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1.
mBio ; 11(4)2020 07 14.
Artigo em Inglês | MEDLINE | ID: mdl-32665276

RESUMO

Inteins, as posttranslational regulatory elements, can tune protein function to environmental changes by conditional protein splicing (CPS). Translated as subdomains interrupting host proteins, inteins splice to scarlessly join flanking sequences (exteins). We used DnaB-intein1 (DnaBi1) from a replicative helicase of Mycobacterium smegmatis to build a kanamycin intein splicing reporter (KISR) that links splicing of DnaBi1 to kanamycin resistance. Using expression in heterologous Escherichia coli, we observed phenotypic classes of various levels of splicing-dependent resistance (SDR) and related these to the insertion position of DnaBi1 within the kanamycin resistance protein (KanR). The KanR-DnaBi1 construct demonstrating the most stringent SDR was used to probe for CPS of DnaB in the native host environment, M. smegmatis We show here that zinc, important during mycobacterial pathogenesis, inhibits DnaB splicing in M. smegmatis Using an in vitro reporter system, we demonstrated that zinc potently and reversibly inhibited DnaBi1 splicing, as well as splicing of a comparable intein from Mycobacterium leprae Finally, in a 1.95 Å crystal structure, we show that zinc inhibits splicing through binding to the very cysteine that initiates the splicing reaction. Together, our results provide compelling support for a model whereby mycobacterial DnaB protein splicing, and thus DNA replication, is responsive to environmental zinc.IMPORTANCE Inteins are present in a large fraction of prokaryotes and localize within conserved proteins, including the mycobacterial replicative helicase DnaB. In addition to their extensive protein engineering applications, inteins have emerged as environmentally responsive posttranslational regulators of the genes that encode them. While several studies have shown compelling evidence of conditional protein splicing (CPS), examination of splicing in the native host of the intein has proven to be challenging. Here, we demonstrated through a number of measures, including the use of a splicing-dependent sensor capable of monitoring intein activity in the native host, that zinc is a potent and reversible inhibitor of mycobacterial DnaB splicing. This work also expands our knowledge of site selection for intein insertion within nonnative proteins, demonstrating that splicing-dependent host protein activation correlates with proximity to the active site. Additionally, we surmise that splicing regulation by zinc has mycobacteriocidal and CPS application potential.


Assuntos
DnaB Helicases/antagonistas & inibidores , Mycobacterium/efeitos dos fármacos , Processamento de Proteína/efeitos dos fármacos , Zinco/farmacologia , Proteínas de Bactérias/antagonistas & inibidores , Proteínas de Bactérias/genética , DnaB Helicases/química , DnaB Helicases/genética , Escherichia coli/genética , Inteínas/genética , Mycobacterium/enzimologia , Mycobacterium/genética , Processamento de Proteína Pós-Traducional
2.
Int J Mycobacteriol ; 4(3): 207-16, 2015 09.
Artigo em Inglês | MEDLINE | ID: mdl-27649868

RESUMO

Mycobacterium aurum (M. aurum) is an environmental mycobacteria that has previously been used in studies of anti-mycobacterial drugs due to its fast growth rate and low pathogenicity. The M. aurum genome has been sequenced and assembled into 46 contigs, with a total length of 6.02Mb containing 5684 annotated protein-coding genes. A phylogenetic analysis using whole genome alignments positioned M. aurum close to Mycobacterium vaccae and Mycobacterium vanbaalenii, within a clade related to fast-growing mycobacteria. Large-scale genomic rearrangements were identified by comparing the M. aurum genome to those of Mycobacterium tuberculosis and Mycobacterium leprae. M. aurum orthologous genes implicated in resistance to anti-tuberculosis drugs in M. tuberculosis were observed. The sequence identity at the DNA level varied from 68.6% for pncA (pyrazinamide drug-related) to 96.2% for rrs (streptomycin, capreomycin). We observed two homologous genes encoding the catalase-peroxidase enzyme (katG) that is associated with resistance to isoniazid. Similarly, two embB homologues were identified in the M. aurum genome. In addition to describing for the first time the genome of M. aurum, this work provides a resource to aid the use of M. aurum in studies to develop improved drugs for the pathogenic mycobacteria M. tuberculosis and M. leprae.


