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<jats:italic>Candida auris<\/jats:italic> show a high prevalence of resistance to Amphotericin B (AmB)\u2014an uncommon trait in most <jats:italic>Candida<\/jats:italic> species. Alterations in ergosterol biosynthesis can contribute to acquired AmB resistance in <jats:styled-content style=\"fixed-case\">\n<jats:italic>C. auris<\/jats:italic>\n<\/jats:styled-content> laboratory strains but are rarely seen in clinical isolates. In this study, we experimentally evolved two drug\u2010susceptible Clade II isolates of <jats:styled-content style=\"fixed-case\">\n<jats:italic>C. auris<\/jats:italic>\n<\/jats:styled-content> to develop AmB resistance. The evolved strains displayed a four to eight fold increase in MIC<jats:sub>50<\/jats:sub> compared to the parental cells. We analyzed changes in their karyotype, genome, lipidome, and transcriptome associated with this acquired resistance. In one lineage, <jats:italic>AOX2<\/jats:italic> was upregulated, and its deletion reversed the AmB resistance phenotype. The <jats:italic>aox2\u0394<\/jats:italic> mutant also failed to evolve AmB resistance under experimental conditions. In the same lineage, restoring the <jats:italic>UPC2<\/jats:italic>\n<jats:sup>\n<jats:italic>S332R<\/jats:italic>\n<\/jats:sup> <jats:italic>and RTG3<\/jats:italic>\n<jats:sup>\n<jats:italic>S101T<\/jats:italic>\n<\/jats:sup> mutations to the wild\u2010type allele restored AmB susceptibility. In another lineage, the ergosterol and sphingolipid pathways were observed to play a critical role, and upregulation of the ERG genes elevated the total sterol content, while significant downregulation of <jats:italic>HSX11<\/jats:italic> (glucosylceramide synthase) resulted in lower levels of glucosylceramides. To our knowledge, this study is the first to show that AmB resistance in <jats:styled-content style=\"fixed-case\">\n<jats:italic>C. auris<\/jats:italic>\n<\/jats:styled-content> can be acquired through mechanisms both dependent on or independent of sterol content modulation, highlighting Aox2 and Upc2 as key regulators of amphotericin resistance.<\/jats:p>","DOI":"10.1111\/mmi.15379","type":"journal-article","created":{"date-parts":[[2025,6,12]],"date-time":"2025-06-12T11:02:03Z","timestamp":1749726123000},"page":"154-172","update-policy":"https:\/\/doi.org\/10.1002\/crossmark_policy","source":"Crossref","is-referenced-by-count":16,"title":["Multi\u2010Omics Analysis of Experimentally Evolved <i>Candida\u00a0auris<\/i> Isolates Reveals Modulation of Sterols, Sphingolipids, and Oxidative Stress in Acquired Amphotericin B Resistance"],"prefix":"10.1111","volume":"124","author":[{"given":"Anshu","family":"Chauhan","sequence":"first","affiliation":[{"name":"Amity Institute of 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