Acetil-Muramil-L-Alanina Amidase/química , N-Acetil-Muramil-L-Alanina Amidase/metabolismo , Sequência de Aminoácidos , Catálise , Domínio Catalítico , Parede Celular/metabolismo , Cristalografia por Raios X/métodos , Dados de Sequência Molecular , Alinhamento de Sequência
15.
Nature ; 501(7468): 512-6, 2013 Sep 26.
Artigo em Inglês | MEDLINE | ID: mdl-24005326

RESUMO

Ubiquitin-mediated targeting of intracellular bacteria to the autophagy pathway is a key innate defence mechanism against invading microbes, including the important human pathogen Mycobacterium tuberculosis. However, the ubiquitin ligases responsible for catalysing ubiquitin chains that surround intracellular bacteria are poorly understood. The parkin protein is a ubiquitin ligase with a well-established role in mitophagy, and mutations in the parkin gene (PARK2) lead to increased susceptibility to Parkinson's disease. Surprisingly, genetic polymorphisms in the PARK2 regulatory region are also associated with increased susceptibility to intracellular bacterial pathogens in humans, including Mycobacterium leprae and Salmonella enterica serovar Typhi, but the function of parkin in immunity has remained unexplored. Here we show that parkin has a role in ubiquitin-mediated autophagy of M. tuberculosis. Both parkin-deficient mice and flies are sensitive to various intracellular bacterial infections, indicating parkin has a conserved role in metazoan innate defence. Moreover, our work reveals an unexpected functional link between mitophagy and infectious disease.


Assuntos
Drosophila melanogaster/imunologia , Drosophila melanogaster/microbiologia , Imunidade Inata/imunologia , Mycobacterium marinum/imunologia , Mycobacterium tuberculosis/imunologia , Salmonella typhimurium/imunologia , Ubiquitina-Proteína Ligases/imunologia , Animais , Autofagia/imunologia , Células da Medula Óssea/microbiologia , Drosophila melanogaster/genética , Drosophila melanogaster/metabolismo , Feminino , Lisina/metabolismo , Macrófagos/microbiologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Mitocôndrias/metabolismo , Mitocôndrias/patologia , Mitofagia , Modelos Imunológicos , Mycobacterium tuberculosis/crescimento & desenvolvimento , Mycobacterium tuberculosis/metabolismo , Poliubiquitina/química , Poliubiquitina/metabolismo , Simbiose/imunologia , Tuberculose/enzimologia , Tuberculose/imunologia , Tuberculose/microbiologia , Tuberculose/patologia , Ubiquitina/análise , Ubiquitina/química , Ubiquitina/metabolismo , Ubiquitina-Proteína Ligases/química , Ubiquitina-Proteína Ligases/deficiência , Ubiquitina-Proteína Ligases/metabolismo
16.
PLoS One ; 7(10): e46862, 2012.
Artigo em Inglês | MEDLINE | ID: mdl-23056492

RESUMO

BACKGROUND: Despite the enormous global burden of tuberculosis (TB), conventional approaches to diagnosis continue to rely on tests that have major drawbacks. The improvement of TB diagnostics relies, not only on good biomarkers, but also upon accurate detection methodologies. The 10-kDa culture filtrate protein (CFP-10) and the 6-kDa early secreted antigen target (ESAT-6) are potent T-cell antigens that are recognised by over 70% of TB patients. Aptamers, a novel sensitive and specific class of detection molecules, has hitherto, not been raised to these relatively TB-specific antigens. METHODS: DNA aptamers that bind to the CFP-10.ESAT-6 heterodimer were isolated. To assess their affinity and specificity to the heterodimer, aptamers were screened using an enzyme-linked oligonucleotide assay (ELONA). One suitable aptamer was evaluated by ELONA using sputum samples obtained from 20 TB patients and 48 control patients (those with latent TB infection, symptomatic non TB patients, and healthy laboratory volunteers). Culture positivity for Mycobacterium tuberculosis (Mtb) served as the reference standard. Accuracy and cut-points were evaluated using ROC curve analysis. RESULTS: Twenty-four out of the 66 aptamers that were isolated bound significantly (p<0.05) to the CFP-10.ESAT-6 heterodimer and six were further evaluated. Their dissociation constant (K(D)) values were in the nanomolar range. One aptamer, designated CSIR 2.11, was evaluated using sputum samples. CSIR 2.11 had sensitivity and specificity of 100% and 68.75% using Youden's index and 35% and 95%, respectively, using a rule-in cut-point. CONCLUSION: This preliminary proof-of-concept study suggests that a diagnosis of active TB using anti-CFP-10.ESAT-6 aptamers applied to human sputum samples is feasible.


