{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,9,25]],"date-time":"2026-09-25T19:25:30Z","timestamp":1790364330258,"version":"4.1.0"},"reference-count":45,"publisher":"MDPI AG","issue":"11","license":[{"start":{"date-parts":[[2021,6,7]],"date-time":"2021-06-07T00:00:00Z","timestamp":1623024000000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"name":"Ministry of Science and Information and  Communications Technologies (ICT)","award":["None"],"award-info":[{"award-number":["None"]}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["Molecules"],"abstract":"<jats:p>Due to lifespan extension and changes in global climate, the increase in mycoses caused by primary and opportunistic fungal pathogens is now a global concern. Despite increasing attention, limited options are available for the treatment of systematic and invasive mycoses, owing to the evolutionary similarity between humans and fungi. Although plants produce a diversity of chemicals to protect themselves from pathogens, the molecular targets and modes of action of these plant-derived chemicals have not been well characterized. Using a reverse genetics approach, the present study revealed that thymol, a monoterpene alcohol from Thymus vulgaris L., (Lamiaceae), exhibits antifungal activity against Cryptococcus neoformans by regulating multiple signaling pathways including calcineurin, unfolded protein response, and HOG (high-osmolarity glycerol) MAPK (mitogen-activated protein kinase) pathways. Thymol treatment reduced the intracellular concentration of Ca2+ by controlling the expression levels of calcium transporter genes in a calcineurin-dependent manner. We demonstrated that thymol decreased N-glycosylation by regulating the expression levels of genes involved in glycan-mediated post-translational modifications. Furthermore, thymol treatment reduced endogenous ergosterol content by decreasing the expression of ergosterol biosynthesis genes in a HOG MAPK pathway-dependent manner. Collectively, this study sheds light on the antifungal mechanisms of thymol against C. neoformans.<\/jats:p>","DOI":"10.3390\/molecules26113476","type":"journal-article","created":{"date-parts":[[2021,6,7]],"date-time":"2021-06-07T13:25:43Z","timestamp":1623072343000},"page":"3476","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":35,"title":["Investigation of Antifungal Mechanisms of Thymol in the Human Fungal Pathogen, Cryptococcus neoformans"],"prefix":"10.3390","volume":"26","author":[{"given":"Kwang-Woo","family":"Jung","sequence":"first","affiliation":[{"name":"Radiation Research Division, Advanced Radiation Technology Institute, Korea Atomic Energy Research Institute, Jeongeup-si 56212, Jeollabuk-do, Korea"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Moon-Soo","family":"Chung","sequence":"additional","affiliation":[{"name":"Radiation Research Division, Advanced Radiation Technology Institute, Korea Atomic Energy Research Institute, Jeongeup-si 56212, Jeollabuk-do, Korea"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-1122-171X","authenticated-orcid":false,"given":"Hyoung-Woo","family":"Bai","sequence":"additional","affiliation":[{"name":"Radiation Research Division, Advanced Radiation Technology Institute, Korea Atomic Energy Research Institute, Jeongeup-si 56212, Jeollabuk-do, Korea"},{"name":"Department of Radiation Science and Technology, University of Science and Technology, Daejeon 34113, Yuseong-gu, Korea"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Byung-Yeoup","family":"Chung","sequence":"additional","affiliation":[{"name":"Radiation Research Division, Advanced Radiation Technology Institute, Korea Atomic Energy Research Institute, Jeongeup-si 