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

Louis S Ates,Moataz Abd El Ghany,Connie R Jiménez,Marion Sparrius,Robert Van Der Weerd,Abdallah M Abdallah,Susanna Commandeur,Rainer Kalscheuer,Wilbert Bitter,Eveline Weerdenburg,Sander R Piersma,Edith N.G Houben,Marina Alber,Roy Ummels,Arnab Pain,Alyaa M Abdel-Haleem,Patrick H Viollier

doi:10.1371/journal.pgen.1005190

Abstract

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.

Highlights

Mycobacterium tuberculosis is one of the most important bacterial pathogens; this pathogen has infected almost thirty percent of the world population and is responsible for 1.4 million deaths annually [1]
We propose a model in which the ESX-5 system is responsible for the transport of cell envelope proteins that are required for PLOS Genetics | DOI:10.1371/journal.pgen
We show that the ESX-5 system is essential for growth of mycobacteria

Summary

Introduction

Mycobacterium tuberculosis is one of the most important bacterial pathogens; this pathogen has infected almost thirty percent of the world population and is responsible for 1.4 million deaths annually [1]. Together with a wide range of other (glyco)lipids, such as lipooligosaccharides (LOSs) [3,4], phthiocerol dimycocerosates (PDIMs) [5,6] and trehalose mycolates [7], form a hydrophobic barrier that is organized as an outer membrane which is microscopically similar to that of Gram-negative bacteria [8,9]. While a compromised outer membrane by the absence of PDIM likely enables a more efficient influx of hydrophobic solutes, MspA is a water-filled channel and allows predominantly diffusion of small hydrophilic molecules. PDIM mutants have been shown, both in this study and by others [6,56], to be more sensitive to both hydrophobic and hydrophilic antimicrobials

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