Mycobacterium tuberculosis H37Rv transcriptional response to antibacterial agents
ABSTRACT: Bacteria commonly adapt to stresses by altering gene expression. To understand the response of M. tuberculosis (MTB) to various antibacterial agents, we performed transcriptomics on MTB bacilli exposed to several test compounds as well as known drugs (capreomycin, cycloserine, ethionamide, isoniazid, kanamycin, moxifloxacin, PA-824, rifampicin, streptomycin). Bacteria were exposed for 16 hrs to various concentrations of each drug (different multiples of the compound's MIC), as noted in the title of each sample. RNA was isolated and applied to arrays provided by TIGR under the NIAID contract N01-AI-15447
Project description:Using cell-based approaches and experimental mouse models for pulmonary TB we unveiled MDSCs as new myeloid populations directly interacting with Mycobacterium tuberculosis (Mtb). MDSCs readily phagocytosed Mtb, released proinflammatory (IL-6, IL-1α) and immunomodulatory (IL-10) cytokines while retaining their suppressive capacity. MDSCs were identified at the site of infection in disease-resistant and -susceptible mice during pulmonary TB. Excessive MDSC accumulation in lungs correlated with elevated surface expression of IL-4Rα and heightened TB lethality. Microarray experiments were performed as dual-color hybridizations on Agilent mouse whole genome catalog 44K arrays. To compensate for dye-specific effects, a dye-reversal color-swap was applied.
Project description:Four cultures (four biological replicates) of each of two strains, wild type H37Rv and a deletion mutant for mce3R gene, were grown in in Middlebrook 7H9 medium supplemented with 0.05% Tween 80, or Middlebrook 7H11, supplemented with albumin 0.5%, dextrose 0.4%, and 0.5% glycerol (M7H9-AD-G). <br>Total RNA was extracted from each culture, dyed with Cy5 and hybridised onto two separated microarrays (technical duplicates). <br>In the second channel a mix of genomic DNA from strains H37Rv and AF2122 was dyed with CY3 and used as a common control in all the microarrays.<br>An statistical analysis of microarray data was performed to detect genes with differential expression between genotypes.
Project description:In this study, we report the identification of a five-locus copper-inducible regulon in Mycobacterium tuberculosis. The identification of a copper responsive regulon unique to pathogenic Mycobacteria suggests copper homeostasis must be maintained during an infection. WT and mutant Mtb cells were grown in Sauton's minimal media to early stationary phase (OD580 = 1.5) and treated with 500 mM copper sulfate (CuSO4) for four hours or the absence
Project description:In this study, we report the identification of a five-locus copper-inducible regulon in Mycobacterium tuberculosis. The identification of a copper responsive regulon unique to pathogenic Mycobacteria suggests copper homeostasis must be maintained during an infection. WT and mutant Mtb cells were grown in Sauton’s minimal media to early stationary phase (OD580 = 1.5) and treated with 500 mM copper sulfate (CuSO4) for four hours or the absence.
Project description:After co-evolving with humans, its only major host, Mycobacterium tuberculosis (Mtb) restrains immune responses well enough to escape eradication, yet elicits enough immunopathology to ensure its transmission. Here, we provide evidence that this balance is regulated in part by a previously uncharacterized, cytosolic, membrane-associated protein with a novel structural fold, encoded by the Mtb gene rv0431. The protein acts by regulating the quantity and quality of Mtb-derived membrane vesicles bearing TLR2 ligands, including the lipoproteins LpqH and SodC. We propose that rv0431 be named virR (vesiculogenesis and immune response regulator). To our knowledge, VirR is the first bacterial protein identified to regulate vesiculogenesis. Transcriptome sequencing of mouse macrophages uninfected, infected with WT Mtb, or infected with rv0431 mutant Mtb.
