ABSTRACT: Transcriptomic analysis of Mtb H37Rv and MtbΔwhiB4 on treatment to 10X Augmentin (AG) i.e. combination of 100 µg/ml of Amoxicillin and 8 µg/ml of Clavulanate and lower Augmentin doses at 1X and 5X
Project description:Global protein alteration in Mycobacterium tuberculosis H37 RV ( Mtb-H37RV) of day 2 (control) and day 16 (persistent) samples (in duplicates) treated with antibiotics 8µg/ml of ciprofloxacin and 0.1 µg/ml of Isoniazid (to mimic persister stage) were accessed using 4 plex iTRAQ label.
Project description:HupB is a 28 kDa in Mycobacterium tuberculosis that is co-expressed with the siderophores mycobactin and carboxymycobactin upon iron limitation. High levels of all the three components are seen in low iron (LI; 0.02 µg Fe / mL) organisms, with negligible expression in high iron organisms (HI; 8 µg Fe / mL). We generated a hupB knock out mutant of M. tuberculosis (H37Rv ∆ hupB) and studied the differential expression of genes upon iron limitation in the WT H37Rv and the mutant. The RNA transcripts of the WT H37Rv, grown under high and low iron conditions of growth were isolated and subjected to microarray analysis to identify the iron-regulated genes and second, the differential expression of genes in iron-limited H37Rv ∆ hupB vs iron-limited WT H37Rv was analysed. Microarray analysis was done commercially by Genotypic Technology (Bangalore, India), an authorised service provider for Agilent Technologies. The study revealed the up-regulation of all the mbt genes of the mycobactin biosynthetic machinery in LI - H37Rv and several other reported iron-regulated genes. The salient feature of this study is the failure of LI - H37Rv ∆ hupB to show any up-regulation of the mbt genes as compared to LI - H37Rv. Among several other genes influenced by HupB, the mutant strain showed low levels of mmpL5 and mmpS5 transcripts, whose expressed products are reported to be associated with siderophore transport and biosynthesis.
Project description:Mtb H37Rv was found to be highly susceptible to ATD-3169 (Redox generating compound). To further understand the mechanism of ATD-3169 action, we performed microarray analysis of Mtb H37Rv exposed to 3µM, 30 µM and 150µM of ATD-3169 for 4h.
Project description:The peritoneal macrophages were infected with Mtb H37Rv for 4 hours, and the miRNA expression profile were analyzed with deep sequencing.
Project description:Gene expression microarray was performed using early log phase culture at 37 degree and 200 rpm followed by RNA extraction from M.tb H37Rv, HigB KO M.tb H37Rv and complemented Hig B KO M.tb H37Rv For gene expression profiling, total RNA was isolated from wild type H37Rv, higB KO mutant strain and complemented strains. The bacterial pellets for M.tb H37Rv wild type, M.tb H37Rv HigB KO mutant strain and complemented strain were resuspended in 1.0 ml TRIzol (Invitrogen Corporation, Carlsbad, CA); mRNA was extracted as per standard protocols and cleaned using RNAeasy columns (Qiagen GmbH, Hilden, Germany). Extracted mRNA (1 μg) was treated with DNase I using the Ambion DNA-free kit (Invitrogen Corporation, Carlsbad, CA) to remove genomic DNA contamination and quantified using Nanodrop 2000c spectrophotometer (Thermo Scientific, USA). The purity and integrity of RNA samples were assessed Agilent 2100 Bio analyzer (Agilent TechnologiesInc., USA). Further, 25ng of RNA was amplified and labeled using Low input Quick Amp WT Labeling kit (Agilent Technologies, USA). The labeled cRNA was purified using RNAeasy columns (Qiagen, USA) and total yields were quantified using Nanodrop 2000c spectrophotometer.
Project description:Expression profile of Mycobacterium tuberculosis H37Rv biofilm as induced by DTT (Reduced) 6mM DTT reduced at 6 mM concentration was added to log phase culture of Mtb H37Rv. After 29 hours RNA was isolated and hybridization was done on microarrays
Project description:The C57B/6 mice were infected with Mtb H37Rv (CFU=200) for 28 days, and the miRNA expression profile from lung tissues were analyzed with deep sequencing.
Project description:Gene expression profiling of Mycobacterium tuberculosis H37Rv in response to compound CDRI-5g (Saquib, M. et al. Eur. J. Med. Chem. 46 (2011) 2217-2223) was studied by administration of compound at 10 µg/ml in broth culture. RNA was extracted at two time points 12 h and 24 h of growth from replicating phase and compound added culture. Replicating phase culture was incubated for the same time and used as a control. Experiment was repeated three times and for each time points triplicate culture was pelleted to isolate total RNA using Qiagen RNA isolation kit.
