Dataset Information

The Mycobacterium tuberculosis transcriptional landscape under genotoxic stress.

ABSTRACT: As an intracellular human pathogen, Mycobacterium tuberculosis (Mtb) is facing multiple stressful stimuli inside the macrophage and the granuloma. Understanding Mtb responses to stress is essential to identify new virulence factors and pathways that play a role in the survival of the tubercle bacillus. The main goal of this study was to map the regulatory networks of differentially expressed (DE) transcripts in Mtb upon various forms of genotoxic stress. We exposed Mtb cells to oxidative (H2O2 or paraquat), nitrosative (DETA/NO), or alkylation (MNNG) stress or mitomycin C, inducing double-strand breaks in the DNA. Total RNA was isolated from treated and untreated cells and subjected to high-throughput deep sequencing. The data generated was analysed to identify DE genes encoding mRNAs, non-coding RNAs (ncRNAs), and the genes potentially targeted by ncRNAs.The most significant transcriptomic alteration with more than 700 DE genes was seen under nitrosative stress. In addition to genes that belong to the replication, recombination and repair (3R) group, mainly found under mitomycin C stress, we identified DE genes important for bacterial virulence and survival, such as genes of the type VII secretion system (T7SS) and the proline-glutamic acid/proline-proline-glutamic acid (PE/PPE) family. By predicting the structures of hypothetical proteins (HPs) encoded by DE genes, we found that some of these HPs might be involved in mycobacterial genome maintenance. We also applied a state-of-the-art method to predict potential target genes of the identified ncRNAs and found that some of these could regulate several genes that might be directly involved in the response to genotoxic stress.Our study reflects the complexity of the response of Mtb in handling genotoxic stress. In addition to genes involved in genome maintenance, other potential key players, such as the members of the T7SS and PE/PPE gene family, were identified. This plethora of responses is detected not only at the level of DE genes encoding mRNAs but also at the level of ncRNAs and their potential targets.

SUBMITTER: Namouchi A

PROVIDER: S-EPMC5057432 | biostudies-literature | 2016 Oct

REPOSITORIES: biostudies-literature

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The Mycobacterium tuberculosis transcriptional landscape under genotoxic stress.

Namouchi Amine A Gómez-Muñoz Marta M Frye Stephan A SA Moen Line Victoria LV Rognes Torbjørn T Tønjum Tone T Balasingham Seetha V SV

BMC genomics 20161010 1

<h4>Background</h4>As an intracellular human pathogen, Mycobacterium tuberculosis (Mtb) is facing multiple stressful stimuli inside the macrophage and the granuloma. Understanding Mtb responses to stress is essential to identify new virulence factors and pathways that play a role in the survival of the tubercle bacillus. The main goal of this study was to map the regulatory networks of differentially expressed (DE) transcripts in Mtb upon various forms of genotoxic stress. We exposed Mtb cells t ...[more]

PMID: 27724857

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Project description:Mycobacterium tuberculosis was grown in Middlebrook 7H9 medium supplemented with 0.4% glycerol, 0.085% NaCL, 0.5% BSA and 0.05% Tyloxapol in roller bottle culture (2 rpm at 37°C). To induce starvation, exponentially growing bacteria were washed and resuspended in PBS supplemented with 0.025% Tyloxapol. Cultures were harvested at 0h, 12h and 24h after starvation induction. After harvesting and lysis, proteins were extracted, reduced, alkylated, and digested using trypsin. The peptides were analysed in DDA mode on a Thermo LTQ Orbitrap XL. Data analysis: Thermo raw files were converted into mzXML format using ProteoWizard. The acquired MS2 spectra were searched with OMSSA (2.1.9), X!Tandem (CYCLONE, 2010.12.01.1), and MyriMatch (2.1.114) against an Mtb H37Rv protein database (TubercuList v2.3, April 2011) additionally containing reversed sequences of all proteins in the database. Search parameters were as follows: semi-tryptic peptides (proteolytic cleavage after lysine and arginine unless followed by proline) and up to two missed cleavages were allowed, mass tolerance of the precursor ions was set to 20 ppm. Carbamidomethylation at cysteines was set as a fixed modification and oxidation at methionines as a variable modification. The output of the search engine was processed using PeptideProphet and iProphet. Only peptides at a false discovery rate of less than 1% were taken into consideration for further analysis. For MS1 based label-free quantification the openMS v1.8 framework was used as described by (Weisser et al., 2013). Signals were normalised on peptide feature level such that the median signal in each sample is the same. Abundances of the three most intense peptides were averaged to get a protein abundance value. The same peptides were used for protein quantification across all samples and proteins with less than three peptides were included. Associated RNA-seq data have been deposited to EBI ArrayExpress with accession number E-MTAB-1616.

Dataset Information

The Mycobacterium tuberculosis transcriptional landscape under genotoxic stress.

Publications

The Mycobacterium tuberculosis transcriptional landscape under genotoxic stress.

Similar Datasets

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