1,912 research outputs found
Platensimycin Activity against Mycobacterial β-Ketoacyl-ACP Synthases
No full textBackground - There is an urgent need for the discovery and development of new drugs against Mycobacterium tuberculosis, the causative agent of tuberculosis, especially due to the recent emergence of multi-drug and extensively-drug resistant strains. Herein, we have examined the susceptibility of mycobacteria to the natural product platensimycin.
Methods and Findings - We have demonstrated that platensimycin has bacteriostatic activity against the fast growing Mycobacterium smegmatis (MIC = 14 µg/ml) and against Mycobacterium tuberculosis (MIC = 12 µg/ml). Growth in the presence of paltensimycin specifically inhibited the biosynthesis of mycolic acids suggesting that the antibiotic targeted the components of the mycolate biosynthesis complex. Given the inhibitory activity of platensimycin against β-ketoacyl-ACP synthases from Staphylococcus aureus, M. tuberculosis KasA, KasB or FabH were overexpressed in M. smegmatis to establish whether these mycobacterial KAS enzymes were targets of platensimycin. In M. smegmatis overexpression of kasA or kasB increased the MIC of the strains from 14 µg/ml, to 30 and 124 µg/ml respectively. However, overexpression of fabH on did not affect the MIC. Additionally, consistent with the overexpression data, in vitro assays using purified proteins demonstrated that platensimycin inhibited Mt-KasA and Mt-KasB, but not Mt-FabH.
Significance - Our results have shown that platensimycin is active against mycobacterial KasA and KasB and is thus an exciting lead compound against M. tuberculosis and the development of new synthetic analogues
EBUS-GS and VBN for GGO lesions
No full textBackground: Endobronchial ultrasonography with guide sheath (EBUS-GS) could be useful for diagnosing ground-glass opacity (GGO) predominant-type lesions in the peripheral lung. Furthermore, several studies have reported that transbronchial biopsy using EBUS-GS and virtual bronchoscopic navigation (VBN) was safe and effective for diagnosing small peripheral lung lesions. Our objectives were to diagnose solitary peripheral GGO predominant-type lesions by transbronchial biopsy using EBUS-GS and VBN under radiographic fluoroscopic guidance, and to evaluate the clinical factors associated with diagnostic yield. Methods: The medical records of 169 patients with GGO predominant-type lesions who underwent transbronchial biopsy using EBUS-GS and VBN under radiographic fluoroscopic guidance were retrospectively reviewed. Results: Endobronchial ultrasonography images could be obtained for 156 (92%) of 169 GGO predominant-type lesions, and 116 (69%) were successfully diagnosed by this method (20 of 31 pure GGO lesions [65%]; 96 of 138 mixed GGO predominant-type lesions [70%]). The mean size of diagnosed lesions was significantly larger than that of nondiagnosed lesions (22 mm versus 18 mm, p < 0.01). Regarding diagnostic yield based on computed tomography sign, cases with presence of a bronchus leading directly to a lesion had significantly higher diagnostic yield than the other lesions (p < 0.01). Conclusions: The addition of VBN to EBUS-GS could be useful in clinical practice for diagnosing GGO predominant-type lesions in the peripheral lung
Modular Approach to Triazole-Linked 1,6-alpha-D-Oligomannosides to the Discovery of Inhibitors of Mycobacterium tuberculosis Cell Wall Synthetase
No full textAiming at developing inhibitors of mannosyltransferases, the enzymes that participate in the biosynthesis of the cell envelope of Mycobacterium tuberculosis, the synthesis of a range of designed triazole-linked 1,6-oligomannosides up to a hexadecamer has been accomplished by a modular approach centered on the Cu(I)-catalyzed azide-alkyne cycloaddition as key process. The efficiency and fidelity of the cycloaddition are substantiated by high yields (76-96%) and exclusive formation of the expected 1,4-disubstituted triazole ring in all oligomer assembling reactions. Key features of oligomers thus prepared are the anomeric carbon-carbon bond of all mannoside residues and the 6-deoxymannoside capping residue. Suitable bioassays with dimer, tetramer, hexamer, octamer, decamer, and hexadecamer showed variable inhibitor activity against mycobacterial alpha-(1,6)-mannosyltransferases, the highest activity (IC50 = 0.14-0.22 mM) being registered with the hexamannoside and octamannoside
Identification of 2-Aminothiazole-4-Carboxylate Derivatives Active against Mycobacterium tuberculosis H37Rv and the beta-Ketoacyl-ACP Synthase mtFabH
No full textBackground: Tuberculosis (TB) is a disease which kills two million people every year and infects approximately over one-third of the world's population. The difficulty in managing tuberculosis is the prolonged treatment duration, the emergence of drug resistance and co-infection with HIV/AIDS. Tuberculosis control requires new drugs that act at novel drug targets to help combat resistant forms of Mycobacterium tuberculosis and reduce treatment duration.Methodology/Principal Findings: Our approach was to modify the naturally occurring and synthetically challenging antibiotic thiolactomycin (TLM) to the more tractable 2-aminothiazole-4-carboxylate scaffold to generate compounds that mimic TLM's novel mode of action. We report here the identification of a series of compounds possessing excellent activity against M. tuberculosis H37Rv and, dissociatively, against the beta-ketoacyl synthase enzyme mtFabH which is targeted by TLM. Specifically, methyl 2-amino-5-benzylthiazole-4-carboxylate was found to inhibit M. tuberculosis H37Rv with an MIC of 0.06 mu g/ml (240 nM), but showed no activity against mtFabH, whereas methyl 2-(2-bromoacetamido)-5-(3-chlorophenyl)thiazole-4-carboxylate inhibited mtFabH with an IC50 of 0.95 +/- 0.05 mu g/ml (2.43 +/- 0.13 mu M) but was not active against the whole cell organism.Conclusions/Significance: These findings clearly identify the 2-aminothiazole-4-carboxylate scaffold as a promising new template towards the discovery of a new class of anti-tubercular agents
Microarray analysis of defined Mycobacterium tuberculosis populations using RNA amplification strategies.
