{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,8,21]],"date-time":"2026-08-21T05:23:45Z","timestamp":1787289825819,"version":"build-2736575974"},"reference-count":47,"publisher":"MDPI AG","issue":"16","license":[{"start":{"date-parts":[[2026,8,19]],"date-time":"2026-08-19T00:00:00Z","timestamp":1787097600000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"DOI":"10.13039\/501100006382","name":"University of Oviedo","doi-asserted-by":"crossref","award":["FUO-19-258"],"award-info":[{"award-number":["FUO-19-258"]}],"id":[{"id":"10.13039\/501100006382","id-type":"DOI","asserted-by":"crossref"}]},{"DOI":"10.13039\/501100006382","name":"University of Oviedo","doi-asserted-by":"crossref","award":["FUO-21-304"],"award-info":[{"award-number":["FUO-21-304"]}],"id":[{"id":"10.13039\/501100006382","id-type":"DOI","asserted-by":"crossref"}]},{"award":["FUO-19-258"],"award-info":[{"award-number":["FUO-19-258"]}],"id":[{"id":"https:\/\/ror.org\/006gksa02","id-type":"ROR","asserted-by":"publisher"}]},{"award":["FUO-21-304"],"award-info":[{"award-number":["FUO-21-304"]}],"id":[{"id":"https:\/\/ror.org\/006gksa02","id-type":"ROR","asserted-by":"publisher"}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["Int. J. Mol. Sci.","IJMS"],"abstract":"<jats:p>Ovotransferrin (OvoTf) is an iron-binding glycoprotein of avian innate immunity with antimicrobial activity, but its antifungal mechanism remains incompletely understood. We examined the candidacidal activity of OvoTf and two derived \u03b3-core-containing peptides, kaliocin-3 (Kal-3) and kaliocin-4 (Kal-4), against Candida albicans. All three agents reduced fungal viability in a concentration-dependent manner, with minimal propidium iodide uptake, indicating a predominantly non-permeabilizing mechanism. Their activity was attenuated by extracellular Na+ and K+, whereas isoosmotic sorbitol did not confer protection. OvoTf induced partial K+ release, and tetraethylammonium increased cell survival. Inhibition of mitochondrial complex I or ATP synthase also reduced susceptibility, although none of the agents detectably suppressed oxygen consumption under the conditions tested. OvoTf, Kal-3, and Kal-4 caused plasma membrane depolarization, ATP accumulation, and impaired glucose-induced proton extrusion. This phenotype is consistent with disruption of Pma1-dependent proton and ion homeostasis, with mitochondrial bioenergetic activity contributing as a downstream requirement rather than constituting the primary target. Neither peptide caused detectable hemolysis; Kal-4 had minimal effects on mammalian cell metabolic viability, whereas Kal-3 reduced MCF-7 metabolic viability at higher concentrations. The activity of \u03b3-core regions in both OvoTf lobes supports their further study as templates for mechanism-guided antifungal design.<\/jats:p>","DOI":"10.3390\/ijms27167415","type":"journal-article","created":{"date-parts":[[2026,8,19]],"date-time":"2026-08-19T13:14:01Z","timestamp":1787145241000},"page":"7415","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":0,"title":["Ovotransferrin and Its \u03b3-Core-Containing Peptides Impair Pma1-Dependent Proton Homeostasis in Candida albicans"],"prefix":"10.3390","volume":"27","author":[{"given":"Victoria","family":"Antu\u00f1a","sequence":"first","affiliation":[{"name":"Laboratory of Oral Microbiology (LMO), University Dental Clinic (CLUO), University of Oviedo, 33006 Oviedo, Asturias, Spain"},{"name":"Health Research Institute of the Principality of Asturias (ISPA), 33011 Oviedo, Asturias, Spain"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Patricia","family":"Fierro","sequence":"additional","affiliation":[{"name":"Laboratory of Oral Microbiology (LMO), University Dental Clinic (CLUO), University of Oviedo, 33006 Oviedo, Asturias, Spain"},{"name":"Primary Care Emergency Service, Cantabrian Health Service, 39000 Santander, Cantabria, Spain"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-2752-0690","authenticated-orcid":false,"given":"Jos\u00e9 