Essential Role of the ESX-5 Secretion System in Outer Membrane Permeability of Pathogenic Mycobacteria.
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.
Department of Medical Microbiology and Infection Control, VU University Medical Center, Amsterdam, the Netherlands.
Louis S
LS
Ates
Department of Medical Microbiology and Infection Control, VU University Medical Center, Amsterdam, the Netherlands.
Roy
R
Ummels
Department of Medical Microbiology and Infection Control, VU University Medical Center, Amsterdam, the Netherlands.
Susanna
S
Commandeur
Robert
R
van de Weerd
Department of Medical Microbiology and Infection Control, VU University Medical Center, Amsterdam, the Netherlands.
Robert
R
van der Weerd
Department of Medical Microbiology and Infection Control, VU University Medical Center, Amsterdam, the Netherlands.
Marion
M
Sparrius
Department of Medical Microbiology and Infection Control, VU University Medical Center, Amsterdam, the Netherlands.
Eveline
E
Weerdenburg
Institute for Medical Microbiology and Hospital Hygiene, Heinrich-Heine-University Düsseldorf, Düsseldorf, Germany.
Marina
M
Alber
Institute for Medical Microbiology and Hospital Hygiene, Heinrich-Heine-University Düsseldorf, Düsseldorf, Germany.
Rainer
R
Kalscheuer
Department of Medical Oncology, OncoProteomics Laboratory, VU University Medical Center, Amsterdam, the Netherlands.
Sander R
SR
Piersma
Biological and Environmental Sciences and Engineering (BESE) division, King Abdullah University of Science and Technology (KAUST), Thuwal, Kingdom of Saudi Arabia.
Abdallah M
AM
Abdallah
Biological and Environmental Sciences and Engineering (BESE) division, King Abdullah University of Science and Technology (KAUST), Thuwal, Kingdom of Saudi Arabia.
Moataz
M
Abd El Ghany
Biological and Environmental Sciences and Engineering (BESE) division, King Abdullah University of Science and Technology (KAUST), Thuwal, Kingdom of Saudi Arabia.
Alyaa M
AM
Abdel-Haleem
Biological and Environmental Sciences and Engineering (BESE) division, King Abdullah University of Science and Technology (KAUST), Thuwal, Kingdom of Saudi Arabia.
Arnab
A
Pain
Department of Medical Oncology, OncoProteomics Laboratory, VU University Medical Center, Amsterdam, the Netherlands.
Connie R
CR
Jiménez
Department of Medical Microbiology and Infection Control, VU University Medical Center, Amsterdam, the Netherlands; Section Molecular Microbiology, Amsterdam Institute of Molecules, Medicine & Systems, Vrije Universiteit Amsterdam, Amsterdam, the Netherlands.
Wilbert
W
Bitter
Section Molecular Microbiology, Amsterdam Institute of Molecules, Medicine & Systems, Vrije Universiteit Amsterdam, Amsterdam, the Netherlands.
Edith N G
EN
Houben
D000667:Ampicillin / Q000494:pharmacology; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism*; D002463:Cell Membrane Permeability; D002853:Chromatography, Liquid; D004251:DNA Transposable Elements; D004269:DNA, Bacterial / Q000235:genetics; D024881:Drug Resistance, Bacterial; D015964:Gene Expression Regulation, Bacterial; D009154:Mutation; D009163:Mycobacterium bovis / Q000235:genetics* / Q000378:metabolism; D019910:Mycobacterium marinum / Q000235:genetics* / Q000378:metabolism; D009169:Mycobacterium tuberculosis / Q000235:genetics / Q000378:metabolism; D017422:Sequence Analysis, DNA; D053719:Tandem Mass Spectrometry; D000069377:Type VII Secretion Systems / Q000235:genetics / Q000378:metabolism*
D001426:Bacterial Proteins; D004251:DNA Transposable Elements; D004269:DNA, Bacterial; D000069377:Type VII Secretion Systems; D000667:Ampicillin