RESUMO
Introduction. ML1899 is conserved in all mycobacterium sp. and is a middle member of mle-ML1898 operon involved in mycolic acid modification.Aim. In the present study attempts were made to characterize ML1899 in detail.Methodology. Bioinformatics tools were used for prediction of active-site residues, antigenic epitopes and a three-dimensional model of protein. The gene was cloned, expressed and purified as His-tagged protein in Escherichia coli for biophysical/biochemical characterization. Recombinant protein was used to treat THP-1 cells to study change in production of nitric oxide (NO), reactive oxygen species (ROS), cytokines and chemokines using flowcytometry/ELISA.Results. In silico analysis predicted ML1899 as a member of α/ß hydrolase family with GXSXG-motif and Ser126, His282, Asp254 as active-site residues that were confirmed by site-directed mutagensis. ML1899 exhibited esterase activity. It hydrolysed pNP-butyrate as optimum substrate at pH 8.0 and 50 °C with 5.56 µM-1 min-1 catalytic efficiency. The enzyme exhibited stability up to 60 °C temperature and between pH 6.0 to 9.0. K m, V max and specific activity of ML1899 were calculated to be 400 µM, 40 µmoles min-1 ml-1 and 27 U mg- 1, respectively. ML1899 also exhibited phospholipase activity. The protein affected the survival of macrophages when treated at higher concentration. ML1899 enhanced ROS/NO production and up-regulated pro-inflammatory cytokines and chemokine including TNF-α, IFN-γ, IL-6 and IL-8 in macrophages. ML1899 was also observed to elicit humoral response in 69â% of leprosy patients.Conclusion. These results suggested that ML1899, an esterase could up-regulate the immune responses in favour of macrophages at a low concentration but kills the THP-1 macrophages cells at a higher concentration.
Assuntos
Proteínas de Bactérias/imunologia , Esterases/imunologia , Hanseníase/microbiologia , Mycobacterium leprae/enzimologia , Sequência de Aminoácidos , Anticorpos Antibacterianos/imunologia , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Citocinas/genética , Citocinas/imunologia , Estabilidade Enzimática , Esterases/química , Esterases/genética , Feminino , Humanos , Concentração de Íons de Hidrogênio , Cinética , Hanseníase/imunologia , Macrófagos/imunologia , Macrófagos/microbiologia , Masculino , Mycobacterium leprae/química , Mycobacterium leprae/genética , Mycobacterium leprae/imunologia , Óxido Nítrico/imunologia , Espécies Reativas de Oxigênio/imunologia , Alinhamento de SequênciaRESUMO
The lipolytic protein LipU was conserved in mycobacterium sp. including M. tuberculosis (MTB LipU) and M. leprae (MLP LipU). The MTB LipU was identified in extracellular fraction and was reported to be essential for the survival of mycobacterium. Therefore to address the problem of drug resistance in pathogen, LipU was selected as a drug target and the viability of finding out some FDA approved drugs as LipU inhibitors in both the cases was explored. Three-dimensional (3D) model structures of MTB LipU and MLP LipU were generated and stabilized through molecular dynamics (MD). FDA approved drugs were screened against these proteins. The result showed that the top-scoring compounds for MTB LipU were Diosmin, Acarbose and Ouabain with the Glide XP score of -12.8, -11.9 and -11.7 kcal/mol, respectively, whereas for MLP LipU protein, Digoxin (-9.2 kcal/mol), Indinavir (-8.2 kcal/mol) and Travoprost (-8.2 kcal/mol) showed highest affinity. These drugs remained bound in the active site pocket of MTB LipU and MLP LipU structure and interaction grew stronger after dynamics. RMSD, RMSF and Rg were found to be persistent throughout the simulation period. Hydrogen bonds along with large number of hydrophobic interactions stabilized the complex structures. Binding free energies obtained through Prime/MM-GBSA were found in the significant range from -63.85 kcal/mol to -34.57 kcal/mol for MTB LipU and -71.33 kcal/mol to -23.91 kcal/mol for MLP LipU. The report suggested high probability of these drugs to demolish the LipU activity and could be probable drug candidates to combat TB and leprosy disease.
