Disabling iron uptake and pilus assembly in uropathogenic Escherichia coli using CRISPR-Cas9: a step toward antivirulence therapy

Disabling iron uptake and pilus assembly in uropathogenic Escherichia coli using CRISPR-Cas9: a step toward antivirulence therapy | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Disabling iron uptake and pilus assembly in uropathogenic Escherichia coli using CRISPR-Cas9: a step toward antivirulence therapy Linu Thomas, Tajo Abraham This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-6502856/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 09 Jul, 2025 Read the published version in Antonie van Leeuwenhoek → Version 1 posted 9 You are reading this latest preprint version Abstract Uropathogenic Escherichia coli (UPEC) is the leading cause of urinary tract infections (UTIs), driven by virulence factors such as iron acquisition systems and adhesive pili. In this study, we employed CRISPR-Cas9-mediated genome editing to functionally inactivate two critical virulence genes— iucD , involved in aerobactin-mediated iron uptake, and papC , encoding the outer membrane usher protein essential for P pilus assembly. Using a clinical UPEC isolate, we introduced premature stop codons via homologous repair templates guided by gene-specific single-guide RNAs. Colony PCR and Sanger sequencing confirmed precise site-specific editing, leading to truncated protein variants. In silico analyses using InterPro and Swiss-Model revealed a complete loss of essential domains in both proteins. Molecular docking studies demonstrated a marked reduction in binding affinities of truncated IucD for NAD(P)H and impaired protein-protein interaction between truncated PapC and PapG. This study highlights the utility of CRISPR-Cas9 as a powerful tool for dissecting bacterial pathogenesis and supports the potential of targeting virulence determinants like iucD and papC as part of an antivirulence strategy for managing UPEC infections. CRISPR-Cas9 gene editing iucD gene papC gene uropathogenic E. coli In silico analyses Full Text Additional Declarations No competing interests reported. Cite Share Download PDF Status: Published Journal Publication published 09 Jul, 2025 Read the published version in Antonie van Leeuwenhoek → Version 1 posted Editorial decision: Revision requested 02 Jun, 2025 Reviews received at journal 02 Jun, 2025 Reviews received at journal 21 May, 2025 Reviewers agreed at journal 14 May, 2025 Reviewers agreed at journal 09 May, 2025 Reviewers invited by journal 24 Apr, 2025 Editor assigned by journal 24 Apr, 2025 Submission checks completed at journal 24 Apr, 2025 First submitted to journal 22 Apr, 2025 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-6502856","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":448413316,"identity":"f11221c1-5787-41c6-b9c1-bcf2c51aa6e3","order_by":0,"name":"Linu Thomas","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA7UlEQVRIiWNgGAWjYBACPmYgwdgAYjI3HGBgsAFxGw/g08IG0nIQrIURpCUNxsCjhQFJC5A4DBbFr4Wd95j0xx020fz9BxsPF7adt1vbfhhoS41NNG6H8aVJHDyTljvjRmLD4Zltt5O3nUkEajmWltuAUwuPmcTBtsO5DTcYGw7zArWYHQBqAbIJa5l//iBIy7lks/MPidSyAWg4UMsBO7MbhG0xtjjblpa7EeQXnnPJCWY3gLYk4PELP/8ZwxuVbTa5884fPvyZp8zO3ux8+sMHH2pscGrBAIlglQnEKgcBe1IUj4JRMApGwcgAANV2Z052dAyQAAAAAElFTkSuQmCC","orcid":"","institution":"Department of Botany, Sir Syed College, Karimbam, Kannur University, Kerala, India","correspondingAuthor":true,"prefix":"","firstName":"Linu","middleName":"","lastName":"Thomas","suffix":""},{"id":448413317,"identity":"17b1c047-34a2-41ac-876a-e1b085c94b16","order_by":1,"name":"Tajo Abraham","email":"","orcid":"","institution":"Department of Botany, Sir Syed College, Karimbam, Kannur University, Kerala, India","correspondingAuthor":false,"prefix":"","firstName":"Tajo","middleName":"","lastName":"Abraham","suffix":""}],"badges":[],"createdAt":"2025-04-22 09:53:05","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-6502856/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-6502856/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1007/s10482-025-02127-3","type":"published","date":"2025-07-09T15:57:45+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":86699421,"identity":"de78dcc5-ae4b-4501-b6c7-bba83e32e4d9","added_by":"auto","created_at":"2025-07-14 16:09:21","extension":"pdf","order_by":1,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":846636,"visible":true,"origin":"","legend":"","description":"","filename":"Manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6502856/v1_covered_b17b7a65-94a4-4d8a-b958-c4f3e3c28025.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Disabling iron uptake and pilus assembly in uropathogenic Escherichia coli using CRISPR-Cas9: a step toward antivirulence therapy","fulltext":[],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":false,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":true,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":true,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"antonie-van-leeuwenhoek","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"anto","sideBox":"Learn more about [Antonie van Leeuwenhoek](https://www.springer.com/journal/10482)","snPcode":"10482","submissionUrl":"https://submission.nature.com/new-submission/10482/3","title":"Antonie van Leeuwenhoek","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"CRISPR-Cas9, gene editing, iucD gene, papC gene, uropathogenic E. coli, In silico analyses","lastPublishedDoi":"10.21203/rs.3.rs-6502856/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-6502856/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eUropathogenic \u003cem\u003eEscherichia coli\u003c/em\u003e (UPEC) is the leading cause of urinary tract infections (UTIs), driven by virulence factors such as iron acquisition systems and adhesive pili. 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