Project description:The numerous sigma factors present in Mycobacterium tuberculosis (MTB) are indicative of adaptability to different environmental conditions. In this report we describe the sigma factor B (sigB) regulon and the phenotypes of a MTB sigB mutant strain exposed to different stresses like SDS and Diamide. This experiment set compares expression profiles between H37Rv wild type and H37Rv sigB null mutant as well as under different stress conditions. Both H37Rv wild type and H37Rv sigB null mutants were treated with either 0.05% SDS or 5mM Diamide for 60 min and their expression profiles were compared with untreated wild type or mutant controls. Biological Replicate
Project description:In previously published work, we identified three Mycobacterium tuberculosis sigma (s) factor genes responding to heat shock (sigB, sigE and sigH ). Two of them (sigB and sigE ) also responded to SDS exposure. As these responses to stress suggested that the s factors encoded by these genes could be involved in pathogenicity, we are studying their role in physiology and virulence. In this work, we characterize a sigE mutant of M. tuberculosis H37Rv. The sigE mutant strain was more sensitive than the wild-type strain to heat shock, SDS and various oxidative stresses. It was also defective in the ability to grow inside both human and murine unactivated macrophages and was more sensitive than the wild-type strain to the killing activity of activated murine macrophages. Using microarray technology and quantitative reverse transcriptionÐpolymerase chain reaction (RTÐPCR), we started to define the sigmaE regulon of M. tuberculosis and its involvement in the global regulation of the stress induced by SDS. We showed the requirement for a functional sigE gene for full expression of sigB and for its induction after SDS exposure but not after heat shock. We also identified several genes that are no longer induced when sigmaE is absent. These genes encode proteins belonging to different classes including transcriptional regulators, enzymes involved in fatty acid degradation and classical heat shock proteins. Keywords: genetic modification design and comparative genome hybridization design 15 samples were analyzed. The quality controls were biological replicate and technical replicate
Project description:Like other bacterial species, Mycobacterium tuberculosis has multiple sigma (s) factors encoded in its genome. In previously published work, we and others have shown that mutations in some of these transcriptional activators render M. tuberculosis sensitive to various environmental stresses and, in some cases, cause attenuated virulence phenotypes. In this paper, we characterize a M. tuberculosis mutant lacking the ECF s factor sigma-H. This mutant was more sensitive than the wild type to heat shock and to various oxidative stresses, but did not show decreased ability to grow inside macrophages. Using quantitative reverse transcription-PCR and microarray technology, we have started to define the sigma-H regulon and its involvement in the global regulation of the response to heat shock and the thiol-specific oxidizing agent diamide. We identified 48 genes whose expression increased after exposure of M. tuberculosis to diamide; out of these, 39 were not induced in the sigH mutant, showing their direct or indirect dependence on sigma-H. Some of these genes encode proteins whose predicted function is related to thiol metabolism, such as thioredoxin, thioredoxin reductase and enzymes involved in cysteine and molybdopterine biosynthesis. Other genes under sigma-H control encode transcriptional regulators such as sigB, sigE, and sigH itself. Keywords: comparative genome hybridization design and genetic modification design 15 samples were analyzed. The quality controls were biological replicate and technical replicate
Project description:Infection with Mycobacterium tuberculosis (Mtb), the bacterium that causes Tuberculosis, remains a global health concern. Both classically and non-classically restricted cytotoxic CD8+ T cells are important to the control of Mtb infection. We and others have demonstrated that the non-classical MHC I molecule HLA-E can present pathogen-derived peptides to CD8+ T cells. In this manuscript, we identified the antigen recognized by an HLA-E-restricted CD8+ T cell clone isolated from an Mtb latently infected individual as a peptide from the Mtb protein, MPT32. Recognition by the CD8+ T cell clone required N-terminal O-linked mannosylation of MPT32 by a mannosyltransferase encoded by the Rv1002c gene. This is the first description of a post-translationally modified Mtb-derived protein antigen presented in the context of an HLA-E specific CD8+ T cell immune response. The identification of an immune response that targets a unique mycobacterial modification is novel and has practical impact in the development of vaccines and diagnostics.
Project description:Tuberculosis (TB) is the deadliest infectious disease worldwide. One obstacle hindering the elimination of TB is our lack of understanding of host-pathogen interactions. Exosomes, naturally loaded with microbial molecules, are circulating markers of TB. Changes in the host protein composition of exosomes from Mycobacterium tuberculosis (Mtb)-infected cells have not been described, can contribute to our understanding of the disease process, and serve as a direct source of biomarkers or as capture targets to enrich for exosomes containing microbial molecules. Here, the protein composition of exosomes from Mtb-infected and uninfected THP-1-derived macrophages was evaluated by tandem-mass-spectrometry and differences in protein abundances were assessed. Our results show that infection with Mtb leads to significant changes in the protein composition of exosomes. Specifically, 41 proteins were significantly more abundant in exosomes from Mtb-infected cells; 63% of these were predicted to be membrane associated. Thus, we used a novel biotinylation strategy to verify protein localization, and confirmed the localization of some of these proteins in the exosomal membrane. Our findings reveal another important scenario where Mtb could be influencing changes in host cells that unveil new features of the host-pathogen interaction and may also be exploited as a source of biomarkers for TB.