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Cited by 325Antifungal resistance and susceptibilityFungal and yeast genetics researchEvolution and Genetic Dynamics

Fitness Trade-offs Restrict the Evolution of Resistance to Amphotericin B

Open access
Vincent, Benjamin Matteson, Lancaster, Alex Kelvin, Scherz-Shouval, Ruth, Whitesell, Luke, Lindquist, Susan·2013·PLoS Biology
DOI 10.1371/journal.pbio.1001692PMC PMC3812114
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Abstract

The evolution of drug resistance in microbial pathogens provides a paradigm for investigating evolutionary dynamics with important consequences for human health. Candida albicans, the leading fungal pathogen of humans, rapidly evolves resistance to two major antifungal classes, the triazoles and echinocandins. In contrast, resistance to the third major antifungal used in the clinic, amphotericin B (AmB), remains extremely rare despite 50 years of use as monotherapy. We sought to understand this long-standing evolutionary puzzle. We used whole genome sequencing of rare AmB-resistant clinical isolates as well as laboratory-evolved strains to identify and investigate mutations that confer AmB resistance in vitro. Resistance to AmB came at a great cost. Mutations that conferred resistance simultaneously created diverse stresses that required high levels of the molecular chaperone Hsp90 for survival, even in the absence of AmB. This requirement stemmed from severe internal stresses caused by the mutations, which drastically diminished tolerance to external stresses from the host. AmB-resistant mutants were hypersensitive to oxidative stress, febrile temperatures, and killing by neutrophils and also had defects in filamentation and tissue invasion. These strains were avirulent in a mouse infection model. Thus, the costs of evolving resistance to AmB limit the emergence of this phenotype in the clinic. Our work provides a vivid example of the ways in which conflicting selective pressures shape evolutionary trajectories and illustrates another mechanism by which the Hsp90 buffer potentiates the emergence of new phenotypes. Developing antibiotics that deliberately create such evolutionary constraints might offer a strategy for limiting the rapid emergence of drug resistance.

AI snapshot

Written from the paper’s full text.

TL;DR

Amphotericin B (AmB) resistance in *Candida* is rare because the mutations that confer resistance impose severe fitness costs, including constitutive stress, Hsp90 dependence, loss of filamentation, hypersensitivity to host stresses, and avirulence in mice. The paper argues that these trade-offs, rather than an inability to mutate, explain why clinical AmB resistance has remained exceptional after decades of use.

Key findings

  • Clinical and laboratory AmB resistance mapped to sterol-pathway loss-of-function mutations: a *TCA2* retrotransposon insertion in *ERG2* in a *C. albicans* clinical isolate; *ERG3* S258F plus a 170-nt *ERG11* deletion in a *C. tropicalis* isolate; and *ERG6* mutations during in vitro evolution. Systematic deletion of seven nonessential late ergosterol genes showed that only *ERG2*, *ERG6*, or combined *ERG3*/*ERG11* loss conferred >3-fold AmB MIC increases; MIC increases were 10-fold for *erg2*, 12-fold for *erg6*, and 20-fold for *erg3 erg11*.
  • AmB-resistant mutants became extremely dependent on Hsp90: Hsp90 inhibitors blocked growth even without AmB and were cytocidal. Hsp90-inhibitor MIC decreases were 18–48-fold for *erg2*, 85–109-fold for *erg6*, and 222–480-fold for *erg3 erg11*, compared with only 2–8-fold or 4–16-fold decreases for other cytotoxic agents.
  • Resistance mutations caused constitutive activation of stress responses and made normally nonessential pathways essential: calcineurin inhibition blocked growth of all AmB-resistant strains, *erg3 erg11* was especially PKC-sensitive, and de novo resistant colonies from *ERG2/erg2* heterozygotes arose at ~10^-5 but were eliminated by geldanamycin or in calcineurin-deficient strains.
  • AmB-resistant mutants suffered severe host-relevant fitness defects: hypersensitivity to 41°C, peroxide, hypochlorous acid, nitric oxide, iron chelation, serum, and neutrophil killing; defective filamentation and endothelial damage; and complete avirulence in mice at a 4×10^6-cell inoculum, with kidney fungal burdens at least three orders of magnitude lower than wild-type or fluconazole-resistant *erg3*.