Assuntos
Antituberculosos/farmacologia , Genoma Bacteriano/genética , Mycobacterium leprae/efeitos dos fármacos , Mycobacterium tuberculosis/efeitos dos fármacos , Mycobacterium/genética , Proteínas de Bactérias/metabolismo , Desenho de Fármacos , Avaliação Pré-Clínica de Medicamentos , Resistência Microbiana a Medicamentos/efeitos dos fármacos , Resistência Microbiana a Medicamentos/genética , Testes de Sensibilidade Microbiana , Mycobacterium/efeitos dos fármacos , Mycobacterium/enzimologia , Mycobacterium/metabolismo , Pentosiltransferases/metabolismo , Peroxidases/metabolismo , Filogenia
3.
BMC Microbiol ; 14: 75, 2014 Mar 24.
Artigo em Inglês | MEDLINE | ID: mdl-24661741

RESUMO

BACKGROUND: Mycobacteria comprise diverse species including non-pathogenic, environmental organisms, animal disease agents and human pathogens, notably Mycobacterium tuberculosis. Considering that the mycobacterial cell wall constitutes a significant barrier to drug penetration, the aim of this study was to conduct a comparative genomics analysis of the repertoire of enzymes involved in peptidoglycan (PG) remodelling to determine the potential of exploiting this area of bacterial metabolism for the discovery of new drug targets. RESULTS: We conducted an in silico analysis of 19 mycobacterial species/clinical strains for the presence of genes encoding resuscitation promoting factors (Rpfs), penicillin binding proteins, endopeptidases, L,D-transpeptidases and N-acetylmuramoyl-L-alanine amidases. Our analysis reveals extensive genetic multiplicity, allowing for classification of mycobacterial species into three main categories, primarily based on their rpf gene complement. These include the M. tuberculosis Complex (MTBC), other pathogenic mycobacteria and environmental species. The complement of these genes within the MTBC and other mycobacterial pathogens is highly conserved. In contrast, environmental strains display significant genetic expansion in most of these gene families. Mycobacterium leprae retains more than one functional gene from each enzyme family, underscoring the importance of genetic multiplicity for PG remodelling. Notably, the highest degree of conservation is observed for N-acetylmuramoyl-L-alanine amidases suggesting that these enzymes are essential for growth and survival. CONCLUSION: PG remodelling enzymes in a range of mycobacterial species are associated with extensive genetic multiplicity, suggesting functional diversification within these families of enzymes to allow organisms to adapt.


Assuntos
Variação Genética , Mycobacterium/enzimologia , Mycobacterium/genética , Peptidoglicano/metabolismo , Biologia Computacional , Sequência Conservada , Microbiologia Ambiental , Genoma Bacteriano , Humanos , Mycobacterium/isolamento & purificação , Infecções por Mycobacterium/microbiologia
4.
PLoS One ; 6(6): e20985, 2011.
Artigo em Inglês | MEDLINE | ID: mdl-21698192

RESUMO

Iron is an essential metal for living organisms but its level must be strictly controlled in cells, because ferrous ion induces toxicity by generating highly active reactive oxygen, hydroxyl radicals, through the Fenton reaction. In addition, ferric ion shows low solubility under physiological conditions. To overcome these obstacles living organisms possess Ferritin superfamily proteins that are distributed in all three domains of life: bacteria, archaea, and eukaryotes. These proteins minimize hydroxyl radical formation by ferroxidase activity that converts Fe(2+) into Fe(3+) and sequesters iron by storing it as a mineral inside a protein cage. In this study, we discovered that mycobacterial DNA-binding protein 1 (MDP1), a histone-like protein, has similar activity to ferritin superfamily proteins. MDP1 prevented the Fenton reaction and protects DNA by the ferroxidase activity. The K(m) values of the ferroxidase activity by MDP1 of Mycobacterium bovis bacillus Calmette-Guérin (BCG-3007c), Mycobacterium tuberculosis (Rv2986c), and Mycobacterium leprae (ML1683; ML-LBP) were 0.292, 0.252, and 0.129 mM, respectively. Furthermore, one MDP1 molecule directly captured 81.4±19.1 iron atoms, suggesting the role of this protein in iron storage. This study describes for the first time a ferroxidase-iron storage protein outside of the ferritin superfamily proteins and the protective role of this bacterial protein from DNA damage.