F.","family":"Fierro","sequence":"additional","affiliation":[{"name":"Laboratory of Oral Microbiology (LMO), University Dental Clinic (CLUO), University of Oviedo, 33006 Oviedo, Asturias, Spain"},{"name":"Health Research Institute of the Principality of Asturias (ISPA), 33011 Oviedo, Asturias, Spain"},{"name":"Department of Functional Biology (Microbiology), Faculty of Medicine, University of Oviedo, 33006 Oviedo, Asturias, Spain"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-9388-8595","authenticated-orcid":false,"given":"\u00c1ngel L.","family":"\u00c1lvarez","sequence":"additional","affiliation":[{"name":"Department of Biochemistry and Molecular Biology, University of Oviedo, 33006 Oviedo, Asturias, Spain"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-7827-981X","authenticated-orcid":false,"given":"Mar\u00eda T.","family":"Andr\u00e9s","sequence":"additional","affiliation":[{"name":"Laboratory of Oral Microbiology (LMO), University Dental Clinic (CLUO), University of Oviedo, 33006 Oviedo, Asturias, Spain"},{"name":"Health Research Institute of the Principality of Asturias (ISPA), 33011 Oviedo, Asturias, Spain"},{"name":"SamerLabs S.L., Asturias Technology Park, 33428 Llanera, Asturias, Spain"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"1968","published-online":{"date-parts":[[2026,8,19]]},"reference":[{"key":"ref_1","first-page":"1","article-title":"Phylogenomic analysis of transferrin family from animals and plants","volume":"17","author":"Bai","year":"2016","journal-title":"Comp. Biochem. Physiol. Part D Genom. Proteom."},{"key":"ref_2","doi-asserted-by":"crossref","first-page":"144810","DOI":"10.1016\/j.ijbiomac.2025.144810","article-title":"Ovotransferrin\u2014Multifunctional protein: Structure, bioactivity, and industrial potential","volume":"317","author":"Pryshchepa","year":"2025","journal-title":"Int. J. Biol. Macromol."},{"key":"ref_3","doi-asserted-by":"crossref","first-page":"218","DOI":"10.1016\/j.bbagen.2011.08.004","article-title":"Physiological roles of ovotransferrin","volume":"1820","author":"Giansanti","year":"2012","journal-title":"Biochim. Biophys. Acta (BBA)-Gen. Subj."},{"key":"ref_4","doi-asserted-by":"crossref","unstructured":"Legros, J., Jan, S., Bonnassie, S., Gautier, M., Croguennec, T., Pezennec, S., Cochet, M.-F., Nau, F., Andrews, S.C., and Baron, F. (2021). The role of ovotransferrin in egg-white antimicrobial activity: A review. Foods, 10.","DOI":"10.3390\/foods10040823"},{"key":"ref_5","doi-asserted-by":"crossref","first-page":"14","DOI":"10.1126\/science.100.2584.14","article-title":"Raw hen egg white and the role of iron in growth inhibition of Shigella dysenteriae, Staphylococcus aureus, Escherichia coli, and Saccharomyces cerevisiae","volume":"100","author":"Schade","year":"1944","journal-title":"Science"},{"key":"ref_6","first-page":"9","article-title":"Identification of the bacteria-inhibiting iron-binding protein of egg white as conalbumin","volume":"11","author":"Alderton","year":"1946","journal-title":"Arch. Biochem."},{"key":"ref_7","doi-asserted-by":"crossref","first-page":"169","DOI":"10.1007\/BF00447027","article-title":"Antifungal activity of ovotransferrin towards genus Candida","volume":"89","author":"Valenti","year":"1985","journal-title":"Mycopathologia"},{"key":"ref_8","doi-asserted-by":"crossref","first-page":"271","DOI":"10.1111\/j.1574-6968.1986.tb01285.x","article-title":"Interaction between lactoferrin and ovotransferrin and Candida cells","volume":"33","author":"Valenti","year":"1986","journal-title":"FEMS Microbiol. Lett."},{"key":"ref_9","doi-asserted-by":"crossref","unstructured":"Legros, J., Bonnassie, S., Cochet, M.-F., Jan, S., Andrews, S.C., and Baron, F. (2025). Impact of ovotransferrin on the membrane integrity of Salmonella Enteritidis under egg-white conditions. Front. Microbiol., 16.","DOI":"10.3389\/fmicb.2025.1539663"},{"key":"ref_10","doi-asserted-by":"crossref","first-page":"359","DOI":"10.1177\/10454411990100030701","article-title":"Oral colonization by Candida albicans","volume":"10","author":"Cannon","year":"1999","journal-title":"Crit. Rev. Oral Biol. Med."