Accumulation of Trehalose-6-Phosphate in Candida auris results in Decreased Echinocandin Resistance and Tolerance by Affecting Cell Wall Chitin Synthesis

Accumulation of Trehalose-6-Phosphate in Candida auris results in Decreased Echinocandin Resistance and Tolerance by Affecting Cell Wall Chitin Synthesis | 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 Accumulation of Trehalose-6-Phosphate in Candida auris results in Decreased Echinocandin Resistance and Tolerance by Affecting Cell Wall Chitin Synthesis Qingjuan Zhu, Sien Van de Velde, Stefanie Wijnants, Hans Carolus, and 11 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-6681474/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 13 Dec, 2025 Read the published version in Nature Communications → Version 1 posted You are reading this latest preprint version Abstract The rise of multidrug-resistant fungal pathogens, like Candida auris, poses a significant public health challenge due to high mortality rates and the limited effectiveness of current treatment options. Echinocandins, targeting β-glucan synthesis, are the first-line therapy for invasive C. auris infections. However, the resistance to this drug class is increasing, underscoring the urgent need for new antifungal targets. This study investigates the role of trehalose biosynthesis in C. auris by either blocking biosynthesis of the intermediate molecule trehalose-6-phosphate (T6P), by disrupting the trehalose-phosphate synthase (Tps1), or blocking biosynthesis of trehalose, by disrupting the trehalose-6-phosphate phosphatase (Tps2). The tps2Δ strain demonstrated heightened susceptibility to echinocandins, while the tps1Δ and tps1Δ tps2Δ strains maintained resistance and tolerance levels comparable to the wild type (WT) strain. Subsequent analysis revealed a link between chitin biosynthesis and T6P levels. In the absence of T6P (in the tps1∆ or tps1∆tps2∆ strains) there was a strong increase in hexokinase activity and accelerated glycolysis, with no significant impact on chitin biosynthesis. However, the tps2Δ strain accumulated high levels of T6P, resulting in the inhibition of hexokinase and a reduced flux of glucose 6-phosphate into the chitin biosynthesis pathway, thereby significantly decreasing cell wall chitin content. The inability to compensate the reduction in β-glucan levels with increased chitin production during echinocandin treatment in the tps2Δ strain, rendered this strain highly susceptible to these drugs. Furthermore, an in vivo systemic infection model demonstrated that the tps2Δ strain exhibited a reduced fungal burden in tissues, with infected mice showing marked improvement during caspofungin treatment. This suggests that Tps2 is a putative target for improving echinocandin treatment and reducing virulence in C. auris. Health sciences/Diseases/Infectious diseases/Fungal infection Biological sciences/Microbiology/Fungi/Fungal biology Biological sciences/Microbiology/Antimicrobials/Antimicrobial resistance Biological sciences/Microbiology/Antimicrobials/Antifungal agents Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Figure 9 Figure 10 Full Text Additional Declarations There is NO Competing Interest. Supplementary Files ZhuSupplementarytableandfigures.docx Supplementary Tables and figures Cite Share Download PDF Status: Published Journal Publication published 13 Dec, 2025 Read the published version in Nature Communications → 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. 