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A small RNA encoded in the Rv2660c locus of Mycobacterium tuberculosis is induced during starvation and infection.

ABSTRACT: Enhanced transcription of the Rv2660c locus in response to starvation of Mycobacterium tuberculosis H37Rv encouraged addition of the predicted Rv2660c protein to an improved vaccine formulation. Using strand-specific RNA sequencing, we show that the up-regulated transcript is in fact a small RNA encoded on the opposite strand to the annotated Rv2660c. The transcript originates within a prophage and is expressed only in strains that carry PhiRv2. The small RNA contains both host and phage sequences and provides a useful biomarker to monitor bacterial starvation during infection and/or non-replicating persistence. Using different approaches we do not find any evidence of Rv2660c at the level of mRNA or protein. Further efforts to understand the mechanism by which Rv2660c improves efficacy of the H56 vaccine are likely to provide insights into the pathology and immunology of tuberculosis.

SUBMITTER: Houghton J

PROVIDER: S-EPMC3861185 | biostudies-literature | 2013

REPOSITORIES: biostudies-literature

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A small RNA encoded in the Rv2660c locus of Mycobacterium tuberculosis is induced during starvation and infection.

Houghton Joanna J Cortes Teresa T Schubert Olga O Rose Graham G Rodgers Angela A De Ste Croix Megan M Aebersold Rudolf R Young Douglas B DB Arnvig Kristine B KB

PloS one 20131212 12

Enhanced transcription of the Rv2660c locus in response to starvation of Mycobacterium tuberculosis H37Rv encouraged addition of the predicted Rv2660c protein to an improved vaccine formulation. Using strand-specific RNA sequencing, we show that the up-regulated transcript is in fact a small RNA encoded on the opposite strand to the annotated Rv2660c. The transcript originates within a prophage and is expressed only in strains that carry PhiRv2. The small RNA contains both host and phage sequenc ...[more]

PMID: 24348997

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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.

Project description:UnlabelledMycobacterium tuberculosis, the etiological agent of tuberculosis, is a Gram-positive bacterium with a unique cell envelope composed of an essential outer membrane. Mycolic acids, which are very-long-chain (up to C100) fatty acids, are the major components of this mycomembrane. The enzymatic pathways involved in the biosynthesis and transport of mycolates are fairly well documented and are the targets of the major antituberculous drugs. In contrast, only fragmented information is available on the expression and regulation of the biosynthesis genes. In this study, we report that the hadA, hadB, and hadC genes, which code for the mycolate biosynthesis dehydratase enzymes, are coexpressed with three genes that encode proteins of the translational apparatus. Consistent with the well-established control of the translation potential by nutrient availability, starvation leads to downregulation of the hadABC genes along with most of the genes required for the synthesis, modification, and transport of mycolates. The downregulation of a subset of the biosynthesis genes is partially dependent on RelMtb, the key enzyme of the stringent response. We also report the phylogenetic evolution scenario that has shaped the current genetic organization, characterized by the coregulation of the hadABC operon with genes of the translational apparatus and with genes required for the modification of the mycolates.ImportanceMycobacterium tuberculosis infects one-third of the human population worldwide, and despite the available therapeutic arsenal, it continues to kill millions of people each year. There is therefore an urgent need to identify new targets and develop a better understanding of how the bacterium is adapting itself to host defenses during infection. A prerequisite of this understanding is knowledge of how this adaptive skill has been implanted by evolution. Nutrient scarcity is an environmental condition the bacterium has to cope with during infection. In many bacteria, adaptation to starvation relies partly on the stringent response. M. tuberculosis's unique outer membrane layer, the mycomembrane, is crucial for its viability and virulence. Despite its being the target of the major antituberculosis drugs, only scattered information exists on how the genes required for biosynthesis of the mycomembrane are expressed and regulated during starvation. This work has addressed this issue as a step toward the identification of new targets in the fight against M. tuberculosis.

Dataset Information

A small RNA encoded in the Rv2660c locus of Mycobacterium tuberculosis is induced during starvation and infection.

Publications

A small RNA encoded in the Rv2660c locus of Mycobacterium tuberculosis is induced during starvation and infection.

Similar Datasets

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