},{"key":"ref_11","unstructured":"World Health Organization (2026, May 20). WHO Fungal Priority Pathogens List to Guide Research, Development and Public Health Action, Available online: https:\/\/www.who.int\/publications\/i\/item\/9789240060241."},{"key":"ref_12","unstructured":"World Health Organization (2026, May 20). Landscape Analysis of Commercially Available and Pipeline In Vitro Diagnostics for Fungal Priority Pathogens, Available online: https:\/\/www.who.int\/publications\/i\/item\/9789240105539."},{"key":"ref_13","doi-asserted-by":"crossref","first-page":"289","DOI":"10.1016\/S0167-4889(97)00132-8","article-title":"Identification of a distinct antibacterial domain within the N-lobe of ovotransferrin","volume":"1401","author":"Ibrahim","year":"1998","journal-title":"Biochim. Biophys. Acta (BBA)-Mol. Cell Res."},{"key":"ref_14","doi-asserted-by":"crossref","first-page":"196","DOI":"10.1016\/S0304-4165(00)00122-7","article-title":"Ovotransferrin antimicrobial peptide (OTAP-92) kills bacteria through a membrane damage mechanism","volume":"1523","author":"Ibrahim","year":"2000","journal-title":"Biochim. Biophys. Acta (BBA)-Gen. Subj."},{"key":"ref_15","doi-asserted-by":"crossref","first-page":"69","DOI":"10.1016\/j.bbrc.2005.03.125","article-title":"Antiviral activity of ovotransferrin-derived peptides","volume":"331","author":"Giansanti","year":"2005","journal-title":"Biochem. Biophys. Res. Commun."},{"key":"ref_16","doi-asserted-by":"crossref","first-page":"109720","DOI":"10.1016\/j.lwt.2020.109720","article-title":"Identification and antimicrobial mechanisms of a novel peptide derived from egg white ovotransferrin hydrolysates","volume":"131","author":"Ma","year":"2020","journal-title":"LWT"},{"key":"ref_17","doi-asserted-by":"crossref","first-page":"2862","DOI":"10.1016\/j.bbamem.2007.07.024","article-title":"The \u03b3-core motif correlates with antimicrobial activity in cysteine-containing kaliocin-1 originating from transferrins","volume":"1768","author":"Yount","year":"2007","journal-title":"Biochim. Biophys. Acta (BBA)-Biomembr."},{"key":"ref_18","doi-asserted-by":"crossref","first-page":"7363","DOI":"10.1073\/pnas.0401567101","article-title":"Multidimensional signatures in antimicrobial peptides","volume":"101","author":"Yount","year":"2004","journal-title":"Proc. Natl. Acad. Sci. USA"},{"key":"ref_19","doi-asserted-by":"crossref","unstructured":"Sagaram, U.S., Pandurangi, R., Kaur, J., Smith, T.J., and Shah, D.M. (2011). Structure\u2013activity determinants in antifungal plant defensins MsDef1 and MtDef4 with different modes of action against Fusarium graminearum. PLoS ONE, 6.","DOI":"10.1371\/journal.pone.0018550"},{"key":"ref_20","doi-asserted-by":"crossref","first-page":"240","DOI":"10.1002\/psc.2479","article-title":"Antimicrobial peptides from plants: Stabilization of the \u03b3-core of a tomato defensin by intramolecular disulfide bond","volume":"19","author":"Avitabile","year":"2013","journal-title":"J. Pept. Sci."},{"key":"ref_21","doi-asserted-by":"crossref","unstructured":"Garrigues, S., Gand\u00eda, M., Borics, A., Marx, F., Manzanares, P., and Marcos, J.F. (2017). Mapping and identification of antifungal peptides in the putative antifungal protein AfpB from the filamentous fungus Penicillium digitatum. Front. Microbiol., 8.","DOI":"10.3389\/fmicb.2017.00592"},{"key":"ref_22","doi-asserted-by":"crossref","unstructured":"Sonderegger, C., V\u00e1radi, G., Galg\u00f3czy, L., Kocsub\u00e9, S., Posch, W., Borics, A., Dubrac, S., T\u00f3th, G.K., Wilflingseder, D., and Marx, F. (2018). The evolutionary conserved \u03b3-core motif influences the anti-Candida activity of the Penicillium chrysogenum antifungal protein PAF. Front. Microbiol., 