Phage Resistance in Vibrio harveyi: Evolutionary strategies and physiological trade-offs

Phage Resistance in Vibrio harveyi: Evolutionary strategies and physiological trade-offs | 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 Phage Resistance in Vibrio harveyi: Evolutionary strategies and physiological trade-offs Pantelis Katharios, Stavros Droubogiannis, Adriana Hotz, Weihong Qi, and 1 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7655806/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 Bacteriophages impose strong selective pressure on bacterial populations, shaping host evolution and driving the emergence of phage-resistant variants. While receptor loss is a well-characterized resistance mechanism, the broader physiological consequences and transcriptional reprogramming associated with resistance remain underexplored, particularly in marine pathogens. Here, we investigate the molecular basis of phage resistance in Vibrio harveyi , an opportunistic aquaculture pathogen, using a combination of time-resolved transcriptomics, whole-genome variant analysis, and phenotypic assays. Exposure of wild-type V. harveyi to the lytic phage Virtus revealed a triphasic transcriptional response: early activation of SOS and stress pathways, mid-phase biosynthetic and translational upregulation, and late-stage repression of metabolism and envelope remodeling consistent with virion maturation. In parallel, we characterized 11 naturally evolved phage-resistant mutants that arose spontaneously under phage selection, identifying three mechanistic categories based on their mutational profiles: PilT -associated mutants, MSHA pilus-associated mutants, and hypermutators with defects in DNA mismatch repair. PilT and MSHA mutants harbored single-point mutations that triggered targeted envelope remodeling and transcriptional shifts. Hypermutators displayed extensive genomic variation and global transcriptional insulation, including persistent SOS activation, suppression of secretion systems, and metabolic downregulation. Despite achieving resistance, several mutants exhibited fitness trade-offs, including impaired growth, attenuated virulence in fish larvae, and altered antibiotic susceptibility. Comparative transcriptomics revealed a shared resistance signature across distinct lineages, marked by consistent upregulation of arginine catabolism and repression of Type VI secretion system components. These findings suggest that structural mutations can act as molecular toggles, globally reprogramming the host’s transcriptional state to resist infection. Our results demonstrate that phage resistance in V. harveyi is a multilayered adaptation involving mutational switching, envelope restructuring, and transcriptional rewiring. This work provides a system-level framework for understanding natural phage resistance and has implications for the development of sustainable phage therapy strategies. Biological sciences/Microbiology/Phage biology Biological sciences/Molecular biology/Transcriptomics Biological sciences/Microbiology/Microbial genetics/Bacterial genes Biological sciences/Microbiology Biological sciences/Evolution Molecular toggle phage resistance Vibrio adaptive evolution Full Text Additional Declarations There is NO Competing Interest. All experiments involving fish larvae were conducted by FELASA-trained personnel from the Institute of Marine Biology, Biotechnology, and Aquaculture at the Hellenic Centre for Marine Research. The Center holds an Operating License for the breeding and use (experimentation) of laboratory animals, registered under EL91-BIObr-03 and EL91-BIOexp-04, respectively. It is important to note that experimentation with fish larvae at the yolk-sac stage, which relies on endogenous feeding, does not fall under the provisions of Directive 2010/63/EU and, therefore, does not require a license. Supplementary Files SupplementaryData.docx Dataset 1 EarlyPhaseInfectionoverWT.xlsx Dataset 2 MiddlePhaseInfectionoverWT.xlsx Dataset 3 LatePhaseInfectionoverWT.xlsx Dataset 4 HypermutatoroverWT.xlsx Dataset 5 MSHAMutantoverWT.xlsx Dataset 6 PilTMutantoverWT.xlsx Dataset 7 Variationanalysis.tsv.xlsx Dataset 8 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-7655806","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":526117674,"identity":"b0edf0a4-0ff9-4389-85b6-fee4b3499132","order_by":0,"name":"Pantelis 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The Center holds an Operating License for the breeding and use (experimentation) of laboratory animals, registered under EL91-BIObr-03 and EL91-BIOexp-04, respectively. It is important to note that experimentation with fish larvae at the yolk-sac stage, which relies on endogenous feeding, does not fall under the provisions of Directive 2010/63/EU and, therefore, does not require a license.