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Structure of the mycobacterial ESX-5 type VII secretion system membrane complex by single-particle analysis

Abstract

Mycobacteria are characterized by their impermeable outer membrane, which is rich in mycolic acids1. To transport substrates across this complex cell envelope, mycobacteria rely on type VII (also known as ESX) secretion systems2. In Mycobacterium tuberculosis, these ESX systems are essential for growth and full virulence and therefore represent an attractive target for anti-tuberculosis drugs3. However, the molecular details underlying type VII secretion are largely unknown, due to a lack of structural information. Here, we report the molecular architecture of the ESX-5 membrane complex from Mycobacterium xenopi determined at 13 Å resolution by electron microscopy. The four core proteins of the ESX-5 complex (EccB5, EccC5, EccD5 and EccE5) assemble with equimolar stoichiometry into an oligomeric assembly that displays six-fold symmetry. This membrane-associated complex seems to be embedded exclusively in the inner membrane, which indicates that additional components are required to translocate substrates across the mycobacterial outer membrane. Furthermore, the extended cytosolic domains of the EccC ATPase, which interact with secretion effectors, are highly flexible, suggesting an as yet unseen mode of substrate interaction. Comparison of our results with known structures of other bacterial secretion systems demonstrates that the architecture of type VII secretion system is fundamentally different, suggesting an alternative secretion mechanism.

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Figure 1: Functional and structural characterization of the M. xenopi ESX-5 complex.
Figure 2: Three-dimensional reconstruction of the ESX-5 complex.
Figure 3: Topology and localization of the individual components of the ESX-5 complex.
Figure 4: Unique features of the mycobacterial T7SS in comparison to other bacterial secretion systems.

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Acknowledgements

This work was generously supported through the Behörde für Wissenschaft, Forschung und Gleichstellung of the city of Hamburg (to T.C.M.) and funded by a VIDI grant from the Netherlands Organization of Scientific Research (NWO) (to E.N.G.H. and C.M.B.). K.S.H.B. was funded by a postdoctoral fellowship from the EMBL Interdisciplinary Postdoc Program (EIPOD) under Marie Curie COFUND actions. The authors thank the EMBL P12 beamline and synchrotron facilities at PETRA III (DESY, Hamburg). The authors thank M. Jeske for assistance with the ATPase assays (EMBL, Heidelberg), S. Bhushan (Nanyang Technological University, Singapore) for initial EM analysis of ESX-5 particles and F. Schluenzen for DESY high-performance cluster support. Samples were recorded at the EM facility of the Vienna Biocenter Core Facilities GmbH (VBCF), Austria. The authors thank S.J. Ludtke (Baylor College of Medicine) for comments on resolution estimation and A. Jakobi and C. Sachse (EMBL, Heidelberg) for providing feedback on the manuscript.

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Contributions

A.H.A.P., E.N.G.H., W.B., T.C.M. and M.W. supervised and supported the project. K.S.H.B., A.H.A.P., E.N.G.H., C.M.B. and L.C. designed experiments. E.N.G.H., R.U., C.M.B. and K.S.H.B. generated the constructs. K.S.H.B., C.M.B. and E.N.G.H. purified the ESX-5 complexes for EM analysis. K.S.H.B. performed gold labelling and ATPase assays. C.M.B. performed secretion analysis and disulfide bond characterization. L.C. and J.M. performed electron microscopy. L.C. collected and processed the negative stain electron microscopy data together with W.L. K.S.H.B. and H.D.T.M. collected and analysed SAXS data with the support of D.I.S. M.M.S. and M.R. conducted the mass-spectrometry analysis. K.S.H.B., A.H.A.P., E.N.G.H., C.M.B., W.B., T.C.M., L.C. and M.W. wrote the paper.

Corresponding authors

Correspondence to Thomas C. Marlovits, Annabel H. A. Parret or Edith N. G. Houben.

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The authors declare no competing financial interests.

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Supplementary Figures 1–9, Supplementary Tables 1–3, Supplementary References. (PDF 3399 kb)

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Beckham, K., Ciccarelli, L., Bunduc, C. et al. Structure of the mycobacterial ESX-5 type VII secretion system membrane complex by single-particle analysis. Nat Microbiol 2, 17047 (2017). https://doi.org/10.1038/nmicrobiol.2017.47

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