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Acquired amphotericin B resistance leads to fitness trade-offs that can be mitigated by compensatory evolution in Candida auris

Nature Microbiology volume 9, pages 3304–3320 (2024)Cite this article

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Abstract

Candida auris is a growing concern due to its resistance to antifungal drugs, particularly amphotericin B (AMB), detected in 30 to 60% of clinical isolates. However, the mechanisms of AMB resistance remain poorly understood. Here we investigated 441 in vitro- and in vivo-evolved C. auris lineages from 4 AMB-susceptible clinical strains of different clades. Genetic and sterol analyses revealed four major types of sterol alterations as a result of clinically rare variations in sterol biosynthesis genes ERG6, NCP1, ERG11, ERG3, HMG1, ERG10 and ERG12. In addition, aneuploidies in chromosomes 4 and 6 emerged during resistance evolution. Fitness trade-off phenotyping and mathematical modelling identified diverse strain- and mechanism-dependent fitness trade-offs. Variation in CDC25 rescued fitness trade-offs, thereby increasing the infection capacity. This possibly contributed to therapy-induced acquired AMB resistance in the clinic. Our findings highlight sterol-modulating mechanisms and fitness trade-off compensation as risks for AMB treatment failure in clinical settings.

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Fig. 1: Experimental evolution and resistance–fitness analysis.
Fig. 2: AMB susceptibility and membrane sterol profile of whole-genome-sequenced strains and constructed ERG gene mutants.
Fig. 3: Fitness trade-offs of four main sterol alteration types.
Fig. 4: Model-based population size estimates of resistant mutants after treatment.
Fig. 5: Resistance and fitness effects of variation in ERG6 and CDC25.

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Data availability

Parental strains of C. auris are available via the CDC & FDA Antibiotic Resistance Isolate Bank (https://wwwn.cdc.gov/arisolatebank/; panel identifier CAU—strain AR0387 and strain AR1097 respectively) or upon request from J.F.M. ([email protected]). Illumina sequences are available via the NCBI Sequence Read Archive (SRA) BioProject PRJNA984918 accession numbers SRR26533546–SRR26533577. AMB susceptibility data of clinical isolates of C. auris from ref. 13 are summarized in supplementary table 1 of that study and were shared by S. R. Lockhart ([email protected]). Source data are provided with this paper. Requests for further information should be directed to and will be fulfilled by the corresponding authors.

Code availability

The code used for mathematical modelling is available via GitHub at https://github.com/GiorgioBoccarella/AMB_paper.

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Acknowledgements

This study was supported by the Fund for Scientific Research Flanders (FWO) under the framework of the Joint Programming Initiative on Antimicrobial Resistance (JPIAMR) fund (project CycleDrug) and by a C3 grant from the Industrial Research Fund of KU Leuven (C3/22/007) granted to P.V.D. H.C., D.S., S.J. and G.B. were supported by FWO PhD fellowships 11D7620N, 11J8122N, 11PRR24N and 1150023N, respectively. H.C. was also supported by a post-doctoral fellowship granted by KU Leuven Internal Funds (PDMT2/23/032). V.B. received funding from the European Union’s Horizon 2020 research and innovation programme under the Marie Skłodowska-Curie grant agreement number 945352. The T.G. group acknowledges support from the Spanish Ministry of Science and Innovation (grant numbers PID2021-126067NB-I00, CPP2021-008552, PCI2022-135066-2 and PDC2022-133266-I00), cofounded by ERDF ‘A way of making Europe’, as well as support from the Catalan Research Agency (AGAUR) (grant number SGR01551), ‘La Caixa’ foundation (grant number LCF/PR/HR21/00737) and Instituto de Salud Carlos III (IMPACT grant IMP/00019 and CIBERINFEC CB21/13/00061-ISCIII-SGEFI/ERDF). P.v.d.B. acknowledges the support of KU Leuven start-up funding (GNM-E1097-STG/21/003). N.C.C. and C.A.C. were supported by the US National Institutes of Health grant 1RO1AI169066. We want to thank C. Pohl-Albertyn and K. Albertyn for providing us with the C. auris clade III strain from South Africa, and J. Parker for advice on sterol identification. We thank the US Centers for Disease Control and Prevention Mycotic Diseases Branch for sharing amphotericin resistance measurements.

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Author notes

  1. These authors contributed equally: Hans Carolus, Dimitrios Sofras.

Authors and Affiliations

  1. Laboratory of Molecular Cell Biology, Department of Biology, KU Leuven, Leuven, Belgium

    Hans Carolus, Dimitrios Sofras, Celia Lobo Romero, Rudy Vergauwen, Saleh Yazdani, Siebe Pierson, Stef Jacobs, Paul Vandecruys, Stefanie Wijnants & Patrick Van Dijck