Assuntos
Dano ao DNA , Ferritinas/fisiologia , Histonas/fisiologia , Mycobacterium/metabolismo , Ceruloplasmina/metabolismo , Mycobacterium/enzimologia , Filogenia , Ligação Proteica
5.
J Biol Chem ; 286(28): 24616-25, 2011 Jul 15.
Artigo em Inglês | MEDLINE | ID: mdl-21592957

RESUMO

Phthiocerol dimycocerosates (PDIMs) and phenolic glycolipids (PGLs) are structurally related lipids noncovalently bound to the outer cell wall layer of Mycobacterium tuberculosis, Mycobacterium leprae, and several opportunistic mycobacterial human pathogens. PDIMs and PGLs are important effectors of virulence. Elucidation of the biosynthesis of these complex lipids will not only expand our understanding of mycobacterial cell wall biosynthesis, but it may also illuminate potential routes to novel therapeutics against mycobacterial infections. We report the construction of an in-frame deletion mutant of tesA (encoding a type II thioesterase) in the opportunistic human pathogen Mycobacterium marinum and the characterization of this mutant and its corresponding complemented strain control in terms of PDIM and PGL production. The growth and antibiotic susceptibility of these strains were also probed and compared with the parental wild-type strain. We show that deletion of tesA leads to a mutant that produces only traces of PDIMs and PGLs, has a slight growth yield increase and displays a substantial hypersusceptibility to several antibiotics. We also provide a robust model for the three-dimensional structure of M. marinum TesA (TesAmm) and demonstrate that a Ser-to-Ala substitution in the predicted catalytic Ser of TesAmm renders a mutant that recapitulates the phenotype of the tesA deletion mutant. Overall, our studies demonstrate a critical role for tesA in mycobacterial biology, advance our understanding of the biosynthesis of an important group of polyketide synthase-derived mycobacterial lipids, and suggest that drugs aimed at blocking PDIM and/or PGL production might synergize with antibiotic therapy in the control of mycobacterial infections.


Assuntos
Parede Celular/enzimologia , Farmacorresistência Bacteriana/fisiologia , Ácido Graxo Sintases/metabolismo , Glicolipídeos/biossíntese , Lipídeos/biossíntese , Mycobacterium/enzimologia , Tioléster Hidrolases/metabolismo , Antibacterianos/química , Antibacterianos/farmacologia , Parede Celular/genética , Desenho de Fármacos , Ácido Graxo Sintases/genética , Deleção de Genes , Glicolipídeos/genética , Humanos , Lipídeos/genética , Mycobacterium/genética , Mycobacterium/patogenicidade , Infecções por Mycobacterium/tratamento farmacológico , Infecções por Mycobacterium/enzimologia , Infecções por Mycobacterium/genética , Tioléster Hidrolases/genética
6.
FEMS Microbiol Lett ; 313(1): 68-74, 2010 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-21039782

RESUMO

ATP synthase is a validated drug target for the treatment of tuberculosis, and ATP synthase inhibitors are promising candidate drugs for the treatment of infections caused by other slow-growing mycobacteria, such as Mycobacterium leprae and Mycobacterium ulcerans. ATP synthase is an essential enzyme in the energy metabolism of Mycobacterium tuberculosis; however, no biochemical data are available to characterize the role of ATP synthase in slow-growing mycobacterial strains. Here, we show that inverted membrane vesicles from the slow-growing model strain Mycobacterium bovis BCG are active in ATP synthesis, but ATP synthase displays no detectable ATP hydrolysis activity and does not set up a proton-motive force (PMF) using ATP as a substrate. Treatment with methanol as well as PMF activation unmasked the ATP hydrolysis activity, indicating that the intrinsic subunit ɛ and inhibitory ADP are responsible for the suppression of hydrolytic activity. These results suggest that the enzyme is needed for the synthesis of ATP, not for the maintenance of the PMF. For the development of new antimycobacterial drugs acting on ATP synthase, screening for ATP synthesis inhibitors, but not for ATP hydrolysis blockers, can be regarded as a promising strategy.


Assuntos
Trifosfato de Adenosina/metabolismo , ATPases Mitocondriais Próton-Translocadoras/metabolismo , Mycobacterium/enzimologia , Mycobacterium/crescimento & desenvolvimento
7.
BMC Microbiol ; 10: 272, 2010 Oct 29.
Artigo em Inglês | MEDLINE | ID: mdl-21029479

