Acquired amphotericin B resistance and fitness trade-off compensation in Candida auris

Abstract

<title>Abstract</title> <italic>Candida auris</italic> is an emergent human fungal pathogen of growing concern due to common drug resistance to all major antifungal drug classes. Although resistance to amphotericin B (AMB) has been detected in 30 to 60% of clinical isolates of <italic>C. auris</italic>, mechanisms of AMB resistance remain poorly characterized. Here we present a large-scale investigation of how AMB resistance can be acquired through genetic adaptation. We typed 441 <italic>in vitro</italic> and <italic>in vivo</italic> evolved <italic>C. auris</italic> lineages from four AMB-susceptible clinical strains of different clades. We show a great diversity of acquired resistance responses with resistance magnitude- and strain-dependent fitness trade-offs. Genotyping and membrane sterol analyses of selected lineages show four major types of membrane sterol alterations. Using a novel, plasmid-based CRISPR-Cas9 allele editing method and Cas9-RNP meditated gene deletions, we show that AMB resistance can be acquired through variation in several sterol biosynthesis regulators including <italic>ERG6, NCP1, ERG11, ERG3, HMG1, ERG10</italic> and <italic>ERG12</italic>. Additionally, we show how aneuploidies in chromosomes 4 and 6 emerge during AMB resistance evolution. By leveraging fitness trade-off phenotyping and mathematical modelling of the <italic>in vivo </italic>environment during treatment, we evaluated the potential of different mechanisms to establish resistant infections and discover a mechanism of fitness trade-off compensation. Variation in <italic>CDC25</italic> substantially enhanced the capacity to establish a resistant infection and may have played a role in facilitating the sole documented clinical case of acquired AMB resistance during treatment in <italic>C. auris</italic>. In summary, our findings show that fitness trade-off compensation along with several sterol modulating mechanisms of acquired AMB resistance represent a potential risk for AMB treatment failure in the clinic.