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

Abstract

The environmentally ubiquitous fungus Mucor circinelloides is a primary cause of the emerging disease mucormycosis. Mucor infection is notable for causing high morbidity and mortality, especially in immunosuppressed patients, while being inherently resistant to the majority of clinically available antifungal drugs. A new, RNA interference (RNAi)-dependent, and reversible epigenetic mechanism of antifungal resistance-epimutation-was recently discovered in M. circinelloides However, the effects of epimutation in a host-pathogen setting were unknown. We employed a systemic, intravenous murine model of Mucor infection to elucidate the potential impact of epimutation in vivo Infection with an epimutant strain resistant to the antifungal agents FK506 and rapamycin revealed that the epimutant-induced drug resistance was stable in vivo in a variety of different organs and tissues. Reversion of the epimutant-induced drug resistance was observed to be more rapid in isolates from the brain than in isolates recovered from the liver, spleen, kidney, or lungs. Importantly, infection with a wild-type strain of Mucor led to increased rates of epimutation after strains were recovered from organs and exposed to FK506 stress in vitro. Once again, this effect was more pronounced in strains recovered from the brain than from other organs. In summary, we report the rapid induction and reversion of RNAi-dependent drug resistance after in vivo passage through a murine model, with pronounced impact in strains recovered from brain. Defining the role played by epimutation in drug resistance and infection advances our understanding of Mucor and other fungal pathogens and may have implications for antifungal therapy.IMPORTANCE 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.

Keywords: antifungal resistance; epigenetics; filamentous fungi.

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 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 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.