Identification of multiple serine hydrolases involved in virulence and cell envelope integrity of Klebsiella pneumoniae

Identification of multiple serine hydrolases involved in virulence and cell envelope integrity of Klebsiella pneumoniae | 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 Article Identification of multiple serine hydrolases involved in virulence and cell envelope integrity of Klebsiella pneumoniae Christian Lentz, Md Jalal Uddin, George Randall, Jiyun Zhu, Tulsi Upadhyay, and 9 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-6281911/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted You are reading this latest preprint version Abstract Klebsiella pneumoniae is a resident of the human gastro-intestinal tract and an opportunistic, critical priority pathogen that can cause severe systemic infections. To overcome emerging multi-drug resistance, discovery and validation of novel targets for developing new treatment options is essential. Here, we explored the highly druggable and functionally diverse enzyme family of serine hydrolases in K. pneumoniae . Using functionalized fluorophosphonate inhibitors as probes we identified 10 serine hydrolases by mass spectrometry-based activity-based protein profiling, 7 of which were previously uncharacterized. Functional validation using transposon mutants deficient in either the putative lysophospholipase PldB, esterase YjfP, or patatin-like phospholipase YchK revealed reduced virulence during Galleria mellonella infection and growth defects in co-culture with human colonic organoids. Mutants deficient in YjfP and the periplasmic protease DegP showed increased susceptibility for serum-killing and an antimicrobial peptide antibiotic, suggesting/confirming a role in maintaining cell envelope integrity. Biochemical characterization and structural analysis of YjfP suggest this protein is an acetyl esterase. This study gives important insights into the molecular mechanisms underlying virulence of K. pneumoniae and its cell physiology at the host interface. It positions several of these hydrolases as anti-virulence or antimicrobial target candidates, inhibition of which might synergize with antibiotics and human immune defenses. Biological sciences/Chemical biology/Enzymes Biological sciences/Microbiology/Bacteria Virulence Chemoproteomics Organoids Galleria mellonella Bacterial colonization Lipase Deacetylase Esterase Full Text Additional Declarations There is NO Competing Interest. Supplementary Files SupplementaryDataset1ProteinsidentifiedinK.pneumoniaeBYMSABPPUddinetal.xlsx Supplementary Data Set 1 SupplementaryDataset2ExcelBLASTpresultsforahomologoftheK.pneumoniaeSHsUddinetal.xlsx Supplementary Data Set 2 UddinetalSI250321.pdf Supplementary information Cite Share Download PDF Status: Under Review Version 1 posted 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. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. 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-6281911","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":435469605,"identity":"7bff8460-e853-4942-b0d1-159ff59c227e","order_by":0,"name":"Christian Lentz","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA/ElEQVRIie2RsYrCQBCGRwLaLKQdEcwrrFh4xYmvsouQyuLKFIek0spe8SUWrrpuZUCbPIBFiqRJZZHrcs1xmxCQKzZpr9ivGNhlPuafXQCH4z+CALqpNVldRnF906eIVhH1mWnQnQ62rU8FBXSO8c+H/PJdvQQLGGUgonQbnB4zKiGdWoekN05M4OwzZhxEUuDgvOEmWDG3KRxDILPLQGkGIHeE3qRRSMYdyqUSuFLaBJM/hMNx0q9oE0wqDRxkTMiQdSt4N8FYiGtFplNcaXxk4ZtOOFl38Y+h91W9bpfqts+z8p38YE8fZRSR9cWeeH9+g/cLDofD4bDzC2/NWqEgFXDMAAAAAElFTkSuQmCC","orcid":"https://orcid.org/0000-0001-7284-2264","institution":"UiT—The Arctic University of Norway","correspondingAuthor":true,"prefix":"","firstName":"Christian","middleName":"","lastName":"Lentz","suffix":""},{"id":435469606,"identity":"b67db5c9-9003-4a32-8672-8ecacd0e8ead","order_by":1,"name":"Md Jalal Uddin","email":"","orcid":"https://orcid.org/0000-0002-3349-7741","institution":"UiT The Arctic University of Norway","correspondingAuthor":false,"prefix":"","firstName":"Md","middleName":"Jalal","lastName":"Uddin","suffix":""},{"id":435469607,"identity":"032b1b9b-69a6-4099-abe0-30a3a6080c51","order_by":2,"name":"George