Gene_locus Report for: myctu-MT1628

BACKGROUND: Multi drug-resistant tuberculosis is a major health threat to humans. Whole genome sequencing of several isoniazid (INH) resistant strains of M. tuberculosis revealed mutations in several genes. Rv1592c was demonstrated as lipolytic enzyme and its expression was up-regulated during isoniazid (INH) treatment. The valine at position 430 of Rv1592c was mutated to alanine frequently in the INH resistant strain of M. tuberculosis. METHODS: In this report, an array of computational approaches was used to understand the role of Val430-Ala mutation in Rv1592c in INH resistance. The impact of mutations on structural stability and degree of INH modification was demonstrated using the molecular dynamics method. The mutation in the Rv1592c gene at V430 position was created by the PCR primer walking method. Mutant and wild type gene was cloned into E. coli-mycobacteria shuttle vector (pVV-16) and expressed in Mycobacterium smegmatis system. The isoniazid susceptibility assay was performed by agar plate culture spot and CFUs count assay. RESULTS: This study demonstrated that the Val430 in Rv1592c makes the part of flap covering the substrate binding cavity. Mutation at Val430-Ala in Rv1592c caused the displacement of the flap region, resulting in uncovering a cavity, which allows accessibility of substrate to the active site cleft. The Val430-Ala mutation in Rv1592c created its structure energetically more stable. RMSD, RMSF and Rg simulation of mutant maintained overall stability throughout the simulation period while the native protein displayed comparatively more fluctuations. Moreover, docking studies showed that INH was bound into the active pocket of the mutant with considerable binding energy (-6.3 kcal/mol). In order to observe constant binding for INH, complexes were simulated for 50 ns. It was observed that after simulation, INH remained bound in the pocket with an increased molecular bonding network with the neighbor amino acid residues. In vitro studies clearly suggested that M. smegmatis expressing mutant has a better survival rate in isoniazid treatment as compared to wild type. CONCLUSION: Overall, this study at the outset suggested that the mutation observed in drug resistant strain provides stability to the Rv1592c protein and increased affinity towards the INH due to flap displacement, leading to the possibility for its modification. In vitro results supported our in silico findings.

The alpha/beta hydrolase fold superfamily is an ancient and widely diversified group of primarily hydrolytic enzymes. In this review, the adaptations of these proteins to the pathogenic lifestyle of Mycobacterium tuberculosis (Mtb), the causative agent of tuberculosis, are examined. Of the 105 alpha/beta hydrolases identified in Mtb, many are associated with lipid metabolism, particularly in the biosynthesis and maintenance of the Mtb's unique cell envelope, as well in the large number of extracellular lipases that are likely responsible for degradation of host lipid material. alpha/beta hydrolase fold proteins are also involved in the evasion and modulation of the immune response, detoxification and metabolic adaptations, including growth, response to acidification of the intracellular environment and dormancy. A striking feature of Mtb's alpha/beta hydrolases is their diversification into virulence-associated niches. It is clear that the alpha/beta hydrolase fold family has made a significant contribution to Mtb's remarkable success as a pathogen.

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 alpha/beta-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, 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 by 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 analyse and characterize unknown hypothetical proteins.

BACKGROUND: Multi drug-resistant tuberculosis is a major health threat to humans. Whole genome sequencing of several isoniazid (INH) resistant strains of M. tuberculosis revealed mutations in several genes. Rv1592c was demonstrated as lipolytic enzyme and its expression was up-regulated during isoniazid (INH) treatment. The valine at position 430 of Rv1592c was mutated to alanine frequently in the INH resistant strain of M. tuberculosis. METHODS: In this report, an array of computational approaches was used to understand the role of Val430-Ala mutation in Rv1592c in INH resistance. The impact of mutations on structural stability and degree of INH modification was demonstrated using the molecular dynamics method. The mutation in the Rv1592c gene at V430 position was created by the PCR primer walking method. Mutant and wild type gene was cloned into E. coli-mycobacteria shuttle vector (pVV-16) and expressed in Mycobacterium smegmatis system. The isoniazid susceptibility assay was performed by agar plate culture spot and CFUs count assay. RESULTS: This study demonstrated that the Val430 in Rv1592c makes the part of flap covering the substrate binding cavity. Mutation at Val430-Ala in Rv1592c caused the displacement of the flap region, resulting in uncovering a cavity, which allows accessibility of substrate to the active site cleft. The Val430-Ala mutation in Rv1592c created its structure energetically more stable. RMSD, RMSF and Rg simulation of mutant maintained overall stability throughout the simulation period while the native protein displayed comparatively more fluctuations. Moreover, docking studies showed that INH was bound into the active pocket of the mutant with considerable binding energy (-6.3 kcal/mol). In order to observe constant binding for INH, complexes were simulated for 50 ns. It was observed that after simulation, INH remained bound in the pocket with an increased molecular bonding network with the neighbor amino acid residues. In vitro studies clearly suggested that M. smegmatis expressing mutant has a better survival rate in isoniazid treatment as compared to wild type. CONCLUSION: Overall, this study at the outset suggested that the mutation observed in drug resistant strain provides stability to the Rv1592c protein and increased affinity towards the INH due to flap displacement, leading to the possibility for its modification. In vitro results supported our in silico findings.