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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-6681474","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":459829778,"identity":"7ea75753-3dc3-4f34-b586-88ce31ea67d6","order_by":0,"name":"Qingjuan Zhu","email":"","orcid":"","institution":"Laboratory of Molecular Cell Biology, Department of Biology, KU Leuven","correspondingAuthor":false,"prefix":"","firstName":"Qingjuan","middleName":"","lastName":"Zhu","suffix":""},{"id":459829779,"identity":"83931086-5239-40aa-9764-1ee2669d01af","order_by":1,"name":"Sien Van de Velde","email":"","orcid":"","institution":"Laboratory of Molecular Cell Biology, Department of Biology, KU Leuven","correspondingAuthor":false,"prefix":"","firstName":"Sien","middleName":"Van","lastName":"de Velde","suffix":""},{"id":459829780,"identity":"6ae8b294-5cb1-4f11-b38c-32650d33a64d","order_by":2,"name":"Stefanie Wijnants","email":"","orcid":"","institution":"Laboratory of Molecular Cell Biology, Department of Biology, KU Leuven","correspondingAuthor":false,"prefix":"","firstName":"Stefanie","middleName":"","lastName":"Wijnants","suffix":""},{"id":459829781,"identity":"62584315-bc75-4a38-928c-bcbc6e126320","order_by":3,"name":"Hans Carolus","email":"","orcid":"https://orcid.org/0000-0003-1507-3475","institution":"Laboratory of Molecular Cell Biology, Department of Biology, KU Leuven","correspondingAuthor":false,"prefix":"","firstName":"Hans","middleName":"","lastName":"Carolus","suffix":""},{"id":459829782,"identity":"12444c40-ab87-4f60-9232-9f7543168142","order_by":4,"name":"Stef Jacobs","email":"","orcid":"https://orcid.org/0000-0001-6535-4051","institution":"Laboratory of Molecular Cell Biology, Department of Biology, KU Leuven","correspondingAuthor":false,"prefix":"","firstName":"Stef","middleName":"","lastName":"Jacobs","suffix":""},{"id":459829783,"identity":"768d8837-a2fb-467c-8ed6-b62be9dd63af","order_by":5,"name":"Dimitrios Sofras","email":"","orcid":"","institution":"KU Leuven","correspondingAuthor":false,"prefix":"","firstName":"Dimitrios","middleName":"","lastName":"Sofras","suffix":""},{"id":459829784,"identity":"c3b943ad-1d04-4373-a987-a5fd456c3e65","order_by":6,"name":"Paul Vandecruys","email":"","orcid":"","institution":"Laboratory of Molecular Cell Biology, Department of Biology, KU Leuven","correspondingAuthor":false,"prefix":"","firstName":"Paul","middleName":"","lastName":"Vandecruys","suffix":""},{"id":459829785,"identity":"04a0df91-81e0-4fb3-a667-5118832f3285","order_by":7,"name":"Odessa Van Goethem","email":"","orcid":"","institution":"KU Leuven","correspondingAuthor":false,"prefix":"","firstName":"Odessa","middleName":"Van","lastName":"Goethem","suffix":""},{"id":459829788,"identity":"31b572c9-88d3-4294-b332-33ea23dd54df","order_by":8,"name":"Regina Feil","email":"","orcid":"https://orcid.org/0000-0002-9936-1337","institution":"Molecular Plant Physiology, Max Planck Institute","correspondingAuthor":false,"prefix":"","firstName":"Regina","middleName":"","lastName":"Feil","suffix":""},{"id":459829786,"identity":"c52f6180-61cf-4143-99b7-d391e776ef8d","order_by":9,"name":"Rudy Vergauwen","email":"","orcid":"","institution":"KU Leuven Laboratory of Molecular Cell Biology","correspondingAuthor":false,"prefix":"","firstName":"Rudy","middleName":"","lastName":"Vergauwen","suffix":""},{"id":459829787,"identity":"ae7c4d8d-02d4-4912-b637-315e215db738","order_by":10,"name":"Wouter Van Genechten","email":"","orcid":"","institution":"KU Leuven Laboratory of Molecular Cell Biology","correspondingAuthor":false,"prefix":"","firstName":"Wouter","middleName":"Van","lastName":"Genechten","suffix":""},{"id":459829794,"identity":"80f415c9-0c2f-496d-9ad3-e02d75b52809","order_by":11,"name":"Wim Van den Ende","email":"","orcid":"","institution":"KU Leuven","correspondingAuthor":false,"prefix":"","firstName":"Wim","middleName":"Van den","lastName":"Ende","suffix":""},{"id":459829797,"identity":"2c6c7cf5-a430-4e4e-a849-d7b7ce66747d","order_by":12,"name":"Uwe Himmelreich","email":"","orcid":"","institution":"KU Leuven","correspondingAuthor":false,"prefix":"","firstName":"Uwe","middleName":"","lastName":"Himmelreich","suffix":""},{"id":459829791,"identity":"bf21f733-8333-445f-b359-66125f9dd3d9","order_by":13,"name":"John E. Lunn","email":"","orcid":"https://orcid.org/0000-0001-8533-3004","institution":"Molecular Plant Physiology, Max Planck Institute","correspondingAuthor":false,"prefix":"","firstName":"John","middleName":"E.","lastName":"Lunn","suffix":""},{"id":459829777,"identity":"e40b45a2-da9d-4efc-9a13-1513be48233b","order_by":14,"name":"Patrick Van Dijck","email":"data:image/png;base64,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","orcid":"https://orcid.org/0000-0002-1542-897X","institution":"Laboratory