Antifungal drug resistance evoked via RNAi-dependent epimutations

doi:10.1038/nature13575

. 2014 Sep 25;513(7519):555-8.

doi: 10.1038/nature13575. Epub 2014 Jul 27.

Antifungal drug resistance evoked via RNAi-dependent epimutations

Affiliations

¹ Department of Molecular Genetics and Microbiology, Duke University Medical Center, Durham, North Carolina 27710, USA.
² 1] Regional Campus of International Excellence "Campus Mare Nostrum", Murcia 30100, Spain [2] Department of Genetics and Microbiology, Faculty of Biology, University of Murcia, Murcia 30100, Spain.
³ 1] Department of Molecular Genetics and Microbiology, Duke University Medical Center, Durham, North Carolina 27710, USA [2] Department of Biostatistics and Bioinformatics, Duke University Medical Center, Durham, North Carolina 27710, USA [3] Duke Center for the Genomics of Microbial Systems, Duke University Medical Center, Durham, North Carolina 27710, USA.
⁴ High-Throughput Sequencing Facility, University of North Carolina, Chapel Hill, North Carolina 27599, USA.
⁵ Department of Genetics and Microbiology, Faculty of Biology, University of Murcia, Murcia 30100, Spain.

PMID: 25079329
PMCID: PMC4177005
DOI: 10.1038/nature13575

Antifungal drug resistance evoked via RNAi-dependent epimutations

Silvia Calo et al. Nature. 2014.

. 2014 Sep 25;513(7519):555-8.

doi: 10.1038/nature13575. Epub 2014 Jul 27.

Affiliations

¹ Department of Molecular Genetics and Microbiology, Duke University Medical Center, Durham, North Carolina 27710, USA.
² 1] Regional Campus of International Excellence "Campus Mare Nostrum", Murcia 30100, Spain [2] Department of Genetics and Microbiology, Faculty of Biology, University of Murcia, Murcia 30100, Spain.
³ 1] Department of Molecular Genetics and Microbiology, Duke University Medical Center, Durham, North Carolina 27710, USA [2] Department of Biostatistics and Bioinformatics, Duke University Medical Center, Durham, North Carolina 27710, USA [3] Duke Center for the Genomics of Microbial Systems, Duke University Medical Center, Durham, North Carolina 27710, USA.
⁴ High-Throughput Sequencing Facility, University of North Carolina, Chapel Hill, North Carolina 27599, USA.
⁵ Department of Genetics and Microbiology, Faculty of Biology, University of Murcia, Murcia 30100, Spain.

PMID: 25079329
PMCID: PMC4177005
DOI: 10.1038/nature13575

Abstract

Microorganisms evolve via a range of mechanisms that may include or involve sexual/parasexual reproduction, mutators, aneuploidy, Hsp90 and even prions. Mechanisms that may seem detrimental can be repurposed to generate diversity. Here we show that the human fungal pathogen Mucor circinelloides develops spontaneous resistance to the antifungal drug FK506 (tacrolimus) via two distinct mechanisms. One involves Mendelian mutations that confer stable drug resistance; the other occurs via an epigenetic RNA interference (RNAi)-mediated pathway resulting in unstable drug resistance. The peptidylprolyl isomerase FKBP12 interacts with FK506 forming a complex that inhibits the protein phosphatase calcineurin. Calcineurin inhibition by FK506 blocks M. circinelloides transition to hyphae and enforces yeast growth. Mutations in the fkbA gene encoding FKBP12 or the calcineurin cnbR or cnaA genes confer FK506 resistance and restore hyphal growth. In parallel, RNAi is spontaneously triggered to silence the fkbA gene, giving rise to drug-resistant epimutants. FK506-resistant epimutants readily reverted to the drug-sensitive wild-type phenotype when grown without exposure to the drug. The establishment of these epimutants is accompanied by generation of abundant fkbA small RNAs and requires the RNAi pathway as well as other factors that constrain or reverse the epimutant state. Silencing involves the generation of a double-stranded RNA trigger intermediate using the fkbA mature mRNA as a template to produce antisense fkbA RNA. This study uncovers a novel epigenetic RNAi-based epimutation mechanism controlling phenotypic plasticity, with possible implications for antimicrobial drug resistance and RNAi-regulatory mechanisms in fungi and other eukaryotes.

