Project description:These data represent the expression patterns of Mycobacterium tuberculosis in progressive hypoxia, nutrient depletion, and in-vivo hollow fiber models of dormancy. The assumptions are that the set of genes that respond to INH treatment during Log phase growth would not be differentially regulated during INH treatment in the dormancy models, and that the overall number of differentially regulated genes would be reduced do to the low metabolic state of the cells. Keywords: Dormancy Model Drug Response Comparison
Project description:A cell-based phenotypic screen for inhibitors of biofilm formation in Mycobacterium tuberculosis (Mtb) identified the small molecule TCA1, which has bactericidal activity against both drug susceptible and drug resistant Mtb, and synergizes with rifampicin (RIF) or isoniazid (INH) in sterilization of Mtb in vitro. In addition, TCA1 has bactericidal activity against non-replicating Mtb in vitro and is efficacious in acute and chronic Mtb infection mouse models, both alone and in combination with INH or RIF. Transcriptional analysis revealed that TCA1 down-regulates genes known to be involved in Mtb dormancy and drug tolerance. Mutagenesis and affinity-based methods identified DprE1 and MoeW, enzymes involved in cell wall and molybdenum cofactor biosynthesis, respectively, as the targets responsible for TCA1’s activity. These in vitro and in vivo results indicate that TCA1functions by a novel mechanism and suggest that it may be the first product of a promising new approach for the development of anti-tuberculosis drugs.
Project description:A cell-based phenotypic screen for inhibitors of biofilm formation in Mycobacterium tuberculosis (Mtb) identified the small molecule TCA1, which has bactericidal activity against both drug susceptible and drug resistant Mtb, and synergizes with rifampicin (RIF) or isoniazid (INH) in sterilization of Mtb in vitro. In addition, TCA1 has bactericidal activity against non-replicating Mtb in vitro and is efficacious in acute and chronic Mtb infection mouse models, both alone and in combination with INH or RIF. Transcriptional analysis revealed that TCA1 down-regulates genes known to be involved in Mtb dormancy and drug tolerance. Mutagenesis and affinity-based methods identified DprE1 and MoeW, enzymes involved in cell wall and molybdenum cofactor biosynthesis, respectively, as the targets responsible for TCA1M-bM-^@M-^Ys activity. These in vitro and in vivo results indicate that TCA1functions by a novel mechanism and suggest that it may be the first product of a promising new approach for the development of anti-tuberculosis drugs. Transcriptional profile of TCA1-treated cells relative to DMSO-treated control. Three biological replicates, third is a dye flip.
Project description:The human pathogen, Mycobacterium tuberculosis, develops a dormant infection, in which organisms survive within the body. We established a unique in vitro dormancy model based on the characterization of drug-resistance to INH and rifampin. M. tuberculosis cells were maintained in controlled and defined multiple stress conditions with low oxygen (5% dissolved oxygen tension), acid (pH 5.) along with glycerol-deprived medium conditions. To monitor gene expression changes in M. tuberculosis in response to the multiple stresses, we performed microarray analysis at the time point of 1, 2, 3, 6, and 12days after treatment. M. tuberculosis adapting to multiple stresses displayed characteristics associated with persistence in vivo, including entry into a non-replicative state and the repression of genes involved in energy regeneration. Under in vitro multiple-stresses, M. tuberculosis significantly modulated gene expression mainly in response to the starvation stresses. Cells exposed to these multiple stress conditions shows significant drug-resistance. Comparison with other in vivo expression profiles demonstrates induction of several common genes for in vitro dormancy conditions. Keywords: Stress response in time course.
Project description:Transcriptional profiling of Mycobacterium tuberculosis H37Rv strains comparing control DMSO treated strains with Linezolid treated strains. Goal was to determine the effects of Linezolid against Mycobacterium tuberculosis H37Rv strains.
Project description:Transcriptional profiling of Mycobacterium tuberculosis H37Rv strains comparing control DMSO treated strains with Lupulone treated strains. Goal was to determine the effects of Lupulone against Mycobacterium tuberculosis H37Rv strains.
Project description:The human pathogen, Mycobacterium tuberculosis, develops a dormant infection, in which organisms survive within the body. We established a unique in vitro dormancy model based on the characterization of drug-resistance to INH and rifampin. M. tuberculosis cells were maintained in controlled and defined multiple stress conditions with low oxygen (5% dissolved oxygen tension), acid (pH 5.) along with glycerol-deprived medium conditions. To monitor gene expression changes in M. tuberculosis in response to the multiple stresses, we performed microarray analysis at the time point of 1, 2, 3, 6, and 12days after treatment. M. tuberculosis adapting to multiple stresses displayed characteristics associated with persistence in vivo, including entry into a non-replicative state and the repression of genes involved in energy regeneration. Under in vitro multiple-stresses, M. tuberculosis significantly modulated gene expression mainly in response to the starvation stresses. Cells exposed to these multiple stress conditions shows significant drug-resistance. Comparison with other in vivo expression profiles demonstrates induction of several common genes for in vitro dormancy conditions. Keywords: Stress response in time course. Samples under multiple stress condition were taken at day 1, 2, 3, 6, and 12 for microarray hybridization. More than two technical replicates per biological samples with Cy3/5 dye-swaps.
Project description:The global protein expression of Mycobacterium tuberculosis H37Rv, responding to VC treatment (5 mM for 24 h), was monitored via tandem mass tag (TMT)-based quantitative proteomic analysis.
Project description:Currently available model organisms such as Mycobacterium smegmatis and Mycobacterium bovis Bacillus Calmette-Guérin (BCG) have significantly contributed to our understanding of tuberculosis (TB) biology, these models have limitations such as differences in genome size, growth rates and virulence. Attenuated Mycobacterium tuberculosis strains may provide more representative, safer models to study M. tuberculosis biology. For example, the M. tuberculosis ΔleuDΔpanCD double auxotroph, has undergone rigorous in vitro and in vivo safety testing. Like other auxotrophic strains, this has subsequently been approved for use in biosafety level (BSL) 2 facilities. Auxotrophic strains have been assessed as models for drug-resistant M. tuberculosis and for studying latent TB. These offer the potential as safe and useful models, but it is important to understand how well these recapitulate salient features of non-attenuated M. tuberculosis. We therefore performed a comprehensive comparison of M. tuberculosis H37Rv and M. tuberculosis ΔleuDΔpanCD. These strains demonstrated similar in vitro and intra-macrophage replication rates, similar responses to anti-TB agents and whole genome sequence conservation. Shotgun proteomics analysis suggested that M. tuberculosis ΔleuDΔpanCD has an increased propensity to enter a dormant state during acid stress, which has been verified using a dual-fluorescent replication reporter assay. Importantly, infection of human peripheral blood mononuclear cells with the 2 strains elicited comparable cytokine production, demonstrating the suitability of M. tuberculosis ΔleuDΔpanCD for immunological assays. We provide comprehensive evidence to support the judicious use of M. tuberculosis ΔleuDΔpanCD as a safe and suitable model organism for M. tuberculosis research, without the need for a BSL3 facility.