RESUMO
One of the reasons for the lengthy tuberculosis (TB) treatment is the difficulty to treat the nonmultiplying mycobacterial subpopulation. In order to assess the ability of (new) TB drugs to target this subpopulation, we need to incorporate dormancy models in our preclinical drug development pipeline. In most available dormancy models, it takes a long time to create a dormant state, and it is difficult to identify and quantify this nonmultiplying condition. The Mycobacterium tuberculosis 18b strain might overcome some of these problems, because it is dependent on streptomycin for growth and becomes nonmultiplying after 10 days of streptomycin starvation but still can be cultured on streptomycin-supplemented culture plates. We developed our 18b dormancy time-kill kinetics model to assess the difference in the activity of isoniazid, rifampin, moxifloxacin, and bedaquiline against log-phase growth compared to the nonmultiplying M. tuberculosis subpopulation by CFU counting, including a novel area under the curve (AUC)-based approach as well as time-to-positivity (TTP) measurements. We observed that isoniazid and moxifloxacin were relatively more potent against replicating bacteria, while rifampin and high-dose bedaquiline were equally effective against both subpopulations. Moreover, the TTP data suggest that including a liquid culture-based method could be of additional value, as it identifies a specific mycobacterial subpopulation that is nonculturable on solid media. In conclusion, the results of our study underline that the time-kill kinetics 18b dormancy model in its current form is a useful tool to assess TB drug potency and thus has its place in the TB drug development pipeline.
Assuntos
Mycobacterium tuberculosis , Preparações Farmacêuticas , Tuberculose , Antituberculosos/farmacologia , Humanos , Isoniazida/farmacologiaRESUMO
Mycobacterial cell walls are complex structures containing a broad range of unusual lipids, glycolipids and other polymers, some of which act as immunomodulators or virulence determinants. Better understanding of the enzymes involved in export processes would enlighten cell wall biogenesis. Bernut et al. () present the findings of a structural and functional investigation of one of the most important transporter families, the MmpL proteins, members of the resistance-nodulation-cell division (RND) superfamily. A Tyr842His missense mutation in the mmpL4a gene was shown to be responsible for the smooth-to-rough morphotype change of the near untreatable opportunistic pathogen Mycobacterium bolletii due to its failure to export a glycopeptidolipid (GPL). This mutation was pleiotropic and markedly increased virulence in infection models. Tyr842 is well conserved in all actinobacterial MmpL proteins suggesting that it is functionally important and this was confirmed by several approaches including replacing the corresponding residue in MmpL3 of Mycobacterium tuberculosis. Structural modelling combined with experimental results showed Tyr842 to be a critical residue for mediating the proton motive force required for GPL export. This mechanistic insight applies to all MmpL proteins and probably to all RND transporters.
Assuntos
Proteínas de Membrana Transportadoras/metabolismo , Mycobacterium tuberculosis/genética , Parede Celular/metabolismo , Glicolipídeos/metabolismo , VirulênciaRESUMO
Mycolic acids are major components of the cell envelope of mycobacteria, such as Mycobacterium tuberculosis, and play an important role in its architecture, impermeability and interaction with the environment. Synthesis of mycolic acids is carried out by two types of fatty acid synthases (FAS) working in concert: type I FAS, a multifunctional enzyme capable of de novo synthesis of medium-chain fatty acids, and type II FAS, responsible for their elongation. In this article we report the identification and characterization of a transcriptional regulator (MabR), whose binding to the FAS-II promoter region was demonstrated in vitro and in vivo. Overexpression and knock-down studies in Mycobacterium smegmatis revealed the repressor nature of MabR, with reduced amounts of FAS-II transcripts and fatty acids in the overproducing strain. Under these conditions, downregulation of fas transcription was also observed, thereby suggesting the existence of cross-talk between the two FAS, mediated by MabR. Finally, the finding that a mabR knock-out mutant could only be obtained in a merodiploid strain of M. smegmatis, confirmed the predicted essentiality, thus implying an essential role for MabR in mycobacterial fatty acid metabolism.