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Conflict of interest statement

The authors declare no competing financial interests.

Figures

**Extended Data Figure 1. *M. circinelloides* can develop resistance to FK506 by two mechanisms, one stable (mutations) and one transient (epimutations)**
a, The WT strain (NRRL3631) grows as hyphae (white, upper left panel) on YPD and as a yeast (yellow, upper center panel) on YPD containing 1 μg/mL of FK506. An FK506-resistant patch that emerged from the southeastern edge of the yeast patch is shown after 10 days of incubation (arrow, upper right panel). Microscopic images corresponding to the culture plates at the top were taken at different incubation periods as indicated and are shown in the lower panels. For yeast growth, cells from the colony were dispersed in water on a microscope slide. The black patch (arrow) in the microscopic image in the lower right panel corresponds to the edge of the compact yeast colony. Images are representative of all of the FK506 resistant isolates obtained (see Supplementary Tables 1, 3, and 5) b, Epimutant strains revert during passage on drug-free media. Y=yeast, H=hyphal. Shaded areas indicate reversion of epimutants to the WT phenotype (yeast growth on YPD supplemented with 1 μg/mL FK506). SM2 strain⁴ harbors an A-to-G substitution (A316G) in the acceptor splice site of intron 2. Darker vertical bars indicate intervals in which some passages are not depicted. c, Reverted epimutant strains from (b) lost their resistance to FK506 and rapamycin (central panels) and remained sensitive to CsA whose mechanism of action does not involve FKBP12 (right panel). The images were taken after 48 hours of incubation at room temperature (~26°C) on YPD or YPD media supplemented with the different drugs. Images are representative of two independent experiments. EM1-S, EM2-S, EM3-S=Epimutants 1, 2, and 3 reverted to restore FK506-sensitivity. fkbAΔ=fkbA null mutant.

Extended Data Figure 2. Epimutations are generated by the RNAi pathway, and are not associated with DNA methylation in *M. circinelloides*
a, Confirmation of the presence of sRNAs in all of the remaining epimutant isolates from the different strains lacking mutations in the *fkbA* and calcineurin genes (*cnaA, cnaB, cnaC*, and *cnbR*) not shown in Figure 2a or Extended Data Fig. 9. The numbers of the isolates correspond to those in Supplementary Table 1. sRNA blots were hybridized with an antisense-specific probe to detect *fkbA* sRNA. 5S rRNA served as a loading control. Abundant sRNAs were detected in all of the strains with the exception of three of the isolates (NRRL3631 isolate 9, R7B isolate 8, and MU410 isolate 26). These isolates also do not show any mutations in the genes analyzed (*fkbA, cnaA, cnaB, cnaC, cnbR*) and the mechanism by which they have developed FK506-resistance remains to be established. The image of the blot in which these three isolates were included is representative of two independent experiments. All of the other blots were generated only once, because a positive signal indicates the presence of sRNA. b, Genomic DNA (~40 μg) from the WT strain (NRRL3631), the three epimutants (1=EM1, 2=EM2, and 3=EM3), and the three reverted strains (1-S=EM1-S, 2-S=EM2-S, and 3-S=EM3-S) was treated with the methylated-DNA-specific restriction enzyme McrBC (NEB) with or without previous treatment with the CpG methyltransferase SsI (NEB), following the manufacturer’s protocols. PCR amplification of the *fkbA* locus (~1.6 kb = 732 bp 5’ upstream *fkbA*, 457 bp *fkbA* ORF and 435 bp 3’ downstream *fkbA*) was carried out using 100 ng of purified DNA. PCR amplification after McrBC treatment yielded similar levels of product as the untreated samples. Virtually no product was obtained by PCR amplification in any of the samples after treatment with the CpG methyltransferase SsI followed by McrBC treatment, indicating that McrBC digested the newly methylated DNA, preventing its amplification. These results indicate that RNAi silencing does not involve DNA methylation of the *fkbA* locus in *M. circinelloides*. Image is representative of two independent experiments.