The alpha/beta hydrolase fold superfamily is an ancient and widely diversified group of primarily hydrolytic enzymes. In this review, the adaptations of these proteins to the pathogenic lifestyle of Mycobacterium tuberculosis (Mtb), the causative agent of tuberculosis, are examined. Of the 105 alpha/beta hydrolases identified in Mtb, many are associated with lipid metabolism, particularly in the biosynthesis and maintenance of the Mtb's unique cell envelope, as well in the large number of extracellular lipases that are likely responsible for degradation of host lipid material. alpha/beta hydrolase fold proteins are also involved in the evasion and modulation of the immune response, detoxification and metabolic adaptations, including growth, response to acidification of the intracellular environment and dormancy. A striking feature of Mtb's alpha/beta hydrolases is their diversification into virulence-associated niches. It is clear that the alpha/beta hydrolase fold family has made a significant contribution to Mtb's remarkable success as a pathogen.

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 alpha/beta-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, 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 by 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 analyse and characterize unknown hypothetical proteins.

To investigate the molecular characteristics of bacillus Calmette-Guerin (BCG) vaccines, the complete genomic sequence of Mycobacterium bovis BCG Tokyo 172 was determined, and the results were compared with those for BCG Pasteur and other M. tuberculosis complex. The genome of BCG Tokyo had a length of 4,371,711bp and contained 4033 genes, including 3950 genes coding for proteins (CDS). There were 18 regions of difference (showing differences of more than 20bp), 20 insertion or deletion (ins/del) mutations of less than 20bp, and 68 SNPs between the two BCG substrains. These findings are useful for better understanding of the genetic differences in BCG substrains due to in vitro evolution of BCG.

To understand the evolution, attenuation, and variable protective efficacy of bacillus Calmette-Guerin (BCG) vaccines, Mycobacterium bovis BCG Pasteur 1173P2 has been subjected to comparative genome and transcriptome analysis. The 4,374,522-bp genome contains 3,954 protein-coding genes, 58 of which are present in two copies as a result of two independent tandem duplications, DU1 and DU2. DU1 is restricted to BCG Pasteur, although four forms of DU2 exist; DU2-I is confined to early BCG vaccines, like BCG Japan, whereas DU2-III and DU2-IV occur in the late vaccines. The glycerol-3-phosphate dehydrogenase gene, glpD2, is one of only three genes common to all four DU2 variants, implying that BCG requires higher levels of this enzyme to grow on glycerol. Further amplification of the DU2 region is ongoing, even within vaccine preparations used to immunize humans. An evolutionary scheme for BCG vaccines was established by analyzing DU2 and other markers. Lesions in genes encoding sigma-factors and pleiotropic transcriptional regulators, like PhoR and Crp, were also uncovered in various BCG strains; together with gene amplification, these affect gene expression levels, immunogenicity, and, possibly, protection against tuberculosis. Furthermore, the combined findings suggest that early BCG vaccines may even be superior to the later ones that are more widely used.

Mycobacterium bovis is the causative agent of tuberculosis in a range of animal species and man, with worldwide annual losses to agriculture of $3 billion. The human burden of tuberculosis caused by the bovine tubercle bacillus is still largely unknown. M. bovis was also the progenitor for the M. bovis bacillus Calmette-Guerin vaccine strain, the most widely used human vaccine. Here we describe the 4,345,492-bp genome sequence of M. bovis AF2122/97 and its comparison with the genomes of Mycobacterium tuberculosis and Mycobacterium leprae. Strikingly, the genome sequence of M. bovis is >99.95% identical to that of M. tuberculosis, but deletion of genetic information has led to a reduced genome size. Comparison with M. leprae reveals a number of common gene losses, suggesting the removal of functional redundancy. Cell wall components and secreted proteins show the greatest variation, indicating their potential role in host-bacillus interactions or immune evasion. Furthermore, there are no genes unique to M. bovis, implying that differential gene expression may be the key to the host tropisms of human and bovine bacilli. The genome sequence therefore offers major insight on the evolution, host preference, and pathobiology of M. bovis.

Countless millions of people have died from tuberculosis, a chronic infectious disease caused by the tubercle bacillus. The complete genome sequence of the best-characterized strain of Mycobacterium tuberculosis, H37Rv, has been determined and analysed in order to improve our understanding of the biology of this slow-growing pathogen and to help the conception of new prophylactic and therapeutic interventions. The genome comprises 4,411,529 base pairs, contains around 4,000 genes, and has a very high guanine + cytosine content that is reflected in the biased amino-acid content of the proteins. M. tuberculosis differs radically from other bacteria in that a very large portion of its coding capacity is devoted to the production of enzymes involved in lipogenesis and lipolysis, and to two new families of glycine-rich proteins with a repetitive structure that may represent a source of antigenic variation.