Randall","email":"","orcid":"","institution":"University of Otago","correspondingAuthor":false,"prefix":"","firstName":"George","middleName":"","lastName":"Randall","suffix":""},{"id":435469608,"identity":"11de1e2b-8cdb-4968-a398-1fbb05b64d93","order_by":3,"name":"Jiyun Zhu","email":"","orcid":"","institution":"Stanford University","correspondingAuthor":false,"prefix":"","firstName":"Jiyun","middleName":"","lastName":"Zhu","suffix":""},{"id":435469609,"identity":"372bb6e1-d19d-41eb-95bc-b054b217d48c","order_by":4,"name":"Tulsi Upadhyay","email":"","orcid":"https://orcid.org/0000-0001-5547-8683","institution":"Stanford University","correspondingAuthor":false,"prefix":"","firstName":"Tulsi","middleName":"","lastName":"Upadhyay","suffix":""},{"id":435469610,"identity":"e0a8ef95-9d28-4ff5-81c4-8364863ae5a9","order_by":5,"name":"Anique Driessen","email":"","orcid":"","institution":"UiT The Arctic University of Norway","correspondingAuthor":false,"prefix":"","firstName":"Anique","middleName":"","lastName":"Driessen","suffix":""},{"id":435469611,"identity":"46a168b3-01e2-4846-9fdc-f7abddfc921c","order_by":6,"name":"Laura van Eijk","email":"","orcid":"https://orcid.org/0009-0007-1613-2269","institution":"UMC Utrecht","correspondingAuthor":false,"prefix":"","firstName":"Laura","middleName":"van","lastName":"Eijk","suffix":""},{"id":435469612,"identity":"511c3fb3-a11f-45e5-a786-5f839e2f31dd","order_by":7,"name":"Paul Stege","email":"","orcid":"","institution":"UMC Utrecht","correspondingAuthor":false,"prefix":"","firstName":"Paul","middleName":"","lastName":"Stege","suffix":""},{"id":435469613,"identity":"13039824-7219-4075-b07d-e3f07e0fd808","order_by":8,"name":"Frerich Masson","email":"","orcid":"","institution":"UMC Utrecht","correspondingAuthor":false,"prefix":"","firstName":"Frerich","middleName":"","lastName":"Masson","suffix":""},{"id":435469614,"identity":"1e5fede6-f1b0-4164-bb77-b58562422ed6","order_by":9,"name":"Marco Viveen","email":"","orcid":"","institution":"UMC Utrecht","correspondingAuthor":false,"prefix":"","firstName":"Marco","middleName":"","lastName":"Viveen","suffix":""},{"id":435469615,"identity":"97422ab0-0976-4fd3-a9a8-26eca85c862d","order_by":10,"name":"Matthew Bogyo","email":"","orcid":"https://orcid.org/0000-0003-3753-4412","institution":"Stanford University","correspondingAuthor":false,"prefix":"","firstName":"Matthew","middleName":"","lastName":"Bogyo","suffix":""},{"id":435469616,"identity":"f7bbaea2-4cc8-4c6d-a64a-d8f867d7e086","order_by":11,"name":"Matthias Fellner","email":"","orcid":"https://orcid.org/0000-0003-3192-6984","institution":"University of Otago","correspondingAuthor":false,"prefix":"","firstName":"Matthias","middleName":"","lastName":"Fellner","suffix":""},{"id":435469617,"identity":"56375243-b4e8-4916-b9c8-12a1e68f2367","order_by":12,"name":"Marcel de Zoete","email":"","orcid":"","institution":"UMC Utrecht","correspondingAuthor":false,"prefix":"","firstName":"Marcel","middleName":"","lastName":"de Zoete","suffix":""},{"id":435469618,"identity":"f8e4b1bb-12d0-4aea-a8cd-523314397b5e","order_by":13,"name":"Mona Johannessen","email":"","orcid":"","institution":"UiT - 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To overcome emerging multi-drug resistance, discovery and validation of novel targets for developing new treatment options is essential. Here, we explored the highly druggable and functionally diverse enzyme family of serine hydrolases in \u003ci\u003eK. pneumoniae\u003c/i\u003e. Using functionalized fluorophosphonate inhibitors as probes we identified 10 serine hydrolases by mass spectrometry-based activity-based protein profiling, 7 of which were previously uncharacterized. Functional validation using transposon mutants deficient in either the putative lysophospholipase PldB, esterase YjfP, or patatin-like phospholipase YchK revealed reduced virulence during \u003ci\u003eGalleria mellonella\u003c/i\u003e infection and growth defects in co-culture with human colonic organoids. 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