Assuntos
Antituberculosos/química , Antituberculosos/farmacologia , Proteínas de Bactérias/antagonistas & inibidores , Proteínas de Bactérias/química , Simulação de Acoplamento Molecular , Simulação de Dinâmica Molecular , Proteínas de Bactérias/genética , Sítios de Ligação , Domínio Catalítico , Avaliação Pré-Clínica de Medicamentos , Humanos , Ligação de Hidrogênio , Interações Hidrofóbicas e Hidrofílicas , Ligantes , Mycobacterium leprae/genética , Mycobacterium tuberculosis/genética , Ligação Proteica , Reprodutibilidade dos TestesRESUMO
Mycobacterium leprae must adopt a metabolic strategy and undergo various metabolic alterations upon infection to survive inside the human body for years in a dormant state. A change in lipid homeostasis upon infection is highly pronounced in Mycobacterium leprae. Lipids play an essential role in the survival and pathogenesis of mycobacteria. Lipids are present in several forms and serve multiple roles from being a source of nutrition, providing rigidity, evading the host immune response to serving as virulence factors, etc. The synthesis and degradation of lipids is a highly regulated process and is the key to future drug designing and diagnosis for mycobacteria. In the current review, an account of the distinct roles served by lipids, the mechanism of their synthesis and degradation has been elucidated.
Assuntos
Hanseníase/microbiologia , Metabolismo dos Lipídeos , Mycobacterium leprae/metabolismo , Animais , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Humanos , Hanseníase/metabolismo , Mycobacterium leprae/genética , Mycobacterium leprae/crescimento & desenvolvimento , Fatores de Virulência/genética , Fatores de Virulência/metabolismoRESUMO
Many mycobacterial proteins involved in lipid metabolism are reported to be essential for survival and pathogenesis of M. tuberculosis. Rv1076 of M. tuberculosis has been annotated as a putative esterase/lipase based on the consensus sequence 'GXSXG'. It is conserved in all the mycobacterial species. Therefore, in the present study we have characterized Rv1076 gene product in detail. The gene rv1076 was expressed in E. coli and purified from inclusion bodies with approx. 40% yield. The protein showed high specific activity with pNP- butyrate as preferred substrate. The enzyme was stable upto 50°C and in pH range of 6-8 i.e. under acidic conditions. Ser-140, Glu-239 and His-269 were confirmed as active site residues using site directed mutagenesis. The specific activity, Km and Vmax of enzyme was determined to be 177Umg-1 protein, 334µM and 262µmolml-1min-1, respectively. Western blot analysis established Rv1076 to be an extracellular protein. Several putative immunodominant epitopes were predicted in Rv1076. Rv1076 elicited strong humoral response in both extrapulmonary and relapsed cases of TB patients. Therefore, we conclude that Rv1076 is a novel secretory esterase of M. tuberculosis which could be a potential immunodominant antigen of M. tuberculosis.
Assuntos
Proteínas de Bactérias/metabolismo , Espaço Extracelular/enzimologia , Imunidade Humoral , Mycobacterium tuberculosis/citologia , Mycobacterium tuberculosis/enzimologia , Anticorpos Antibacterianos/imunologia , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Proteínas de Bactérias/imunologia , Clonagem Molecular , Sequência Conservada , Humanos , Concentração de Íons de Hidrogênio , Cinética , Modelos Moleculares , Mycobacterium tuberculosis/genética , Mycobacterium tuberculosis/fisiologia , Conformação Proteica , Transporte Proteico , Temperatura , Tuberculose Pulmonar/sangue , Tuberculose Pulmonar/imunologiaRESUMO
The functional aspect of several mycobacterium proteins annotated as hypothetical are yet to be discovered. In the present investigation, in silico approaches were used to predict the biological function of some of the unknown Mtb proteins, which were further validated by wet lab experiments. After screening thousands of Mtb proteins, functionally unknown hypothetical proteins Rv0421c, Rv0519c, Rv0774c, Rv1191, Rv1592c, and Rv3591c were chosen on the basis of their importance in Mtb life cycle. All these proteins posses the α/ß-hydrolase topological fold, characteristic of lipases/esterases, with serine, aspartate, and histidine as the putative members of the catalytic triad. The catalytic serine is located in pentapeptide motif "GXSXG" and oxyanion residue is in dipeptide motif HG. To further support our observation, molecular docking was performed with conventional synthetic lipolytic substrates (pNP-esterss) and specific lipase/esterase inhibitors (tetrahydrolipstatin and phenylmethanesulfonyl fluoride (PMSF)). Significant docking score and strong interaction of substrates/inhibitors with these proteins revealed that these could be possible lipases/esterases. To validate the in silico studies, these genes were cloned from Mtb genome and the proteins were over-expressed in pQE-30/Escherichia coli M15 system. The expressed proteins were purified to homogeneity and enzymatic activity was determined using pNP esters as substrate. The enzyme activity of recombinant proteins was inhibited by tetrahydrolipstatin and PMSF pre-treatment. Outcome of the present investigation provided a basic platform to analyze and characterize unknown hypothetical proteins.