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CryoEM structure of the Vibrio cholerae Type IV competence pilus secretin PilQ

Weaver, Sara J. and Sazinsky, Matthew H. and Dalia, Triana N. and Dalia, Ankur B. and Jensen, Grant J. (2020) CryoEM structure of the Vibrio cholerae Type IV competence pilus secretin PilQ. . (Unpublished) https://resolver.caltech.edu/CaltechAUTHORS:20200305-083306543

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Abstract

Natural transformation is the process by which bacteria take up genetic material from their environment and integrate it into their genome by homologous recombination. It represents one mode of horizontal gene transfer and contributes to the spread of traits like antibiotic resistance. In Vibrio cholerae, the Type IV competence pilus is thought to facilitate natural transformation by extending from the cell surface, binding to exogenous DNA, and retracting to thread this DNA through the outer membrane secretin, PilQ. A lack of structural information has hindered our understanding of this process, however. Here, we solved the first ever high-resolution structure of a Type IV competence pilus secretin. A functional tagged allele of VcPilQ purified from native V. cholerae cells was used to determine the cryoEM structure of the PilQ secretin in amphipol to ~2.7 Å. This structure highlights for the first time key differences in the architecture of the Type IV competence pilus secretin from the Type II and Type III Secretin System secretins. Based on our cryoEM structure, we designed a series of mutants to interrogate the mechanism of PilQ. These experiments provide insight into the channel that DNA likely traverses to promote the spread of antibiotic resistance via horizontal gene transfer by natural transformation. We prove that it is possible to reduce pilus biogenesis and natural transformation by sealing the gate, suggesting VcPilQ as a new drug target.


Item Type:Report or Paper (Discussion Paper)
Related URLs:
URLURL TypeDescription
https://doi.org/10.1101/2020.03.03.975896DOIDiscussion Paper
https://resolver.caltech.edu/CaltechAUTHORS:20200210-091000513Related ItemConference Abstract
ORCID:
AuthorORCID
Weaver, Sara J.0000-0001-7753-6215
Sazinsky, Matthew H.0000-0001-7550-5116
Dalia, Triana N.0000-0001-7614-1377
Dalia, Ankur B.0000-0003-2203-1230
Jensen, Grant J.0000-0003-1556-4864
Additional Information:The copyright holder for this preprint is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under a CC-BY-NC-ND 4.0 International license. Posted March 04, 2020. Cryo Electron microscopy was performed in the Beckman Institute Resource Center for Transmission Electron Microscopy at Caltech. Dr. Songye Chen and Dr. Andrey Malyutin assisted with data collection. Dr. Spiros D. Garbis, Dr. Annie Moradian, Dr. Michael Sweredoski, and Dr. Brett Lomenick at the Caltech Proteome Exploration Laboratory (PEL) performed and analyzed mass spectrometry results. Dr. Naima Sharaf, Jeffery Lai, and Prof. Doug Rees provided invaluable advice on membrane protein biochemistry and instrumentation. Jane Ding and Welison Floriano provided computational support. Dr. Debnath Ghosal, Dr. Mohammed Kaplan, Dr. Davi Ortega, Dr. Catherine Oikonomou, Dr. Lauren Ann Metskas, Dr. Christopher Barnes, Claudia Jette, and Andrew Schacht provided feedback and advice. This work was supported in part by grant R35GM128674 from the National Institutes of Health to A.B.D. Author Contributions: S.J.W. conceptualized the project, expressed and purified the protein, prepared samples for cryoEM, collected cryoEM data, processed cryoEM data, assisted in atomic model building, interpreted results, designed figures, and wrote the paper. M.S. purified protein, assisted with cryoEM sample prep and data collection, built the atomic model, interpreted results, and provided feedback on the paper. T.D. engineered the V. cholerae constructs, performed microbial assays, and interpreted results. A.D. conceptualized the project, obtained funding, engineered the V. cholerae constructs, performed microbial assays, interpreted results, designed figures, and provided feedback on the paper. G.J.J. conceptualized the project, obtained funding, and provided feedback on the paper. The authors declare no competing interests.
Funders:
Funding AgencyGrant Number
NIHR35GM128674
Record Number:CaltechAUTHORS:20200305-083306543
Persistent URL:https://resolver.caltech.edu/CaltechAUTHORS:20200305-083306543
Official Citation:CryoEM structure of the Vibrio cholerae Type IV competence pilus secretin PilQ. Sara J. Weaver, Matthew H. Sazinsky, Triana N. Dalia, Ankur B. Dalia, Grant J. Jensen. bioRxiv 2020.03.03.975896; doi: https://doi.org/10.1101/2020.03.03.975896
Usage Policy:No commercial reproduction, distribution, display or performance rights in this work are provided.
ID Code:101719
Collection:CaltechAUTHORS
Deposited By: Tony Diaz
Deposited On:05 Mar 2020 17:17
Last Modified:11 May 2020 17:57

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