Assuntos
Aptâmeros de Nucleotídeos/metabolismo , DNA de Cadeia Simples/metabolismo , Mycobacterium tuberculosis/isolamento & purificação , Técnica de Seleção de Aptâmeros/métodos , Escarro/microbiologia , Antígenos de Bactérias/química , Antígenos de Bactérias/metabolismo , Aptâmeros de Nucleotídeos/química , Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Ligação Competitiva , DNA de Cadeia Simples/química , Humanos , Mycobacterium tuberculosis/metabolismo , Conformação de Ácido Nucleico , Multimerização Proteica , Estrutura Quaternária de Proteína , Especificidade por Substrato
17.
Glycobiology ; 22(8): 1118-27, 2012 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-22534567

RESUMO

Mannose-capped lipoarabinomannan (ManLAM) is a complex lipoglycan abundantly present in the Mycobacterium tuberculosis cell envelope. Many biological properties have been ascribed to ManLAM, from directly interacting with the host and participating in the intracellular survival of M. tuberculosis, to triggering innate and adaptive immune responses, including the activation of CD1b-restricted T cells. Due to its structural complexity, ManLAM is considered a heterogeneous population of molecules which may explain its different biological properties. The presence of various modifications such as fatty acids, succinates, lactates, phosphoinositides and methylthioxylose in ManLAM have proven to correlate directly with its biological activity and may potentially be involved in the interactions between CD1b and the T cell population. To further delineate the specific ManLAM epitopes involved in CD1b-restricted T cell recognition, and their potential roles in mediating immune responses in M. tuberculosis infection, we established a method to resolve ManLAM into eight different isoforms based on their different isoelectric values. Our results show that a ManLAM isoform with an isoelectric value of 5.8 was the most potent in stimulating the production of interferon-γ in different CD1b-restricted T-cell lines. Compositional analyses of these isoforms of ManLAM revealed a direct relationship between the overall charge of the ManLAM molecule and its capacity to be presented to T cells via the CD1 compartment.


Assuntos
Antígenos CD1/metabolismo , Lipopolissacarídeos/metabolismo , Manose/metabolismo , Mycobacterium tuberculosis/metabolismo , Linfócitos T/metabolismo , Tuberculose/metabolismo , Antígenos CD1/imunologia , Proliferação de Células , Células Dendríticas/citologia , Células Dendríticas/imunologia , Células Dendríticas/metabolismo , Humanos , Interferon gama/metabolismo , Ponto Isoelétrico , Hanseníase/imunologia , Hanseníase/metabolismo , Lipopolissacarídeos/imunologia , Mycobacterium tuberculosis/imunologia , Mycobacterium tuberculosis/isolamento & purificação , Fosfatos/metabolismo , Isoformas de Proteínas , Succinatos/metabolismo , Linfócitos T/imunologia , Tuberculose/imunologia , Tuberculose/microbiologia
18.
Chem Biol Drug Des ; 79(6): 1056-62, 2012 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-22405030

RESUMO

Tuberculosis is the second leading infectious killer with 9 million new cases in 2009. Extensive use of pathogen's lipid metabolism especially in utilizing the host lipids and virulence highlights the importance of exported lipid-catabolizing enzymes. Current study aims to emphasize the importance of Rv0183, an exported monoacylglycerol lipase, involved in metabolizing the host cell membrane lipids. Sequence analysis and homology modeling shows Rv0183 is highly conserved throughout mycobacterial species even in Mycobacterium leprae and also significantly divergent from mammalian lipases. Additionally, employing virtual screening using NCI diversity set and ZINC database with criteria of molecules with higher predicted free energy of binding toward Rv0183 than human lipase, potential inhibitors have been identified for Rv0183. A tautomer of ZINC13451138, known inhibitor for HIV-1 integrase is the best hit with difference in free energy of binding of 8.72 kcal/mol. The sequence and structure analysis were helpful in identifying the ligand binding sites and molecular function of the mycobacterial specific monoacylglycerol lipase. Rv0183 represents a suitable and promising drug target and is also a step towards understanding dormancy development and reactivation, thereby addressing pathogen's drug resistance. Experimental studies on the discovered potential inhibitors in this virtual screen should further validate the therapeutic utility of Rv0183.


