Journal article

A Key Motif in the Cholesterol-Dependent Cytolysins Reveals a Large Family of Related Proteins

Jordan C Evans, Bronte A Johnstone, Sara L Lawrence, Craig J Morton, Michelle P Christie, Michael W Parker, Rodney K Tweten

mBio | AMER SOC MICROBIOLOGY | Published : 2020

Abstract

The cholesterol-dependent cytolysins (CDCs) are bacterial, β-barrel, pore-forming toxins. A central enigma of the pore-forming mechanism is how completion of the prepore is sensed to initiate its conversion to the pore. We identified a motif that is conserved between the CDCs and a diverse family of nearly 300 uncharacterized proteins present in over 220 species that span at least 10 bacterial and 2 eukaryotic phyla. Except for this motif, these proteins exhibit little similarity to the CDCs at the primary structure level. Studies herein show this motif is a critical component of the sensor that initiates the prepore-to-pore transition in the CDCs. We further show by crystallography, single ..

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Grants

Awarded by NIH NIAID


Awarded by Australian Research Council


Awarded by National Health and Medical Research Council of Australia Research Fellow


Funding Acknowledgements

This work was supported by a grant from the NIH NIAID (1R37AI037657) to R.K.T. and by grants from the Australian Research Council to M.W.P. and C.J.M. (DP160101874, DP200102871). Funding from the Victorian Government Operational Infrastructure Support Scheme to St Vincent's Institute is acknowledged. M.W.P. is a National Health and Medical Research Council of Australia Research Fellow (APP1117183). B.A.J. is supported by an Australian Government Research Training Program (RTP) Scholarship. This research was partly undertaken at the Australian Synchrotron, part of the Australian Nuclear Science and Technology Organization, and made use of the ACRF Detector on the MX2 beamline. We thank the beamline staff for their assistance. We thank Andrew Leis, Associate Sergey Rubanov, and Eric Hanssen from the Bio21 Advanced Microscopy Facility, Bio21 Institute (University of Melbourne). The technical support of Patricia Parrish and Elizabeth Caldwell is appreciated. Elizabethkingia anophelis strain AG1 was a generous gift from Jiannong Xu (New Mexico State University). J.C.E. designed and performed the experiments in Table 1 and Figures 1 to 5 to characterize the conserved motif, and so was assigned first position in the co-first authors. S.L.L. expressed, purified, crystallized, and collected X-ray diffraction data for the crystal structure. S.L.L. performed SDS-AGE analysis and S.L.L. and B.A.J. took negative stain EM images of CDCL/CDCLS. B.A.J. collected X-ray diffraction data and performed the single particle EM studies on CDCLS and CDCL/CDCLS pore structures. Therefore, B.A.J. and S.L.L. were assigned second and third positions in the co-first authors. C.J.M. constructed the PFO pore model and solved the CDCL crystal structure and so was assigned the fourth co-first author position. M.P.C. performed negative stain EM imaging of PFO mutants. R.K.T. and M.W.P. supervised the biochemical analysis and structural biology components of the work, respectively. All authors contributed to the preparation and writing of the manuscript.