No full textBACKGROUND: The amplification of bacterial RNA is required if complex host-pathogen interactions are to be studied where the recovery of bacterial RNA is limited. Here, using a whole genome Mycobacterium tuberculosis microarray to measure cross-genome representation of amplified mRNA populations, we have investigated two approaches to RNA amplification using different priming strategies. The first using oligo-dT primers after polyadenylation of the bacterial RNA, the second using a set of mycobacterial amplification-directed primers both linked to T7 polymerase in vitro run off transcription.
RESULTS: The reproducibility, sensitivity, and the representational bias introduced by these amplification systems were examined by contrasting expression profiles of the amplified products from inputs of 500, 50 and 5 ng total M. tuberculosis RNA with unamplified RNA from the same source. In addition, as a direct measure of the effectiveness of bacterial amplification for identifying biologically relevant changes in gene expression, a model M. tuberculosis system of microaerophilic growth and non-replicating persistence was used to assess the capability of amplified RNA microarray comparisons. Mycobacterial RNA was reproducibly amplified using both methods from as little as 5 ng total RNA (~equivalent to 2 x 105 bacilli). Differential gene expression patterns observed with unamplified RNA in the switch from aerobic to microaerophilic growth were also reflected in the amplified expression profiles using both methods.
CONCLUSION: Here we describe two reproducible methods of bacterial RNA amplification that will allow previously intractable host-pathogen interactions during bacterial infection to be explored at the whole genome level by RNA profiling
Thiacetazone, an antitubercular drug that inhibits cyclopropanation of cell wall mycolic acids in mycobacteria
No full textBackground. Mycolic acids are a complex mixture of branched, long-chain fatty acids, representing key components of the highly hydrophobic mycobacterial cell wall. Pathogenic mycobacteria carry mycolic acid sub-types that contain cyclopropane rings. Double bonds at specific sites on mycolic acid precursors are modified by the action of cyclopropane mycolic acid synthases (CMASs). The latter belong to a family of S-adenosyl-methionine-dependent methyl transferases, of which several have been well studied in Mycobacterium tuberculosis, namely, MmaA1 through A4, PcaA and CmaA2. Cyclopropanated mycolic acids are key factors participating in cell envelope permeability, host immunomodulation and persistence of M. tuberculosis. While several antitubercular agents inhibit mycolic acid synthesis, to date, the CMASs have not been shown to be drug targets. Methodology/Principle Findings. We have employed various complementary approaches to show that the antitubercular drug, thiacetazone (TAC), and its chemical analogues, inhibit mycolic acid cyclopropanation. Dramatic changes in the content and ratio of mycolic acids in the vaccine strainMycobacterium bovis BCG, as well as in the related pathogenic speciesMycobacterium marinum were observed after treatment with the drugs. Combination of thin layer chromatography, mass spectrometry and Nuclear Magnetic Resonance (NMR) analyses of mycolic acids purified fromdrug-treated mycobacteria showed a significant loss of cyclopropanation in both the a- and oxygenated mycolate sub-types. Additionally, High-Resolution Magic Angle Spinning (HR-MAS) NMR analyses on whole cells was used to detect cell wall-associated mycolates and to quantify the cyclopropanation status of the cell envelope. Further, overexpression of cmaA2, mmaA2 or pcaA in mycobacteria partially reversed the effects of TAC and its analogue on mycolic acid cyclopropanation, suggesting that the drugs act directly on CMASs. Conclusions/Significance. This is a first report on them echanism of action of TAC, demonstrating the CMASs as its cellular targets in mycobacteria. The implications of this study may be important for the design of alternative strategies for tuberculosis treatment
Re-emergence of tuberculosis: strategies and treatment
No full textTuberculosis (TB) was declared a global emergency by the WHO in 1993. Approximately one third of the world\u27s population is infected with Mycobacterium tuberculosis. Despite the availability of effective chemotherapy, 3.5 million TB deaths occur each year. In addition to this human cost, TB also represents a significant economic burden for developing countries. The worldwide spread of TB is mainly caused by the emergence of multidrug-resistant (MDR) M. tuberculosis strains, thus underlining the need for novel antimycobacterial drugs with improved efficacy. Research in TB chemotherapy, as well as in vaccine development, has entered an intense phase in an attempt to combat this disease