Methods

  • Whole-genome sequencing (~50-fold coverage) of two independent AmB-resistant clinical isolates—one *C. albicans* and one *C. tropicalis*—plus the AmB-sensitive *C. albicans* reference SC5314; reads were aligned with BWA and variants called with GATK, followed by IGV inspection, loss-of-heterozygosity visualization, and sliding-window heterozygosity analysis.
  • In vitro evolution of *C. albicans* ATCC-10231 by serial passage seven times into twofold-increasing AmB concentrations, with whole-genome sequencing of parental and passage strains; targeted deletion of all seven nonessential late ergosterol-pathway genes in *C. albicans*; MIC/growth assays, sterol spectrophotometry, qRT-PCR stress profiling, neutrophil killing assays, filamentation assays, endothelial LDH cytotoxicity assays, and a murine tail-vein infection model.
  • Main analyses included broth microdilution MIC80 measurements, spot assays, alamar blue growth quantification, Student’s t tests, and histology/CFU burden from mouse kidneys.

Limitations

  • Stated by the paper: further transformants could not be obtained in the AmB-resistant mutants for technical reasons, precluding complementation of the phenotype; the authors note that sequencing many more AmB-sensitive and resistant isolates would be necessary to establish that selective sweeps fixed the mutations; and direct CFU comparisons between mouse infection groups cannot be made because wild-type, *erg3*, and mutant strains survived for different periods before sacrifice.
  • Stated by the paper: other explanations for the rarity of AmB resistance may also be relevant, including that lipid-targeting drugs are not susceptible to resistance via drug-binding-pocket substitutions or efflux.
  • Evident from the text: virulence testing used one AmB-resistant clinical isolate plus laboratory mutants, so the avirulence of other clinical AmB-resistant isolates was inferred rather than directly tested.

Model-written summary. Check it against the paper before relying on it.

Integrity signals

Registry

Crossref · 10.1371/journal.pbio.1001692

Cites

61 of 61 references verified0 citation errors · 0 not checkable

Statistics

Nothing to recompute.

Claims the paper makes

0 of 24 cited sources checked · 0 supported · 0 flagged

  • Findingown data

    AmB-resistant mutants were generally more sensitive than wild-type cells to many cytotoxic compounds, with MIC decreases of 2- to 8-fold in erg2 or erg6 mutants and 4- to 16-fold in the erg3 erg11 mutant.

    “AmB-resistant mutants were, indeed, generally more sensitive than wild-type cells to many of the cytotoxic compounds. The decrease from the wild-type in the MIC of any of these cytotoxic agents ranged from 2- to 8-fold in the erg2 or erg6 mutants and 4- to 16-fold in the erg3 erg11 mutant (Figure S4A–C).”
    Quote in the paperSubstantial claim
  • Findingown data

    The erg2 mutants had a 10-fold increase in AmB MIC.

    “For the erg2 mutants this was a 10-fold increase in MIC (Figure 1D)”
    Quote in the paperSubstantial claim
  • Findingown data

    The erg3 erg11 strains showed dramatically increased sensitivity to PKC inhibition.

    “The erg3 erg11 strains were slightly less sensitive to calcineurin inhibitors, but showed a dramatically increased sensitivity to PKC inhibition.”
    Quote in the paperSubstantial claim
  • Findingown data

    Among all seven nonessential genes in the latter half of the ergosterol biosynthesis pathway, only deletion of ERG2, ERG6, or of ERG3 and ERG11 together conferred more than a 3-fold increase in AmB MIC.

    “Only the deletion of ERG2, ERG6, or of ERG3 and ERG11 together conferred more than a 3-fold increase in the AmB MIC (Figure 1D).”
    Quote in the paperSubstantial claim
  • Findingown data

    Low concentrations of either Hsp90 inhibitor completely blocked the growth of all AmB-resistant strains, even in the absence of AmB.

    “Surprisingly, however, low concentrations of either of the Hsp90 inhibitors completely blocked the growth of all of the AmB-resistant strains, even in the absence of AmB (Figures 2A–D and S4B).”
    Quote in the paperSubstantial claim
    • Rests on an assumption: The Hsp90 inhibitors geldanamycin and radicicol are highly specific for Hsp90.
  • May combine two claims. 31
  • The quote only partly states this claim. 4
  • Classified as probably not a substantial claim. 31

  • May combine two claims.

    Findingown data

    The C. albicans AmB-resistant isolate has high heterozygosity across its genome, with only two small regions of homozygosity, one of which includes the ERG2 transposon insertion.

    “Whole-genome analysis of polymorphisms indicated that the strain carried a high level of heterozygosity across its entire genome, with only two small regions of homozygosity. Strikingly, one of these included the transposon insertion in ERG2 (Figure S1B).”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Findingown data

    The AmB-sensitive, fluconazole-resistant erg3 mutant did not show dramatic upregulation of any of the responses tested, but only a weak induction of several iron starvation genes.

