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. 2020 Jan 8;11(1):127.
doi: 10.1038/s41467-019-13890-z.

5-fluorocytosine resistance is associated with hypermutation and alterations in capsule biosynthesis in Cryptococcus

Affiliations

5-fluorocytosine resistance is associated with hypermutation and alterations in capsule biosynthesis in Cryptococcus

R Blake Billmyre et al. Nat Commun. .

Abstract

Patients infected with the fungal pathogen Cryptococcus are most effectively treated with a combination of 5-fluorocytosine (5FC) and amphotericin B. 5FC acts as a prodrug, which is converted into toxic 5-fluorouracil (5FU) upon uptake into fungal cells. However, the pathogen frequently develops resistance through unclear mechanisms. Here we show that resistance to 5FC in Cryptococcus deuterogattii is acquired more frequently in isolates with defects in DNA mismatch repair that confer an elevated mutation rate. We use whole genome sequencing of 16 independent isolates to identify mutations associated with 5FC resistance in vitro. We find mutations in known resistance genes (FUR1 and FCY2) and in a gene UXS1, previously shown to encode an enzyme that converts UDP-glucuronic acid to UDP-xylose for capsule biosynthesis, but not known to play a role in 5FC metabolism. Mutations in UXS1 lead to accumulation of UDP-glucuronic acid and alterations in nucleotide metabolism, which appear to suppress toxicity of both 5FC and its toxic derivative 5FU.

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Conflict of interest statement

The authors declare no competing interests.

Figures

Fig. 1
Fig. 1. 5FC resistance is enhanced by defects in mismatch repair.
a Swab assays were conducted using both the wildtype R265 strain and two independent msh2Δ::NEO mutants to test for the ability to generate resistance to 5FC. All three strains developed resistance; however, the mismatch repair mutants generated resistant isolates at a higher frequency. b A fluctuation assay was conducted to compare 5FC resistance quantitatively between wildtype R265 and two independent msh2Δ::NEO mutants. Mutation rate was normalized to the wildtype strain. Red is used to indicate wildtype strains while green indicates msh2 deletion strains. Both mutator strains showed a greater than 15-fold increase in the rate of resistance. Data shown are the mean of 10 replicates and error bars indicate 95% confidence intervals.
Fig. 2
Fig. 2. Exposure to 5FC generates an adaptive advantage for mutator strains.
Competition experiments between a tester strain with a neomycin resistance marker and a wildtype R265 strain. (Strains used: SEC501, RBB17, RBB18). Overnight cultures were mixed 1:1 and then used to inoculate a second overnight culture in liquid YNB with and without 5FC. All three marked strains showed a slight growth defect in comparison to the unmarked strain in nonselective media but only the hypermutator strains demonstrated a dramatic growth advantage when grown in YNB + 5FC. Boxplots show minimum, first quartile, median, third quartile, and maximum values. Points represent the results from three individual replicates and are summarized by the box plot. The R265 NEOR vs wildtype competition is gray, while the two independent msh2Δ::NEO vs wildtype competitions are dark and light blue.
Fig. 3
Fig. 3. 5FC resistant mutants are cross-resistant to 5FU.
a Isolates that were selected based on growth on 5FC media were patched to YNB, YNB plus 5FC, and YNB plus 5FU. Each plate has parental and fur1 mutant controls in the top row. Hypermutator controls have occasional resistant colonies that emerged in the growth patch. Sanger and Illumina sequencing revealed that 12 of 29 isolates had sustained mutations in FUR1. b Schematic showing the predicted domains encoded by the UXS1 gene, as well as the location and number of mutations identified. Frame-shift alleles are shown in red and missense are shown in blue.
Fig. 4
Fig. 4. uxs1 mutants mediate 5FC resistance through a xylosylation-independent mechanism.
a KN99 deletion strains from the C. neoformans deletion collection show that deletion of UXS1 confers resistance to 5FC and 5FU. The RBB18-2 strain carrying a fur1 mutation is resistant to 5FC and 5FU although more weakly to 5FU. The R265-3 strain carrying a fur1 mutation is completely resistant to both drugs. b Spot dilution assay on YNB, YNB plus 5FC, and YNB plus 5FU demonstrating overexpression of UXS1 driven by the actin promoter does not confer increased sensitivity to 5FC or 5FU. c Spot dilution assays on YNB, YNB plus 5FC, and YNB plus 5FU demonstrating that mutants deficient in UDP-xylose transport (uxt1Δ, uxt2Δ, uxt1Δ uxt2Δ) and xylose transferase mutants (cxt1Δ, cxt2Δ, cxt1Δ cxt2Δ) show no change in 5FC and 5FU sensitivity. d Spot dilution assay on YPD, YPD plus 5FC, and YPD plus 5FU showing that ugd1 mutants are viable on rich YPD media but retain sensitivity to 5FC and 5FU. In addition, ugd1 uxs1 double mutants retain sensitivity to 5FC and 5FU like a ugd1 single mutant rather than gain resistance like the uxs1 single mutant.
Fig. 5
Fig. 5. Model of inhibition of 5FC/5FU toxicity by uxs1 mutation.
Potential mechanisms by which uxs1 mutations may confer resistance to both 5FC and 5FU. Mutation of uxs1 causes an accumulation of UDP-glucuronic acid, the product of Ugd1, which either impairs production of toxic fluoridated molecules or rescues inhibition of the targets of those fluoridated molecules, such as thymidylate synthase. Protein names are in red for those where mutations were found in this study.

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