56212, Jeollabuk-do, Korea"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Sungbeom","family":"Lee","sequence":"additional","affiliation":[{"name":"Radiation Research Division, Advanced Radiation Technology Institute, Korea Atomic Energy Research Institute, Jeongeup-si 56212, Jeollabuk-do, Korea"},{"name":"Department of Radiation Science and Technology, University of Science and Technology, Daejeon 34113, Yuseong-gu, Korea"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"1968","published-online":{"date-parts":[[2021,6,7]]},"reference":[{"key":"ref_1","doi-asserted-by":"crossref","unstructured":"Bongomin, F., Gago, S., Oladele, R.O., and Denning, D.W. (2017). Global and Multi-National Prevalence of Fungal Diseases-Estimate Precision. J. Fungi, 3.","DOI":"10.3390\/jof3040057"},{"key":"ref_2","doi-asserted-by":"crossref","first-page":"36","DOI":"10.3389\/fmicb.2017.00036","article-title":"Antifungal Therapy: New Advances in the Understanding and Treatment of Mycosis","volume":"8","author":"Scorzoni","year":"2017","journal-title":"Front. Microbiol."},{"key":"ref_3","doi-asserted-by":"crossref","first-page":"719","DOI":"10.1038\/nrd3074","article-title":"An insight into the antifungal pipeline: Selected new molecules and beyond","volume":"9","author":"Casadevall","year":"2010","journal-title":"Nat. Rev. Drug Discov."},{"key":"ref_4","doi-asserted-by":"crossref","first-page":"S3","DOI":"10.1016\/j.amjmed.2011.11.001","article-title":"Antifungal drug resistance: Mechanisms, epidemiology, and consequences for treatment","volume":"125","author":"Pfaller","year":"2012","journal-title":"Am. J. Med."},{"key":"ref_5","doi-asserted-by":"crossref","first-page":"1191","DOI":"10.2307\/3869817","article-title":"Two Classes of Plant Antibiotics: Phytoalexins versus \u201cPhytoanticipins\u201d","volume":"6","author":"VanEtten","year":"1994","journal-title":"Plant Cell"},{"key":"ref_6","doi-asserted-by":"crossref","first-page":"253","DOI":"10.1016\/S0031-9422(00)00450-7","article-title":"Plant-fungal interactions: The search for phytoalexins and other antifungal compounds from higher plants","volume":"56","author":"Grayer","year":"2001","journal-title":"Phytochemistry"},{"key":"ref_7","doi-asserted-by":"crossref","first-page":"728065","DOI":"10.1155\/2012\/728065","article-title":"Essential oils composition and antioxidant properties of three Thymus species","volume":"2012","author":"Amiri","year":"2012","journal-title":"Evid.-Based Complement. Altern. Med. eCAM"},{"key":"ref_8","first-page":"1688","article-title":"Thymol, thyme, and other plant sources: Health and potential uses. Phytother","volume":"32","author":"Salehi","year":"2018","journal-title":"Res. PTR"},{"key":"ref_9","doi-asserted-by":"crossref","first-page":"191","DOI":"10.1016\/S0378-1097(03)00890-5","article-title":"Susceptibility of methicillin-resistant staphylococci to oregano essential oil, carvacrol and thymol","volume":"230","author":"Nostro","year":"2004","journal-title":"FEMS Microbiol. Lett."},{"key":"ref_10","doi-asserted-by":"crossref","first-page":"448","DOI":"10.1046\/j.1472-765X.2003.01427.x","article-title":"Activity of natural antimicrobial compounds against Escherichia coli and Salmonella enterica serovar typhimurium","volume":"37","author":"Olasupo","year":"2003","journal-title":"Lett. Appl. Microbiol."