RESUMO

BACKGROUND: Rhomboids are ubiquitous proteins with diverse functions in all life kingdoms, and are emerging as important factors in the biology of some pathogenic apicomplexa and Providencia stuartii. Although prokaryotic genomes contain one rhomboid, actinobacteria can have two or more copies whose sequences have not been analyzed for the presence putative rhomboid catalytic signatures. We report detailed phylogenetic and genomic analyses devoted to prokaryotic rhomboids of an important genus, Mycobacterium. RESULTS: Many mycobacterial genomes contained two phylogenetically distinct active rhomboids orthologous to Rv0110 (rhomboid protease 1) and Rv1337 (rhomboid protease 2) of Mycobacterium tuberculosis H37Rv, which were acquired independently. There was a genome-wide conservation and organization of the orthologs of Rv1337 arranged in proximity with glutamate racemase (mur1), while the orthologs of Rv0110 appeared evolutionary unstable and were lost in Mycobacterium leprae and the Mycobacterium avium complex. The orthologs of Rv0110 clustered with eukaryotic rhomboids and contained eukaryotic motifs, suggesting a possible common lineage. A novel nonsense mutation at the Trp73 codon split the rhomboid of Mycobacterium avium subsp. Paratuberculosis into two hypothetical proteins (MAP2425c and MAP2426c) that are identical to MAV_1554 of Mycobacterium avium. Mycobacterial rhomboids contain putative rhomboid catalytic signatures, with the protease active site stabilized by Phenylalanine. The topology and transmembrane helices of the Rv0110 orthologs were similar to those of eukaryotic secretase rhomboids, while those of Rv1337 orthologs were unique. Transcription assays indicated that both mycobacterial rhomboids are possibly expressed. CONCLUSIONS: Mycobacterial rhomboids are active rhomboid proteases with different evolutionary history. The Rv0110 (rhomboid protease 1) orthologs represent prokaryotic rhomboids whose progenitor may be the ancestors of eukaryotic rhomboids. The Rv1337 (rhomboid protease 2) orthologs appear more stable and are conserved nearly in all mycobacteria, possibly alluding to their importance in mycobacteria. MAP2425c and MAP2426c provide the first evidence for a split homologous rhomboid, contrasting whole orthologs of genetically related species. Although valuable insights to the roles of rhomboids are provided, the data herein only lays a foundation for future investigations for the roles of rhomboids in mycobacteria.


Assuntos
Proteínas de Bactérias/genética , Genoma Bacteriano , Mycobacterium/classificação , Mycobacterium/enzimologia , Peptídeo Hidrolases/genética , Filogenia , Sequência de Aminoácidos , Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Genômica , Humanos , Dados de Sequência Molecular , Mycobacterium/química , Mycobacterium/genética , Infecções por Mycobacterium/microbiologia , Peptídeo Hidrolases/química , Peptídeo Hidrolases/metabolismo , Homologia de Sequência de Aminoácidos
8.
Microb Pathog ; 43(5-6): 173-8, 2007.
Artigo em Inglês | MEDLINE | ID: mdl-17611072

RESUMO

Although proteases are recognized as important virulent factors in pathogenic microorganisms, little information is available so far regarding the potential role of these enzymes in diseases caused by mycobacteria. Here we use bioinformatic tools to compare the protease-coding genes present in the genome of Mycobacterium leprae, Mycobacterium tuberculosis, Mycobacterium bovis and Mycobacterium avium paratuberculosis. This analysis allowed a review of the nomenclature of the protease family present in mycobacteria. A special attention was devoted to the 'decaying genome' of M. leprae where a relatively high level of conservation of protease-coding genes was observed when compared to other genes families. A total of 39 genes out of the 49 found in M. bovis were identified in M. leprae. Of relevance, a core of well-conserved 38 protease genes shared by the four species was defined. This set of proteases is probably essential for survival in the host and disease outcome and may constitute novel targets for drug development leading to a more effective control of mycobacterial diseases.


Assuntos
Genômica , Mycobacterium/genética , Peptídeo Hidrolases/genética , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Biologia Computacional , Genoma Bacteriano , Mycobacterium/enzimologia , Mycobacterium/patogenicidade , Peptídeo Hidrolases/metabolismo
9.
J Microbiol Methods ; 68(1): 32-9, 2007 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-16839634

RESUMO

Mycobacterium spp. possess a complex cell envelope that consists of a plasma membrane, a peptidoglycan-arabinogalactan complex which in turn is esterified by mycolic acids that form with other non-bound lipids an asymmetric permeability barrier and an outer layer, also called a capsule in the case of pathogenic species. In order to investigate the functional roles of the cell envelope components, especially those of the major pathogens Mycobacterium tuberculosis and Mycobacterium leprae, it is necessary to fractionate the envelope by breaking the unusual wall that covers these bacteria. To this aim we first compared the efficiency of high pressure (cell disrupter/French press) with those of pathogen-compatible breakage methods such as sonication, bead beater and lysozyme treatment using the non-pathogenic Mycobacterium smegmatis. When the distribution of various specific markers of the cell envelope compartments, which include mycolic acids, arabinose, NADH oxidase activity, cell wall and cytosolic proteins, were determined sonication combined with lysozyme treatment was found to be the best option. The protocol of subcellular fractionation was then validated for pathogenic species by applying the method to Mycobacterium bovis BCG cells, an attenuated strain of the M. tuberculosis complex.