    “Intriguingly, the AmB-sensitive, fluconazole-resistant erg3 mutant did not show dramatic upregulation of any of the responses tested, but only a weak induction of several iron starvation genes.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    Sterol extracts from the wild-type laboratory strain exhibited the double-peaked spectrum characteristic of conjugated C5-C6 and C7-C8 bonds, while extracts from both laboratory mutants and clinical isolates did not.

    “Extracts from the wild-type laboratory strain exhibited the double-peaked spectrum between 240 and 300 nm characteristic of the conjugated bonds (Figure 1C). Extracts from both the laboratory mutants and the clinical isolates did not.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    Modest inhibition of Hsp90 completely blocked fluconazole resistance without affecting growth of the fluconazole-resistant isolate in the absence of fluconazole.

    “As previously described, modest inhibition of Hsp90 completely blocked fluconazole resistance (Figure 2A, middle panel). It did not affect growth of the fluconazole-resistant isolate in the absence of fluconazole (Figure 2A, top panel).”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Findingown data

    Hog1, but not calcineurin, is required for wild-type levels of AmB tolerance in the absence of ergosterol biosynthesis mutations.

    “Hog1, but not calcineurin, is required for wild-type levels of AmB tolerance in the absence of ergosterol biosynthesis mutations.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    The decreases in MIC of the four Hsp90 inhibitors were 18- to 48-fold for erg2 mutants, 85- to 109-fold for erg6 mutants, and 222- to 480-fold for erg3 erg11 mutants.

    “But the decreases in MIC of the four Hsp90 inhibitors were 18- to 48-fold for the erg2 mutants, 85- to 109-fold for the erg6 mutants, and 222- to 480-fold for the erg3 erg11 mutants (Figure S4B–C).”
    Quote in the paperSubstantial claim

  • May combine two claims. The quote only partly states this claim.

    Findingown data

    AmB-resistant mutants exhibited constitutive activation of diverse stress responses, including iron starvation pathways (most strongly in erg3/erg11 and erg6 mutants), general plasma membrane and oxidative stress genes, and calcineurin targets (most strongly in lab strains).

    “The AmB-resistant mutants indeed exhibited a constitutive activation of diverse stress responses (Figure 3A, left panel).”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    Treatment with either calcineurin inhibitor inhibited growth of all AmB-resistant strains, with complete growth inhibition of erg2 and erg6 mutants.

    “Treatment with either calcineurin inhibitor inhibited growth of all of the Amphotericin-resistant strains, with complete growth inhibition of erg2 and erg6 mutants (Figure 3B).”
    Quote in the paperSubstantial claim

  • May combine two claims. The quote only partly states this claim.

    Findingown data

    In vitro evolution of C. albicans ATCC-10231 under gradually increasing AmB produced a trajectory of mutations in ERG6: the parental strain was heterozygous for a Glu70Stop premature stop codon; an Asp180Gly mutation appeared in the other allele at the second step; the Q70Stop allele increased to a 3:1 ratio at the third step; and a loss of heterozygosity resulted in homozygosity of the nonsense.

    “Alignment of genome sequences and algorithmic detection of novel polymorphisms emerging within the series revealed a trajectory of mutations in the ERG6 (ORF19.1631) gene, encoding Δ(24)-sterol C-methyltransferase (Figure 1B).”
    • Also stated in: § Laboratory Evolution of AmB Resistance Implicates ERG6 Mutations · plain
      “Finally, a loss of heterozygosity event, unique to the left arm chromosome 3 where ERG6 resides, resulted in homozygosity of the nonsense allele (Figure S3A–B).”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    The fluconazole-resistant erg3 mutant exhibited a mild delay in hyphal protrusion but still formed substantial filaments.

    “The fluconazole-resistant erg3 mutant exhibited a mild delay in hyphal protrusion but still formed substantial filaments.”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Findingown data

    The erg6 and erg3 erg11 strains and clinical isolates were entirely unable to form hyphal extensions, and remained mainly in the yeast form.

    “The erg6 and erg3 erg11 strains and clinical isolates were entirely unable to form hyphal extensions, and remained mainly in the yeast form.”
    Quote in the paperSubstantial claim

  • The quote only partly states this claim.

    Findingown data

    Wild-type strains exhibited long and robust filaments after 4 h of incubation in 10% fetal bovine serum in RPMI.

