{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,11,4]],"date-time":"2026-11-04T21:54:55Z","timestamp":1793829295398,"version":"4.6.1"},"reference-count":41,"publisher":"Oxford University Press (OUP)","issue":"7","license":[{"start":{"date-parts":[[2018,3,15]],"date-time":"2018-03-15T00:00:00Z","timestamp":1521072000000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/academic.oup.com\/journals\/pages\/open_access\/funder_policies\/chorus\/standard_publication_model"}],"funder":[{"DOI":"10.13039\/100000060","name":"National Institute of Allergy and Infectious Diseases","doi-asserted-by":"publisher","id":[{"id":"10.13039\/100000060","id-type":"DOI","asserted-by":"publisher"}]},{"DOI":"10.13039\/100000002","name":"NIH","doi-asserted-by":"publisher","id":[{"id":"10.13039\/100000002","id-type":"DOI","asserted-by":"publisher"}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":[],"published-print":{"date-parts":[[2018,10,1]]},"abstract":"<jats:title>Abstract<\/jats:title>\n               <jats:p>Cryptococcosis is an opportunistic fungal infection caused by members of the two sibling species complexes: Cryptococcus neoformans and Cryptococcus gattii. Flucytosine (5FC) is one of the most widely used antifungals against Cryptococcus spp., yet very few studies have looked at the molecular mechanisms responsible for 5FC resistance in this pathogen. In this study, we examined 11 C. gattii clinical isolates of the major molecular type VGIII based on differential 5FC susceptibility and asked whether there were genomic changes in the key genes involved in flucytosine metabolism. Susceptibility assays and sequencing analysis revealed an association between a point mutation in the cytosine deaminase gene (FCY1) and 5FC resistance in two of the studied 5FC resistant C. gattii VGIII clinical isolates, B9322 and JS5. This mutation results in the replacement of arginine for histidine at position 29 and occurs within a variable stretch of amino acids. Heterologous expression of FCY1 and spot sensitivity assays, however, demonstrated that this point mutation did not have any effect on FCY1 activities and was not responsible for 5FC resistance. Comparative sequence analysis further showed that no changes in the amino acid sequence and no genomic alterations were observed within 1\u00a0kb of the upstream and downstream sequences of either cytosine permeases (FCY2-4) or uracil phosphoribosyltransferase (FUR1) genes in 5FC resistant and 5FC susceptible C. gattii VGIII isolates. The herein obtained results suggest that the observed 5FC resistance in the isolates B9322 and JS5 is due to changes in unknown protein(s) or pathway(s) that regulate flucytosine metabolism.<\/jats:p>","DOI":"10.1093\/mmy\/myx135","type":"journal-article","created":{"date-parts":[[2018,2,28]],"date-time":"2018-02-28T04:12:21Z","timestamp":1519791141000},"page":"857-867","source":"Crossref","is-referenced-by-count":24,"title":["Flucytosine resistance in <i>Cryptococcus gattii<\/i> is indirectly mediated by the FCY2-FCY1-FUR1 pathway"],"prefix":"10.1093","volume":"56","author":[{"given":"Kiem","family":"Vu","sequence":"first","affiliation":[{"name":"Department of Pharmacology, University of California , Davis, California, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"suffix":"III","given":"George R","family":"Thompson","sequence":"additional","affiliation":[{"name":"Department of Medical Microbiology and Immunology, University of California , Davis, California, USA"},{"name":"Department of Internal Medicine, Division of Infectious Diseases, University of California Davis Medical Center , Davis, California, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Chandler C","family":"Roe","sequence":"additional","affiliation":[{"name":"Translational Genomics Research Institute , Flagstaff, Arizona, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Jane E","family":"Sykes","sequence":"additional","affiliation":[{"name":"Department of Medicine and