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Cited by 205Antifungal resistance and susceptibilityFungal and yeast genetics researchFungal Infections and Studies

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Drug-Resistant Epimutants Exhibit Organ-Specific Stability and Induction during Murine Infections Caused by the Human Fungal Pathogen Mucor circinelloides

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Chang, Zanetta, Heitman, Joseph·2019·mBio
DOI 10.1128/mbio.02579-19PMC PMC6831780

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Abstract

The emerging fungal pathogen Mucor circinelloides causes a severe infection, mucormycosis, which leads to considerable morbidity and mortality. Treatment of Mucor infection is challenging because Mucor is inherently resistant to nearly all clinical antifungal agents. An RNAi-dependent and reversible mechanism of antifungal resistance, epimutation, was recently reported for Mucor . Epimutation has not been studied in vivo , and it was unclear whether it would contribute to antifungal resistance observed clinically. We demonstrate that epimutation can both be induced and reverted after in vivo passage through a mouse; rates of both induction and reversion are higher after brain infection than after infection of other organs (liver, spleen, kidneys, or lungs). Elucidating the roles played by epimutation in drug resistance and infection will improve our understanding of Mucor and other fungal pathogens and may have implications for antifungal treatment.

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Crossref · 10.1128/mbio.02579-19

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FIG 1 Retro-orbital and tail vein injections produce equivalent outcomes. BALB/c mice were infected with 1.25 × 106 spores of M. circinelloides f. circinelloides strain 1006PhL through either the retro-orbital or tail vein route. There were five mice in each group. (A) The two methods of infection produce equivalent mortality. (B) The retro-orbital method of infection produces equivalent homogenized fungal burdens in randomly sampled moribund mice (N = 2). (C) Histopathological analysis of brains from mice infected with spores by retro-orbital and tail vein injection, stained with GMS (Gomori methenamine-silver) reveals abundant fungal elements from both routes of infection.
FIG 1 Retro-orbital and tail vein injections produce equivalent outcomes. BALB/c mice were infected with 1.25 × 106 spores of M. circinelloides f. circinelloides strain 1006PhL through either the retro-orbital or tail vein route. There were five mice in each group. (A) The two methods of infection produce equivalent mortality. (B) The retro-orbital method of infection produces equivalent homogenized fungal burdens in randomly sampled moribund mice (N = 2). (C) Histopathological analysis of brains from mice infected with spores by retro-orbital and tail vein injection, stained with GMS (Gomori methenamine-silver) reveals abundant fungal elements from both routes of infection.CC BY · PubMed Central
FIG 2 FK506 epimutants revert in an organ-specific manner after in vivo passage. fkbA epimutant and wild-type M. circinelloides f. circinelloides strains were recovered from multiple organs after in vivo infection/passage in one mouse each. Strains were recovered after the mouse was moribund, 4 days postinfection. Strain SCV522 is a fkbA epimutant. Strain 1006PhL is the wild-type (WT) strain. (A) Representative images of colonies recovered from the brains and livers of mice infected with either strain SCV522 or 1006PhL and patched onto YPD with and without FK506. All colonies patched on nonselective YPD medium grew as hyphae. FK506-sensitive strains patched on YPD + FK506 medium grew as smaller yeast colonies. Loss of FK506 resistance from the epimutant strain was observed in the brain, but not in the liver. (B) Quantification of the percentage of FK506 resistance by organ, after in vivo passage. There were 150 colonies per organ; however, fewer than 150 colonies were recovered from some organs (see Table S1 in the supplemental material). The Spores column shows SCV522 and 1006PhL spores plated without in vivo passage. FK506 resistance was below the limit of detection in all strains derived from wild-type 1006PhL infection. (C) sRNA hybridization of representative sensitive (S) and resistant (R) strains randomly selected from SCV522-infected mouse after passage. All resistant isolates continued to express sRNA against fkbA, whereas reverted, FK506-sensitive isolates did not. 5S rRNA was stained with ethidium bromide (EtBr) and served as the loading control. epi, epimutant; nt, nucleotides.
FIG 2 FK506 epimutants revert in an organ-specific manner after in vivo passage. fkbA epimutant and wild-type M. circinelloides f. circinelloides strains were recovered from multiple organs after in vivo infection/passage in one mouse each. Strains were recovered after the mouse was moribund, 4 days postinfection. Strain SCV522 is a fkbA epimutant. Strain 1006PhL is the wild-type (WT) strain. (A) Representative images of colonies recovered from the brains and livers of mice infected with either strain SCV522 or 1006PhL and patched onto YPD with and without FK506. All colonies patched on nonselective YPD medium grew as hyphae. FK506-sensitive strains patched on YPD + FK506 medium grew as smaller yeast colonies. Loss of FK506 resistance from the epimutant strain was observed in the brain, but not in the liver. (B) Quantification of the percentage of FK506 resistance by organ, after in vivo passage. There were 150 colonies per organ; however, fewer than 150 colonies were recovered from some organs (see Table S1 in the supplemental material). The Spores column shows SCV522 and 1006PhL spores plated without in vivo passage. FK506 resistance was below the limit of detection in all strains derived from wild-type 1006PhL infection. (C) sRNA hybridization of representative sensitive (S) and resistant (R) strains randomly selected from SCV522-infected mouse after passage. All resistant isolates continued to express sRNA against fkbA, whereas reverted, FK506-sensitive isolates did not. 5S rRNA was stained with ethidium bromide (EtBr) and served as the loading control. epi, epimutant; nt, nucleotides.CC BY · PubMed Central
FIG 3 Organ-specific development of FK506 resistance after in vivo passage. Mice (N = 3) were infected with the wild-type 1006PhL strain; colonies were recovered on YPD + FK506 medium when mice were moribund, 4 days postinfection. Five days after recovery, resistant and sensitive colonies were counted. (A) Percentage of FK506 resistance by organ, showing higher rates of resistance in the brain. 1006Phl spores from strains without in vivo passage were plated and are shown in the graph. Significance was determined via one-way ANOVA (P = 0.0035) with posthoc Tukey’s multiple-comparison test. (B) sRNA hybridization analysis against fkbA demonstrated the development of sRNA expression against fkbA in strains from all organs and spore conditions. SCV522 is the fkbA epimutant. 1006PhL is the wild-type strain. Strains isolated from each individual mouse are identified by the color of the label (blue, green, or red). Two of the 27 resistant (R) strains and all three of the sensitive (S) strains (one from each mouse) showed no evidence of sRNA expression against fkbA.
FIG 3 Organ-specific development of FK506 resistance after in vivo passage. Mice (N = 3) were infected with the wild-type 1006PhL strain; colonies were recovered on YPD + FK506 medium when mice were moribund, 4 days postinfection. Five days after recovery, resistant and sensitive colonies were counted. (A) Percentage of FK506 resistance by organ, showing higher rates of resistance in the brain. 1006Phl spores from strains without in vivo passage were plated and are shown in the graph. Significance was determined via one-way ANOVA (P = 0.0035) with posthoc Tukey’s multiple-comparison test. (B) sRNA hybridization analysis against fkbA demonstrated the development of sRNA expression against fkbA in strains from all organs and spore conditions. SCV522 is the fkbA epimutant. 1006PhL is the wild-type strain. Strains isolated from each individual mouse are identified by the color of the label (blue, green, or red). Two of the 27 resistant (R) strains and all three of the sensitive (S) strains (one from each mouse) showed no evidence of sRNA expression against fkbA.CC BY · PubMed Central