Assuntos
Antituberculosos/química , Proteínas de Bactérias/antagonistas & inibidores , Ácidos Carboxílicos/farmacologia , Cicloexanos/farmacologia , Metabolismo dos Lipídeos/efeitos dos fármacos , Mycobacterium tuberculosis/metabolismo , Fenóis/farmacologia , Antituberculosos/farmacologia , Proteínas de Bactérias/metabolismo , Sítios de Ligação , Ácidos Carboxílicos/química , Cicloexanos/química , Bases de Dados Factuais , Humanos , Ligação de Hidrogênio , Simulação de Dinâmica Molecular , Monoacilglicerol Lipases/química , Monoacilglicerol Lipases/metabolismo , Mycobacterium tuberculosis/efeitos dos fármacos , Fenóis/química , Ligação Proteica , Estrutura Terciária de Proteína , Termodinâmica
19.
J Biol Chem ; 286(34): 29993-30002, 2011 Aug 26.
Artigo em Inglês | MEDLINE | ID: mdl-21730061

RESUMO

Mycobacterium tuberculosis encodes five type VII secretion systems that are responsible for exporting a number of proteins, including members of the Esx family, which have been linked to tuberculosis pathogenesis and survival within host cells. The gene cluster encoding ESX-3 is regulated by the availability of iron and zinc, and secreted protein products such as the EsxG·EsxH complex have been associated with metal ion acquisition. EsxG and EsxH have previously been shown to form a stable 1:1 heterodimeric complex, and here we report the solution structure of the complex, which features a core four-helix bundle decorated at both ends by long, highly flexible, N- and C-terminal arms that contain a number of highly conserved residues. Despite clear similarities in the overall backbone fold to the EsxA·EsxB complex, the structure reveals some striking differences in surface features, including a potential protein interaction site on the surface of the EsxG·EsxH complex. EsxG·EsxH was also found to contain a specific Zn(2+) binding site formed from a cluster of histidine residues on EsxH, which are conserved across obligate mycobacterial pathogens including M. tuberculosis and Mycobacterium leprae. This site may reflect an essential role in zinc ion acquisition or point to Zn(2+)-dependent regulation of its interaction with functional partner proteins. Overall, the surface features of both the EsxG·EsxH and the EsxA·EsxB complexes suggest functions mediated via interactions with one or more target protein partners.


Assuntos
Proteínas de Bactérias/química , Sistemas de Secreção Bacterianos , Complexos Multiproteicos/química , Mycobacterium tuberculosis/química , Proteínas de Bactérias/metabolismo , Humanos , Ferro/química , Ferro/metabolismo , Complexos Multiproteicos/metabolismo , Mycobacterium leprae/química , Mycobacterium leprae/metabolismo , Mycobacterium tuberculosis/metabolismo , Estrutura Quaternária de Proteína , Estrutura Terciária de Proteína , Células U937 , Zinco/química , Zinco/metabolismo
20.
J Lipid Res ; 52(2): 272-7, 2011 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-21076119

RESUMO

The cell wall of mycobacteria includes a thick, robust, and highly impermeable outer membrane made from long-chain mycolic acids. These outer membranes form a primary layer of protection for mycobacteria and directly contribute to the virulence of diseases such as tuberculosis and leprosy. We have formed in vitro planar membranes using pure mycolic acids on circular apertures 20 to 90 µm in diameter. We find these membranes to be long lived and highly resistant to irreversible electroporation, demonstrating their general strength. Insertion of the outer membrane channel MspA into the membranes was observed indicating that the artificial mycolic acid membranes are suitable for controlled studies of the mycobacterial outer membrane and can be used in nanopore DNA translocation experiments.


Assuntos
Lipídeos de Membrana/química , Membranas Artificiais , Mycobacterium tuberculosis/metabolismo , Ácidos Micólicos/química , 1,2-Dipalmitoilfosfatidilcolina/química , Permeabilidade da Membrana Celular , Concentração de Íons de Hidrogênio , Nanoporos , Porinas/química
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