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Antifungal drug resistance evoked via RNAi-dependent epimutations

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 calcineurin1. Calcineurin inhibition by FK506 blocks M. circinelloides transition to hyphae and enforces yeast growth2. 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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Figure 1: RNAi-dependent epimutations confer FK506 resistance in M. circinelloides.
Figure 2: Epimutant strains express abundant sRNA antisense to fkbA.

Accession codes

Primary accessions

GenBank/EMBL/DDBJ

Gene Expression Omnibus

Data deposits

Sequences for the fkbA gene from WT strain NRRL3631 and epimutant strains (EM1, EM2 and EM3) were deposited in GenBank with accession numbers KF203228, KF203229, KF203230 and KF203231. Raw data from high-throughput sRNA sequencing of WT, epimutant and revertant strains have been deposited in NCBI’s Gene Expression Omnibus and are accessible through accession number GSE56353.

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Acknowledgements

We thank R. Skalsky and V. Ponnusamy for technical support and J. Wöstemeyer for trisporic acid. We thank B. Cullen, T. Petes, B. Billmyre, M. Feretzaki, J. Kingsbury and V. Ponnusamy for critical reading. This work was supported by NIH grants R37 AI39115-17, R01 AI50438-10, R01 CA154499-04 and the Spanish MICINN BFU2009-07220 and MINECO BFU2012-32246, co-financed by FEDER.

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Authors

Contributions

S.C., C.S.-W., S.T.M., R.M.R.-V., M.E.C. and J.H. designed experiments, interpreted data and wrote the paper. S.C., C.S.-W., R.J.B., S.C.L. and F.E.N. performed experiments. P.M. sequenced the sRNA library. J.A.G. analysed deep-sequencing data. S.T.M., R.M.R.-V., M.E.C. and J.H. provided materials.

Corresponding author

Correspondence to Joseph Heitman.

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Competing interests

The authors declare no competing financial interests.

Extended data figures and tables

Extended Data Figure 1 M. circinelloides can develop resistance to FK506 by two mechanisms, one stable (mutation) and one transient (epimutation).

a, The wild-type strain (NRRL3631) grows as hyphae (white, upper left panel) on YPD and as a yeast (yellow, upper centre panel) on YPD containing 1 μg ml−1 of FK506. An FK506-resistant patch that emerged from the south-eastern 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 wild-type phenotype (yeast growth on YPD supplemented with 1 μg ml−1 FK506). SM2 strain4 harbours 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 h 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 and 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 Fig. 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 analysed (fkbA, cnaA, cnaB, cnaC and cnbR) and the mechanism by which they have developed FK506-resistance remains to be established. C+, EM1. 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 wild-type strain (NRRL3631), the three epimutants (1, EM1; 2, EM2; 3, EM3), and the three reverted strains (1-S, EM1-S; 2-S, EM2-S; 3-S, EM3-S) was treated with the methylated-DNA-specific restriction enzyme McrBC (NEB) with or without previous treatment with the CpG methyltransferase SssI (NEB), following the manufacturer’s protocols. PCR amplification of the fkbA locus (1.6 kilobases = 732 base pairs (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 SssI 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 neighbouring 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 poly(A) tails of both mRNAs 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 FK506r strains that lacked Mendelian mutations isolated from the two independent patAΔ. sRNA blots were generated once because a positive 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′ polyadenylated. 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 nucleotides upstream of the STOP codon. The 3′ end is located 40 nucleotides 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 fkbA sRNAs were detected by high-throughput sequencing in the wild-type strain.

In the wild-type strain, the total count of antisense sRNA complementary to fkbA is extremely low (sixteen reads), and below the detection limit of northern blot. All but one 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). Sequence logos summarizing all sixteen reads are shown, with one logo for each size antisense read observed (x axis). The unit ‘bits’ on the y axis of the sequence logos indicate how much the frequency of a base in each position departs from the expected random distribution. 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. Positions in the logo with zero bits indicate a random distribution of bases. Bit values at or near two indicate that the same base is always present in that position. Bit values of one indicate that two bases have equal distribution in the position. Note that because the total number of fkbA antisense reads in the wild-type strain is very small, they are probably not representative of the true distribution of sRNAs, so the logos probably 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 wild-type strain.

Epimutant strains (EM1, EM2 and EM3) that had reverted to an FK506-sensitive wild-type phenotype (yeast growth in the presence of FK506) after several passages on drug-free media were exposed a second time to 1 μg ml−1 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 FK506r 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 and 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 wild type, which suggests that no mutations arose promoting epimutation. In addition, because rdrp1 mutations enhance epimutation frequency and stability, the rdrp1 gene was sequenced in EM3 and found to be wild-type with no mutations. P values were obtained based on a Fisher’s exact probability test for a 2 by 2 contingency table, comparing each of the mutant strains individually versus the wild-type strain NRRL3631.

Extended Data Figure 8 sRNAs were detected by high-throughput sequencing in the epimutant strains, but not in the wild-type 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 sRNAs 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 five 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 count distribution for the fkbA gene is represented with a heavy grey 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 the fkbA gene, and were not detected by sRNA blots (data not shown). WT, strain NRRL3631; EM1-R, EM2-R and 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 that the isolate was dcl1Δ by PCR. ago2 (MU416) and ago3 (MU414) null mutants showed a frequency of epimutation similar to the wild-type 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 wild type 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 wild type, 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 analysed, 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. The northern blot was first probed for antisense RNA to avoid residual signal from fkbA mRNA. patA expression was not affected in any the fkbAΔ or the RNAi mutant strains, but as expected was absent in the two independent patAΔ 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 FK506r and fkbA silencing.

a, The indicated Mucor strains, plus M. circinelloides f. lusitanicus (Mcl) wild-type strain used as a control, were incubated on YPD media for 3 days (top panel) and on YPD supplemented with 1 μg ml−1 of FK506 for up to 10 days (lower panels). The M. circinelloides f. lusitanicus and 1006PhL strains grew as a yeast colony until FK506r 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 several 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 FK506r patches. The M. circinelloides f. griseocyanus 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 FK506r 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 FK506r 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 analysed.

Supplementary information

Supplementary Information

The Supplementary Information contains 8 Tables that were too long to be included as Extended Data. Each table contains a small legend to explain the content included. These tables include the following information: Tables 1, 3 and 5: lists of the mutations found in the fkbA and calcineurin genes sequenced in every strain/isolate used for this study Table 2: 5′ and 3′ RACE sequences addressed in the manuscript Table 4: frequency of epimutants/mutants after exposure to stress conditions (experiment carried out to answer comments from the reviewers) Table 6: primers used in this study Table 7: adapter sequences used for sRNA libraries Table 8: basic library data (PDF 652 kb)

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Calo, S., Shertz-Wall, C., Lee, S. et al. Antifungal drug resistance evoked via RNAi-dependent epimutations. Nature 513, 555–558 (2014). https://doi.org/10.1038/nature13575

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