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Using the SpyTag/SpyCatcher system to determine the path of substrates through the mycobacterial type VII secretion system

Y Ding(1,2) C M Bunduc(1,2,3,4,5) W Bitter(1,2,6) E NG Houben(1,2)

1:Section Molecular Microbiology, Amsterdam Institute for Life and Environment (A-Life), Vrije Universiteit, Amsterdam, The Netherlands; 2:Section Molecular Microbiology, Amsterdam Institute of Molecular and Life Sciences (AIMMS), Vrije Universiteit, Amsterdam, The Netherlands; 3:Centre for Structural Systems Biology, Hamburg, Germany; 4:Institute of Structural and Systems Biology, University Medical Center Hamburg-Eppendorf, Hamburg, Germany; 5:German Electron Synchrotron Centre, Hamburg, Germany; 6:Department of Medical Microbiology and Infection Control, Amsterdam UMC, Amsterdam, The Netherlands

Mycobacterium tuberculosis (Mtb) possesses a highly impermeable cell envelope (CE), which is composed of an inner membrane (IM) and a specific mycolic acid-containing outer membrane (OM). Mtb strictly depends on extracellular proteins to survive and cause infection, as a portion of these proteins mediate crucial host-pathogen interactions. The majority of secreted proteins are exported by type VII secretion systems (T7SSs), of which ESX-1, ESX-2, ESX-3, ESX-4 and ESX-5 are present in Mtb. How T7SSs mediate export of proteins while maintaining the permeability barrier of the CE, important for survival during infection, remains unknown. Recently, the high-resolution structure of the ESX-5 inner membrane (IM) complex of Mtb has been solved. As the structure reveals a closed channel that resides solely in the IM, the path of substrates through the channel and the remaining layers of the CE remain unclear. In this project we use the SpyTag/SpyCatcher system, a highly versatile technology for irreversible conjugation of recombinant proteins, to determine the path of T7SS substrates through the CE. We use this system (i) to lock substrates in the secretion channel, after which we will co-purify this translocation intermediate with membrane complex for structural analysis, and (ii) to determine whether substrates face the periplasmic space during export. Understanding the mechanism of protein transport through the impermeable mycobacterial CE will provide important insights into the functionality of this complex structure and for the exploitation of T7SSs for better anti-tuberculosis treatments.

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