Gene_locus Report for: myctu-yc88

Rv1288, a conserved hypothetical protein of M. tuberculosis (M.tb), was recently characterized as two-domain esterase enzyme by in silico study. In the present study, Rv1288 and its domains (Est and Lyt) were cloned individually from M.tb into E. coli for expression and purification. The purified rRv1288 and rEst proteins exhibited lipolytic activity with medium chain length esters as optimum substrates, while Lyt domain did not show enzymatic activity. However, presence of Lyt domain resulted in enhanced rate of protein aggregation at higher temperature. Both rRv1288 and rEst followed the similar patterns of substrate specificity, temperature and pH activity. Site directed mutagenesis confirmed the Ser-294, Asp-391 and His-425 as catalytic site residues. Rv1288 was found to be present in cell wall fraction of M.tb H37Ra. Peptidoglycan binding activity of Rv1288 and its domains demonstrated that the Lyt domain is essential for anchoring protein to the cell wall. Expression of rv1288 was up regulated in M.tb under nutrient starved condition. Over expression of rv1288 in surrogate host M. smegmatis led to change in colony morphology, enhanced pellicle and aggregate formation that might be linked with the changed lipid composition of bacterial cell wall. Cell wall of M. smegmatis expressing rv1288 had higher amount of lipids, with a significant increase in trehalose dimycolate content. Rv1288 also leads to increase in drug resistance of M. smegmatis. Rv1288 also enhanced the intracellular survival of M. smegmatis in Raw264.7 cell line. Overall, this study suggested that Rv1288, a cell wall localized carboxyl hydrolase with mycolyl-transferase activity, modulated the cell wall lipids to favor the survival of bacteria under stress condition.

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.

BACKGROUND: Death toll due to tuberculosis is still rising day by day. Whole genome sequence of Mycobacterium tuberculosis has provided a platform to conduct research in order to identify the probable drug target. OBJECTIVES: Out of 4000 gene products of M. tuberculosis, approximately 40% of proteins are annotated as hypothetical. Identifying and characterizing these proteins could provide a new prescriptive for developing new TB drugs. Rv1288, a protein of M. tuberculosis H37Rv has been annotated as a hypothetical protein in database. Attempt has been made to assign a meaningful role to rv1288 gene product in M. tuberculosis life cycle. METHODS: A homology 3D structure of both domains was separately generated and assigned as Rv1288LytE and Rv1288est. Molecular simulation of Rv1288est was carried out for proper structure analysis. To further confirm the predictive role of Rv1288 in mycobacterium life cycle, molecular docking was performed. N-acetyl glucosamine, a major constituent of cell wall was docked with LytE domain, whereas, esterase domain was docked with lipolytic substrate, pNP-ester derivatives and inhibitors THL/PMSF. RESULTS: In-silico analysis revealed that Rv1288 is a two domain protein, an N-terminal LytE domain containing three consecutive LysM motifs and a C-terminal esterase domain of esterase D family. LytE domain has the property to bind N-acetyl glucosamine moieties of peptidoglycan, a major component of cell wall. Detailed in-silico sequence analysis revealed that this LytE domain may help in positioning the esterase domain to the cell wall of mycobacterium. Esterase domain comprised a tetrapeptide motif HGGG, a pentapeptide sequence motif GxSxG and conserved amino acid residues Ser-141, Asp-238 and His-272 which constitute a catalytic triad characteristic of other hormone sensitive lipases/ esterases. Docking studies suggested that THL and PMSF could be the potent inhibitors for Rv1288 protein. CONCLUSION: In the present investigation, we bioinformatically confirmed that Rv1288 is most likely a LytE domain containing lipolytic enzyme showing similarity to hormone sensitive lipases/esterases.