Assuntos
Fracionamento Celular/métodos , Mycobacterium/química , Carboidratos/análise , Parede Celular/química , Parede Celular/enzimologia , Lipídeos de Membrana/análise , Complexos Multienzimáticos/análise , Mycobacterium/enzimologia , Ácidos Micólicos/análise , NADH NADPH Oxirredutases/análise , Sonicação , Frações Subcelulares/química , Frações Subcelulares/enzimologia
10.
J Bacteriol ; 185(23): 6870-82, 2003 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-14617651

RESUMO

Mycobacterium ulcerans causes Buruli ulcer, the third most prevalent mycobacterial infection of immunocompetent humans after tuberculosis and leprosy. Recent work has shown that the production by M. ulcerans of mycolactone, a novel polyketide, may partly explain the pathogenesis of Buruli ulcer. To search for the genetic basis of virulence in M. ulcerans, we took advantage of the close genetic relationship between M. ulcerans and Mycobacterium marinum by performing genomic suppressive subtractive hybridization of M. ulcerans with M. marinum. We identified several DNA fragments specific to M. ulcerans, in particular, a type I polyketide synthase locus with a highly repetitive modular arrangement. We postulate that this locus is responsible for the synthesis of mycolactone in M. ulcerans.


Assuntos
Epotilonas/genética , Complexos Multienzimáticos/genética , Mycobacterium/genética , Sequência de Aminoácidos , Toxinas Bacterianas , Southern Blotting , Epotilonas/análise , Humanos , Macrolídeos , Dados de Sequência Molecular , Complexos Multienzimáticos/análise , Mycobacterium/enzimologia , Hibridização de Ácido Nucleico/métodos , Reação em Cadeia da Polimerase , Alinhamento de Sequência
11.
Chem Biol ; 9(7): 767-76, 2002 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-12144918

RESUMO

Analysis of the genomes of M. tuberculosis, M. leprae, M. smegmatis, and M. avium has revealed a large family of genes homologous to known sulfotransferases. Despite reports detailing a suite of sulfated glycolipids in many mycobacteria, a corresponding family of sulfotransferase genes remains uncharacterized. Here, a sequence-based analysis of newly discovered mycobacterial sulfotransferase genes, named stf1-stf10, is presented. Interestingly, two sulfotransferase genes are highly similar to mammalian sulfotransferases, increasing the list of mycobacterial eukaryotic-like protein families. The sulfotransferases join an equally complex family of mycobacterial sulfatases: a large family of sulfatase genes has been found in all of the mycobacterial genomes examined. As sulfated molecules are common mediators of cell-cell interactions, the sulfotransferases and sulfatases may be involved in regulating host-pathogen interactions.


Assuntos
Mycobacterium/enzimologia , Mycobacterium/genética , Sulfatases/genética , Sulfotransferases/genética , Sequência de Aminoácidos , Animais , Humanos , Dados de Sequência Molecular , Filogenia , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , Sulfatases/metabolismo , Sulfatos/química , Sulfatos/metabolismo , Sulfotransferases/metabolismo
12.
Arch Microbiol ; 176(5): 381-5, 2001 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-11702081

RESUMO

Mycobacterium sp. Pyr-1 produces an enzyme with nitroreductase activity that reduces 1-nitropyrene and 4-nitrobenzoic acid to the corresponding aromatic amines. This enzyme was constitutive and required NADH; and its activity was enhanced by FAD. It was inhibited by antimycin A, dicumarol, and o-iodosobenzoic acid; and it was inactivated by ammonium sulfate precipitation. After purification to homogeneity, the protein produced a single band on native and SDS-polyacrylamide gels and had a single amino-terminal sequence. The N-terminal amino acid sequence was identical to the corresponding sequences of the lipoamide dehydrogenases of M. leprae, M. tuberculosis and Corynebacterium glutamicum. The amino-terminal sequence was also similar to lipoamide dehydrogenases from M. smegmatis and several other bacteria. The amino acid sequence of an internal peptide (12 of 13 amino acids) was nearly identical to the corresponding sequences of lipoamide dehydrogenases from M. leprae and M. tuberculosis and was similar to those of C. glutamicum, Streptomyces coelicolor and S. seoulensis. The data show that a unique lipoamide dehydrogenase in Mycobacterium sp. Pyr-1, which differs from classic (Type I) bacterial nitroreductases, reduces aromatic nitro compounds to aromatic amines.


