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. 2020 May 5;117(18):9973-9980.
doi: 10.1073/pnas.2001451117. Epub 2020 Apr 17.

Transposon mobilization in the human fungal pathogen Cryptococcus is mutagenic during infection and promotes drug resistance in vitro

Asiya Gusa  1 , Jonathan D Williams  1 , Jang-Eun Cho  1 , Anna Floyd Averette  1 , Sheng Sun  1 , Eva Mei Shouse  1 , Joseph Heitman  1 , J Andrew Alspaugh  1   2 , Sue Jinks-Robertson  3
Affiliations

Transposon mobilization in the human fungal pathogen Cryptococcus is mutagenic during infection and promotes drug resistance in vitro

Asiya Gusa et al. Proc Natl Acad Sci U S A. .

Abstract

When transitioning from the environment, pathogenic microorganisms must adapt rapidly to survive in hostile host conditions. This is especially true for environmental fungi that cause opportunistic infections in immunocompromised patients since these microbes are not well adapted human pathogens. Cryptococcus species are yeastlike fungi that cause lethal infections, especially in HIV-infected patients. Using Cryptococcus deneoformans in a murine model of infection, we examined contributors to drug resistance and demonstrated that transposon mutagenesis drives the development of 5-fluoroorotic acid (5FOA) resistance. Inactivation of target genes URA3 or URA5 primarily reflected the insertion of two transposable elements (TEs): the T1 DNA transposon and the TCN12 retrotransposon. Consistent with in vivo results, increased rates of mutagenesis and resistance to 5FOA and the antifungal drugs rapamycin/FK506 (rap/FK506) and 5-fluorocytosine (5FC) were found when Cryptococcus was incubated at 37° compared to 30° in vitro, a condition that mimics the temperature shift that occurs during the environment-to-host transition. Inactivation of the RNA interference (RNAi) pathway, which suppresses TE movement in many organisms, was not sufficient to elevate TE movement at 30° to the level observed at 37°. We propose that temperature-dependent TE mobilization in Cryptococcus is an important mechanism that enhances microbial adaptation and promotes pathogenesis and drug resistance in the human host.

Keywords: Cryptococcus; drug resistance; fungal pathogen; temperature; transposons.

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Conflict of interest statement

Competing interest statement: A.C. and J.H. are coauthors on a forthcoming position paper. They have not collaborated directly.

Figures

Fig. 1.
Fig. 1.
PCR amplification of URA5 in representative 5FOA-resistant mutants showing T1, TCN12 (full-length), and TCN12 solo long terminal repeat (LTR) insertions. (A) Amplification from genomic DNA of mutants recovered from the lungs of mouse 3. (B) Amplification from genomic DNA of mutants isolated after growth at 30° or 37° in vitro. (C) Illustration of the T1 DNA transposon with 11-bp terminal inverted repeats and the TCN12 retrotransposon with the gag-pol-polyprotein and 150-bp LTR direct repeats. The integrase (INT) and reverse transcriptase (RT) open reading frames are indicated. Amplification of a wild-type (WT)-sized URA5 band in addition to the larger band diagnostic of TE insertion presumably reflects loss of the TE in a subpopulation of cells during nonselective growth prior to isolation of genomic DNA.
Fig. 2.
Fig. 2.
TE insertions (T1 and TCN12) in URA3 and URA5 of 5FOA-resistant mutants recovered from mice (M1–M8; color coded by organ) and inoculum cultures (C1–C7; green font). C7 was used to infect both M7 and M8. Highlighted in gray are TE insertions from mouse 3 and culture 3. The organ from which each mutant was recovered is indicated: lungs (L, blue), kidneys (K, red), brain (B, purple). Arrows represent the forward or reverse integration of each TE. Uppercase letters are exon sequences, and lowercase letters are intron sequences. Underlined sequences indicate those sequences that are duplicated following TE integration. For some of the TCN12 insertions, only a solo LTR was detected.
Fig. 3.
Fig. 3.
Temperature-dependent mutagenesis is driven by TE insertions. (A) Drug-resistance rates for 5FOA, rap/FK506, and 5FC when growth before drug selection was at either 30° (blue) or 37° (red) in vitro. (B) Insertion rates of individual TEs into URA3 or URA5 (URA3/5). (C) Distribution of T1 and TCN12 insertions in URA3, URA5, and FRR1 in drug-resistant mutants isolated from 37° cultures; the distribution at each locus is significantly different (P < 0.001 by χ2 contingency test). (D) Insertion rates of T1 and TCN12 into the FRR1 gene. Error bars are 95% confidence intervals (CIs); rates are statistically different if error bars do not overlap. Asterisks indicate no insertions were detected; the rate shown was calculated assuming one event.
Fig. 4.
Fig. 4.
RNAi is not responsible for temperature-dependent control of TE movement. Insertion rates of T1 and TCN12 into URA3/5 in WT- and RNAi-deficient (rdp1Δ) strains grown at 30° (blue) or 37° (red). Error bars are 95% CIs; rates are statistically different if error bars do not overlap. Asterisks indicate no insertions were detected; the rate shown was calculated assuming one event.
Fig. 5.
Fig. 5.
The 5FOA resistance rates and TE insertion rates in clinical and environmental isolates. (A) 5FOA resistance rates for clinical strains NIH12, 528, AD7-71, and environmental strain NIH433 grown at 30° (blue) or 37° (red). The ratios of the rates at the two temperatures are indicated by the brackets. (B) Insertion rates of TEs into URA3/5 in clinical isolates NIH12 and 528. Error bars are 95% CIs; rates are statistically different if error bars do not overlap. Asterisks indicate no insertions were detected; the rate shown was calculated assuming one event.
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