**Extended Data Figure 3. mRNAs from *fkbA* and its neighboring gene *patA* overlap in their 3’ regions by 92 bp**
a, The *fkbA* and *patA* genes are convergently oriented. The intergenic region is only 40 bp, and the mRNA overlap of their 3’ UTR regions spans 92 nucleotides. b, Alignment of the overlapping *fkbA* and *patA* 3’ regions based on 3’ RACE analysis. The direction of the transcripts are the same as in the upper figure, where the *patA* transcript is 5’ to 3’ end (top sequence) and the *fkbA* transcript is in the opposite orientation (3’ to 5’, bottom sequence). The polyA tails of both mRNA are shown.

**Extended Data Figure 4. *patA* expression to generate overlapping RNA molecules is not necessary for *fkbA* silencing**
a, Two independent *patA* null mutants (M1=MU434 and M2=MU435) were generated by homologous recombination, employing *pyrG* as the selectable marker. The *patA* ORF was replaced with the *pyrG* gene after electroporation of protoplasts with a gene deletion cassette, generated by overlap PCR, containing the selectable marker *pyrG* flanked by 5’ upstream and 3’ downstream sequences flanking the *patA* ORF. Almost 400 bp from the 3’ end of *patA* were preserved to keep intact the 3’UTR of *fkbA*. PCRs from 5’ and 3’ junctions (P1/P3 and P4/P2 respectively), the *patA* ORF (P5/P6), and spanning the *patA* and *fkbA* loci (P1/P2) were performed to confirm the deletion of the *patA* ORF and correct insertion of the *pyrG* disruption cassette (bottom). Image is representative of two independent experiments. 3’RACE assays were performed to verify that the *pyrG* 3’UTR and *fkbA* 3’UTR do not overlap in the *patA* null mutants (See Supplementary Table 2). b, Confirmation of the presence of sRNAs in epimutants derived from two independent *patA* null mutants. The numbers of the isolates correspond to those in Supplementary Table 1. An antisense-specific probe was used to detect *fkbA* sRNAs by northern blot. 5S rRNA served as a loading control. Abundant sRNAs complementary to *fkbA* were detected in all of the FK506^R strains that lacked Mendelian mutations isolated from the two independent *patA*Δ. sRNA blots were generated once because apositive signal indicates the presence of sRNA.

**Extended Data Figure 5. *fkbA* antisense RNA is complementary to mature *fkbA* RNA**
The complete sequence of the antisense RNA was determined based on 5’ and 3’ RACE analyses (bottom sequence) and compared to the *fkbA* DNA (top sequence). These analyses indicate the antisense RNA is 5’ capped and 3’ poly-adenylated. The *fkbA* introns were absent in the antisense sequence and the 3’ end matched the beginning of the 5’ UTR found on *fkbA* mRNA by 5’ RACE analysis, indicating that mature spliced *fkbA* RNA is used as a template by an RdRP to generate the complementary strand. The antisense RNA 5’ end is located 7 nt upstream of the STOP codon. The 3’ end is located 40 nt upstream of the ATG codon. The *fkbA* DNA sequence includes the sequenced 5’ and 3’ UTR regions in blue and the introns in black. The *fkbA* coding region is indicated in red. The ATG start and TAA stop codons are shown in green boxes.

**Extended Data Figure 6. Very few sRNAs were detected by high-throughput sequencing in the WT strain**
The normalized number of reads of each antisense sRNA complementary to *fkbA* is extremely low in the WT strain, and below the detection limit of northern blot (numbers expressed in reads per million). Almost all of the antisense sRNA detected have a uridine at the 5’ terminus, features typically found in sRNA that interact with Argonaute proteins, suggesting that they may represent authentic sRNAs but are present at insufficient levels to trigger RNAi silencing. Sense sRNA does not show any bias (data not shown). The height of a letter represents the frequency with which the base is observed in that position, and the total height of the letters in a position indicates how strong the bias is for specific bases in that position. Note that because the total number of *fkbA* antisense reads in the WT strain is very small, they are not probably representative of the true distribution of sRNAs, so the logos are likely to overestimate any sRNA sequence bias.