Epidemiology, School of Veterinary Medicine, University of California , Davis, California, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Elizabeth M","family":"Dreibe","sequence":"additional","affiliation":[{"name":"Translational Genomics Research Institute , Flagstaff, Arizona, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Shawn R","family":"Lockhart","sequence":"additional","affiliation":[{"name":"Mycotic Diseases Branch, Centers for Disease Control and Prevention , Atlanta, Georgia USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Wieland","family":"Meyer","sequence":"additional","affiliation":[{"name":"Molecular Mycology Research Laboratory, Center for Infectious Diseases and Microbiology, Marie Bashir Institute for Emerging Infectious Diseases and Biosecurity, Westmead Clinical School, Sydney Medical School, Westmead Hospital, The University of Sydney, Westmead Institute for Medical Research , Sydney, Australia"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"David M","family":"Engelthaler","sequence":"additional","affiliation":[{"name":"Translational Genomics Research Institute , Flagstaff, Arizona, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Angie","family":"Gelli","sequence":"additional","affiliation":[{"name":"Department of Pharmacology, University of California , Davis, California, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"286","published-online":{"date-parts":[[2018,3,15]]},"reference":[{"key":"2024022608422149100_bib1","doi-asserted-by":"crossref","first-page":"1185","DOI":"10.3201\/eid1508.081384","article-title":"Spread of Cryptococcus gattii into Pacific Northwest region of the United States","volume":"15","author":"Datta","year":"2009","journal-title":"Emerg Infect Dis"},{"key":"2024022608422149100_bib2","doi-asserted-by":"crossref","first-page":"792","DOI":"10.1111\/cmi.12590","article-title":"Cryptococcal therapies and drug targets: the old, the new and the promising","volume":"18","author":"Coelho","year":"2016","journal-title":"Cell Microbiol"},{"key":"2024022608422149100_bib3","doi-asserted-by":"crossref","first-page":"291","DOI":"10.1086\/649858","article-title":"Clinical practice guidelines for the management of cryptococcal disease: 2010 update by the Infectious Diseases Society of America","volume":"50","author":"Perfect","year":"2010","journal-title":"Clin Infect Dis"},{"key":"2024022608422149100_bib4","doi-asserted-by":"crossref","first-page":"1291","DOI":"10.1056\/NEJMoa1110404","article-title":"Combination Antifungal Therapy for Cryptococcal Meningitis","volume":"368","author":"Day","year":"2013","journal-title":"N Engl J Med"},{"key":"2024022608422149100_bib5","doi-asserted-by":"crossref","first-page":"276","DOI":"10.1007\/PL00011265","article-title":"Flucytosine primary resistance in Candida species and Cryptococcus neoformans","volume":"20","author":"Cuenca-Estrella","year":"2001","journal-title":"Eur J Clin Microbiol Infect Dis"},{"key":"2024022608422149100_bib6","doi-asserted-by":"crossref","first-page":"4377","DOI":"10.1128\/AAC.48.11.4377-4386.2004","article-title":"Molecular mechanisms of primary resistance to flucytosine in Candida albicans","volume":"48","author":"Hope","year":"2004","journal-title":"Antimicrob Agents Chemother"},{"key":"2024022608422149100_bib7","doi-asserted-by":"crossref","first-page":"662","DOI":"10.1086\/313749","article-title":"Practice guidelines for the treatment of candidiasis. Infectious Diseases Society of America","volume":"30","author":"Rex","year":"2000","journal-title":"Clin Infect Dis"},{"key":"2024022608422149100_bib8","doi-asserted-by":"crossref","first-page":"45","DOI":"10.3109\/10408418709104447","article-title":"The genetic basis of resistance to 5-fluorocytosine in Candida species and Cryptococcus neoformans","volume":"15","author":"Whelan","year":"1987","journal-title":"Crit Rev Microbiol"},{"key":"2024022608422149100_bib9","doi-asserted-by":"crossref","first-page":"649","DOI":"10.1128\/AAC.3.6.649","article-title":"5-fluorocytosine