Assuntos
Proteínas de Bactérias/metabolismo , Ácido Graxo Sintase Tipo II/metabolismo , Ácidos Graxos/biossíntese , Metabolismo dos Lipídeos , Mycobacterium smegmatis/genética , Proteínas de Bactérias/genética , Sítios de Ligação , Ácido Graxo Sintase Tipo II/genética , Regulação Bacteriana da Expressão Gênica , Genes Reguladores , Mycobacterium smegmatis/enzimologia , Óperon , Regiões Promotoras GenéticasRESUMO
The study of tissue-derived Mycobacterium leprae provides insights to the immunopathology of leprosy and helps identify broad molecular features necessary for mycobacterial parasitism. A major membrane protein (MMP-II) of in vivo-derived M. leprae previously recognized (Hunter, S.W., B. Rivoire, V. Mehra, B.R. Bloom, and P.J. Brennan. 1990. J. Biol. Chem. 265:14065) was purified from extracts of the organism and partial amino acid sequence obtained. This information allowed recognition, within one of the cosmids that encompass the entire M. leprae genome, of a complete gene, bfr, encoding a protein of subunit size 18.2 kD. The amino acid sequence deduced from the major membrane protein II (MMP-II) gene revealed considerable homology to several bacterioferritins. Analysis of the native protein demonstrated the iron content, absorption spectrum, and large native molecular mass (380 kD) of several known bacterioferritins. The ferroxidase-center residues typical of ferritins were conserved in the M. leprae product. Oligonucleotides derived from the amino acid sequence of M. leprae bacterioferritin enabled amplification of much of the MMP-II gene and the detection of homologous sequences in Mycobacterium paratuberculosis, Mycobacterium avium, Mycobacterium tuberculosis, Mycobacterium intracellulare, and Mycobacterium scrofulaceum. The role of this iron-rich protein in the virulence of M. leprae is discussed.
Assuntos
Proteínas de Bactérias/isolamento & purificação , Grupo dos Citocromos b/isolamento & purificação , Ferritinas/isolamento & purificação , Mycobacterium leprae/química , Sequência de Aminoácidos , Sequência de Bases , Grupo dos Citocromos b/química , Grupo dos Citocromos b/genética , Ferritinas/química , Ferritinas/genética , Dados de Sequência Molecular , Peso Molecular , Reação em Cadeia da PolimeraseRESUMO
OBJECTIVES: Antimicrobial resistance (AMR) is a priority for surveillance in bacterial infections. For leprosy, AMR has not been assessed because Mycobacterium leprae does not grow in vitro. We aim to obtain AMR data using molecular detection of resistance genes and to conduct a prospective open survey of resistance to antileprosy drugs in countries where leprosy is endemic through a WHO surveillance network. METHODS: From 2009 to 2015, multi-bacillary leprosy cases at sentinel sites of 19 countries were studied for resistance to rifampicin, dapsone and ofloxacin by PCR sequencing of the drug-resistance-determining regions of the genes rpoB, folP1 and gyrA. RESULTS: Among 1932 (1143 relapse and 789 new) cases studied, 154 (8.0%) M. leprae strains were found with mutations conferring resistance showing 182 resistance traits (74 for rifampicin, 87 for dapsone and 21 for ofloxacin). Twenty cases showed rifampicin and dapsone resistance, four showed ofloxacin and dapsone resistance, but no cases were resistant to rifampicin and ofloxacin. Rifampicin resistance was observed among relapse (58/1143, 5.1%) and new (16/789, 2.0%) cases in 12 countries. India, Brazil and Colombia reported more than five rifampicin-resistant cases. CONCLUSIONS: This is the first study reporting global data on AMR in leprosy. Rifampicin resistance emerged, stressing the need for expansion of surveillance. This is also a call for vigilance on the global use of antimicrobial agents, because ofloxacin resistance probably developed in relation to the general intake of antibiotics for other infections as it is not part of the multidrug combination used to treat leprosy.