    “After 4 h of incubation, wild-type strains exhibited long and robust filaments (Figure 5A).”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    Histological analysis demonstrated healthy kidneys with some signs of resolving acute inflammation.

    “Histological analysis demonstrated healthy kidneys with some signs of resolving acute inflammation, suggesting that innate immune attack may have contributed to the clearance of these strains (Figure 6C).”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    Mice infected with the erg3 mutant had extremely high kidney fungal burdens, filamentous fungal morphology, and extensive kidney necrosis at the time of death.

    “At the time of death, mice infected with this mutant had extremely high kidney fungal burdens, filamentous fungal morphology, and extensive kidney necrosis.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    Wild-type infection resulted in high viable fungal burden in the kidney, extensive filamentous fungal morphology, and moderate tissue damage.

    “Necropsy revealed high viable fungal burden in the kidney, extensive filamentous fungal morphology, and moderate tissue damage (Figure 6B–C).”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    Some mice showed mild weight loss in the first day after inoculation, but within a few days all infected mice recovered and appeared healthy.

    “Some mice showed mild weight loss in the first day after inoculation. But within a few days all infected mice recovered and appeared healthy.”
    Quote in the paperSubstantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisecitation

    In other fungal pathogens, rare AmB-resistant isolates have been identified that lack ERG2 or ERG6.

    “In other fungal pathogens, sterol analysis of rare AmB-resistant isolates has identified resistant strains lacking ERG2, encoding C-8 sterol isomerase, and ERG6, encoding C-24 sterol methyltransferase [22]–[24].”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [22]–[24]

      Not checked in the free run.

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisecitation

    The emergence and maintenance of resistance to triazole and echinocandin antifungals critically depends on Hsp90.

    “We previously reported that the emergence and maintenance of resistance to triazole and echinocandin antifungals critically depends on the molecular chaperone Hsp90 (ORF19.6515) [25].”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [25] LE Cowen, 2005, Hsp90 potentiates the rapid evolution of new traits: drug resistance in diverse fungi, Science source

      Not checked in the free run.

  • Classified as probably not a substantial claim. May combine two claims.

    Premisecitation

    Binding of amphotericin B to ergosterol induces pore formation in the plasma membrane and results in rapid cell death.

    “Among other effects, this binding induces pore formation in the plasma membrane and results in rapid cell death.”
    Quote in the paperSubstantial claim0 of 1 sources checked
    • cites [7],[8]

      Not checked in the free run.

  • May combine two claims.

    Interpretationreasoning

    ERG2, ERG6, and ERG3/ERG11 are the most critical mutations for AmB resistance because they have all been detected in the clinic and validated in the laboratory.

    “While other mutations conferring resistance to AmB may exist, these three are the most critical, as they have all been detected in the clinic and validated in the laboratory.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Premisecitation

    Mild compromise of Hsp90 function prevents the de novo emergence of resistance to triazoles and reverses resistance of strains that had previously evolved triazole and echinocandin resistance in the clinic.

    “In pathogenic fungi, we have shown that mild compromise of Hsp90 function prevents the de novo emergence of resistance to triazoles and, in fact, reverses the resistance of strains of which had previously evolved triazole and echinocandin resistance in the clinic [25],[29],[30].”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [25],[29],[30]

      Not checked in the free run.

  • Classified as probably not a substantial claim.

    Premisecitation

    Hsp90 promotes the maturation of diverse metastable signal transduction proteins, including kinases, phosphatases, and ubiquitin ligases (Hsp90 clients), which function in many stress response pathways.

    “Hsp90 promotes the maturation of a diverse array of metastable signal transduction proteins, including kinases, phosphatases, and ubiquitin ligases (known as Hsp90 clients) [32]. These function in many stress response pathways.”
    Quote in the paperSubstantial claim0 of 1 sources checked
    • cites [32] M Taipale, 2012, Quantitative analysis of HSP90-client interactions reveals principles of substrate recognition, Cell source

      Not checked in the free run.

  • Classified as probably not a substantial claim. May combine two claims.

    Premisecitation

    Hsp90 constitutes ~1% of total cellular protein and acts as a protein homeostasis buffer; its activity can be reduced up to 10-fold without impairing normal growth.

    “Hsp90 is one of the most abundant proteins in eukaryotic cells, constituting ∼1% of total cellular protein, and acts as a protein homeostasis buffer. Although Hsp90 is an essential protein, its activity can be reduced up to 10-fold without impairing normal growth [25]–[27].”
    Quote in the paperSubstantial claim0 of 1 sources checked
    • cites [25]–[27]

      Not checked in the free run.

  • May combine two claims.