},{"key":"ref_11","doi-asserted-by":"crossref","first-page":"502","DOI":"10.1111\/j.1439-0507.2007.01412.x","article-title":"Inhibitory activity of thymol against the formation and viability of Candida albicans hyphae","volume":"50","author":"Braga","year":"2007","journal-title":"Mycoses"},{"key":"ref_12","doi-asserted-by":"crossref","unstructured":"Shen, Q., Zhou, W., Li, H., Hu, L., and Mo, H. (2016). ROS Involves the Fungicidal Actions of Thymol against Spores of Aspergillus flavus via the Induction of Nitric Oxide. PLoS ONE, 11.","DOI":"10.1371\/journal.pone.0155647"},{"key":"ref_13","doi-asserted-by":"crossref","first-page":"2611","DOI":"10.1007\/s13197-019-03747-0","article-title":"Antifungal activity of thymol and carvacrol against postharvest pathogens Botrytis cinerea","volume":"56","author":"Zhang","year":"2019","journal-title":"J. Food Sci. Technol."},{"key":"ref_14","doi-asserted-by":"crossref","first-page":"14418","DOI":"10.3390\/molecules171214418","article-title":"Antifungal activity of Thymus vulgaris L. essential oil and its constituent phytochemicals against Rhizopus oryzae: Interaction with ergosterol","volume":"17","author":"Lima","year":"2012","journal-title":"Molecules"},{"key":"ref_15","doi-asserted-by":"crossref","first-page":"2630","DOI":"10.1080\/14786419.2018.1547296","article-title":"Antifungal activity study of the monoterpene thymol against Cryptococcus neoformans","volume":"34","author":"Teixeira","year":"2020","journal-title":"Nat. Prod. Res."},{"key":"ref_16","doi-asserted-by":"crossref","first-page":"873","DOI":"10.1016\/S1473-3099(17)30243-8","article-title":"Global burden of disease of HIV-associated cryptococcal meningitis: An updated analysis","volume":"17","author":"Rajasingham","year":"2017","journal-title":"Lancet Infect. Dis."},{"key":"ref_17","doi-asserted-by":"crossref","first-page":"69","DOI":"10.1146\/annurev.micro.60.080805.142102","article-title":"The biology of the Cryptococcus neoformans species complex","volume":"60","author":"Lin","year":"2006","journal-title":"Annu. Rev. Microbiol."},{"key":"ref_18","doi-asserted-by":"crossref","first-page":"753","DOI":"10.1038\/nrmicro1245","article-title":"Deciphering the model pathogenic fungus Cryptococcus neoformans","volume":"3","author":"Idnurm","year":"2005","journal-title":"Nat. Rev. Microbiol."},{"key":"ref_19","doi-asserted-by":"crossref","first-page":"e59","DOI":"10.1002\/cpmc.59","article-title":"Genetic Manipulation of Cryptococcus neoformans","volume":"50","author":"Jung","year":"2018","journal-title":"Curr. Protoc. Microbiol."},{"key":"ref_20","doi-asserted-by":"crossref","first-page":"2285","DOI":"10.1091\/mbc.e04-11-0987","article-title":"Specialization of the HOG pathway and its impact on differentiation and virulence of Cryptococcus neoformans","volume":"16","author":"Bahn","year":"2005","journal-title":"Mol. Biol. Cell"},{"key":"ref_21","doi-asserted-by":"crossref","first-page":"1377","DOI":"10.1046\/j.1365-2958.2003.03508.x","article-title":"The Cryptococcus neoformans MAP kinase Mpk1 regulates cell integrity in response to antifungal drugs and loss of calcineurin function","volume":"48","author":"Kraus","year":"2003","journal-title":"Mol. Microbiol."},{"key":"ref_22","doi-asserted-by":"crossref","first-page":"2576","DOI":"10.1093\/emboj\/16.10.2576","article-title":"Calcineurin is required for virulence of Cryptococcus neoformans","volume":"16","author":"Odom","year":"1997","journal-title":"EMBO J."},{"key":"ref_23","doi-asserted-by":"crossref","unstructured":"Cheon, S.A., Jung, K.W., Chen, Y.L., Heitman, J., Bahn, Y.S., and Kang, H.A. (2011). Unique evolution of the UPR pathway with a novel bZIP transcription factor, Hxl1, for controlling pathogenicity of Cryptococcus neoformans. PLoS Pathog., 7.","DOI":"10.1371\/journal.ppat.1002177"},{"key":"ref_24","doi-asserted-by":"crossref","first-page":"352","DOI":"10.1046\/j.1365-2958.2000.01852.x","article-title":"RAS1 regulates filamentation, mating and growth at high temperature of Cryptococcus neoformans","volume":"36","author":"Alspaugh","year":"2000","journal-title":"Mol. Microbiol."