Assuntos
Antibacterianos/uso terapêutico , Farmacorresistência Bacteriana/genética , Hanseníase/epidemiologia , Mycobacterium leprae/efeitos dos fármacos , Mycobacterium leprae/genética , Antibacterianos/efeitos adversos , Proteínas de Bactérias/genética , Biópsia por Agulha , Brasil/epidemiologia , Colômbia/epidemiologia , DNA Girase/genética , Dapsona/uso terapêutico , Doenças Endêmicas/estatística & dados numéricos , Monitoramento Epidemiológico , Saúde Global , Humanos , Índia/epidemiologia , Hanseníase/diagnóstico , Hanseníase/tratamento farmacológico , Hanseníase/microbiologia , Testes de Sensibilidade Microbiana , Mutação , Ofloxacino/uso terapêutico , Reação em Cadeia da Polimerase , Estudos Prospectivos , Recidiva , Rifampina/uso terapêutico , Vigilância de Evento Sentinela , Pele/microbiologia , Pele/patologia , Inquéritos e Questionários , Organização Mundial da SaúdeRESUMO
Genes for functional Ser/Thr protein kinases (STPKs) are ubiquitous in prokaryotic genomes, but little is known about their physiological substrates and their actual involvement in bacterial signal transduction pathways. We report here the identification of GarA (Rv1827), a Forkhead-associated (FHA) domain-containing protein, as a putative physiological substrate of PknB, an essential Ser/Thr protein kinase from Mycobacterium tuberculosis. Using a global proteomic approach, GarA was found to be the best detectable substrate of the PknB catalytic domain in non-denatured whole-cell protein extracts from M. tuberculosis and the saprophyte Mycobacterium smegmatis. Enzymological and binding studies of the recombinant proteins demonstrate that docking interactions between the activation loop of PknB and the C-terminal FHA domain of GarA are required to enable efficient phosphorylation at a single N-terminal threonine residue, Thr22, of the substrate. The predicted amino acid sequence of the garA gene, including both the N-terminal phosphorylation motif and the FHA domain, is strongly conserved in mycobacteria and other related actinomycetes, suggesting a functional role of GarA in putative STPK-mediated signal transduction pathways. The ensuing model of PknB-GarA interactions suggests a substrate recruitment mechanism that might apply to other mycobacterial kinases bearing multiple phosphorylation sites in their activation loops.
Assuntos
Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Mycobacterium tuberculosis/química , Proteínas Quinases/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Sequência de Aminoácidos , Fosforilação , Ligação Proteica , Proteínas Serina-Treonina Quinases/fisiologia , Proteômica/métodos , Transdução de Sinais , Especificidade por SubstratoRESUMO
Genomics is providing us with a mass of information about the biochemistry, physiology and pathogenesis of Mycobacterium tuberculosis and Mycobacterium leprae. Comparison of the two genome sequences is mutually enriching and indicates that the M. leprae genome appears to have undergone shrinkage and large-scale gene inactivation, which may account for the exceptionally slow growth of this organism.
Assuntos
Genoma Bacteriano , Mycobacterium leprae/genética , Mycobacterium tuberculosis/genéticaRESUMO
Since the publication of the complete genome sequence of Mycobacterium tuberculosis in 1998, there has been a marked intensification and diversification of activities in the field of tuberculosis research. Among the areas that have advanced spectacularly are comparative genomics, functional genomics-notably the study of the transcriptome and proteome - and cell envelope biogenesis, especially as it relates to the mechanism of action of antimycobacterial drugs.
Assuntos
Genes Bacterianos , Genoma Bacteriano , Mycobacterium tuberculosis/genética , Tuberculose/microbiologia , Variação Antigênica , Humanos , Mycobacterium tuberculosis/metabolismo , Mycobacterium tuberculosis/patogenicidade , Proteoma , Transcrição Gênica , Tuberculose/metabolismo , Tuberculose/patologiaRESUMO
During the last decade, great advances have been made in the study of bacterial genomes which is perhaps better described by the term bacterial genomics. The application of powerful techniques, such as pulsed-field gel electrophoresis of macro-restriction fragments of genomic DNA, has freed the characterisation of the chromosomes of many bacteria from the constraints imposed by classical genetic analysis. It is now possible to analyse the genome of virtually every microorganism by direct molecular methods and to construct detailed physical and gene maps. In this review, the various practical approaches are compared and contrasted, and some of the emerging themes of bacterial genomics, such as the size, shape, number and organisation of chromosomes are discussed.