**Extended Data Figure 7. Reverted strains (EM1-S, EM2-S, and EM3-S) exposed to a second round of FK506 selection undergo epimutations at the same frequency as the WT strain**
Epimutant strains (EM1, EM2, and EM3) that had reverted to an FK506 sensitive WT phenotype (yeast growth in the presence of FK506) after several passages on drug-free media were exposed a second time to 1 μg/mL FK506 to ascertain if genomic mutations had occurred that enhance epimutant formation. a, The numbers of the isolates correspond to those in Supplementary Table 1. The new FK506^R isolates that lacked a Mendelian mutation in any of the target genes showed abundant sRNAs complementary to *fkbA* based on northern blot of sRNA hybridized with an *fkbA* antisense-specific probe. 5S rRNA served as a loading control. Images are representative of two independent experiments. EM1-S, EM2-S, EM3-S =Epimutants 1, 2, and 3 reverted to restored FK506-sensitivity. C+=EM1 before reversion of FK506-resistance. b, The frequency of epimutation was similar or lower in the reverted epimutant strains compared to the WT, which argues against mutations that arose promoting epimutation. In addition, because *rdrp1* mutations enhance epimutation frequency and stability, the *rdrp1* gene was sequenced in EM3 and found to be WT with no mutations.p-values were obtained based on a Fisher Exact Probability Test for a 2×2 Contingency Table, comparing each of the mutant strains individually versus the WT strain NRRL3631.

Extended Data Figure 8. sRNA were detected by high-throughput sequencing in the epimutant strains, but not in the WT and reverted strains. This pattern was not conserved in any other loci in the genome
a, sRNAs were found to span exon-exon junctions. Antisense and sense sRNA from EM1 that span intron 1 are shown at the top and bottom respectively. The numbers on the left are the normalized read counts (reads per million) for each specific sRNA. Only sRNAs with 5 or more read counts are shown. The reference sequence is in red and sRNAs spanning the intron are in black. b, Distribution of antisense sRNA for the *fkbA* gene across the different strains and the 50 genes with sRNA patterns most closely correlated with *fkbA*. sRNA read counts distribution for the *fkbA* gene is represented with a heavy gray line (left panel). The 50 genes with the closest correlated pattern are represented with thin colored lines. The bottom part of the left panel has been expanded to elucidate the read count patterns of the correlated genes (right panel). While some of these genes show an apparent similarity to the pattern of sRNA in the *fkbA* silenced and revertant strains, the levels of read counts are in most cases ~100-fold lower than that for *fkbA* gene, and were not detected by sRNA blots (data not shown). WT=strain NRRL3631. EM1-R, EM2-R, EM3-R=Epimutants 1, 2, and 3 resistant to FK506. EM1-S and EM3-S=Epimutants 1 and 3 reverted to restored FK506-sensitivity.

**Extended Data Figure 9. sRNA and antisense *fkbA* RNA detection in the different mutant strains lacking RNAi pathway components**
a, The numbers of the isolates correspond to those in Supplementary Table 1. No epimutants (absence of sRNA complementary to *fkbA*) were found in the *dcl2* (MU410), *dcl1* (MU407), *ago1* (MU413, MU426), or *rdrp2* (MU420, MU428) null mutants based on sRNA northern blots hybridized with an *fkbA* antisense-specific probe. Only one epimutant was recovered in the second independent *dcl1* mutant (MU406). We reconfirmed this result (sRNA blot, no *fkbA* mutation) and also validated the isolate was *dcl1*Δ by PCR. *ago2* (MU416) and *ago3* (MU414) null mutants showed a frequency of epimutation similar to the WT strain (R7B). The *rdrp1* null mutant (MU419) showed an elevated epimutation frequency. The conclusion that Dcl2, Dcl1, Ago1, and RdRP2 are required for epimutation is supported by the congruence of phenotype, and the analysis of two independent null mutants each for *ago1, rdrp2*, and *dcl1*. R7B served as WT for this experiment as all of the RNAi silencing mutants were generated in this background. 5S rRNA served as a loading control. C+=EM1 strain. Images from *dicer* mutant blots are representative of two independent experiments. The remaining blots were generated once. b, Total RNA was isolated from the WT, *fkbA* mutant, *patA* mutant, R7B and the indicated RNAi pathway mutants, and 50 μg of total RNA were used to ensure signals could be detected from all of the mRNA analyzed, as the level of *patA* expression is low. All of the silencing mutant strains have the same level of expression of the *fkbA* antisense RNA based on northern blot. Antisense- and sense-specific probes were used to detect antisense and sense *fkbA* RNA respectively. The northern blot was first probed for antisense RNA to avoid residual signal from *fkbA* mRNA. *patA* expression was not affected in any the *fkbA*Δ or the RNAi mutant strains, but as expected was absent in the two independent *patA*Δ strains. *actin* served as a loading control. Images are representative of three independent experiments.