of Molecular Cell Biology, Department of Biology, KU Leuven","correspondingAuthor":true,"prefix":"","firstName":"Patrick","middleName":"Van","lastName":"Dijck","suffix":""}],"badges":[],"createdAt":"2025-05-16 14:16:30","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-6681474/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-6681474/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1038/s41467-025-67022-x","type":"published","date":"2025-12-13T05:00:00+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":83280826,"identity":"65adbfcc-ada0-41b2-bf61-38c8bd351be8","added_by":"auto","created_at":"2025-05-22 10:16:48","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":1909977,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eGrowth and stress resistance phenotypes of \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eC. auris \u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003etrehalose pathway mutants. (A) \u003c/strong\u003eGrowth curves of WT, \u003cem\u003etps1\u003c/em\u003e1, \u003cem\u003etps2\u003c/em\u003e1 , and \u003cem\u003etps1\u003c/em\u003e1 \u003cem\u003etps2\u003c/em\u003e1 strains in liquid RPMI 1640 medium supplemented with 0.2% and 2% glucose at 37°C. Optical density at 600 nm was measured over 48 hours. Colour and symbol legends for different strains are given in C. Data shown is based on the average of two technical repeats for each strain. \u003cstrong\u003e(B) \u003c/strong\u003eGrowth evaluation after 48h on RPMI 1640 medium at 37°C, 39°C, and 42°C. Colour and symbol legends for different strains are given in C. Data shown is based on the average of two technical repeats for each strain. \u003cstrong\u003e(C) \u003c/strong\u003eBDA depict the relative growth as a function of stressor concentration in RPMI 1640 (pH 7, 2% glucose) after 48h of incubation. The stress-inducing compounds used were CFW, CR, SDS, NaCl and H2O2. Error bars represent the standerd error of the mean (SEM) of two technical repeats per strains. \u003cstrong\u003e(D) \u003c/strong\u003eHeatmap that represents the RR\u003csup\u003eAUC\u003c/sup\u003e of the deletion strains for the same stressors, compared to the WT strain. The colour scale determines the difference in RR\u003csup\u003eAUC\u003c/sup\u003e between the deletion strains and the WT strain. This calculated value is depicted on the heatmap.\u003c/p\u003e","description":"","filename":"Figure1.png","url":"https://assets-eu.researchsquare.com/files/rs-6681474/v1/257b50a6a21580cac91d4105.png"},{"id":83281618,"identity":"ee51d45b-a71e-4192-9667-ce23c3ca6aff","added_by":"auto","created_at":"2025-05-22 10:24:48","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":4632790,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eAntifungal susceptibility testing (AFST) of WT and mutant strains. (A) \u003c/strong\u003eBDA for caspofungin (CAS), micafungin (MCF), anidulafungin (AND), amphotericin B (AMB) and fluconazole (FLU) for the WT, \u003cem\u003etps1\u003c/em\u003eΔ, \u003cem\u003etps2\u003c/em\u003eΔ and \u003cem\u003etps1\u003c/em\u003eΔ \u003cem\u003etps2\u003c/em\u003eΔ strains. Error bars indicate standard deviation based on two technical replicates. \u003cstrong\u003e(B) \u003c/strong\u003eHeatmap that represents the RR\u003csup\u003eAUC\u003c/sup\u003e of the deletion strains for antifungal drugs, compared to the WT strain. The colour scale determines the difference in RR\u003csup\u003eAUC\u003c/sup\u003e between the deletion strains and the WT strain. This calculated value is depicted on the heatmap. \u003cstrong\u003e(C) \u003c/strong\u003eEtest analysis at 37°C (48 hours) showing an inhibition ellipse in regions of the medium with high concentrations of antifungal drugs where the cells are unable to grow. The MIC value (in µg/ml) is read as the lowest concentration at which the border of the elliptical growth inhibition zone intercepted the strip, and is indicated in the top-right corner of each image.