},{"key":"ref_25","doi-asserted-by":"crossref","first-page":"75","DOI":"10.1128\/EC.1.1.75-84.2002","article-title":"Adenylyl cyclase functions downstream of the G\u03b1 protein Gpa1 and controls mating and pathogenicity of Cryptococcus neoformans","volume":"1","author":"Alspaugh","year":"2002","journal-title":"Eukaryot. Cell"},{"key":"ref_26","doi-asserted-by":"crossref","unstructured":"Jung, K.W., Yang, D.H., Kim, M.K., Seo, H.S., Lim, S., and Bahn, Y.S. (2016). Unraveling Fungal Radiation Resistance Regulatory Networks through the Genome-Wide Transcriptome and Genetic Analyses of Cryptococcus neoformans. mBio, 7.","DOI":"10.1128\/mBio.01483-16"},{"key":"ref_27","doi-asserted-by":"crossref","unstructured":"Jung, K.W., Lee, Y., Huh, E.Y., Lee, S.C., Lim, S., and Bahn, Y.S. (2019). Rad53- and Chk1-Dependent DNA Damage Response Pathways Cooperatively Promote Fungal Pathogenesis and Modulate Antifungal Drug Susceptibility. mBio, 10.","DOI":"10.1128\/mBio.01726-18"},{"key":"ref_28","doi-asserted-by":"crossref","first-page":"5062","DOI":"10.1128\/AAC.01050-10","article-title":"Mechanism of antifungal activity of terpenoid phenols resembles calcium stress and inhibition of the TOR pathway","volume":"54","author":"Rao","year":"2010","journal-title":"Antimicrob. Agents Chemother."},{"key":"ref_29","doi-asserted-by":"crossref","first-page":"4853","DOI":"10.1111\/febs.12458","article-title":"The calcium transporter Pmc1 provides Ca2+ tolerance and influences the progression of murine cryptococcal infection","volume":"280","author":"Kmetzsch","year":"2013","journal-title":"FEBS J."},{"key":"ref_30","doi-asserted-by":"crossref","unstructured":"Park, H.S., Chow, E.W., Fu, C., Soderblom, E.J., Moseley, M.A., Heitman, J., and Cardenas, M.E. (2016). Calcineurin Targets Involved in Stress Survival and Fungal Virulence. PLoS Pathog., 12.","DOI":"10.1371\/journal.ppat.1005873"},{"key":"ref_31","doi-asserted-by":"crossref","first-page":"681","DOI":"10.1111\/j.1432-1033.1991.tb15959.x","article-title":"Yeast carboxypeptidase Y requires glycosylation for efficient intracellular transport, but not for vacuolar sorting, in vivo stability, or activity","volume":"197","author":"Winther","year":"1991","journal-title":"Eur. J. Biochem."},{"key":"ref_32","doi-asserted-by":"crossref","first-page":"1197","DOI":"10.1128\/EC.00120-09","article-title":"Remodeling of global transcription patterns of Cryptococcus neoformans genes mediated by the stress-activated HOG signaling pathways","volume":"8","author":"Ko","year":"2009","journal-title":"Eukaryot. Cell"},{"key":"ref_33","doi-asserted-by":"crossref","first-page":"87","DOI":"10.1016\/S1567-1356(03)00126-0","article-title":"Combined overexpression of genes of the ergosterol biosynthetic pathway leads to accumulation of sterols in Saccharomyces cerevisiae","volume":"4","author":"Veen","year":"2003","journal-title":"FEMS Yeast Res."},{"key":"ref_34","doi-asserted-by":"crossref","first-page":"1002","DOI":"10.3390\/molecules17011002","article-title":"Antifungal activity of eugenol analogues. Influence of different substituents and studies on mechanism of action","volume":"17","author":"Carrasco","year":"2012","journal-title":"Molecules"},{"key":"ref_35","doi-asserted-by":"crossref","first-page":"4584","DOI":"10.1128\/AAC.00551-15","article-title":"The Monoterpene Carvacrol Generates Endoplasmic Reticulum Stress in the Pathogenic Fungus Candida albicans","volume":"59","author":"Chaillot","year":"2015","journal-title":"Antimicrob. Agents Chemother."