resistance in Cryptococcus neoformans","volume":"3","author":"Block","year":"1973","journal-title":"Antimicrob Agents Chemother"},{"key":"2024022608422149100_bib10","doi-asserted-by":"crossref","first-page":"53","DOI":"10.1128\/EC.05236-11","article-title":"A flucytosine-responsive Mbp1\/Swi4-like protein, Mbs1, plays pleiotropic roles in antifungal drug resistance, stress response, and virulence of Cryptococcus neoformans","volume":"11","author":"Song","year":"2012","journal-title":"Eukaryotic cell"},{"key":"2024022608422149100_bib11","doi-asserted-by":"crossref","first-page":"e01464","DOI":"10.1128\/mBio.01464-14","article-title":"Cryptococcus gattii in North American Pacific Northwest: whole-population genome analysis provides insights into species evolution and dispersal","volume":"5","author":"Engelthaler","year":"2014","journal-title":"MBio"},{"key":"2024022608422149100_bib12","doi-asserted-by":"crossref","first-page":"e0004861","DOI":"10.1371\/journal.pntd.0004861","article-title":"MLST and whole-genome-based population analysis of Cryptococcus gattii VGIII links clinical, veterinary and environmental strains, and reveals divergent serotype specific sub-populations and distant ancestors","volume":"10","author":"Firacative","year":"2016","journal-title":"PLoS Negl Trop Dis"},{"key":"2024022608422149100_bib13","doi-asserted-by":"crossref","first-page":"e74737","DOI":"10.1371\/journal.pone.0074737","article-title":"Cryptococcus gattii in the United States: genotypic diversity of human and veterinary isolates","volume":"8","author":"Lockhart","year":"2013","journal-title":"PLoS One"},{"key":"2024022608422149100_bib14","doi-asserted-by":"crossref","first-page":"2061","DOI":"10.1128\/JCM.03392-13","article-title":"Antifungal drug susceptibility and phylogenetic diversity among Cryptococcus isolates from dogs and cats in North America","volume":"52","author":"Singer","year":"2014","journal-title":"J Clin Microbiol"},{"key":"2024022608422149100_bib15","doi-asserted-by":"crossref","first-page":"539","DOI":"10.1128\/JCM.01505-09","article-title":"Correlation of genotype and in vitro susceptibilities of Cryptococcus gattii strains from the Pacific Northwest of the United States","volume":"48","author":"Iqbal","year":"2010","journal-title":"J Clin Microbiol"},{"key":"2024022608422149100_bib16","doi-asserted-by":"crossref","first-page":"e1002205","DOI":"10.1371\/journal.ppat.1002205","article-title":"A diverse population of Cryptococcus gattii molecular type VGIII in southern Californian HIV\/AIDS patients","volume":"7","author":"Byrnes","year":"2011","journal-title":"PLoS Pathog"},{"key":"2024022608422149100_bib17","article-title":"Reference method for broth dilution antifungal susceptibility testing of yeasts","volume-title":"CLSI document M27-A3","author":"CLSI","year":"2008"},{"key":"2024022608422149100_bib18","doi-asserted-by":"crossref","first-page":"119","DOI":"10.1016\/0378-1119(95)00037-7","article-title":"Yeast vectors for the controlled expression of heterologous proteins in different genetic backgrounds","volume":"156","author":"Mumberg","year":"1995","journal-title":"Gene"},{"key":"2024022608422149100_bib19","first-page":"107","article-title":"Yeast transformation by the LiAc\/SS carrier DNA\/PEG method","volume":"313","author":"Goetz","year":"2006","journal-title":"Methods Mol Biol"},{"key":"2024022608422149100_bib20","doi-asserted-by":"crossref","first-page":"38","DOI":"10.1006\/fgbi.1999.1180","article-title":"Gene disruption by biolistic transformation in serotype D strains of Cryptococcus neoformans","volume":"29","author":"Davidson","year":"2000","journal-title":"Fungal Genet Biol"},{"key":"2024022608422149100_bib21","doi-asserted-by":"crossref","first-page":"4733","DOI":"10.1128\/AAC.00605-10","article-title":"Mutational analysis of flucytosine resistance in Candida glabrata","volume":"54","author":"Edlind","year":"2010","journal-title":"Antimicrob Agents