Rv1288, a conserved hypothetical protein of M. tuberculosis (M.tb), was recently characterized as two-domain esterase enzyme by in silico study. In the present study, Rv1288 and its domains (Est and Lyt) were cloned individually from M.tb into E. coli for expression and purification. The purified rRv1288 and rEst proteins exhibited lipolytic activity with medium chain length esters as optimum substrates, while Lyt domain did not show enzymatic activity. However, presence of Lyt domain resulted in enhanced rate of protein aggregation at higher temperature. Both rRv1288 and rEst followed the similar patterns of substrate specificity, temperature and pH activity. Site directed mutagenesis confirmed the Ser-294, Asp-391 and His-425 as catalytic site residues. Rv1288 was found to be present in cell wall fraction of M.tb H37Ra. Peptidoglycan binding activity of Rv1288 and its domains demonstrated that the Lyt domain is essential for anchoring protein to the cell wall. Expression of rv1288 was up regulated in M.tb under nutrient starved condition. Over expression of rv1288 in surrogate host M. smegmatis led to change in colony morphology, enhanced pellicle and aggregate formation that might be linked with the changed lipid composition of bacterial cell wall. Cell wall of M. smegmatis expressing rv1288 had higher amount of lipids, with a significant increase in trehalose dimycolate content. Rv1288 also leads to increase in drug resistance of M. smegmatis. Rv1288 also enhanced the intracellular survival of M. smegmatis in Raw264.7 cell line. Overall, this study suggested that Rv1288, a cell wall localized carboxyl hydrolase with mycolyl-transferase activity, modulated the cell wall lipids to favor the survival of bacteria under stress condition.

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.

BACKGROUND: Death toll due to tuberculosis is still rising day by day. Whole genome sequence of Mycobacterium tuberculosis has provided a platform to conduct research in order to identify the probable drug target. OBJECTIVES: Out of 4000 gene products of M. tuberculosis, approximately 40% of proteins are annotated as hypothetical. Identifying and characterizing these proteins could provide a new prescriptive for developing new TB drugs. Rv1288, a protein of M. tuberculosis H37Rv has been annotated as a hypothetical protein in database. Attempt has been made to assign a meaningful role to rv1288 gene product in M. tuberculosis life cycle. METHODS: A homology 3D structure of both domains was separately generated and assigned as Rv1288LytE and Rv1288est. Molecular simulation of Rv1288est was carried out for proper structure analysis. To further confirm the predictive role of Rv1288 in mycobacterium life cycle, molecular docking was performed. N-acetyl glucosamine, a major constituent of cell wall was docked with LytE domain, whereas, esterase domain was docked with lipolytic substrate, pNP-ester derivatives and inhibitors THL/PMSF. RESULTS: In-silico analysis revealed that Rv1288 is a two domain protein, an N-terminal LytE domain containing three consecutive LysM motifs and a C-terminal esterase domain of esterase D family. LytE domain has the property to bind N-acetyl glucosamine moieties of peptidoglycan, a major component of cell wall. Detailed in-silico sequence analysis revealed that this LytE domain may help in positioning the esterase domain to the cell wall of mycobacterium. Esterase domain comprised a tetrapeptide motif HGGG, a pentapeptide sequence motif GxSxG and conserved amino acid residues Ser-141, Asp-238 and His-272 which constitute a catalytic triad characteristic of other hormone sensitive lipases/ esterases. Docking studies suggested that THL and PMSF could be the potent inhibitors for Rv1288 protein. CONCLUSION: In the present investigation, we bioinformatically confirmed that Rv1288 is most likely a LytE domain containing lipolytic enzyme showing similarity to hormone sensitive lipases/esterases.

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.

Virulence and immunity are poorly understood in Mycobacterium tuberculosis. We sequenced the complete genome of the M. tuberculosis clinical strain CDC1551 and performed a whole-genome comparison with the laboratory strain H37Rv in order to identify polymorphic sequences with potential relevance to disease pathogenesis, immunity, and evolution. We found large-sequence and single-nucleotide polymorphisms in numerous genes. Polymorphic loci included a phospholipase C, a membrane lipoprotein, members of an adenylate cyclase gene family, and members of the PE/PPE gene family, some of which have been implicated in virulence or the host immune response. Several gene families, including the PE/PPE gene family, also had significantly higher synonymous and nonsynonymous substitution frequencies compared to the genome as a whole. We tested a large sample of M. tuberculosis clinical isolates for a subset of the large-sequence and single-nucleotide polymorphisms and found widespread genetic variability at many of these loci. We performed phylogenetic and epidemiological analysis to investigate the evolutionary relationships among isolates and the origins of specific polymorphic loci. A number of these polymorphisms appear to have occurred multiple times as independent events, suggesting that these changes may be under selective pressure. Together, these results demonstrate that polymorphisms among M. tuberculosis strains are more extensive than initially anticipated, and genetic variation may have an important role in disease pathogenesis and immunity.

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.