The PhoP-dependent ncRNA Mcr7 Modulates the TAT Secretion System in Mycobacterium Tuberculosis

PLoS Pathog. 2014 May 29;10(5):e1004183. doi: 10.1371/journal.ppat.1004183. eCollection 2014 May.

Abstract

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 through proteins like EspR, which controls Esx1 function, via regulation of the espACD operon. 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 an 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.

MeSH terms

  • Animals
  • Bacterial Proteins / genetics*
  • Bacterial Proteins / metabolism
  • Gene Expression Regulation, Bacterial / physiology
  • Gene Products, tat / metabolism
  • Humans
  • Membrane Transport Proteins / biosynthesis*
  • Mice, Inbred C57BL
  • Mutation / genetics
  • Mycobacterium tuberculosis / genetics
  • Mycobacterium tuberculosis / metabolism*
  • Mycobacterium tuberculosis / pathogenicity
  • Operon / genetics
  • Proteomics / methods
  • RNA, Untranslated / genetics
  • RNA, Untranslated / metabolism*
  • Virulence
  • beta-Lactamases / metabolism

Substances

  • Bacterial Proteins
  • Gene Products, tat
  • Membrane Transport Proteins
  • RNA, Untranslated
  • beta-Lactamases

Grant support

This work was supported by grant BIO2011–23555 from Spanish Ministry of Economy and Competitiveness, grant FP7 European NEWTBVAC 241745, Swiss National Science Foundation under grant 31003A–140778 and SystemsX. LS was supported by grant BES-2009-013037 from Spanish Ministry of Economy and Competitiveness. JGA was supported by Juan de la Cierva Programme (reference JCI-2009-03799) from Spanish Ministry of Science and Innovation. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.