Assuntos
Bactérias/genética , Genoma Bacteriano , Mapeamento Cromossômico/métodos , Cromossomos Bacterianos/ultraestrutura , Eletroforese em Gel de Campo Pulsado , Biblioteca GênicaRESUMO
A rare EcoRI restriction fragment length polymorphism (RFLP) in the 3' end of the human c-mos locus has been identified in DNA from patients with breast tumors, esophageal carcinomas and leukemias. Until now, this RFLP has not been found in normal populations, suggesting that its presence may reflect some cancer susceptibility. To characterize this RFLP, we have isolated both alleles of the c-mos locus from DNA of a breast cancer patient and determined the nucleotide sequence of the polymorphic region. Our results show that this RFLP is due to a single nucleotide substitution (T instead of C), resulting in the disappearance of EcoRI site.
Assuntos
Neoplasias da Mama/genética , Oncogenes , Polimorfismo Genético , Polimorfismo de Fragmento de Restrição , Proteínas Proto-Oncogênicas/genética , Alelos , Sequência de Bases , Humanos , Dados de Sequência Molecular , MutaçãoRESUMO
Tuberculosis has resurged during the past decade in many industrialized countries, and strains of Mycobacterium tuberculosis that are resistant to one or more of the main antituberculous drugs are emerging. The molecular basis of mycobacterial drug resistance is now beginning to be understood. Resistance derives from mutations in chromosomal genes leading to overproduction, alteration or loss of the drug target.
Assuntos
Isoniazida/farmacologia , Mycobacterium tuberculosis/efeitos dos fármacos , Rifampina/farmacologia , Resistência Microbiana a Medicamentos/genética , Resistência a Múltiplos Medicamentos , Mutação/genéticaRESUMO
Comparative genomics, and related technologies, are helping to unravel the molecular basis of the pathogenesis, host range, evolution and phenotypic differences of the slow-growing mycobacteria. In the highly conserved Mycobacterium tuberculosis complex, where single-nucleotide polymorphisms are rare, insertion and deletion events (InDels) are the principal source of genome plasticity. InDels result from recombinational or insertion sequence (IS)-mediated events, expansion of repetitive DNA sequences, or replication errors based on repetitive motifs that remove blocks of genes or contract coding sequences. Comparative genomic analyses also suggest that loss of genes is part of the ongoing evolution of the slow-growing mycobacterial pathogens and might also explain how the vaccine strain BCG became attenuated.
Assuntos
Evolução Molecular , Genômica , Mycobacterium/genética , Mycobacterium/patogenicidade , Tuberculose/microbiologia , Sequência de Aminoácidos , Biologia Computacional , Genoma Bacteriano , Humanos , Dados de Sequência Molecular , Mycobacterium/classificação , Análise de Sequência com Séries de Oligonucleotídeos , FilogeniaRESUMO
The complete nucleotide sequence and genomic organization of human papillomavirus type 18, associated with cervical cancer, has been established. A detailed comparative analysis was undertaken leading to the identification of a number of features specific for genital papillomaviruses and the construction of a phylogenetic tree. Genital papillomaviruses differ from other human and animal papillomaviruses as they possess a longer E1 open reading frame (ORF) and have a characteristic control region. Phylogenetically, HPV 18 is located between the benign genital viruses, HPV 6 and HPV 11, and the malignant isolates, HPV 16 and HPV 33, and may represent an evolutionary intermediate among oncogenic papillomaviruses. Viral gene products known to be involved in cellular transformation are those of ORFs E5, E6 and E7. Significant sequence variation was found between the E6 to E7 regions of different integrated forms of HPV 18. On re-examination of the E6 primary structures we noticed that the gene has evolved by successive duplications of a unit encoding 33 amino acids, which include a Cys-X-X-Cys motif. Furthermore, the E7 gene product has apparently evolved in the same manner and is related to E6. Both gene products bear a striking resemblance to the transcriptional factor IIIA of Xenopus laevis, the prototype of a new class of nucleic acid binding proteins.