Assuntos
Di-Hidrolipoamida Desidrogenase/química , Mycobacterium/enzimologia , Nitrorredutases/isolamento & purificação , Nitrorredutases/metabolismo , Sequência de Aminoácidos , Di-Hidrolipoamida Desidrogenase/metabolismo , Dados de Sequência Molecular , Mycobacterium/crescimento & desenvolvimento , Nitrorredutases/química
13.
Nucleic Acids Res ; 29(21): 4310-8, 2001 Nov 01.
Artigo em Inglês | MEDLINE | ID: mdl-11691918

RESUMO

A survey of a vast range of mycobacterial strains led us to discover a new Pps1 intein allele in Mycobacterium gastri which differs from those of Mycobacterium tuberculosis and Mycobacterium leprae in both its sequence and insertion site. While little is known about Pps1, except that it belongs to the YC24 family of ABC transporters, we show that, unlike the other inteins described so far from Eubacteria, the MgaPps1 intein possesses a specific endonuclease activity. The intein is the first eubacterial intein to be characterised as an endonuclease. Like other intein endonucleases, its minimal sequence for recognition and cleavage is quite large, with 22 bp spanning the Pps1-c site. The fact that an active endonuclease is found among the mycobacterial inteins supports the concept of a cyclical model of invasion by horizontal transfer of these genes, followed by degeneration and loss until a new invasion event, thus explaining their long-term persistence in closely related eubacterial species.


Assuntos
Transportadores de Cassetes de Ligação de ATP/metabolismo , Alelos , Proteínas de Bactérias/metabolismo , Endonucleases/metabolismo , Mycobacterium/enzimologia , Mycobacterium/genética , Transportadores de Cassetes de Ligação de ATP/genética , Transportadores de Cassetes de Ligação de ATP/isolamento & purificação , Sequência de Aminoácidos , Proteínas de Bactérias/genética , Proteínas de Bactérias/isolamento & purificação , Sequência de Bases , Tampões (Química) , Clonagem Molecular , Sequência Conservada , Endonucleases/genética , Endonucleases/isolamento & purificação , Evolução Molecular , Transferência Genética Horizontal/genética , Genes Bacterianos/genética , Concentração de Íons de Hidrogênio , Modelos Genéticos , Dados de Sequência Molecular , Recombinação Genética/genética , Alinhamento de Sequência , Especificidade por Substrato
14.
Microbiology (Reading) ; 146 ( Pt 1): 199-208, 2000 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-10658666

RESUMO

Trehalose is present as a free disaccharide in the cytoplasm of mycobacteria and as a component of cell-wall glycolipids implicated in tissue damage associated with mycobacterial infection. To obtain an overview of trehalose metabolism, we analysed data from the Mycobacterium tuberculosis genome project and identified ORFs with homology to genes encoding enzymes from three trehalose biosynthesis pathways previously characterized in other bacteria. Functional assays using mycobacterial extracts and recombinant enzymes derived from these ORFs demonstrated that mycobacteria can produce trehalose from glucose 6-phosphate and UDP-glucose (the OtsA-OtsB pathway) from glycogen-like alpha(1-->4)-linked glucose polymers (the TreY-TreZ pathway) and from maltose (the TreS pathway). Each of the pathways was found to be active in both rapid-growing Mycobacterium smegmatis and slow-growing Mycobacterium bovis BCG. The presence of a disrupted treZ gene in Mycobacterium leprae suggests that this pathway is not functional in this organism. The presence of multiple biosynthetic pathways indicates that trehalose plays an important role in mycobacterial physiology.


Assuntos
Mycobacterium/metabolismo , Trealose/biossíntese , Genoma Bacteriano , Glucanos/metabolismo , Glucose-6-Fosfato/metabolismo , Maltose/metabolismo , Mycobacterium/enzimologia , Mycobacterium/genética , Mycobacterium tuberculosis/genética , Pressão Osmótica , Proteínas Recombinantes/metabolismo , Análise de Sequência de DNA , Homologia de Sequência de Aminoácidos
15.
Drugs ; 58 Suppl 2: 19-22, 1999.
Artigo em Inglês | MEDLINE | ID: mdl-10553700

