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:Transcriptional profiling of Mycobacterium tuberculosis CDC1551 and 6-azasteroid resistant mutants ASR1 and ASR2 with treatment of 6-azasteroids, 4a and 6a, for 6 hours.
Project description:Transcription profile of WT compare to sigma factor sigI deletion mutant at OD1 and OD2. This microarray is simple comparison of wild type strain of M. tuberculosis CDC1551 and its sigma factor gene deletion mutant (sigI) at OD1 and OD2, respectively.
Project description:Transcriptional profiling of Mycobacterium tuberculosis CDC1551 comparing control butyric acid-treated cells with 20mM db-cAMP treated cells after 2hrs of treatment with shaking at 200rpm at 37C.
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:Mechanisms governing Mycobacterium tuberculosis acid-fastness and its capacity to induce long-term infections remain unknown. Serine/Threonine phosphorylation represents an emerging theme allowing mycobacteria to adapt their cell envelope structure/composition in response to environmental changes. We addressed whether phosphorylation of KasB, a mycolic acid biosynthetic enzyme, modulates M. tuberculosis pathogenicity. Phosphorylation of KasB occurred at Thr334 and Thr336 in vitro and in mycobacteria. A mutant strain bearing an kasB_T334D/T336D allele, mimicking constitutive KasB phosphorylation, was generated by specialized linkage transduction. This resulted in shortened mycolic acids and the lack of trans-cyclopropanation. Structural/modeling analyses revealed Thr334 and Thr336 in the vicinity of the catalytic triad, implying that phosphorylation of these residues impaired KasB activity. Importantly, the phosphomimetic strain lost acid-fast staining and was more attenuated than a kasB deletion mutant in immunocompetent and immunodeficient mice. The absence of lung pathology and mortality infers this mutant to represent a valuable vaccine candidate. This work emphasizes the critical role of Ser/Thr kinase-dependent signaling in controlling mycolic acid elongation, acid-fastness, virulence and has important clinical implications for diagnosis of latent infections. Transcriptome of kasB null, phosphoablative, and phosphomimetic mutants compared to parental. Triplicate 10ml cultures of M. tuberculosis CDC1551 and kasB null, phosphoablative, and phosphomimetic mutants were grown to OD 1.0 and harvested for transcriptional profiling.
Project description:Transcriptional profiling of Mycobacterium tuberculosis CDC1551 comparing control butyric acid-treated cells with 20mM db-cAMP treated cells after 2hrs of treatment with shaking at 200rpm at 37C. Two-condition experiment, butyric acid (Control) Vs db-cAMP treated cells (Test). Biological replicates: 3 control, 3 test. One replicate per array.
Project description:Proteomics on whole cell lysates from Mycobacterium tuberculosis CDC1551, a ppe38-ppe71 deletion mutant and a complemented strain grown in modified sautons medium till mid logarithmic phase
Project description:A M. tuberculosis transposon library was used to infect WT and iNOS-/- mice. Surviving mutants were recovered from spleens, genomic DNA was extracted, and labeled probes were synthesized from transposon ends. Probes from each WT mouse were hybridized with probes from a similar iNOS-/- mouse. Two-condition experiment, Growth in WT vs. iNOS-/- mice. Biological replicates: TraSH probe made from 5 wild type and 5 iNOS-/- mice after 3 weeks of infection and from 8 wild type and 8 iNOS-/- mice after 4 weeks of infection. Technical replicates: TraSH probe was synthesized twice from each mouse and dyes were swapped. One array contains probe from one iNOS-/- and one WT mouse. Each biological replicate has two arrays, representing technical replicate dye swaps from one iNOS-/- and one WT mouse.
Project description:Supernatants of M. tuberculosis CDC1551, a ppe38-ppe71 knock out mutant and a complemented strain containing tween was used for mass spectrometry and proteomics study to identify the differential regulation of proteins between these strains.