\u003c/p\u003e","description":"","filename":"Figure2.png","url":"https://assets-eu.researchsquare.com/files/rs-6681474/v1/4669594721e8c20d47c431fb.png"},{"id":83281616,"identity":"1cdcf518-06ec-4a39-95d2-b795b7485917","added_by":"auto","created_at":"2025-05-22 10:24:48","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":260181,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eIntracellular T6P and trehalose concentrations in response to caspofungin exposure. (A) \u003c/strong\u003eQuantification of intracellular T6P levels in exponentially growing cells treated with or without caspofungin for 60 minutes. \u003cstrong\u003e(B) \u003c/strong\u003eQuantification of intracellular trehalose levels under the same experimental conditions (ww, wet weight). Average T6P and trehalose levels with SEM are shown of two experiments using three independent transformants of each strain. Statistical analysis was conducted by two-way ANOVA with Bonferroni correction.\u003c/p\u003e","description":"","filename":"Figure3.png","url":"https://assets-eu.researchsquare.com/files/rs-6681474/v1/cfdb26707f22854a43664a0a.png"},{"id":83280828,"identity":"392698f5-7163-4a8e-a1a9-e65dd0059f8e","added_by":"auto","created_at":"2025-05-22 10:16:48","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":1680373,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eFunctional roles of \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eTPS2 \u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003eacross \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eC. auris \u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003eclades. (A) \u003c/strong\u003ePhylogenetic analysis of Tps2 orthologs across \u003cem\u003eC. albicans\u003c/em\u003e, \u003cem\u003eC. haemulonii\u003c/em\u003e, \u003cem\u003eC. pseudohaemulonii\u003c/em\u003e, \u003cem\u003eC. duobushaemulonii \u003c/em\u003eand five \u003cem\u003eC. auris \u003c/em\u003eclades. \u003cstrong\u003e(B) \u003c/strong\u003eBDA assay showing the drug (caspofungin, micafungin and anidulafungin) susceptibility and stress (SDS, CFW and CR) susceptibility of clade I \u003cem\u003etps2\u003c/em\u003eΔ, clade III WT, clade III \u003cem\u003etps2\u003c/em\u003eΔ, clade IV WT, clade IV \u003cem\u003etps2\u003c/em\u003eΔ, clade V WT, and clade V \u003cem\u003etps2\u003c/em\u003eΔ strains. \u003cstrong\u003e(C) \u003c/strong\u003eHeatmap that represents the RR\u003csup\u003eAUC\u003c/sup\u003e of the deletion strains for the same stressors, compared to the WT strain. The colour scale determines the difference in RR\u003csup\u003eAUC\u003c/sup\u003e between the deletion strains and the WT strain. This calculated value is depicted on the heatmap.\u003c/p\u003e","description":"","filename":"Figure4.png","url":"https://assets-eu.researchsquare.com/files/rs-6681474/v1/864b2ddda8cf13b54e47207c.png"},{"id":83280836,"identity":"6c36100b-c88f-4a43-bb4d-aba575b13fab","added_by":"auto","created_at":"2025-05-22 10:16:48","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":1922447,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eDeletion of \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eTPS2 \u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003eresults in reduced chitin content in the \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eC. auris \u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003ecell wall. (A) \u003c/strong\u003eConfocal microscopy of cell wall components in \u003cem\u003eC. auris \u003c/em\u003eWT and mutant strains. Triple-staining of chitin, β-glucans and mannans fluorescently labelled with CFW, Alexa Fluor 488 and Concanavalin A-Alexa fluor 488 antibodies, respectively. Differential Interface Contrast (DIC) and fluorescent pictures are shown. Higher fluorescent colour intensity reflects an increase in the respective cell wall component. Laser exposure time was kept constant for each individual species. Images were processed using Fiji software. Scale bars represent 10 µm. \u003cstrong\u003e(B) \u003c/strong\u003eMeasurement of total cell-wall, β-glucan, and chitin contents in \u003cem\u003eC. auris \u003c/em\u003ecells as described in Material and Methods. Data represent the results of at least three independent experiments with SEM. Statistical analysis was conducted by two-way ANOVA with Bonferroni correction: **, P \u0026lt; 0.01.