Chemother"},{"key":"2024022608422149100_bib22","doi-asserted-by":"crossref","first-page":"961","DOI":"10.1016\/S0969-2126(03)00153-9","article-title":"The 1.14 A crystal structure of yeast cytosine deaminase: evolution of nucleotide salvage enzymes and implications for genetic chemotherapy","volume":"11","author":"Ireton","year":"2003","journal-title":"Structure"},{"key":"2024022608422149100_bib23","doi-asserted-by":"crossref","first-page":"19111","DOI":"10.1074\/jbc.M300874200","article-title":"Crystal structure of yeast cytosine deaminase. Insights into enzyme mechanism and evolution","volume":"278","author":"Ko","year":"2003","journal-title":"J Biol Chem"},{"key":"2024022608422149100_bib24","doi-asserted-by":"crossref","first-page":"2982","DOI":"10.1128\/AAC.00880-08","article-title":"Nonsense and missense mutations in FCY2 and FCY1 genes are responsible for flucytosine resistance and flucytosine-fluconazole cross-resistance in clinical isolates of Candida lusitaniae","volume":"53","author":"Florent","year":"2009","journal-title":"Antimicrob Agents Chemother"},{"key":"2024022608422149100_bib25","doi-asserted-by":"crossref","first-page":"12766","DOI":"10.1038\/ncomms12766","article-title":"Systematic functional analysis of kinases in the fungal pathogen Cryptococcus neoformans","volume":"7","author":"Lee","year":"2016","journal-title":"Nat Commun"},{"key":"2024022608422149100_bib26","doi-asserted-by":"crossref","first-page":"369","DOI":"10.1128\/AAC.00824-06","article-title":"Molecular mechanism of flucytosine resistance in Candida lusitaniae: contribution of the FCY2, FCY1, and FUR1 genes to 5-fluorouracil and fluconazole cross-resistance","volume":"51","author":"Papon","year":"2007","journal-title":"Antimicrob Agents Chemother"},{"key":"2024022608422149100_bib27","doi-asserted-by":"crossref","first-page":"4678","DOI":"10.1128\/AAC.00607-09","article-title":"A Ser29Leu substitution in the cytosine deaminase Fca1p is responsible for clade-specific flucytosine resistance in Candida dubliniensis","volume":"53","author":"McManus","year":"2009","journal-title":"Antimicrob Agents Chemother"},{"key":"2024022608422149100_bib28","doi-asserted-by":"crossref","first-page":"707","DOI":"10.1002\/yea.1387","article-title":"Various cytosine\/adenine permease homologues are involved in the toxicity of 5-fluorocytosine in Saccharomyces cerevisiae","volume":"23","author":"Paluszynski","year":"2006","journal-title":"Yeast"},{"key":"2024022608422149100_bib29","doi-asserted-by":"crossref","first-page":"1","DOI":"10.1007\/s002940050169","article-title":"Characterization of the Saccharomyces cerevisiae FCY1 gene encoding cytosine deaminase and its homologue FCA1 of Candida albicans","volume":"31","author":"Erbs","year":"1997","journal-title":"Current Genetics"},{"key":"2024022608422149100_bib30","doi-asserted-by":"crossref","first-page":"585","DOI":"10.1111\/j.1365-2958.1990.tb00627.x","article-title":"The purine-cytosine permease gene of Saccharomyces cerevisiae: primary structure and deduced protein sequence of the FCY2 gene product","volume":"4","author":"Weber","year":"1990","journal-title":"Mol Microbiol"},{"key":"2024022608422149100_bib31","doi-asserted-by":"crossref","first-page":"149","DOI":"10.1016\/0378-1119(90)90026-N","article-title":"The FUR1 gene of Saccharomyces cerevisiae: cloning, structure and expression of wild-type and mutant alleles","volume":"88","author":"Kern","year":"1990","journal-title":"Gene"},{"key":"2024022608422149100_bib32","doi-asserted-by":"crossref","first-page":"2163","DOI":"10.1128\/JCM.43.5.2163-2167.2005","article-title":"Global trends in the antifungal susceptibility of Cryptococcus neoformans (1990 to 2004)","volume":"43","author":"Pfaller","year":"2005","journal-title":"J Clin Microbiol"},{"key":"2024022608422149100_bib33","doi-asserted-by":"crossref","first-page":"5139","DOI":"10.1128\/AAC.00746-10","article-title":"In vitro antifungal susceptibilities and amplified fragment length polymorphism genotyping of a worldwide collection of 350 clinical, veterinary, and environmental Cryptococcus gattii isolates","volume":"54","author":"Hagen","year":"2010","journal-title":"Antimicrob. Agents Chemother."