Composition of the type VII secretion system membrane complex

Authors

  • Edith N. G. Houben,

    Corresponding author
    1. Department of Molecular Microbiology, Institute of Molecular Cell Biology, VU University Amsterdam, Amsterdam, The Netherlands
    • Department of Medical Microbiology and Infection Control, VU University Medical Center, Amsterdam, The Netherlands
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    • Have contributed equally to this work.
  • Jovanka Bestebroer,

    1. Department of Medical Microbiology and Infection Control, VU University Medical Center, Amsterdam, The Netherlands
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    • Have contributed equally to this work.
    • Current address: Department Medical Microbiology, University Medical Center Utrecht, Utrecht, The Netherlands.
  • Roy Ummels,

    1. Department of Medical Microbiology and Infection Control, VU University Medical Center, Amsterdam, The Netherlands
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  • Louis Wilson,

    1. Department of Infectious Diseases, Leiden University Medical Center, Leiden, The Netherlands
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  • Sander R. Piersma,

    1. Department of Medical Oncology, OncoProteomics Laboratory, VU University Medical Center, Amsterdam, The Netherlands
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  • Connie R. Jiménez,

    1. Department of Medical Oncology, OncoProteomics Laboratory, VU University Medical Center, Amsterdam, The Netherlands
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  • Tom H. M. Ottenhoff,

    1. Department of Infectious Diseases, Leiden University Medical Center, Leiden, The Netherlands
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  • Joen Luirink,

    1. Department of Molecular Microbiology, Institute of Molecular Cell Biology, VU University Amsterdam, Amsterdam, The Netherlands
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  • Wilbert Bitter

    1. Department of Medical Microbiology and Infection Control, VU University Medical Center, Amsterdam, The Netherlands
    2. Department of Molecular Microbiology, Institute of Molecular Cell Biology, VU University Amsterdam, Amsterdam, The Netherlands
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For correspondence. E-mail e.houben@vumc.nl; Tel. (+31) 20 4448296; Fax (+31) 20 4448318.

Summary

Pathogenic mycobacteria require type VII secretion (T7S) systems to transport virulence factors across their complex cell envelope. These bacteria have up to five of these systems, termed ESX-1 to ESX-5. Here, we show that ESX-5 of Mycobacterium tuberculosis mediates the secretion of EsxN, PPE and PE_PGRS proteins, indicating that ESX-5 is a major secretion pathway in this important pathogen. Using the ESX-5 system of Mycobacterium marinum and Mycobacterium bovis BCG as a model, we have purified and analysed the T7S membrane complex under native conditions. blue native-PAGE and immunoprecipitation experiments showed that the ESX-5 membrane complex of both species has a size of ∼ 1500 kDa and is composed of four conserved membrane proteins, i.e. EccB5, EccC5, EccD5 and EccE5. Subsequent limited proteolysis suggests that EccC5 and EccE5 mostly reside on the periphery of the complex. We also observed that EccC5 and EccD5 expression is essential for the formation of a stable membrane complex. These are the first data on a T7S membrane complex and, given the high conservation of its components, our data can likely be generalized to most mycobacterial T7S systems.

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