    Interpretationown data

    The hypersensitivity of AmB-resistant strains to Hsp90 inhibitors cannot simply be attributed to a general increase in drug accumulation; rather, their growth critically depends on maintaining very high levels of Hsp90 function even in the absence of AmB.

    “Thus, the hypersensitivity of AmB-resistant strains to Hsp90 inhibitors cannot simply be attributed to a general increase in drug accumulation. Rather, the growth of these mutants must critically depend on maintaining very high levels of Hsp90 function even in the absence of AmB.”
    Quote in the paperSubstantial claim
    • Rests on an assumption: The Hsp90 inhibitors geldanamycin and radicicol are highly specific for Hsp90.

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Study design and conductnone given

    For stress assays in Figures 4B-E and S5B-C, data represent the mean of six wells pooled from experiments performed on two separate days.

    “For stress assays (Figures 4B–E and S5B–C), data represent the mean of six wells, pooled from experiments performed on two separate days.”
    Quote in the paperNot a substantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

    Study design and conductnone given

    Endothelial cytotoxicity results are pooled from two experiments, with six replicate wells per experiment.

    “Results pooled from two experiments are displayed (six replicate wells per experiment).”
    Quote in the paperNot a substantial claim

  • Classified as probably not a substantial claim.

    Study design and conductnone given

    Control wells lacking C. albicans were included to verify that the treatment does not quantify growth of neutrophil cells.

    “Control wells lacking C. albicans were included to verify that this treatment does not quantify growth of the neutrophil cells.”
    Quote in the paperSubstantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

    Study design and conductown data

    Neutrophil killing assay results are from three separate plates, with each mutant strain's growth presented as a fraction of wild-type growth from the same plate.

    “Results from three separate plates are shown; growth of each mutant strain is presented as a fraction of the wild-type growth from the same plate.”
    Quote in the paperNot a substantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Study design and conductnone given

    Equal weights of cell pellets were used across strains for sterol extraction.

    “Equal weights of cell pellets were used across strains.”
    Quote in the paperNot a substantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Study design and conductnone given

    Each qRT-PCR measurement was obtained from an average of four wells, including two biological replicates and two technical replicates.

    “Each measurement was obtained from an average of four wells, including two biological replicates and two technical replicates.”
    Quote in the paperNot a substantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Study design and conductreasoning

    RPMI-agar plates were used within 6 h of pouring because of the potential instability of AmB and peroxides.

    “All RPMI-agar plates were used within 6 h of pouring due to the potential instability of AmB and peroxides; it is recommended to test a range of AmB concentrations in agar plates due to potential chemical instability.”
    Quote in the paperNot a substantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisecitation

    Pharmacological inhibition of Hsp90 is now being explored as a strategy to forestall the emergence of resistance in diverse malignancies.

    “Indeed, pharmacological inhibition of Hsp90 is now being explored as a strategy to forestall the emergence of resistance in diverse malignancies [28].”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [28] L Whitesell, 2012, Inhibiting HSP90 to treat cancer: a strategy in evolution, Curr Mol Med source

      Not checked in the free run.

  • Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims. The quote only partly states this claim.

    Study design and conductnone given

    qRT-PCR expression analysis used the comparative ΔCt method with normalization to four internal control genes (TDH3, ACT1, TEF3, and RPP2B), and the mean of the four normalizations was used.

    “using normalization to four internal control genes: TDH3, ACT1, TEF3, and RPP2B; the mean value obtained from the four normalizations was used.”
    Quote in the paperNot a substantial claim

  • Classified as probably not a substantial claim.

    Premisecitation

    Hsp90 plays a critical role in the evolution of drug resistance in Candida and Aspergillus.

    “As previously reported, Hsp90 plays a critical role in the evolution of drug resistance in Candida and Aspergillus [25].”
    Quote in the paperSubstantial claim0 of 1 sources checked
    • cites [25] LE Cowen, 2005, Hsp90 potentiates the rapid evolution of new traits: drug resistance in diverse fungi, Science source

      Not checked in the free run.

  • Classified as probably not a substantial claim.

    Study design and conductnone given

    For susceptibility to AmB and stress response inhibitors, growth scores were normalized to the wild-type strain in the absence of AmB (or DMSO negative control).

    “For susceptibility to AmB and stress response inhibitors (Figures 1D, 3B–C, and S5A), growth scores were determined by normalization of the values for each sample to the values obtained for the wild-type strain in the absence of AmB (or in the DMSO negative control for Figure 3B).”
    Quote in the paperSubstantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Study design and conductown data

    The in vitro gradual selection of AmB resistance was repeated in three independent selections.