Cytidine diphosphate-diacylglycerol synthesis in Mycobacterium smegmatis
No full textRecent studies have demonstrated that, during infection of macrophages by mycobacteria, phospholipids (PLs) are released from the mycobacterial cell wall within infected macrophages and transported out of this compartment into intracellular vesicles. The release of these PLs may have functions that influence the outcome of mycobacterial infections. Despite their important role, little is known about the biosynthesis of PLs in mycobacteria. In all organisms, PL biosynthesis begins with acylation of sn-glycerol 3-phosphate to form phosphatidic acid (PA), which is then converted to the central liponucleotide intermediate, cytidine diphosphate-diacylglycerol (CDP-DAG) via the CDP-DAG synthase (CDS). The present work examines CDS activity in Mycobacterium smegmatis extracts, with regard to subcellular localization, pH dependence, bivalent and univalent cation requirement, substrate specificity and regulation by nucleotides. We show that CDS activity, which is mainly found within the cytoplasmic membrane, is Mg2+-dependent and activated by K+ ions. Among PAs containing saturated fatty acids, dipalmitoyl-PA is the preferred substrate [K-m = 0.23+/-0.03 mM for Triton X-100 (v/v)/PA in the ratio 5: 1]. Moreover, CDS activity is inhibited by the reaction products PPi (IC50 = 1.5 mM), CDP-DAG (IC50 = 0.3 mM) and the nucleotides ATP, UTP and GTP. This study contributes to the delineation of PL biosynthesis in mycobacteria
Characterization and regulation of inositol monophosphatase activity in Mycobacterium smegmatis
No full textMycobacterium tuberculosis and related members of the genus Mycobacterium contain a number of inositol-based lipids, such as phosphatidylinositol mannosides, lipomannan and lipoarabinomannan. The synthesis of phosphatidylinositol in M. smegmatis is essential for growth and myo-inositol is a key metabolite for mycobacteria. Little is known about the biosynthesis of inositol in mycobacteria and the only known de novo pathway for myo-inositol biosynthesis involves a two-step process. First, cyclization of glucose 6-phosphate to afford myo-inositol I-phosphate via inositol-1-phosphate synthase and, secondly, dephosphorylation of myo-inositol 1-phosphate by inositol monophosphatase (IMP) to afford myo-inositol. The following report examines IMP activity in M. smegmatis extracts, with regard to pH dependence, bivalent cation requirement, univalent cation inhibition, regulation by growth and carbon source. We show that IMP activity, which is optimal at the end of the exponential growth phase in Sauton\u27s medium, is Mg2+-dependent. Moreover, IMP activity is inhibited by Li+ and Na+, with Li+ also being able to inhibit growth of M. smegmatis in vivo. This study represents a first step in the delineation of myo-inositol biosynthesis in mycobacteria
Biosynthesis of the galactan component of the mycobacterial cell wall
No full textThe structural core of the cell walls of Mycobacterium spp. consists of peptidoglycan bound by a linker unit (-α-L-Rhap-(1→3)-D-GlcNAc-P-) to a galactofuran, which in turn is attached to arabinofuran and mycolic acids. The sequence of reactions leading to the biogenesis of this complex starts with the formation of the linker unit on a polyprenyl-P to produce polyprenyl-P-P-GlcNAc-Rha (Mikusova, K., Mikus, M., Besra, G. S., Hancock, I., and Brennan, P. J. (1996) J. Biol. Chem. 271, 7820-7828). We now establish that formation of the galactofuran takes place on this intermediate with UDP-Galf as the Galf donor presented in the form of UDP-Galp and UDP-Galp mutase (the glf gene product) and is catalyzed by galactofuranosyl transferases, one of which, the Mycobacterium tuberculosis H37Rv3808c gene product, has been identified. Evidence is also presented for the growth of the arabinofuran on this polyprenyl-P-P-linker unit-galactan intermediate catalyzed by unidentified arabinosyl transferases, with decaprenyl-P-Araf or 5-P-ribosyl-PP as the Araf donor. The product of these steps, the lipid-linked-LU-galactan-arabinan has been partially characterized in terms of its heterogeneity, size, and composition. Biosynthesis of the major components of mycobacterial cell walls is proving to be extremely complex. However, partial definition of arabinogalactan synthesis, the site of action of several major anti-tuberculosis drugs, facilitates the present day thrust for new drugs to counteract multiple drug-resistant tuberculosis
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