**Extended Data Figure 10. *M. circinelloides* f. *circinelloides* (*Mcc*), and *M. circinelloides* f. *griseocyanus* (*Mcg*) strains were tested for generation of FK506^R and *fkbA* silencing**
a, The indicated *Mucor* strains, plus *M. circinelloides* f. *lusitanicus* (*Mcl*) WT strain used as a control, were incubated on YPD media for 3 days (top panel) and on YPD supplemented with 1 μg/mL of FK506 for up to 10 days (lower panels). The *Mcl* and 1006PhL strains grew as a yeast colony until FK506^R sectors started to grow as hyphae. The Mucho strain grew as a yeast colony for several days, and in some of the plates a resistant patch appeared, but after 7-8 days the colonies developed aerial hyphae on top of the yeast colony, producing FK506 sensitive spores that grew as yeast after falling on the media, preventing the development of more FK506^R patches. The *Mcg* strain was more sensitive to FK506 without a visible colony until day 6 when the spores started to germinate as a mixture of yeast and hyphae that did not produce any FK506^R growth. Images are representative of ~40 independent colonies from each strain. b, Confirmation of the presence of sRNAs in epimutants derived from one of the two pathogenic *M. circinelloides f. circinelloides* strains. The numbers of the isolates correspond to those in Supplementary Table 5. An antisense strain-specific probe was used to detect *fkbA* sRNAs by northern blot from both strains (using 30 μg of sRNA). 5S rRNA served as a loading control. Abundant sRNAs complementary to *fkbA* were detected in all of the FK506^R strains that lacked Mendelian mutations isolated from the 1006PhL strain, but not from the Mucho strain. Images from the lower blots are representative of two independent experiments. Images from the upper blots were generated once since sRNA positive signals were detected from all samples analyzed.

Figure 1. RNAi-dependent epimutations confer FK506-resistance in *M. circinelloides*
a, WT, *fkbA* mutant (*fkbA*Δ), and epimutant strains were grown on YPD media alone or supplemented with FK506, rapamycin, or cyclosporin A (CsA). Images representative of several independent experiments. **b-c**, The epimutant strains EM1, EM2, and EM3 were grown on YPD media with FK506 (FK506 lanes), or YPD drug-free media (R lanes). The reverted strains EM1-S, EM2-S, and EM3-S (S lanes) were grown in YPD media and whole cell protein and RNA extracts were prepared. b, Equivalent protein amounts (120 μg) were resolved by SDS-PAGE and analyzed by western blot with an anti-*S. cerevisiae*-FKBP12 antibody. AnS. cerevisiae extract (Sc) was included as control for antibody specificity; tubulin served as loading control. Images representative of seven independent experiments. c, 50 μg total mRNA was analyzed by northern blot employing probes specific for *fkbA, act1* (loading control), and antisense *fkbA* mRNAs. Images representative of six independent experiments for the *fkbA* and *act1* probes, and two for the antisense *fkbA* probe.

