Essential Role of the ESX-5 Secretion System in Outer Membrane Permeability of Pathogenic Mycobacteria

PLoS Genet. 2015 May 4;11(5):e1005190. doi: 10.1371/journal.pgen.1005190. eCollection 2015 May.


Mycobacteria possess different type VII secretion (T7S) systems to secrete proteins across their unusual cell envelope. One of these systems, ESX-5, is only present in slow-growing mycobacteria and responsible for the secretion of multiple substrates. However, the role of ESX-5 substrates in growth and/or virulence is largely unknown. In this study, we show that esx-5 is essential for growth of both Mycobacterium marinum and Mycobacterium bovis. Remarkably, this essentiality can be rescued by increasing the permeability of the outer membrane, either by altering its lipid composition or by the introduction of the heterologous porin MspA. Mutagenesis of the first nucleotide-binding domain of the membrane ATPase EccC5 prevented both ESX-5-dependent secretion and bacterial growth, but did not affect ESX-5 complex assembly. This suggests that the rescuing effect is not due to pores formed by the ESX-5 membrane complex, but caused by ESX-5 activity. Subsequent proteomic analysis to identify crucial ESX-5 substrates confirmed that all detectable PE and PPE proteins in the cell surface and cell envelope fractions were routed through ESX-5. Additionally, saturated transposon-directed insertion-site sequencing (TraDIS) was applied to both wild-type M. marinum cells and cells expressing mspA to identify genes that are not essential anymore in the presence of MspA. This analysis confirmed the importance of esx-5, but we could not identify essential ESX-5 substrates, indicating that multiple of these substrates are together responsible for the essentiality. Finally, examination of phenotypes on defined carbon sources revealed that an esx-5 mutant is strongly impaired in the uptake and utilization of hydrophobic carbon sources. Based on these data, we propose a model in which the ESX-5 system is responsible for the transport of cell envelope proteins that are required for nutrient uptake. These proteins might in this way compensate for the lack of MspA-like porins in slow-growing mycobacteria.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Ampicillin / pharmacology
  • Bacterial Proteins / genetics
  • Bacterial Proteins / metabolism*
  • Cell Membrane Permeability
  • Chromatography, Liquid
  • DNA Transposable Elements
  • DNA, Bacterial / genetics
  • Drug Resistance, Bacterial
  • Gene Expression Regulation, Bacterial*
  • Mutation
  • Mycobacterium bovis / genetics*
  • Mycobacterium bovis / metabolism
  • Mycobacterium marinum / genetics*
  • Mycobacterium marinum / metabolism
  • Mycobacterium tuberculosis / genetics
  • Mycobacterium tuberculosis / metabolism
  • Sequence Analysis, DNA
  • Tandem Mass Spectrometry
  • Type VII Secretion Systems / genetics
  • Type VII Secretion Systems / metabolism*


  • Bacterial Proteins
  • DNA Transposable Elements
  • DNA, Bacterial
  • Type VII Secretion Systems
  • Ampicillin

Grant support

This work was funded by a VIDI grant from the Netherlands Organization of Scientific Research (ENGH), by the Jürgen Manchot Foundation (RK and MA) and by the Innovative Medicines Initiative Joint Undertaking under grant agreement n°115337, resources of which are composed of financial contribution from the European Union’s Seventh Framework Programme (FP7/2007–2013) and EFPIA companies’ in kind contribution (SC). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.