    “The process was repeated in three independent selections.”
    Quote in the paperNot a substantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

    Study design and conductnone given

    For susceptibility assays in Figures 1D, 3B-C, and S5A, each condition was tested in duplicate and repeated on at least two different days.

    “For these assays (Figures 1D, 3B–C, and S5A), each condition was tested in duplicate and repeated on at least two different days.”
    Quote in the paperNot a substantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisecitation

    Advances in structural biology and medicinal chemistry have enabled the design of enzyme inhibitors that are less susceptible to resistance mediated by point mutations or by drug efflux.

    “Advances in structural biology and medicinal chemistry have enabled the design of enzyme inhibitors that are less susceptible to resistance mediated by point mutations or by drug efflux [47]–[50].”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [47]–[50]

      Not checked in the free run.

  • May combine two claims.

    Premisecitation

    Treatment failure is linked to other factors, including the inability of the drug to penetrate certain niches of the body, dose-limiting renal toxicity, or the underlying disease of the patient.

    “Instead, treatment failure is linked to other factors, including the inability of the drug to penetrate certain niches of the body, dose-limiting renal toxicity, or the underlying disease of the patient [6],[7],[9].”
    Quote in the paperSubstantial claim0 of 1 sources checked
    • cites [6],[7],[9]

      Not checked in the free run.

  • Classified as probably not a substantial claim.

    Study design and conductnone given

    Control wells were inoculated in identical conditions but without neutrophils added.

    “Control wells were inoculated in the identical conditions but without neutrophils added.”
    Quote in the paperSubstantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Study design and conductnone given

    A veterinary pathologist was consulted for histological analysis in the murine model.

    “A veterinary pathologist was consulted for histological analysis.”
    Quote in the paperNot a substantial claim

  • Classified as probably not a substantial claim. May combine two claims.

    Study design and conductnone given

    Proper insertion of the auxotrophic marker and loss of the endogenous gene were confirmed by PCR.

    “Proper insertion of the auxotrophic marker and loss of the endogenous gene were confirmed by PCR.”
    Quote in the paperSubstantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Interpretationreasoning

    Hsp90's role in supporting stress responses operates very broadly in the evolution of phenotypic diversity.

    “Given its conservation, we suggest its role in supporting stress responses operates very broadly in the evolution of phenotypic diversity, allowing organisms to acquire mutations that confer novel phenotypes but simultaneously create stresses that otherwise would not be tolerated.”
    Quote in the paperNot a substantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

    Study design and conductnone given

    Each Candida strain in the murine systemic infection model was tested in two independent experiments performed at different times, and data were pooled.

    “Each strain was tested in two independent experiments (performed at different times), and data were pooled.”
    Quote in the paperNot a substantial claim

  • Classified as probably not a substantial claim.

    Study design and conductnone given

    For stress assays, growth scores were calculated by dividing mutant growth by wild-type growth in each stress condition.

    “here, growth scores were calculated by dividing the growth value of the mutant by the growth value of the wild type in each particular stress condition.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Interpretationown data

    Several deletion or loss of function mutations can readily arise in Candida and confer resistance to AmB in vitro.

    “Nevertheless, our work indicates that several deletion or loss of function mutations can readily arise in Candida and confer resistance to AmB in vitro.”
    Quote in the paperSubstantial claim

  • Classified as probably not a substantial claim. May combine two claims.

    Study design and conductreasoning

    Four knockout strains were compared for phenotypic concordance in filamentation and stress resistance to minimize the effect of secondary mutations, with additional data on a second mutant strain for key mutants.

    “These four strains were compared for phenotypic concordance in filamentation and stress resistance to minimize the effect of secondary mutations; additional data on a second mutant strain for key mutants is presented in Figure S5.”
    Quote in the paperSubstantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Study design and conductnone given

    The sliding-window heterozygosity analysis used only SNPs of quality 1,000 or more.

    “Using only SNPs of quality 1,000 or more, we then computed the “base ratio” at each position by dividing the count of reads for the minor allele base by the total number of reads for both bases at that SNP position”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Interpretationown data

    The resistant strains failed to tolerate the host environment or immune attack and colonize the kidney, let alone damage it.

    “Thus, the resistant strains failed to tolerate the host environment or immune attack and colonize this organ, let alone damage it.”
    Quote in the paperSubstantial claim

  • Classified as probably not a substantial claim.

    Study design and conductnone given

    In the neutrophil killing assay, relative growth was measured by dividing values in the presence of neutrophils by values in their absence for each strain.