},{"key":"ref_36","doi-asserted-by":"crossref","unstructured":"Ansari, M.A., Fatima, Z., and Hameed, S. (2016). Anticandidal Effect and Mechanisms of Monoterpenoid, Perillyl Alcohol against Candida albicans. PLoS ONE, 11.","DOI":"10.1371\/journal.pone.0162465"},{"key":"ref_37","doi-asserted-by":"crossref","first-page":"41","DOI":"10.1007\/s10096-010-1050-8","article-title":"Fungicidal activity of thymol and carvacrol by disrupting ergosterol biosynthesis and membrane integrity against Candida. Eur","volume":"30","author":"Ahmad","year":"2011","journal-title":"J. Clin. Microbiol. Infect. Dis."},{"key":"ref_38","doi-asserted-by":"crossref","first-page":"630","DOI":"10.1111\/mmi.12388","article-title":"Sulphiredoxin plays peroxiredoxin-dependent and -independent roles via the HOG signalling pathway in Cryptococcus neoformans and contributes to fungal virulence","volume":"90","author":"Upadhya","year":"2013","journal-title":"Mol. Microbiol."},{"key":"ref_39","doi-asserted-by":"crossref","unstructured":"Tang, X., Shao, Y.L., Tang, Y.J., and Zhou, W.W. (2018). Antifungal Activity of Essential oil Compounds (Geraniol and Citral) and Inhibitory Mechanisms on Grain Pathogens (Aspergillus flavus and Aspergillus ochraceus). Molecules, 23.","DOI":"10.3390\/molecules23092108"},{"key":"ref_40","doi-asserted-by":"crossref","first-page":"826","DOI":"10.3109\/13693786.2013.795664","article-title":"Thymol antifungal mode of action involves telomerase inhibition","volume":"51","author":"Darvishi","year":"2013","journal-title":"Med. Mycol."},{"key":"ref_41","doi-asserted-by":"crossref","first-page":"1149","DOI":"10.1091\/mbc.9.5.1149","article-title":"The medial-Golgi ion pump Pmr1 supplies the yeast secretory pathway with Ca2+ and Mn2+ required for glycosylation, sorting, and endoplasmic reticulum-associated protein degradation","volume":"9","author":"Durr","year":"1998","journal-title":"Mol. Biol. Cell"},{"key":"ref_42","doi-asserted-by":"crossref","first-page":"24282","DOI":"10.1038\/srep24282","article-title":"Yeast Gdt1 is a Golgi-localized calcium transporter required for stress-induced calcium signaling and protein glycosylation","volume":"6","author":"Colinet","year":"2016","journal-title":"Sci. Rep."},{"key":"ref_43","doi-asserted-by":"crossref","unstructured":"Gunes-Bayir, A., Kocyigit, A., Guler, E.M., and Dadak, A. (2020). In Vitro Hormetic Effect Investigation of Thymol on Human Fibroblast and Gastric Adenocarcinoma Cells. Molecules, 25.","DOI":"10.3390\/molecules25143270"},{"key":"ref_44","doi-asserted-by":"crossref","first-page":"357","DOI":"10.4014\/jmb.1609.09036","article-title":"Molecular Characterization of Adenylyl Cyclase Complex Proteins Using Versatile Protein-Tagging Plasmid Systems in Cryptococcus neoformans","volume":"27","author":"So","year":"2017","journal-title":"J. Microbiol. Biotechnol."},{"key":"ref_45","doi-asserted-by":"crossref","first-page":"10104","DOI":"10.1038\/srep10104","article-title":"Effects of zinc transporters on Cryptococcus gattii virulence","volume":"5","author":"Diehl","year":"2015","journal-title":"Sci. Rep."}],"container-title":["Molecules"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/www.mdpi.com\/1420-3049\/26\/11\/3476\/pdf","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2025,10,11]],"date-time":"2025-10-11T06:11:49Z","timestamp":1760163109000},"score":1,"resource":{"primary":{"URL":"https:\/\/www.mdpi.com\/1420-3049\/26\/11\/3476"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2021,6,7]]},"references-count":45,"journal-issue":{"issue":"11","published-online":{"date-parts":[[2021,6]]}},"alternative-id":["molecules26113476"],"URL":"https:\/\/doi.org\/10.3390\/molecules26113476","relation":{},"ISSN":["1420-3049"],"issn-type":[{"value":"1420-3049","type":"electronic"}],"subject":[],"published":{"date-parts":[[2021,6,7]]}}}