Probing the role of glycocalyx in host-microbe interactions with systematic modification of the glycomic surface

Probing the role of glycocalyx in host-microbe interactions with systematic modification of the glycomic surface | 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 Probing the role of glycocalyx in host-microbe interactions with systematic modification of the glycomic surface Carlito Lebrilla, Ying Sheng, Sheryl Joyce Grijaldo-Alvarez, Yixuan Xie, and 1 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-3919137/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 Glycosylation is a conserved post-translational modification by which oligosaccharides are attached to a macromolecule, typically proteins and lipids found on the cell surface. In particular, protein glycosylation play an important role in host-microbe interactions which can further help in describing microbial pathogenicity, recognition by the host immune system and host susceptibility to infections. Glycan-mediated host-microbe interactions were empirically investigated using glycan arrays; however, characterizing the cellular glycocalyx via in situ binding with cells has not been explored. In this research, we established a metabolic-engineered host cell model that generates controlled glycan structure phenotypes on the cell surface. Specifically, we produced host cell surfaces that were primarily fucosylated, sialylated, undecorated complex-type structures, and high mannose-type structures. The manipulated host cell N-glycomes were characterized and confirmed by liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis. To validate glycans involved during infection, we employed adherence assays which showed fucosylated N-glycans on the colon cell line HCT116 cell surface significantly increased the number of adhered Salmonella enterica serovar Typhimurium (S. Typhi) compared to the other glycan subtypes. Furthermore, we showed that adherence of S. Typhi to HCT116 cells could be blocked by co-incubation with free fucose monosaccharides. The results proved that fucose residues on host cells bind with S. Typhi during bacterial infection. Proteomic analysis showed that the glycoengineering did not change the abundances of membrane proteins, indicating that host protein expression did not contribute to the increased adherence of S. Typhi. Meanwhile, glycoproteomic analysis yielded site-specific N-glycosylation information. Glycopeptides were identified and quantified using a standard glycoproteomic workflow. Collectively, our results suggested the importance of glycans in host-microbe interactions and provided a novel insight into the significance of host glycome during pathogenesis. Biological sciences/Biochemistry/Glycobiology Biological sciences/Biochemistry/Glycomics Biological sciences/Microbiology/Bacteria/Bacterial host response Full Text Additional Declarations There is NO Competing Interest. 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. 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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-3919137","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":271599575,"identity":"8d95c13f-869f-4d36-aaf5-f01a37456d23","order_by":0,"name":"Carlito Lebrilla","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA70lEQVRIiWNgGAWjYDACCeYGZiApw8fAfICBgQ2MGBh48GphBGvhYWNgSyBJCwNQC48BVD0BLfyzGxsfF+6x4GFjP/NN4keZXWIf/wHGB2/b8Fhy52Cz8YxnQIfx5G6T7DmXnNgmkcBsOBePFgOJxDZpngMgv+Ruk2ZsYzZmk2Bgk+bFr6X9N1gL/5tnQC31xmz8B9h/E9DSxgzWIpHDBtRyWI6NIYGNGZ8WkF+kZ4C1PDO27Dl3XI5NIrFZcs453Fr4Zzcf/FxwoE6Onz/54Y0fZdU88v2HD354U4ZbCzbA2ECa+lEwCkbBKBgFGAAABetEV//TDZ0AAAAASUVORK5CYII=","orcid":"https://orcid.org/0000-0001-7190-5323","institution":"University of California Davis","correspondingAuthor":true,"prefix":"","firstName":"Carlito","middleName":"","lastName":"Lebrilla","suffix":""},{"id":271599576,"identity":"344d7a03-d8e2-4749-801b-4be6f6f47d57","order_by":1,"name":"Ying Sheng","email":"","orcid":"https://orcid.org/0000-0002-6002-9783","institution":"University of California, Davis","correspondingAuthor":false,"prefix":"","firstName":"Ying","middleName":"","lastName":"Sheng","suffix":""},{"id":271599577,"identity":"ca960555-f213-43c5-8080-4e435d1fca67","order_by":2,"name":"Sheryl Joyce Grijaldo-Alvarez","email":"","orcid":"https://orcid.org/0000-0001-7822-6462","institution":"University of California Davis","correspondingAuthor":false,"prefix":"","firstName":"Sheryl","middleName":"Joyce","lastName":"Grijaldo-Alvarez","suffix":""},{"id":271599578,"identity":"9aed657c-37f3-47ee-89c3-c6640877378e","order_by":3,"name":"Yixuan Xie","email":"","orcid":"https://orcid.org/0000-0002-8512-6053","institution":"University of California, Davis","correspondingAuthor":false,"prefix":"","firstName":"Yixuan","middleName":"","lastName":"Xie","suffix":""},{"id":271599579,"identity":"534dd22e-c61e-4a10-a888-87f39783b22d","order_by":4,"name":"Maurice Wong","email":"","orcid":"https://orcid.org/0000-0003-3851-2928","institution":"University of California, Davis","correspondingAuthor":false,"prefix":"","firstName":"Maurice","middleName":"","lastName":"Wong","suffix":""}],"badges":[],"createdAt":"2024-02-02 00:35:20","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-3919137/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-3919137/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":53045029,"identity":"33f061c8-8883-43d3-a95f-f1a7538cc521","added_by":"auto","created_at":"2024-03-20 02:39:13","extension":"pdf","order_by":1,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1040165,"visible":true,"origin":"","legend":"","description":"","filename":"FinalManuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-3919137/v1_covered_e50af65a-6fed-456c-bd23-7bd5bb3d29d8.pdf"}],"financialInterests":"There is \u003cb\u003eNO\u003c/b\u003e Competing Interest.","formattedTitle":"Probing the role of glycocalyx in host-microbe interactions with systematic modification of the glycomic surface","fulltext":[],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":false,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":true,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":true,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"nature-portfolio","isNatureJournal":true,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"","title":"Nature Portfolio","twitterHandle":"","acdcEnabled":false,"dfaEnabled":false,"editorialSystem":"ejp","reportingPortfolio":"","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"","lastPublishedDoi":"10.21203/rs.3.rs-3919137/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-3919137/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"Glycosylation is a conserved post-translational modification by which oligosaccharides are attached to a macromolecule, typically proteins and lipids found on the cell surface. 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To validate glycans involved during infection, we employed adherence assays which showed fucosylated N-glycans on the colon cell line HCT116 cell surface significantly increased the number of adhered Salmonella enterica serovar Typhimurium (S. Typhi) compared to the other glycan subtypes. Furthermore, we showed that adherence of S. Typhi to HCT116 cells could be blocked by co-incubation with free fucose monosaccharides. The results proved that fucose residues on host cells bind with S. Typhi during bacterial infection. Proteomic analysis showed that the glycoengineering did not change the abundances of membrane proteins, indicating that host protein expression did not contribute to the increased adherence of S. Typhi. Meanwhile, glycoproteomic analysis yielded site-specific N-glycosylation information. Glycopeptides were identified and quantified using a standard glycoproteomic workflow. 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