    “Relative growth was measured by dividing values obtained in the presence of neutrophils by those obtained in their absence for each strain.”
    Quote in the paperSubstantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Study design and conductnone given

    All experimental procedures were carried out according to NIH guidelines and MIT protocols for the ethical treatment of animals.

    “All experimental procedures were carried out according to NIH guidelines and MIT protocols for the ethical treatment of animals.”
    Quote in the paperNot a substantial claim

  • Classified as probably not a substantial claim. May combine two claims.

    Premisecitation

    Hsp90 allows drug-resistant mutants to survive stresses imposed by triazoles and echinocandins, but is not required to tolerate the mutations conferring resistance to those drugs alone.

    “Hsp90 allows drug-resistant mutants to survive stresses imposed by triazoles and echinocandins, but is not required to tolerate the mutations conferring resistance to those drugs alone.”
    Quote in the paperSubstantial claim0 of 1 sources checked
    • cites [25] LE Cowen, 2005, Hsp90 potentiates the rapid evolution of new traits: drug resistance in diverse fungi, Science source

      Not checked in the free run.

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Study design and conductnone given

    In the murine systemic infection model, inocula were counted by hemocytometer and plating of dilutions.

    “counted by hemocytometer and plating of dilutions”
    Quote in the paperNot a substantial claim
53 of 53

  • The paper relies on this without support.

    No systematic analysis of AmB resistance mutations in Candida using matched isogenic strains has been performed, and the consequences of these mutations for Candida biology and pathogenicity remain largely unexplored.

    “However, there has been no systematic analysis of AmB resistance mutations in Candida using matched isogenic strains. More importantly, the consequences of these mutations upon the biology and pathogenicity of Candida remain largely unexplored.”

  • The paper relies on this without support.

    AmB-sensitive parental strains from which rare AmB-resistant isolates evolved are not available.

    “As AmB-sensitive parental strains from which rare AmB-resistant isolates evolved are not available, the identification of mutations conferring resistance has proven challenging.”

  • The paper relies on this without support.

    The Hsp90 inhibitors geldanamycin and radicicol are highly specific for Hsp90.

    “taking advantage of two structurally unrelated natural products with high specificity for Hsp90 (geldanamycin and radicicol).”

  • The paper relies on this without support.

    Hsp90-dependent stress-response pathways are not essential for growth of wild-type strains.

    “Hsp90-dependent stress-response pathways are not essential for growth of wild-type strains.”

  • The paper relies on this without support.

    Resistance to AmB rarely evolves during the treatment of infected patients.

    “Although we have successfully validated the ability of several ergosterol biosynthesis mutations to confer resistance to AmB in vitro, resistance rarely evolves during the treatment of infected patients.”

  • The paper relies on this without support.

    AmB remains as effective as ever 50 years after its introduction.

    “Yet, 50 years after its introduction, AmB remains as effective as ever.”
6 of 6

Claims other papers make about this one

Each is checked against this paper’s own text: does it say what the citing paper says it does?

  • Resistance to amphotericin B is very rare in yeast species due to highly deleterious fitness costs.

    “Resistance to AMB is very rare in yeast species due to highly deleterious fitness costs13.”

    From Candida auris skin tropism and antifungal resistance are mediated by carbonic anhydrase Nce103 (2025) · cites this paper as [13]

  • Amphotericin B resistance may arise from alterations in the ergosterol biosynthesis pathway, causing depletion of ergosterol and accumulation of alternate sterols.

    “Resistance may arise from alterations in the ergosterol biosynthesis pathway, causing depletion of ergosterol and the accumulation of alternate sterols.”

    From Molecular mechanisms governing antifungal drug resistance (2023) · cites this paper as [45]

  • In Candida species, amphotericin B resistance has been associated with mutations in several ergosterol biosynthesis genes, including ERG2, ERG3, ERG6, and ERG11.

    “In Candida species, amphotericin B resistance has been associated with mutations in several ergosterol biosynthesis genes, including ERG2, ERG3, ERG6, and ERG1142,45–47.”

    From Molecular mechanisms governing antifungal drug resistance (2023) · cites this paper as [45]

  • Mutations that confer polyene resistance simultaneously create severe cellular stress, resulting in hypersensitivity to oxidative stress, febrile temperatures, and killing by neutrophils.

    “However, such mutations that confer polyene resistance simultaneously create severe cellular stress, which results in hypersensitivity to oxidative stress, febrile temperatures, and killing by neutrophils45.”