Assuntos
DNA Viral , Genes Virais , Papillomaviridae/genética , Sequência de Aminoácidos , Sequência de Bases , Papillomaviridae/classificação , Filogenia , Biossíntese de Proteínas , Proteínas Virais/classificaçãoRESUMO
The molecular organization of the malT region of the Escherichia coli K-12 chromosome has been elucidated by nucleotide sequence studies. A single open reading frame of 901 codons comprises the malT gene which is separated by a repetitive extragenic palindromic unit from an unidentified gene, orfX, divergently oriented with respect to malT. The predicted Mr of the MalT protein is 102988, making it the largest transcriptional regulatory protein yet described in E. coli. By deleting in vitro the 3'-end of the gene or constructing malT-lacZ gene fusions, it was found that the integrity of the C-terminus of MalT is indispensable for the activity of the protein. Furthermore, it was found that truncated MalT proteins lacking up to 300 amino acids at the C-terminus blocked the activity of the wild type protein. No sequence homology can be found either with the other activators known in E. coli or with the other proteins of the maltose regulon.
Assuntos
Escherichia coli/genética , Genes Bacterianos , Genes , Maltose/metabolismo , Sequência de Aminoácidos , Proteínas de Bactérias/genética , Sequência de Bases , Mapeamento Cromossômico , Códon , Hibridização de Ácido Nucleico , Plasmídeos , Homologia de Sequência do Ácido Nucleico , Transcrição GênicaRESUMO
The LexA-regulated sulA (sfiA) gene of Escherichia coli encodes an unstable protein which inhibits cell division. By determining the nucleotide sequences of the corresponding genes from the related bacteria Salmonella typhimurium, Enterobacter aerogenes and Serratia marcescens it was found that the regulatory region and the LexA binding site (SOS box) have been better conserved during evolution than the coding sequence. The N terminus of the SulA protein [amino acid (aa) residues 1-30] has diverged extensively during the evolution of Enterobacteriaceae, whereas the central region (aa residues 31-149) has been well conserved. At the C terminus a sequence showing some homology to the N protein of phage lambda was detected that may represent a recognition site for the Lon protease, which is known to degrade both polypeptides. When expressed in E. coli, the foreign sulA genes did not block cell division suggesting that their products are inactive. This may indicate that the N terminus of the SulA protein is involved in recognizing the cell division apparatus.
Assuntos
Evolução Biológica , Reparo do DNA , Enterobacteriaceae/genética , Proteínas de Escherichia coli , Genes Bacterianos , Resposta SOS em Genética , Sequência de Aminoácidos , Proteínas de Bactérias/genética , Sequência de Bases , Divisão Celular , Códon , Enterobacter/genética , Escherichia coli/genética , Genes , Genes Reguladores , Salmonella typhimurium/genética , Serratia marcescens/genéticaRESUMO
We have previously described sigma A and sigma B and their structural genes, mysA and mysB, respectively, in Mycobacterium smegmatis. We have now sequenced the corresponding regions in the M. tuberculosis and M. leprae chromosomes, and have found the two homologous genes. The chromosomal linkage and the deduced amino acid (aa) sequences of the two genes show very high similarity in the three species of mycobacteria. We also report the finding of two other open reading frames (ORF) in these clusters. orfX, which has an unknown function, is located between mysA and mysB. The other ORF, located downstream from mysB, encodes a homolog of DtxR, the iron regulatory protein from Corynebacterium diphtheriae (Cd).
Assuntos
Genoma Bacteriano , Mycobacterium leprae/genética , Mycobacterium tuberculosis/genética , Fator sigma/genética , Sequência de Aminoácidos , Sequência de Bases , Mapeamento Cromossômico , Dados de Sequência Molecular , Família Multigênica , Alinhamento de SequênciaRESUMO
Mycobacterium tuberculosis, the scourge of humanity, is one of the most successful and scientifically challenging pathogens of all time. To catalyse the conception of new prophylactic and therapeutic interventions against tuberculosis, and to enhance our understanding of the biology of the tubercle bacillus, the complete genome sequence of the most widely used strain, H37Rv, has been determined. Bioinformatic analysis led to the identification of approximately 4000 genes in the 4.41 Mb genome sequence and provided fresh insight into the biochemistry, physiology. genetics and immunology of this much-feared bacterium. Genomic information is centralised in TubercuList (http://www.pasteur.fr/Bio/TubercuList/).