RESUMO

The fluoroquinolones have been shown to be active in vitro against many mycobacterial species, including most strains of Mycobacterium tuberculosis complex and M. fortuitum, and some strains of M. kansasii, M. avium-intracellulare (MAI) complex and M. leprae. Ciprofloxacin, ofloxacin and sparfloxacin are the best studied of these agents to date, and are among the most active of this group against M. tuberculosis and other mycobacteria. Treatment of patients with multidrug-resistant pulmonary tuberculosis using ofloxacin has resulted in the selection of quinolone-resistant mutants in a few patients. Many strains of MAI, however, are resistant to fluoroquinolones, and structure-activity relationships and DNA gyrase studies have been undertaken to identify the moieties associated with activity and the lack thereof. The genetic and molecular basis of quinolone resistance in mycobacteria has revealed both the recent progress made in these areas and the limitations of the quinolones against this genus. Considerable progress will need to be made in resolving these issues in order for the quinolones to become clinically useful antimycobacterial agents.


Assuntos
Anti-Infecciosos/farmacologia , Mycobacterium/efeitos dos fármacos , 4-Quinolonas , Animais , Anti-Infecciosos/uso terapêutico , Fluoroquinolonas , Humanos , Mycobacterium/enzimologia , Mycobacterium/genética , Infecções por Mycobacterium/tratamento farmacológico , Infecções por Mycobacterium/microbiologia
16.
Antimicrob Agents Chemother ; 42(8): 2084-8, 1998 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-9687411

RESUMO

The in vitro activities of seven quinolones and the sequences of the quinolone resistance-determining regions (QRDR) in the A and B subunits of DNA gyrase were determined for 14 mycobacterial species. On the basis of quinolone activity, quinolones were arranged from that with the greatest to that with the least activity as follows: sparfloxacin, levofloxacin, ciprofloxacin, ofloxacin, pefloxacin, flumequine, and nalidixic acid. Based on MICs, the species could be organized into three groups: resistant (Mycobacterium avium, M. intracellulare, M. marinum, M. chelonae, M. abscessus [ofloxacin MICs, >/=8 microg/ml]), moderately susceptible (M. tuberculosis, M. bovis BCG, M. kansasii, M. leprae, M. fortuitum third biovariant, M. smegmatis [ofloxacin MICs, 0.5 to 1 microg/ml]), and susceptible (M. fortuitum, M. peregrinum, M. aurum [ofloxacin MICs,

Assuntos
Anti-Infecciosos/farmacologia , Proteínas de Bactérias/química , DNA Topoisomerases Tipo II/química , Mycobacterium/efeitos dos fármacos , Sequência de Aminoácidos , Sequência de Bases , DNA Topoisomerases Tipo II/genética , Fluoroquinolonas , Testes de Sensibilidade Microbiana , Dados de Sequência Molecular , Mycobacterium/enzimologia
17.
Vaccine ; 16(13): 1344-8, 1998 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-9682400

RESUMO

The growth of Mycobacterium w, a candidate strain for leprosy vaccine in submerged culture, was inhibited by the presence of over 40% oxygen saturation in the medium. Intracellular levels of superoxide dismutase and catalase were very low in the beginning. However, under controlled oxygenation, these levels increased with time. The augmentations of these antioxidant enzymes were associated with the elevated oxygen consumption by the culture. By maintaining the oxygen level below 20% during 6-day culture, it was possible to grow Mycobacterium w in five production batches up to a cell density of 3.7 +/- 0.70 x 10(9) bacilli ml-1. The shelf life of the vaccine produced in different batches was more than 2 years, both at 4 degrees C and at 26 degrees C. This provides a cost-effective, unit culture technology for the production of this candidate leprosy vaccine from a nonpathogenic organism, which will facilitate the widespread use of the vaccine.


Assuntos
Vacinas Bacterianas , Hanseníase/prevenção & controle , Mycobacterium/metabolismo , Consumo de Oxigênio , Vacinas Bacterianas/imunologia , Vacinas Bacterianas/uso terapêutico , Técnicas Bacteriológicas , Reatores Biológicos , Catalase/metabolismo , Estabilidade de Medicamentos , Armazenamento de Medicamentos , Mycobacterium/enzimologia , Mycobacterium/crescimento & desenvolvimento , Superóxido Dismutase/metabolismo
18.
Eur J Biochem ; 251(3): 795-803, 1998 Feb 01.
Artigo em Inglês | MEDLINE | ID: mdl-9490054

