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Identification of gene fusion events in Mycobacterium tuberculosis that encode novel chimeric proteins

ABSTRACT: Mycobacterium tuberculosis is a facultative intracellular pathogen, responsible for causing tuberculosis. The harsh environment in which M. tuberculosis survives requires this pathogen to maintain an evolutionary advantage. However, the apparent absence of horizontal gene transfer in M. tuberculosis imposes restrictions in the ways by which evolution can occur. Large scale changes in the genome can be introduced through genome reduction, recombination events and structural variation. Here, we identify a functional chimeric protein in the ppe38-71 locus, the absence of which is known to have an impact on protein secretion and virulence. To examine whether this approach was used more often by this pathogen we further develop software that detects potential gene fusion events from multigene deletions using whole-genome sequencing data. With this software we could identify a number of other putative gene-fusion events within the genomes of M. tuberculosis isolates. We were able to demonstrate the expression of one of these gene fusions at the protein level using mass spectrometry. Therefore, gene fusions may provide an additional means of evolution for M. tuberculosis in its natural environment whereby novel proteins and functions can arise.

INSTRUMENT(S): Orbitrap Fusion

ORGANISM(S): Mycobacterium Tuberculosis H37rv

SUBMITTER: Samantha Sampson

LAB HEAD: Samantha Sampson

PROVIDER: PXD017298 | Pride | 2020-05-12

REPOSITORIES: Pride

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Project description:The PhoPR two-component system is essential for virulence in Mycobacterium tuberculosis where it controls expression of approximately 2% of the genes, including those for the ESX-1 secretion apparatus, a major virulence determinant. Mutations in phoP lead to compromised production of pathogen-specific cell wall components and attenuation both ex vivo and in vivo. Using antibodies against the native protein in ChIP-seq experiments (chromatin immunoprecipitation followed by high-throughput sequencing) we demonstrated that PhoP binds to at least 35 loci on the M. tuberculosis genome. The PhoP regulon comprises several transcriptional regulators as well as genes for polyketide synthases and PE/PPE proteins. Integration of ChIP-seq results with high-resolution transcriptomic analysis (RNA-seq) revealed that PhoP controls 30 genes directly, whilst regulatory cascades are responsible for signal amplification and downstream effects. The most prominent site of PhoP regulation was located in the intergenic region between rv2395 and PE_PGRS41, where the mcr7 gene codes for a small non-coding RNA (ncRNA). Northern blot experiments confirmed the absence of Mcr7 in the M. tuberculosis phoP mutant as well as low-level expression of the ncRNA in M. tuberculosis complex members other than M. tuberculosis. By means of genetic and proteomic analyses we demonstrated that Mcr7 modulates translation of the tatC mRNA thereby impacting the activity of the Twin Arginine Translocation (Tat) protein secretion apparatus. As a result, secretion of the immunodominant Ag85 complex and the beta-lactamase BlaC is affected, among others. Mcr7, the first ncRNA of M. tuberculosis whose function has been established, therefore represents a missing link between the PhoPR two-component system and the downstream functions necessary for successful infection of the host. ChiP-Seq: M. tuberculosis H37Rv (GC1237) cultures grown in vitro to exponential phase. Two wild type samples plus one isogenic phoP mutant as control

Project description:The PhoPR two-component system is essential for virulence in Mycobacterium tuberculosis where it controls expression of approximately 2% of the genes, including those for the ESX-1 secretion apparatus, a major virulence determinant. Mutations in phoP lead to compromised production of pathogen-specific cell wall components and attenuation both ex vivo and in vivo. Using antibodies against the native protein in ChIP-seq experiments (chromatin immunoprecipitation followed by high-throughput sequencing) we demonstrated that PhoP binds to at least 35 loci on the M. tuberculosis genome. The PhoP regulon comprises several transcriptional regulators as well as genes for polyketide synthases and PE/PPE proteins. Integration of ChIP-seq results with high-resolution transcriptomic analysis (RNA-seq) revealed that PhoP controls 30 genes directly, whilst regulatory cascades are responsible for signal amplification and downstream effects. The most prominent site of PhoP regulation was located in the intergenic region between rv2395 and PE_PGRS41, where the mcr7 gene codes for a small non-coding RNA (ncRNA). Northern blot experiments confirmed the absence of Mcr7 in the M. tuberculosis phoP mutant as well as low-level expression of the ncRNA in M. tuberculosis complex members other than M. tuberculosis. By means of genetic and proteomic analyses we demonstrated that Mcr7 modulates translation of the tatC mRNA thereby impacting the activity of the Twin Arginine Translocation (Tat) protein secretion apparatus. As a result, secretion of the immunodominant Ag85 complex and the beta-lactamase BlaC is affected, among others. Mcr7, the first ncRNA of M. tuberculosis whose function has been established, therefore represents a missing link between the PhoPR two-component system and the downstream functions necessary for successful infection of the host. RNA-Seq: M. tuberculosis H37Rv (GC1237) cultures grown in vitro to exponential phase. One wild type sample plus one isogenic phoP mutant sample

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Identification of gene fusion events in Mycobacterium tuberculosis that encode novel chimeric proteins

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