Assuntos
DNA Bacteriano/análise , Genoma Bacteriano , Mycobacterium tuberculosis/genética , Análise de Sequência de DNA/métodos , Análise de Sequência de DNA/tendênciasRESUMO
Two channels were observed in extracts of whole Mycobacterium bovis BCG cells using organic solvents and detergents. The channels derived from organic solvent treatment had a single-channel conductance of about 4.0 nS in 1 M KCl in lipid bilayer membranes with properties similar to those of the channels discovered previously in Mycobacterium smegmatis and Mycobacterium chelonae. The channel was in its open configuration only at low transmembrane potentials. At higher voltages it switched to closed states that were almost impermeable for ions. Lipid bilayer experiments in the presence of detergent extracts of whole cells revealed another channel with a single-channel conductance of only 780 pS in 1 M KCl. Our results indicate that the mycolic acid layer of M. bovis BCG contains two channels, one is cation-selective and its permeability properties can be finely controlled by cell wall asymmetry or potentials. The other one is anion-selective, has a rather small single-channel conductance and is voltage-insensitive. The concentration of channel-forming proteins in the cell wall seems to be small, which is in agreement with the low cell wall permeability for hydrophilic solutes.
Assuntos
Canais Iônicos/fisiologia , Mycobacterium bovis/fisiologia , Ânions/metabolismo , Cátions/metabolismo , Eletrofisiologia , Mycobacterium bovis/ultraestruturaRESUMO
Mycobacterial genomics has uncovered a novel regulatory gene, oxyS, belonging to the LysR family. There is extensive similarity in the DNA-binding domain of OxyS with that of OxyR, the oxidative stress response protein of many bacteria. Since the oxyR gene of Mycobacterium tuberculosis has been multiply inactivated during evolution it was conceivable that some of its functions could be effected by OxyS. It is shown here that OxyS is produced at low levels and that there are at least three different oxyS alleles present in clinical isolates of M. tuberculosis that are susceptible or resistant to isoniazid. Overproduction or depletion of OxyS did not affect susceptibility to isoniazid but increasing the concentration of the regulator lowered levels of the alkyl hydroperoxide reductase, AhpC, and rendered the tubercle bacillus more susceptible to organic hydroperoxides.
Assuntos
Proteínas de Bactérias/metabolismo , Regulação Bacteriana da Expressão Gênica , Mycobacterium tuberculosis/efeitos dos fármacos , Estresse Oxidativo/fisiologia , Peróxidos/farmacologia , Proteínas Repressoras/metabolismo , Fatores de Transcrição/metabolismo , Sequência de Aminoácidos , Antituberculosos/farmacologia , Proteínas de Bactérias/genética , Resistência Microbiana a Medicamentos , Humanos , Isoniazida/farmacologia , Dados de Sequência Molecular , Mycobacterium tuberculosis/genética , Mycobacterium tuberculosis/fisiologia , Peroxidases/genética , Peroxidases/metabolismo , Peroxirredoxinas , Proteínas Repressoras/química , Proteínas Repressoras/genética , Fatores de Transcrição/química , Fatores de Transcrição/genéticaRESUMO
The imminent completion of the genome sequence of Mycobacterium bovis will reveal the genetic blueprint for this most successful pathogen. Comparative analysis with the genome sequences of M. tuberculosis and M. bovis BCG promises to expose the genetic basis for the phenotypic differences between the tubercle bacilli, offering unparalleled insight into the virulence factors of the M. tuberculosis complex. Initial analysis of the sequence data has already revealed a novel deletion from M. bovis, as well as identifying variation in members of the PPE family of proteins. As the study of bacterial pathogenicity enters the postgenomic phase, the genome sequence of M. bovis promises to serve as a cornerstone of mycobacterial genetics.