Gene_locus Report for: myctu-yt28

With the high number of patients infected by tuberculosis and the sharp increase of drug-resistant tuberculosis cases, developing new drugs to fight this disease has become increasingly urgent. In this context, analogs of the naturally occurring enolphosphates Cyclipostins and Cyclophostin (CyC analogs) offer new therapeutic opportunities. The CyC analogs display potent activity both in vitro and in infected macrophages against several pathogenic mycobacteria including Mycobacterium tuberculosis and Mycobacterium abscessus. Interestingly, these CyC inhibitors target several enzymes with active-site serine or cysteine residues that play key roles in mycobacterial lipid and cell wall metabolism. Among them, TesA, a putative thioesterase involved in the synthesis of phthiocerol dimycocerosates (PDIMs) and phenolic glycolipids (PGLs), has been identified. These two lipids (PDIM and PGL) are non-covalently bound to the outer cell wall in several human pathogenic mycobacteria and are important virulence factors. Herein, we used biochemical and structural approaches to validate TesA as an effective pharmacological target of the CyC analogs. We confirmed both thioesterase and esterase activities of TesA, and showed that the most active inhibitor CyC17 binds covalently to the catalytic Ser104 residue leading to a total loss of enzyme activity. These data were supported by the X-ray structure, obtained at a 2.6-A resolution, of a complex in which CyC17 is bound to TesA. Our study provides evidence that CyC17 inhibits the activity of TesA, thus paving the way to a new strategy for impairing the PDIM and PGL biosynthesis, potentially decreasing the virulence of associated mycobacterial species.

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.

With the high number of patients infected by tuberculosis and the sharp increase of drug-resistant tuberculosis cases, developing new drugs to fight this disease has become increasingly urgent. In this context, analogs of the naturally occurring enolphosphates Cyclipostins and Cyclophostin (CyC analogs) offer new therapeutic opportunities. The CyC analogs display potent activity both in vitro and in infected macrophages against several pathogenic mycobacteria including Mycobacterium tuberculosis and Mycobacterium abscessus. Interestingly, these CyC inhibitors target several enzymes with active-site serine or cysteine residues that play key roles in mycobacterial lipid and cell wall metabolism. Among them, TesA, a putative thioesterase involved in the synthesis of phthiocerol dimycocerosates (PDIMs) and phenolic glycolipids (PGLs), has been identified. These two lipids (PDIM and PGL) are non-covalently bound to the outer cell wall in several human pathogenic mycobacteria and are important virulence factors. Herein, we used biochemical and structural approaches to validate TesA as an effective pharmacological target of the CyC analogs. We confirmed both thioesterase and esterase activities of TesA, and showed that the most active inhibitor CyC17 binds covalently to the catalytic Ser104 residue leading to a total loss of enzyme activity. These data were supported by the X-ray structure, obtained at a 2.6-A resolution, of a complex in which CyC17 is bound to TesA. Our study provides evidence that CyC17 inhibits the activity of TesA, thus paving the way to a new strategy for impairing the PDIM and PGL biosynthesis, potentially decreasing the virulence of associated mycobacterial species.

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.

A new class of Cyclophostin and Cyclipostins (CyC) analogs have been investigated against Mycobacterium tuberculosis H37Rv (M. tb) grown either in broth medium or inside macrophages. Our compounds displayed a diversity of action by acting either on extracellular M. tb bacterial growth only, or both intracellularly on infected macrophages as well as extracellularly on bacterial growth with very low toxicity towards host macrophages. Among the eight potential CyCs identified, CyC 17 exhibited the best extracellular antitubercular activity (MIC50 = 500 nM). This compound was selected and further used in a competitive labelling/enrichment assay against the activity-based probe Desthiobiotin-FP in order to identify its putative target(s). This approach, combined with mass spectrometry, identified 23 potential candidates, most of them being serine or cysteine enzymes involved in M. tb lipid metabolism and/or in cell wall biosynthesis. Among them, Ag85A, CaeA and HsaD, have previously been reported as essential for in vitro growth of M. tb and/or survival and persistence in macrophages. Overall, our findings support the assumption that CyC 17 may thus represent a novel class of multi-target inhibitor leading to the arrest of M. tb growth through a cumulative inhibition of a large number of Ser- and Cys-containing enzymes participating in important physiological processes.

Infection of the zebrafish with Mycobacterium marinum is regarded as a well-established experimental model to study the pathogenicity of Mycobacterium tuberculosis. Herein, a M. marinum transposon mutant library was screened for attenuated M. marinum phenotypes using a Dictyostelium discoideum assay. In one attenuated mutant, the transposon was located within tesA, encoding a putative type II thioesterase. Thin-layer chromatography analyses indicated that the tesA::Tn mutant failed to produce two major cell wall-associated lipids. Mass spectrometry and nuclear magnetic resonance clearly established the nature of missing lipids as phthioglycol diphthioceranates and phenolic glycolipids, respectively, indicating that TesA is required for the synthesis of both lipids. When injected into the zebrafish embryo bloodstream, the mutant was found to be highly attenuated, thus validating the performance and relevance of the Dictyostelium screen. Consistent with these in vivo findings, tesA::Tn exhibited increased permeability defects in vitro, which may explain its failure to survive in host macrophages. Unexpectedly, virulence was retained when bacteria were injected into the notochord. Histological and ultrastructural studies of the infected notochord revealed the presence of actively proliferating mycobacteria, leading to larval death. This work presents for the first time the notochord as a compartment highly susceptible to mycobacterial infection.

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.

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.

AIMS: Phthiocerol dimycocerosate (PDIM) waxes and other lipids are necessary for successful Mycobacterium tuberculosis infection, although the exact role of PDIM in host-pathogen interactions remains unclear. In this study, we investigated the contribution of tesA, drrB, pks6 and pks11 genes in complex lipid biosynthesis in M. tuberculosis. METHODS AND RESULTS: Four mutants were selected from M. tuberculosis H37Rv transposon mutant library. The transposon insertion sites were confirmed to be within the M. tuberculosis open reading frames for tesA (a probable thioesterase), drrB (predicted ABC transporter), pks11 (putative chalcone synthase) and pks6 (polyketide synthase). The first three of these transposon mutants were unable to generate PDIM and the fourth lacked novel polar lipids. CONCLUSIONS: Mycobacterium tuberculosis can be cultivated in vitro without the involvement of certain lipid synthesis genes, which may be necessary for in vivo pathogenicity. SIGNIFICANCE AND IMPACT OF THE STUDY: The use of transposon mutants is a new functional genomic approach for the eventual definition of the mycobacterial 'lipidome'.

Polyketide synthases (PKSs) of Mycobacterium tuberculosis are increasingly being seen as producers of virulence factors that are important for pathogenesis by the bacterium. Thus, the phenolphthiocerol synthase PKS cluster of M. tuberculosis is responsible, in part, for the synthesis of a virulence determinant called phthiocerol dimycocerosate (PDIM). Here, we provide evidence that the PpsE protein, which is part of that cluster, interacts with the type II thioesterase TesA of M. tuberculosis. The interaction was demonstrated by employing a two-hybrid system, and confirmed using a GST (glutathione S-transferase) pull-down' assay after both proteins had been purified to homogeneity. Based on the present findings, a revised model for the processing of polyketides during the synthesis of PDIM is presented.

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.