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Metadata

Authors
Chang, Z., & Heitman, J.
Affiliations
Duke University (US)
Corresponding author
Heitman, Joseph
ORCID
Venue
mBio
Publisher
American Society for Microbiology
Published
2019-12-24
Volume
10
Issue
6
ISSN
  • 2150-7511 (ISSN-L)
  • 2161-2129
DOI
10.1128/mbio.02579-19
PMC
PMC6831780
OpenAlex
W2985567791
Cited by
202
References
43
Citations by year
2026: 21 · 2025: 16 · 2024: 22 · 2023: 30 · 2022: 42 · 2021: 39 · 2020: 30 · 2019: 2
FWCI
21.85
Type
journal article
Language
en
Open access
Licence
Locations
  • mBio (publishedVersion, cc-by)
  • bioRxiv (Cold Spring Harbor Laboratory) (acceptedVersion, cc-by-nc-nd)
  • Europe PMC (PubMed Central) (publishedVersion, cc-by)
  • PubMed Central (publishedVersion, cc-by)
  • PubMed (publishedVersion)
  • DOAJ (DOAJ: Directory of Open Access Journals) (submittedVersion)
  • DOAJ (DOAJ: Directory of Open Access Journals) (submittedVersion, cc-by-sa)
Funders
HHS | National Institutes of Health (AI39115-21, AI50113-15, AI104533-05, AI112595-04)
Topics
  • Health Sciences › Medicine › Infectious Diseases › Antifungal resistance and susceptibility
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Fungal and yeast genetics research
  • Health Sciences › Medicine › Infectious Diseases › Fungal Infections and Studies
Keywords
Mucor circinelloides, mucormycosis, epimutations, antifungal resistance, brain infection, fungal pathogens
MeSH terms
Animals, Antifungal Agents, Disease Models, Animal, Humans, Male, Mice, Inbred BALB C, Mucor, Mucormycosis, Tacrolimus, Sirolimus, Drug Resistance, Fungal, RNA Interference, Epigenesis, Genetic, Mice

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