Transcritpome analysis of Mycobacterium tuberculosis (Mtb) mutants deficient in ergothioneine, or mycothiol production
ABSTRACT: This experiment was performed to determine deferentially regulated genes in the absence of redox buffer ergothioneine (EGT), or mycothiol (MSH). We had used two Mtb mutants namely egtA:Tn and egtD:Tn which are deficient in EGT production ( Transposon Mutant of EGT biosynthesis pathway); In addition, we also used Mtb mshA mutant, which does not produce MSH ( Deletion mutant of Mycothiol biosynthesis ). For microarray analysis, RNA was obtained from exponentially growing (OD 600 nm - 0.6-0.8) CDC1551 (wild type), mshA mutant, egtA:Tn and egtD:Tn strains cultured in 7H9 medium supplemented with ADST (Albumin Dextrose Saline Tyloxapol) using RNApro (MP Biomedicals, USA) as per manufacturer’s instructions. The quality of RNA was examined by running the samples on Experion gene chips (BioRad).
The mechanisms by which Mycobacterium tuberculosis (Mtb) maintains metabolic equilibrium to survive during infection and upon exposure to antimycobacterial drugs are poorly characterized. Ergothioneine (EGT) and mycothiol (MSH) are the major redox buffers present in Mtb, but the contribution of EGT to Mtb redox homeostasis and virulence remains unknown. We report that Mtb WhiB3, a 4Fe-4S redox sensor protein, regulates EGT production and maintains bioenergetic homeostasis. We show that central c ...[more]
Project description:Following phagocytosis by macrophages, Mycobacterium tuberculosis (Mtb) senses the intracellular environment and remodels its gene expression for growth in the phagosome. Abramovitch et.al. in this current study identified an Acid and Phagosome Regulated (aprABC) locus that is unique to the Mtb complex and whose gene expression is induced during growth in acidic environments in vitro and in macrophages. The authors propose a model where phoP senses the acidic pH of the phagosome and induces aprABC expression to fine-tune processes unique for intracellular adaptation of Mtb complex bacteria. This study uses microarray analyses to compare transcriptional responses of wild type Mycobacterium tuberculosis (CDC1551) to aprABC locus deletion mutants and the phoP transposon mutant. The bacteria were grown to early log phase in vented T-75 standing flasks containing 12 mL of pH 7.0 7H9 OADC medium. Transcript levels of the wild type bacteria were compared to the following mutants: aprABC null, aprBC null, aprC null, phoP::Tn mutant.
Project description:The purpose of this study was to examine how ethoxzolamide modulates gene M. tuberculosis gene expression at acidic pH. We observed that ethoxzolamide downregulates genes of the PhoPR regulon. Mtb strain CDC1551 was grown at 37C in T-25 vented, standing tissue culture flasks in 8 ml of buffered 7H9 medium seeded an initial OD of 0.2. Three conditions were examined with two biological replicates: 1) DMSO treated, pH 7.0, 2) DMSO treated, pH 5.7, 3) 40 µM ethoxzolamide treated, pH 5.7. The CDC1551(phoP::Tn) mutant was grown in a similar manner and treated with DMSO at pH 7.0 and 5.7. Following six days incubation, total bacterial RNA was extracted and analyzed as previously described.
Project description:Sigma factor E (SigE) controls the expression of genes that are essential for Mtb virulence. In this work, we have identified the SigE regulon during infection of macrophages Our results indicate that SigE regulates the expression of genes involved in the maintenance of Mtb cell envelope integrity and function (i.e., detoxification and secretion). Keywords: strains comparison We compared the global gene expression of the H37Rv strain and the sigE mutant strain of Mtb in different conditions: growing exponentially in liquid media; after 2h of incubation in RPMI; or after 24 hours of infection of human macrophage-like THP-1 cells.
Project description:To characterize the role of Rv0516c in the osmotic stress response of Mycobacterium tuberculosis (Mtb), we performed transcriptional profiling of CDC1551 Rv0516c::Tn following treatment with 140 mM NaCl for 1 hr relative to an untreated control. CDC1551 Rv0516c::Tn was treated with 140 mM NaCl for 1 h and compared to the same strain treated with 0 mM NaCl for 0 h. 4 biological replicates, independently grown and harvested. One replicate per array.