  2. Evolutionary Modelling Group, Department of Microbial and Molecular Systems, KU Leuven, Leuven, Belgium

    Giorgio Boccarella & Pieter van den Berg

  3. Broad Institute of MIT and Harvard, Cambridge, MA, USA

    Poppy Sephton-Clark & Christina A. Cuomo

  4. Barcelona Supercomputing Centre (BSC-CNS), Barcelona, Spain

    Vladislav Biriukov & Toni Gabaldón

  5. Institute for Research in Biomedicine (IRB), Barcelona Institute of Science and Technology, Barcelona, Spain

    Vladislav Biriukov & Toni Gabaldón

  6. Molecular Microbiology and Immunology Department, Brown University, Providence, RI, USA

    Nicholas C. Cauldron & Christina A. Cuomo

  7. Centre of Expertise in Mycology, Radboudumc/CWZ, Nijmegen, The Netherlands

    Jacques F. Meis

  8. Institute of Translational Research, CECAD, University of Cologne, Cologne, Germany

    Jacques F. Meis

  9. Catalan Institution for Research and Advanced Studies (ICREA), Barcelona, Spain

    Toni Gabaldón

  10. CIBER de Enfermedades Infecciosas, Instituto de Salud Carlos III, Madrid, Spain

    Toni Gabaldón

  11. Evolutionary Modelling Group, Department of Biology, KU Leuven, Leuven, Belgium

    Pieter van den Berg

  12. St. Jude Children’s Research Hospital, Memphis, TN, USA

    Jeffrey M. Rybak

  13. KU Leuven One Health Institute, KU Leuven, Leuven, Belgium

    Patrick Van Dijck

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  1. Hans Carolus
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  2. Dimitrios Sofras
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H.C. conceptualized, designed and supervised the study, provided funding, carried out experiments, analysed the data, and wrote the paper. D.S. carried out experiments, analysed the data and wrote the paper. G.B. designed and carried out mathematical modelling. P.S.-C., V.B. and N.C.C. aided in sequence data analysis. C.L.R. aided in phenotypic screening and sterol profiling, R.V. aided in sterol profiling and analysis, S.Y. aided in murine infections and treatment, S.P. aided in in vitro experimental evolution assays, S.J. aided in mutation rate analysis and P.V. aided in ploidy determination. S.W. provided conceptual insights, J.F.M. provided strains, and J.M.R. provided plasmids and aided in the design for strain construction. T.G., P.v.d.B., C.A.C. and P.V.D. provided funding and supervised the study. All authors edited and/or approved the paper.

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Correspondence to Hans Carolus or Patrick Van Dijck.

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Nature Microbiology thanks Jan-Ulrich Kreft, Rebecca Shapiro, Clement Tsui, Jianping Xu and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.

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Extended data

Extended Data Fig. 1 Strain selection and genotyping.

a) Selection of in vitro and in vivo evolved isolates for genotyping based on resistance [RR(AUC) > 1] and susceptibility ranges (BDA MIC range 2 ≤ MIC < 16 µg/ml and ETEST® MIC range of 1.5 ≤ MIC < 32 µg/ml) observed in clinical AMB resistant C. auris strains. Pie chart shows proportions of strains containing ERG6, ERG11 and/or ERG3 variation in 50 selected strains (targeted sequencing and WGS). Details of all genotyped strains can be found in Supplementary Table 2 (Supplementary). b) Graphic representation of variations found in genes involved in ergosterol biosynthesis in selected WGS strains. Lollipop pictograms indicate mutations, indels or frameshifts (nucleotide deletion or insertion), brackets indicate larger deletions (more details see Table 1). A partial translocation in NCP1 in strain 14 is not displayed here but is shown separately in Supplementary Fig. 3 (Supplementary). Colors of lolipops indicate clade origin (legend see Fig. 1b,/c). Functional domains are annotated as GHMPK(N/C): galacto-homoserine, mevalonate and phosphomevalonate kinases (N/C)-terminal ___domain, SSD: sterol sensing ___domain, HMG-CoA R: HMG-CoA reductase catalytic ___domain, ACAT(N/C)D: acetyl-coenzyme A acetyltransferases (N/C)-terminal ___domain, FNOS: flavodoxin/NO synthase, BD: binding ___domain, FAH: fatty acid hydrοxylase, SAM: S-adenosyl-methionine, SMC: Sterol methyltransferase C-terminal, a: active site, b: conserved site, g: iron binding site (C465), d: SAM binding residues (129-135,152,153,179-181). c–f) Sequencing read coverage of Clade I (C), III (D), IV (E) and V (F) strains. Relative coverage is plotted per chromosome position based on a fully assembled genome of each clade as described in the Methods section. Columns represent the different chromosomes (Chr) and rows indicate different strains. Strain 1, 10, 18 and 30 are the parental strains of Clade I, III, IV and V respectively, indicated as ‘wt’ (wild type).

Source data

Extended Data Fig. 2 Azole susceptibility of WGS strains.

Table listing selected evolved strains (numbers) and their respective ERG-genotype (details see Table 1). The four major membrane sterol alteration types (1-4) are indicated by numbered boxes on the left, colored based on the typical sterol present for that type (see Fig. 2). The heatmap on the left shows relative growth (%) in different concentrations of fluconazole (FLU) and posaconazole (POS) in a BDA assay, averaged over 2 technical repeats. The heatmaps on the right show the relative (to parental strain) FLU and POS resistance (RR), based on AUC and MIC as measured by BDA. # detailed information about the partial translocation of NCP1 in strain 14 can be found in Supplementary Fig. 3 (Supplementary). *Chr6 contains ERG27. **Chr4 contains ERG6, ERG2, ERG7 and ERG12.

Source data

Supplementary information

Supplementary Information

Supplementary Figs. 1–13 and Supplementary Tables 1–7.

Reporting Summary

Source data

Source Data Figs. 1–5 and Extended Data Figs. 1 and 2

Source data for Figs 1–5 and Extended Data Figs. 1 and 2 can be found in the different tabs of this Excel sheet.

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Carolus, H., Sofras, D., Boccarella, G. et al. Acquired amphotericin B resistance leads to fitness trade-offs that can be mitigated by compensatory evolution in Candida auris. Nat Microbiol 9, 3304–3320 (2024). https://doi.org/10.1038/s41564-024-01854-z

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