\u003c/p\u003e","description":"","filename":"Figure5.png","url":"https://assets-eu.researchsquare.com/files/rs-6681474/v1/f9a0376b10439ea2b7b0bf7b.png"},{"id":83280833,"identity":"a5b6b563-50ee-474c-ae3b-15e89dc8f28b","added_by":"auto","created_at":"2025-05-22 10:16:48","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":1081075,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eBasal expression levels of genes involved in fungal cell wall biosynthesis in WT and mutant strains. (A) \u003c/strong\u003eSchematic of the biosynthetic pathways of chitin and β-(1,3) glucan in fungi. Transcript levels of \u003cstrong\u003e(B) \u003c/strong\u003echitin synthase genes (\u003cem\u003eCHS1\u003c/em\u003e, \u003cem\u003eCHS2\u003c/em\u003e, \u003cem\u003eCHS3\u003c/em\u003e, and \u003cem\u003eCHS8\u003c/em\u003e), \u003cstrong\u003e(C) \u003c/strong\u003eglutamine-fructose-6-phosphate aminotransferase gene (\u003cem\u003eGFA1\u003c/em\u003e), and \u003cstrong\u003e(D) \u003c/strong\u003eβ-glucan synthase genes (\u003cem\u003eFKS1 \u003c/em\u003eand \u003cem\u003eFKS2\u003c/em\u003e) were quantified by qPCR from cells harvested at the exponential growth phase. Relative gene expression levels are presented as SEM from two independent experiments, each performed with three biological replicates. Values were normalized to the average expression in WT samples. Statistical significance was assessed using one-way ANOVA with Bonferroni correction on log2-transformed data (*p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001).\u003c/p\u003e","description":"","filename":"Figure6.png","url":"https://assets-eu.researchsquare.com/files/rs-6681474/v1/05054ca85b96ae1c8c87632c.png"},{"id":83280830,"identity":"0860fd4a-c26b-472c-b1f4-d90db40173f3","added_by":"auto","created_at":"2025-05-22 10:16:48","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":1253913,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eT6P plays a role in glucose metabolism. (A) \u003c/strong\u003eSchematic of glucose metabolism and its integration with trehalose biosynthesis, glycolysis, and cell wall precursor pathways. \u003cstrong\u003e(B) \u003c/strong\u003eKinase activity on glucose. The cells were grown in RPMI 1640 medium with 100 mM glucose before proteins were isolated and kinase activity was measured. The values of the deletion strains are normalized relative to the WT strain which has 100% kinase activity. The average of three independent experiments each consisting of three biological repeats and three technical repeats is shown and the error bars represent the SEM. Statistical analysis was done by using an one-way ANOVA test with Bonferroni correction (*p ≤ 0.05, **p ≤ 0.01). \u003cstrong\u003e(C-F) \u003c/strong\u003e\u003csup\u003e13\u003c/sup\u003eC NMR spectral analysis of glucose utilization and metabolites in the studied strains after incubation with [1-\u003csup\u003e13\u003c/sup\u003eC]glucose at 37°C. Quantification was based on \u003csup\u003e13\u003c/sup\u003eC signal intensities relative to the initial glucose signal. The total signal from [1-\u003csup\u003e13\u003c/sup\u003eC]glucose at the first time point (t=15min) was set to 100%, and the NMR signals of downstream metabolites were expressed as a percentage of this initial reference signal. This relative quantification reflects \u003csup\u003e13\u003c/sup\u003eC-label redistribution over time and does not represent absolute metabolite concentrations in the medium. ‘Relative Glucose Signal %’ referes to the relative \u003csup\u003e13\u003c/sup\u003eC signal intensity (in %) of the initial alpha + beta [1-\u003csup\u003e13\u003c/sup\u003eC] glucose signal for the relative quantification of glucose utilization and glycerol, mannitol and ethanol synthesis.