9.","DOI":"10.3389\/fmicb.2018.01655"},{"key":"ref_23","doi-asserted-by":"crossref","unstructured":"T\u00f3th, L., V\u00e1radi, G., Boros, \u00c9., Borics, A., Ficze, H., Nagy, I., T\u00f3th, G.K., R\u00e1khely, G., Marx, F., and Galg\u00f3czy, L. (2020). Biofungicidal potential of Neosartorya (Aspergillus) fischeri antifungal protein NFAP and novel synthetic \u03b3-core peptides. Front. Microbiol., 11.","DOI":"10.3389\/fmicb.2020.00820"},{"key":"ref_24","doi-asserted-by":"crossref","first-page":"7206","DOI":"10.1021\/acsomega.3c09377","article-title":"Rational design of antifungal peptides based on the \u03b3-core motif of a Neosartorya (Aspergillus) fischeri antifungal protein to improve structural integrity, efficacy, and spectrum","volume":"9","author":"Bende","year":"2024","journal-title":"ACS Omega"},{"key":"ref_25","doi-asserted-by":"crossref","first-page":"6693","DOI":"10.1007\/s12602-025-10890-y","article-title":"Screening the \u03b3-core motif peptides of ascomycetous antifungal proteins for antifungal activity and potential therapeutic applicability","volume":"18","author":"Karemera","year":"2026","journal-title":"Probiotics Antimicrob. Proteins"},{"key":"ref_26","doi-asserted-by":"crossref","unstructured":"Slezina, M.P., and Odintsova, T.I. (2026). Antimicrobial potential of defensin-derived \u03b3-core peptides of Thinopyrum elongatum (Host) D.R. Dewey as Bio-inspired Pesticides. Int. J. Mol. Sci., 27.","DOI":"10.3390\/ijms27104219"},{"key":"ref_27","doi-asserted-by":"crossref","first-page":"4206","DOI":"10.1128\/AAC.03130-15","article-title":"Antifungal mechanism of action of lactoferrin: Identification of H+-ATPase (P3A-type) as a new apoptotic-cell membrane receptor","volume":"60","author":"Fierro","year":"2016","journal-title":"Antimicrob. Agents Chemother."},{"key":"ref_28","doi-asserted-by":"crossref","unstructured":"Andr\u00e9s, M.T., Acosta-Zald\u00edvar, M., Gonz\u00e1lez-Seisdedos, J., and Fierro, J.F. (2019). Cytosolic acidification is the first transduction signal of lactoferrin-induced regulated cell death pathway. Int. J. Mol. Sci., 20.","DOI":"10.3390\/ijms20235838"},{"key":"ref_29","doi-asserted-by":"crossref","first-page":"309","DOI":"10.1016\/j.ijbiomac.2022.01.075","article-title":"A review on lactoferrin as a proton pump inhibitor","volume":"202","author":"Fierro","year":"2022","journal-title":"Int. J. Biol. Macromol."},{"key":"ref_30","doi-asserted-by":"crossref","unstructured":"Andr\u00e9s, M.T., Fierro, P., Antu\u00f1a, V., and Fierro, J.F. (2024). The antimicrobial activity of human defensins at physiological non-permeabilizing concentrations is caused by the inhibition of the plasma membrane H+-ATPases. Int. J. Mol. Sci., 25.","DOI":"10.3390\/ijms25137335"},{"key":"ref_31","doi-asserted-by":"crossref","unstructured":"Andr\u00e9s, M.T., Yount, N.Y., Acosta-Zald\u00edvar, M., Yeaman, M.R., and Fierro, J.F. (2024). The archetypal gamma-core motif of antimicrobial Cys-rich peptides inhibits H+-ATPases in target pathogens. Int. J. Mol. Sci., 25.","DOI":"10.3390\/ijms25179672"},{"key":"ref_32","doi-asserted-by":"crossref","first-page":"20309","DOI":"10.1074\/jbc.M710470200","article-title":"Vacuolar and plasma membrane proton pumps collaborate to achieve cytosolic pH homeostasis in yeast","volume":"283","author":"Kane","year":"2008","journal-title":"J. Biol. Chem."},{"key":"ref_33","doi-asserted-by":"crossref","first-page":"933","DOI":"10.1016\/j.bbagen.2011.03.011","article-title":"Intracellular pH is a tightly controlled signal in yeast","volume":"1810","author":"Orij","year":"2011","journal-title":"Biochim. Biophys. Acta (BBA)-Gen. Subj."},{"key":"ref_34","doi-asserted-by":"crossref","first-page":"33","DOI":"10.1007\/978-3-319-25304-6_3","article-title":"Proton transport and pH control in fungi","volume":"892","author":"Kane","year":"2016","journal-title":"Adv. Exp. Med. Biol."},{"key":"ref_35","doi-asserted-by":"crossref","first-page":"209","DOI":"10.3109\/10408419409114555","article-title":"Fungal plasma membrane proton pumps as promising new antifungal targets","volume":"20","author":"Monk","year":"1994","journal-title":"Crit. Rev. Microbiol."