RESUMO

We have refined the X-ray structures of two site-directed mutants of the iron-dependent superoxide dismutase (SOD) from Mycobacterium tuberculosis. These mutations which affect residue 145 in the enzyme (H145Q and H145E) were designed to alter its metal-ion specificity. This residue is either Gln or His in homologous SOD enzymes and has previously been shown to play a role in active-site interactions since its side-chain helps to coordinate the metal ion via a solvent molecule which is thought to be a hydroxide ion. The mutations were based on the observation that in the closely homologous manganese dependent SOD from Mycobacterium leprae, the only significant difference from the M. tuberculosis SOD within 10 A of the metal-binding site is the substitution of Gln for His at position 145. Hence an H145Q mutant of the M. tuberculosis (TB) SOD was engineered to investigate this residue's role in metal ion dependence and an isosteric H145E mutant was also expressed. The X-ray structures of the H145Q and H145E mutants have been solved at resolutions of 4.0 A and 2.5 A, respectively, confirming that neither mutation has any gross effects on the conformation of the enzyme or the structure of the active site. The residue substitutions are accommodated in the enzyme's three-dimensional structure by small local conformational changes. Peroxide inhibition experiments and atomic absorption spectroscopy establish surprisingly the H145E mutant SOD has manganese bound to it whereas the H145Q mutant SOD retains iron as the active-site metal. This alteration in metal specificity may reflect on the preference of manganese ions for anionic ligands.


Assuntos
Ferro/metabolismo , Mycobacterium tuberculosis/enzimologia , Conformação Proteica , Superóxido Dismutase/química , Sequência de Aminoácidos , Sítios de Ligação , Cristalografia por Raios X/métodos , Ferro/farmacologia , Modelos Moleculares , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Mycobacterium/enzimologia , Mutação Puntual , Reação em Cadeia da Polimerase , Estrutura Terciária de Proteína , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/química , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , Superóxido Dismutase/biossíntese
20.
J Biol Chem ; 272(27): 16741-5, 1997 Jul 04.
Artigo em Inglês | MEDLINE | ID: mdl-9201977

RESUMO

Surface-exposed unusual lipids containing phthiocerol and phenolphthiocerol are found only in the cell wall of slow-growing pathogenic mycobacteria and are thought to play important roles in host-pathogen interaction. The enzymology and molecular genetics of biosynthesis of phthiocerol and phenolphthiocerol are unknown. We postulate the domain organization of a set of multifunctional enzymes and a cluster of genes (pps) that would encode these enzymes for the biosynthesis of phthiocerol and phenolphthiocerol. A cosmid containing the postulated pps gene cluster was identified by screening a genomic library of Mycobacterium bovis BCG with the postulated homologous domains from mycocerosic acid synthase and fatty acid synthase genes as probes. Homologous cosmids were also identified in the genomic libraries of Mycobacterium tuberculosis and Mycobacterium leprae. M. bovis BCG was transformed with a pps disruption construct, made from the BCG cosmid by introducing the hygromycin resistance gene as the positive-selectable marker and the sacB gene as the counter-selectable marker. Gene disruption by homologous recombination with double crossover was confirmed by polymerase chain reaction and Southern hybridization. Chromatographic analysis showed that the phenolphthiocerol derivative, mycoside B, and phthiocerol dimycocerosates were not produced by the gene knockout mutants. This result confirms the identity of the pps genes. With the identification of the pps gene clusters in both M. tuberculosis and M. leprae, it should be possible to test the postulated roles of these unique lipids in tuberculosis and leprosy.


Assuntos
Parede Celular/metabolismo , Lipídeos de Membrana/genética , Mycobacterium/genética , Ceras/metabolismo , Acil Coenzima A/metabolismo , Aciltransferases/genética , Aciltransferases/metabolismo , Alelos , Cromatografia em Camada Delgada , Cosmídeos/genética , Cosmídeos/metabolismo , Eletroforese em Gel de Poliacrilamida , Ácido Graxo Sintases/genética , Ácido Graxo Sintases/metabolismo , Álcoois Graxos/metabolismo , Deleção de Genes , Glicolipídeos/metabolismo , Lipídeos de Membrana/biossíntese , Modelos Químicos , Modelos Genéticos , Dados de Sequência Molecular , Complexos Multienzimáticos/genética , Complexos Multienzimáticos/metabolismo , Mutagênese Insercional , Mycobacterium/enzimologia , Mycobacterium/patogenicidade , Mycobacterium bovis/enzimologia , Mycobacterium bovis/genética , Mycobacterium bovis/patogenicidade , Mycobacterium leprae/enzimologia , Mycobacterium leprae/genética , Mycobacterium leprae/patogenicidade , Mycobacterium tuberculosis/enzimologia , Mycobacterium tuberculosis/genética , Mycobacterium tuberculosis/patogenicidade , Virulência/genética
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