@article{bc6df7ad7d7745ac8e0ba23ebcb89073,
title = "A key motif in the cholesterol-dependent cytolysins reveals a large family of related proteins",
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 particle analysis, and biochemical studies of one of these CDC-like (CDCL) proteins from Elizabethkingia anophelis, a commensal of the malarial mosquito midgut, that a high degree of structural similarity exists between the CDC and CDCL monomer structures and both form large oligomeric pore complexes. Furthermore, the conserved motif in the E. anophelis CDCL crystal structure occupies a nearly identical position and makes similar contacts to those observed in the structure of the archetype CDC, perfringolysin O (PFO). This suggests a common function in the CDCs and CDCLs and may explain why only this motif is conserved in the CDCLs. Hence, these studies identify a critical component of the sensor involved in initiating the prepore-to-pore transition in the CDCs, which is conserved in a large and diverse group of distant relatives of the CDCs. IMPORTANCE The cholesterol-dependent cytolysins{\textquoteright} pore-forming mechanism relies on the ability to sense the completion of the oligomeric prepore structure and initiate the insertion of the β-barrel pore from the assembled prepore structure. These studies show that a conserved motif is an important component of the sensor that triggers the prepore-to-pore transition and that it is conserved in a large family of previously unidentified CDC-like proteins, the genes for which are present in a vast array of microbial species that span most terrestrial environments, as well as most animal and human microbiomes. These studies establish the foundation for future investigations that will probe the contribution of this large family of CDC-like proteins to microbial survival and human disease.",
keywords = "Bacteroides, Bacteroidetes, Chryseobacterium, Deinococcus, Elizabethkingia, MACPF, Pore-forming, Thalassiosira oceanica, Toxin",
author = "Evans, {Jordan C.} and Johnstone, {Bronte A.} and Lawrence, {Sara L.} and Morton, {Craig J.} and Christie, {Michelle P.} and Parker, {Michael W.} and Tweten, {Rodney K.}",
note = "Funding Information: 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{\textquoteright}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. Funding Information: 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. Publisher Copyright: {\textcopyright} 2020 Evans et al.",
year = "2020",
month = sep,
doi = "10.1128/mBio.02351-20",
language = "English",
volume = "11",
journal = "mBio",
issn = "2161-2129",
publisher = "American Society for Microbiology",
number = "5",
}