\u003c/p\u003e","formattedTitle":"Phage Resistance in Vibrio harveyi: Evolutionary strategies and physiological trade-offs","fulltext":[],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":false,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":true,"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":"Molecular toggle, phage resistance, Vibrio, adaptive evolution","lastPublishedDoi":"10.21203/rs.3.rs-7655806/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7655806/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eBacteriophages impose strong selective pressure on bacterial populations, shaping host evolution and driving the emergence of phage-resistant variants. While receptor loss is a well-characterized resistance mechanism, the broader physiological consequences and transcriptional reprogramming associated with resistance remain underexplored, particularly in marine pathogens. Here, we investigate the molecular basis of phage resistance in \u003cem\u003eVibrio harveyi\u003c/em\u003e, an opportunistic aquaculture pathogen, using a combination of time-resolved transcriptomics, whole-genome variant analysis, and phenotypic assays. Exposure of wild-type \u003cem\u003eV. harveyi\u003c/em\u003e to the lytic phage Virtus revealed a triphasic transcriptional response: early activation of SOS and stress pathways, mid-phase biosynthetic and translational upregulation, and late-stage repression of metabolism and envelope remodeling consistent with virion maturation. In parallel, we characterized 11 naturally evolved phage-resistant mutants that arose spontaneously under phage selection, identifying three mechanistic categories based on their mutational profiles: \u003cem\u003ePilT\u003c/em\u003e-associated mutants, MSHA pilus-associated mutants, and hypermutators with defects in DNA mismatch repair. \u003cem\u003ePilT\u003c/em\u003e and MSHA mutants harbored single-point mutations that triggered targeted envelope remodeling and transcriptional shifts. Hypermutators displayed extensive genomic variation and global transcriptional insulation, including persistent SOS activation, suppression of secretion systems, and metabolic downregulation. Despite achieving resistance, several mutants exhibited fitness trade-offs, including impaired growth, attenuated virulence in fish larvae, and altered antibiotic susceptibility. Comparative transcriptomics revealed a shared resistance signature across distinct lineages, marked by consistent upregulation of arginine catabolism and repression of Type VI secretion system components. These findings suggest that structural mutations can act as molecular toggles, globally reprogramming the host\u0026rsquo;s transcriptional state to resist infection. Our results demonstrate that phage resistance in \u003cem\u003eV. harveyi\u003c/em\u003e is a multilayered adaptation involving mutational switching, envelope restructuring, and transcriptional rewiring. This work provides a system-level framework for understanding natural phage resistance and has implications for the development of sustainable phage therapy strategies.\u003c/p\u003e","manuscriptTitle":"Phage Resistance in Vibrio harveyi: Evolutionary strategies and physiological trade-offs","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-10-10 04:20:37","doi":"10.21203/rs.3.rs-7655806/v1","editorialEvents":[],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"communications-biology","isNatureJournal":true,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"commsbio","sideBox":"Learn more about [Communications Biology](http://www.nature.com/commsbio/)","snPcode":"","submissionUrl":"","title":"Communications Biology","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"ejp","reportingPortfolio":"Communications Series","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"833dc008-a422-463a-a07f-1cc941c6aaf0","owner":[],"postedDate":"October 10th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[{"id":55919572,"name":"Biological sciences/Microbiology/Phage biology"},{"id":55919573,"name":"Biological sciences/Molecular biology/Transcriptomics"},{"id":55919574,"name":"Biological sciences/Microbiology/Microbial genetics/Bacterial genes"},{"id":55919575,"name":"Biological sciences/Microbiology"},{"id":55919576,"name":"Biological sciences/Evolution"}],"tags":[],"updatedAt":"2026-04-29T09:42:04+00:00","versionOfRecord":[],"versionCreatedAt":"2025-10-10 04:20:37","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-7655806","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7655806","identity":"rs-7655806","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}