},{"key":"2024022608422149100_bib34","doi-asserted-by":"crossref","first-page":"5898","DOI":"10.1128\/AAC.01115-12","article-title":"Cryptococcus neoformans-Cryptococcus gattii species complex: an international study of wild-type susceptibility endpoint distributions and epidemiological cutoff values for fluconazole, itraconazole, posaconazole, and voriconazole","volume":"56","author":"Espinel-Ingroff","year":"2012","journal-title":"Antimicrob Agents Chemother"},{"key":"2024022608422149100_bib35","doi-asserted-by":"crossref","first-page":"2606","DOI":"10.1128\/AAC.00048-11","article-title":"Trends in antifungal drug susceptibility of Cryptococcus neoformans isolates obtained through population-based surveillance in South Africa in 2002\u20132003 and 2007\u20132008","volume":"55","author":"Govender","year":"2011","journal-title":"Antimicrob Agents Chemother"},{"key":"2024022608422149100_bib36","first-page":"675213","article-title":"Global molecular epidemiology of Cryptococcus neoformans and Cryptococcus gattii: an atlas of the molecular types","author":"Cogliati","year":"2013","journal-title":"Scientifica (Cairo)"},{"key":"2024022608422149100_bib37","doi-asserted-by":"crossref","first-page":"16","DOI":"10.1016\/j.fgb.2015.02.009","article-title":"Recognition of seven species in the Cryptococcus gattii\/Cryptococcus neoformans species complex","volume":"78","author":"Hagen","year":"2015","journal-title":"Fungal Genet Biol"},{"key":"2024022608422149100_bib38","doi-asserted-by":"crossref","first-page":"e00357","DOI":"10.1128\/mSphere.00357-16","article-title":"The case for adopting the \u201cspecies complex\u201d nomenclature for the etiologic agents of cryptococcosis","volume":"2","author":"Kwon-Chung","year":"2017","journal-title":"mSphere"},{"key":"2024022608422149100_bib39","doi-asserted-by":"crossref","first-page":"1608","DOI":"10.1091\/mbc.01-10-0472","article-title":"Discrimination between paralogs using microarray analysis: application to the Yap1p and Yap2p transcriptional networks","volume":"13","author":"Cohen","year":"2002","journal-title":"Mol Biol Cell"},{"key":"2024022608422149100_bib40","doi-asserted-by":"crossref","first-page":"2506","DOI":"10.1128\/AAC.01777-10","article-title":"Loss of heterozygosity of FCY2 leading to the development of flucytosine resistance in Candida tropicalis","volume":"55","author":"Chen","year":"2011","journal-title":"Antimicrob Agents Chemother"},{"key":"2024022608422149100_bib41","doi-asserted-by":"crossref","first-page":"e0135110","DOI":"10.1371\/journal.pone.0135110","article-title":"New mechanisms of flucytosine resistance in C. glabrata unveiled by a Chemogenomics analysis in S. cerevisiae","volume":"10","author":"Costa","year":"2015","journal-title":"PloS one"}],"container-title":["Medical Mycology"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/academic.oup.com\/mmy\/article-pdf\/56\/7\/857\/56755598\/mmy_56_7_857.pdf","content-type":"application\/pdf","content-version":"vor","intended-application":"syndication"},{"URL":"https:\/\/academic.oup.com\/mmy\/article-pdf\/56\/7\/857\/56755598\/mmy_56_7_857.pdf","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2024,2,26]],"date-time":"2024-02-26T08:43:00Z","timestamp":1708936980000},"score":1,"resource":{"primary":{"URL":"https:\/\/academic.oup.com\/mmy\/article\/56\/7\/857\/4938061"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2018,3,15]]},"references-count":41,"journal-issue":{"issue":"7","published-print":{"date-parts":[[2018,10,1]]}},"URL":"https:\/\/doi.org\/10.1093\/mmy\/myx135","relation":{},"ISSN":["1460-2709","1369-3786"],"issn-type":[{"value":"1460-2709","type":"electronic"},{"value":"1369-3786","type":"print"}],"subject":[],"published-other":{"date-parts":[[2018,10]]},"published":{"date-parts":[[2018,3,15]]}}}