},{"key":"ref_36","doi-asserted-by":"crossref","first-page":"57","DOI":"10.1128\/AAC.49.1.57-70.2005","article-title":"Surface-active fungicidal D-peptide inhibitors of the plasma membrane proton pump that block azole resistance","volume":"49","author":"Monk","year":"2005","journal-title":"Antimicrob. Agents Chemother."},{"key":"ref_37","doi-asserted-by":"crossref","first-page":"e00032-17","DOI":"10.1128\/AAC.00032-17","article-title":"Identification of antifungal H+-ATPase inhibitors with effect on plasma membrane potential","volume":"61","author":"Kjellerup","year":"2017","journal-title":"Antimicrob. Agents Chemother."},{"key":"ref_38","doi-asserted-by":"crossref","unstructured":"Childers, D.S., Raziunaite, I., Avelar, G.M., Mackie, J., Budge, S., Stead, D., Gow, N.A.R., Lenardon, M.D., Ballou, E.R., and MacCallum, D.M. (2016). The rewiring of ubiquitination targets in a pathogenic yeast promotes metabolic flexibility, host colonization and virulence. PLoS Pathog., 12.","DOI":"10.1371\/journal.ppat.1005566"},{"key":"ref_39","doi-asserted-by":"crossref","first-page":"343","DOI":"10.1016\/j.ijbiomac.2020.12.224","article-title":"Lactoferrin perturbs lipid rafts and requires integrity of Pma1p\u2013lipid rafts association to exert its antifungal activity against Saccharomyces cerevisiae","volume":"171","author":"Chaves","year":"2021","journal-title":"Int. J. Biol. Macromol."},{"key":"ref_40","doi-asserted-by":"crossref","first-page":"4081","DOI":"10.1128\/AAC.01597-07","article-title":"Human lactoferrin induces apoptosis-like cell death in Candida albicans: Critical role of K+-channel-mediated K+ efflux","volume":"52","author":"Fierro","year":"2008","journal-title":"Antimicrob. Agents Chemother."},{"key":"ref_41","doi-asserted-by":"crossref","first-page":"3251","DOI":"10.1128\/IAI.71.6.3251-3260.2003","article-title":"Killing of Candida albicans by human salivary histatin 5 is modulated, but not determined, by the potassium channel TOK1","volume":"71","author":"Baev","year":"2003","journal-title":"Infect. Immun."},{"key":"ref_42","doi-asserted-by":"crossref","first-page":"139","DOI":"10.1152\/advan.00029.2004","article-title":"Generation of resting membrane potential","volume":"28","author":"Wright","year":"2004","journal-title":"Adv. Physiol. Educ."},{"key":"ref_43","doi-asserted-by":"crossref","first-page":"4550","DOI":"10.1128\/iai.63.11.4550-4552.1995","article-title":"Giardicidal activity of lactoferrin and N-terminal peptides","volume":"63","author":"Turchany","year":"1995","journal-title":"Infect. Immun."},{"key":"ref_44","doi-asserted-by":"crossref","first-page":"2583","DOI":"10.1128\/AAC.49.7.2583-2588.2005","article-title":"Different anti-Candida activities of two human lactoferrin-derived peptides, Lfpep and kaliocin-1","volume":"49","author":"Fierro","year":"2005","journal-title":"Antimicrob. Agents Chemother."},{"key":"ref_45","doi-asserted-by":"crossref","first-page":"1319","DOI":"10.1107\/S0907444998004417","article-title":"Structure of human apolactoferrin at 2.0 \u00c5 resolution. Refinement and analysis of ligand-induced conformational change","volume":"54","author":"Jameson","year":"1998","journal-title":"Acta Crystallogr. Sect. D Biol. Crystallogr."},{"key":"ref_46","doi-asserted-by":"crossref","first-page":"28445","DOI":"10.1074\/jbc.274.40.28445","article-title":"Crystal structure of hen apo-ovotransferrin. Both lobes adopt an open conformation upon loss of iron","volume":"274","author":"Kurokawa","year":"1999","journal-title":"J. Biol. Chem."},{"key":"ref_47","doi-asserted-by":"crossref","unstructured":"Wesseling, C.M.J., Wood, T.M., Bertheussen, K., Lok, S., and Martin, N.I. (2021). Thrombin-derived peptides potentiate the activity of Gram-positive-specific antibiotics against Gram-negative bacteria. 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