\u003c/p\u003e","description":"","filename":"Figure7.png","url":"https://assets-eu.researchsquare.com/files/rs-6681474/v1/f1e40bcf6ab12804ee2a60b7.png"},{"id":83280837,"identity":"9268d898-8772-49c6-8513-c311f0fe6ff7","added_by":"auto","created_at":"2025-05-22 10:16:48","extension":"png","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":743538,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eExogenous glucosamine supplementation restore susceptibility to caspofungin. (A) \u003c/strong\u003eGrowth heatmap of the \u003cem\u003etps2\u003c/em\u003eΔ strain during a BDA with varying caspofungin concentrations and different levels of exogenous glucosamine supplementation. \u003cstrong\u003e(B) \u003c/strong\u003eEtest analysis at 37°C (48 hours) showing an inhibition ellipse in regions of the medium with high concentrations of caspofungin where the cells are unable to grow. The MIC value (in µg/mL) is read as the lowest concentration at which the border of the elliptical growth inhibition zone intercepted the strip, and is indicated in the top-right corner of each image.\u003c/p\u003e","description":"","filename":"Figure8.png","url":"https://assets-eu.researchsquare.com/files/rs-6681474/v1/80f5c1b7267b568fb79614c2.png"},{"id":83280835,"identity":"ee369e7f-8bfe-4490-ad25-00d8d2ad22f4","added_by":"auto","created_at":"2025-05-22 10:16:48","extension":"png","order_by":9,"title":"Figure 9","display":"","copyAsset":false,"role":"figure","size":533458,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eFungal burden in the target organs of mice infected with \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eC. auris \u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003estrains. (A) \u003c/strong\u003eSchematic representation of the experiment processes for the mice infection model. \u003cstrong\u003e(B) \u003c/strong\u003eThe CFUs in untreated (PBS) and treated (with 5 mg/kg/24h caspofungin) mice were calculated per gram organ tissue. Asterix above the bars represent statistically significant differences compared to WT strain (t-test; p\u0026lt;0.05).\u003c/p\u003e","description":"","filename":"Figure9.png","url":"https://assets-eu.researchsquare.com/files/rs-6681474/v1/617ad86ee11c2bdfe4d38677.png"},{"id":83281617,"identity":"9bb7b1da-1578-47a8-b8a1-1f3c1a79c05e","added_by":"auto","created_at":"2025-05-22 10:24:48","extension":"png","order_by":10,"title":"Figure 10","display":"","copyAsset":false,"role":"figure","size":1075249,"visible":true,"origin":"","legend":"\u003cp\u003eSchematic representation of the initial glucose metabolism in fungi and its connection to trehalose, chitin and β-(1,3) glucan biosynthesis pathway.\u003c/p\u003e","description":"","filename":"Figure10.png","url":"https://assets-eu.researchsquare.com/files/rs-6681474/v1/b7d32119533e525afde9ba19.png"},{"id":99934544,"identity":"d24f4070-6562-4922-bcd4-cf2150924254","added_by":"auto","created_at":"2026-01-10 08:07:57","extension":"pdf","order_by":1,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":4283030,"visible":true,"origin":"","legend":"Article File","description":"","filename":"ZhuAurisFinal.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6681474/v1_covered_c9383f4b-3f39-41b9-b7b2-0b93af5cee44.pdf"},{"id":83281619,"identity":"f9c4c0cf-2735-4c03-bab9-d591b6ff1cb8","added_by":"auto","created_at":"2025-05-22 10:24:48","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":3211554,"visible":true,"origin":"","legend":"Supplementary Tables and figures","description":"","filename":"ZhuSupplementarytableandfigures.docx","url":"https://assets-eu.researchsquare.com/files/rs-6681474/v1/b064d825e5b5183a3bba5202.docx"}],"financialInterests":"There is \u003cb\u003eNO\u003c/b\u003e Competing Interest.","formattedTitle":"\u003cp\u003eAccumulation of Trehalose-6-Phosphate in \u003cem\u003eCandida\u003c/em\u003e \u003cem\u003eauris\u003c/em\u003e results in Decreased Echinocandin Resistance and Tolerance by Affecting Cell Wall Chitin Synthesis\u003c/p\u003e","fulltext":[],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":false,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":true,"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":"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-6681474/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-6681474/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"The