Project description:During lung infection Mycobacterium tuberculosis (Mtb) resides in macrophages and subverts the bactericidal mechanisms of these professional phagocytes. In this work we have analyzed by DNA microarray technique the global transcription profile of Mtb infecting primary human macrophages in order to identify putative bacterial pathogenic factors that can be relevant for the intracellular survival of Mtb. Keywords: time course We compared the global gene expression of the H37Rv strain of Mtb after 4 hours and 24 hours of infection of human macrophage-like THP-1 cells with the gene expression profile of the strain growing exponentially in broth cultures.
Project description:The gene expression profiles of Mtb after treatment at the minimal inhibitory concentration (MIC) or 4 X MIC at an early stage (up to 6 hours) was compared to untreated Mtb. Our experiment is designed to in order to ensure that the primary effects (0-6h) of the drugs and any dose (1X MIC and 4X MIC) responses would be captured.
Project description:We sequenced mRNA from Mtb that had been treated with 5 mM nitrate or was untreated in standard 7H9/ADNaCl medium. This generated the first analysis of gene expression following mycobacterial nitrate respiration. Examination of mRNA levels in Mtb following nitrate respiration, which results in the production of nitrite.
Project description:M. tuberculosis thioredoxin reductase (TrxB2) is essential for Mtb physiology and pathogenesis. To gain insight into its biological functions, we generated the TrxB2-TetON-tetO mutants, in which TrxB2 is partially depleted in the absence of atc without impairing bacterial viability. We grew TrxB2-TetON-tetO-WT in 7H9 medium with 400 ng/ml atc, until the OD reached 0.5~0.6. Then Mtb was washed with 7H9 medium 3 times and suspended in 7H9 medium with or without atc. Then samples were taken 24, 48, 72 and 120 hrs later.
Project description:Background: Conflicting results have been reported about the role of the two-component sensor and transcriptional regulator DosS/DosR, controlling the expression of the dormancy DosR regulon, for in vivo virulence of M. tuberculosis. Here, we have used a new approach to further analyze the relevance of the dosRS system, by driving DosR (Rv3133c) expression under the control of a constitutive promoter (phsp60). Methodology/Principal Findings: M. tuberculosis H37Rv constitutively expressing the transcriptional regulator DosR (Mtb::DosR) was compared to wild type M. tuberculosis (Mtb+/+) for in vitro growth kinetics and expression of the target genes of the DosR dormancy regulon, for in vivo virulence and for immunogenicity in mice. Under aerobic conditions, hsp60-driven DosR induced the expression of 28 out of 39 tested DosR regulon genes. In vitro growth characteristics were comparable for both strains, but Mtb::DosR showed an attenuated in vivo phenotype in immunocompetent mice, as indicated by reduced bacterial replication, reduced pulmonary immunopathology, reduced cachexia and significantly prolonged survival time as compared Mtb+/+. In immunodeficient SCID mice, Mtb::DosR was fully virulent. RT-qPCR analysis revealed a strong and comparable pulmonary TNF-?? and IL-23 expression following intratracheal infection, whereas IL-12 and IL-17 expression was slightly higher with wild type Mtb+/+. Finally, mice persistently infected with Mtb::DosR for 8 months showed five to tenfold higher lung IFN-?? responses against ten of the 48 DosR regulon encoded antigens (Rv1733c, Rv1734, Rv1738, Rv1996, Rv1997, Rv2029c, Rv2623, Rv2627c, Rv2628 and Rv3127) than mice actively infected with Mtb+/+. In spleen however, DosR regulon encoded antigen specific IFN-?? responses were similar in both groups. Conclusions/Significance. Collectively, these results suggest that increased DosR regulon encoded antigen specific pulmonary T cell responses are responsible for the attenuated phenotype of Mtb::DosR and that infection with Mtb::DosR could be used as a new animal model for studying key aspects of latent tuberculosis. Set of arrays that are part of repeated experiments