Figure 2. Epimutant strains express abundant sRNA antisense to *fkbA*
a, sRNA were extracted from WT, epimutants (FK506 and R lanes), and reverted strains (S lanes) after growth in YPD media alone (R, S lanes) or with FK506 (FK506 lanes). sRNAs (25μg) were analyzed by sRNA blot employing an antisense-specific probe for the *fkbA* gene or a probe for *5S rRNA* (loading control). Images representative of three independent experiments. b, The presence of sense and antisense *fkbA* sRNA was analyzed by high-throughput sequencing in WT, epimutants, and two revertant strains (EM1-S, EM3-S). sRNA amount is expressed in reads per million, and they are distributed along the *fkbA* ORF (bottom) c, Analysis of size and first nucleotide (inset) of antisense sRNAs. The representation corresponds to data obtained from isolate EM1-R. Similar results were observed for the EM2-R and EM3-R epimutants.

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References

1. Liu J, et al. Calcineurin is a common target of cyclophilin-cyclosporin A and FKBP-FK506 complexes. Cell. 1991;66:807–815. - PubMed
1. Lee SC, Li A, Calo S, Heitman J. Calcineurin plays key roles in the dimorphic transition and virulence of the human pathogenic zygomycete Mucor circinelloides. PLOS Pathog. 2013;9:e1003625. - PMC - PubMed
1. Orlowski M. Mucor dimorphism. Microbiol Rev. 1991;55:234–258. - PMC - PubMed
1. Bastidas RJ, Shertz CA, Lee SC, Heitman J, Cardenas ME. Rapamycin exerts antifungal activity in vitro and in vivo against Mucor circinelloides via FKBP12-dependent inhibition of Tor. Eukaryotic Cell. 2012;11:270–281. - PMC - PubMed
1. Heitman J, Movva NR, Hall MN. Targets for cell cycle arrest by the immunosuppressant rapamycin in yeast. Science. 1991;253:905–909. - PubMed

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[1] Liu J, et al. Calcineurin is a common target of cyclophilin-cyclosporin A and FKBP-FK506 complexes. Cell. 1991;66:807–815. - PubMed

[2] Liu J, et al. Calcineurin is a common target of cyclophilin-cyclosporin A and FKBP-FK506 complexes. Cell. 1991;66:807–815. - PubMed

[3] Lee SC, Li A, Calo S, Heitman J. Calcineurin plays key roles in the dimorphic transition and virulence of the human pathogenic zygomycete Mucor circinelloides. PLOS Pathog. 2013;9:e1003625. - PMC - PubMed

[4] Lee SC, Li A, Calo S, Heitman J. Calcineurin plays key roles in the dimorphic transition and virulence of the human pathogenic zygomycete Mucor circinelloides. PLOS Pathog. 2013;9:e1003625. - PMC - PubMed

[5] Orlowski M. Mucor dimorphism. Microbiol Rev. 1991;55:234–258. - PMC - PubMed

[6] Orlowski M. Mucor dimorphism. Microbiol Rev. 1991;55:234–258. - PMC - PubMed

[7] Bastidas RJ, Shertz CA, Lee SC, Heitman J, Cardenas ME. Rapamycin exerts antifungal activity in vitro and in vivo against Mucor circinelloides via FKBP12-dependent inhibition of Tor. Eukaryotic Cell. 2012;11:270–281. - PMC - PubMed

[8] Bastidas RJ, Shertz CA, Lee SC, Heitman J, Cardenas ME. Rapamycin exerts antifungal activity in vitro and in vivo against Mucor circinelloides via FKBP12-dependent inhibition of Tor. Eukaryotic Cell. 2012;11:270–281. - PMC - PubMed

[9] Heitman J, Movva NR, Hall MN. Targets for cell cycle arrest by the immunosuppressant rapamycin in yeast. Science. 1991;253:905–909. - PubMed

[10] Heitman J, Movva NR, Hall MN. Targets for cell cycle arrest by the immunosuppressant rapamycin in yeast. Science. 1991;253:905–909. - PubMed

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Antifungal drug resistance evoked via RNAi-dependent epimutations

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Antifungal drug resistance evoked via RNAi-dependent epimutations

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