    From Molecular mechanisms governing antifungal drug resistance (2023) · cites this paper as [45]

  • Severe fitness trade-offs disfavour the acquisition of resistance to the polyene amphotericin B in multiple fungal species, explaining why resistance to this antifungal class remains extremely limited in the laboratory and clinic.

    “Severe fitness trade-offs disfavour the acquisition of resistance to the polyene amphotericin B in multiple fungal species, explaining why resistance to this antifungal class remains extremely limited in the laboratory and clinic45.”

    From Molecular mechanisms governing antifungal drug resistance (2023) · cites this paper as [45]

Self-check

Percentages, sums and reported differences re-computed from the paper’s own numbers.

Each contradiction between two passages listed here was confirmed by an independent check.

  • Endothelial cytotoxicity analysis uses conflicting t-test descriptions

    Major

    The Figure 5B caption says the comparison used a paired Student's t test, while the assay methods say it used an unpaired Student's t test.

  • Wild-type kidney damage is described as both moderate and severe

    Minor

    The results describe moderate tissue damage in kidneys from wild-type-infected mice, but the Figure 6C caption describes severe kidney pathology in the wild-type group.

2 of 2

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Related

Authors
Vincent, B. M., Lancaster, A. K., Scherz-Shouval, R., Whitesell, L., & Lindquist, S.
Affiliations
  • Whitehead Institute for Biomedical Research (US)
  • Howard Hughes Medical Institute (US)
Corresponding author
Lindquist, Susan
ORCID
Venue
PLoS Biology
Publisher
Public Library of Science (PLoS)
Published
2013-10-29
Volume
11
Issue
10
Pages
e1001692
ISSN
  • 1544-9173 (ISSN-L)
  • 1545-7885
DOI
10.1371/journal.pbio.1001692
PMC
PMC3812114
OpenAlex
W2153898589
Semantic Scholar
75e34b869f34a9b7be1cbad20751b6800eace175
Cited by
322
References
61
Citations by year
2026: 24 · 2025: 25 · 2024: 35 · 2023: 27 · 2022: 20 · 2021: 34 · 2020: 28 · 2019: 31 · 2018: 22 · 2017: 26 · 2016: 17 · 2015: 21 · 2014: 11 · 2013: 1
FWCI
12.35
Type
journal article
Language
en
Open access
Licence
Locations
  • PLoS Biology (publishedVersion, cc-by)
  • Europe PMC (PubMed Central) (publishedVersion, cc-by)
  • PubMed Central (publishedVersion, cc-by)
  • PubMed (publishedVersion)
  • DSpace@MIT (Massachusetts Institute of Technology) (submittedVersion, cc-by)
  • DOAJ (DOAJ: Directory of Open Access Journals) (submittedVersion, cc-by-sa)
  • Figshare (submittedVersion, cc-by)
  • PLoS eBooks
Funders
  • National Science Foundation
  • Howard Hughes Medical Institute
  • G. Harold and Leila Y. Mathers Foundation
Topics
  • Health Sciences › Medicine › Infectious Diseases › Antifungal resistance and susceptibility
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Fungal and yeast genetics research
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Genetics › Evolution and Genetic Dynamics
Keywords
amphotericin B resistance, Candida albicans, antifungal resistance, oxidative stress sensitivity, neutrophil killing, tissue invasion, avirulence, experimental evolution, clinical isolates, evolutionary constraints
MeSH terms
Amphotericin B, Animals, Candida albicans, Candidiasis, Disease Models, Animal, Ergosterol, Biological Evolution, Fungal Proteins, Humans, Microbial Sensitivity Tests, Mutation, Stress, Physiological, Virulence, Reproducibility of Results, HSP90 Heat-Shock Proteins, Drug Resistance, Fungal, Microbial Viability, Mice, Host-Pathogen Interactions, Genetic Fitness

About these checks

Registry, retraction, Cites and statistics results are computed, not judged, with one exception: a printed reference that no index lists is read once by a model before it counts as a citation error. Claims are extracted by a model and kept only when their quote appears verbatim in the text; each cited source is then checked, with a model’s help, for whether it says what the claim relies on. Those verdicts can be wrong — read the claim’s quote and the cited source before relying on one.

Claims read by deepseek-v4.1-flash under definition version 3 (extract-v3) on 2026-10-09.

Classified by jev-1.13-20260917 (gold-guideline-v0.2) on 2026-10-09.

Statistics and arithmetic are recomputed, not judged.

Confidence bands are shown only for fields calibrated on our annotated test set. None is calibrated yet, so no band is shown.

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