rise of multidrug-resistant fungal pathogens, like Candida auris, poses a significant public health challenge due to high mortality rates and the limited effectiveness of current treatment options. Echinocandins, targeting β-glucan synthesis, are the first-line therapy for invasive C. auris infections. However, the resistance to this drug class is increasing, underscoring the urgent need for new antifungal targets. This study investigates the role of trehalose biosynthesis in C. auris by either blocking biosynthesis of the intermediate molecule trehalose-6-phosphate (T6P), by disrupting the trehalose-phosphate synthase (Tps1), or blocking biosynthesis of trehalose, by disrupting the trehalose-6-phosphate phosphatase (Tps2). The tps2Δ strain demonstrated heightened susceptibility to echinocandins, while the tps1Δ and tps1Δ tps2Δ strains maintained resistance and tolerance levels comparable to the wild type (WT) strain. Subsequent analysis revealed a link between chitin biosynthesis and T6P levels. In the absence of T6P (in the tps1∆ or tps1∆tps2∆ strains) there was a strong increase in hexokinase activity and accelerated glycolysis, with no significant impact on chitin biosynthesis. However, the tps2Δ strain accumulated high levels of T6P, resulting in the inhibition of hexokinase and a reduced flux of glucose 6-phosphate into the chitin biosynthesis pathway, thereby significantly decreasing cell wall chitin content. The inability to compensate the reduction in β-glucan levels with increased chitin production during echinocandin treatment in the tps2Δ strain, rendered this strain highly susceptible to these drugs. Furthermore, an in vivo systemic infection model demonstrated that the tps2Δ strain exhibited a reduced fungal burden in tissues, with infected mice showing marked improvement during caspofungin treatment. This suggests that Tps2 is a putative target for improving echinocandin treatment and reducing virulence in C. auris.","manuscriptTitle":"Accumulation of Trehalose-6-Phosphate in Candida auris results in Decreased Echinocandin Resistance and Tolerance by Affecting Cell Wall Chitin Synthesis","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-05-22 10:16:43","doi":"10.21203/rs.3.rs-6681474/v1","editorialEvents":[],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"nature-communications","isNatureJournal":true,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"NCOMMS","sideBox":"Learn more about [Nature Communications](http://www.nature.com/ncomms/)","snPcode":"","submissionUrl":"https://mts-ncomms.nature.com/","title":"Nature Communications","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"ejp","reportingPortfolio":"Nature Communications","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"ef3afd27-487d-43e0-ba25-a84211eaf1b9","owner":[],"postedDate":"May 22nd, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[{"id":48900309,"name":"Health sciences/Diseases/Infectious diseases/Fungal infection"},{"id":48900310,"name":"Biological sciences/Microbiology/Fungi/Fungal biology"},{"id":48900311,"name":"Biological sciences/Microbiology/Antimicrobials/Antimicrobial resistance"},{"id":48900312,"name":"Biological sciences/Microbiology/Antimicrobials/Antifungal agents"}],"tags":[],"updatedAt":"2026-01-10T08:07:37+00:00","versionOfRecord":{"articleIdentity":"rs-6681474","link":"https://doi.org/10.1038/s41467-025-67022-x","journal":{"identity":"nature-communications","isVorOnly":false,"title":"Nature Communications"},"publishedOn":"2025-12-13 05:00:00","publishedOnDateReadable":"December 13th, 2025"},"versionCreatedAt":"2025-05-22 10:16:43","video":"","vorDoi":"10.1038/s41467-025-67022-x","vorDoiUrl":"https://doi.org/10.1038/s41467-025-67022-x","workflowStages":[]},"version":"v1","identity":"rs-6681474","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-6681474","identity":"rs-6681474","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}