A non-canonical RNA degradation pathway suppresses RNAi-dependent epimutations in the human fungal pathogen Mucor circinelloides

Calo, Silvia; Nicolás, Francisco E.; Lee, Soo Chan; Vila, Ana; Cervantes, María; Torres‐Martínez, Santiago; Ruiz‐Vázquez, Rosa M.; Cárdenas, María E.; Heitman, Joseph

doi:10.1371/journal.pgen.1006686

Cited by 57 publications

(114 citation statements)

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“…Surprisingly, none of the mutants carried Mendelian mutations in genes for the calcineurin A or B subunit, FKBP12, or two subtypes of cyclophilin A ( cypA and cypB ) (data not shown). In addition, the mutants did not carry epimutations in the cypA and cypB genes, in which small RNA blots did not reveal any small RNAs derived from the cypA and cypB genes (51) (data not shown). Therefore, we hypothesized that the CSR mutants were also calcineurin bypass mutants that do not require calcineurin activity for hyphal growth.…”

Section: Resultsmentioning

confidence: 95%

“…One of the common ways through which most pathogenic microbes become resistant to drugs is having mutations in the drug-receptor, in this case FKBP12 or catalytic A subunit or regulatory B subunits of calcineurin (27). Calo et al have shown that Mucor can be transiently resistant to FK506 by triggering an RNA interference specific to the gene encoding FKBP12, resulting in silencing of the drug target gene (51, 52). In this study, we identified a novel resistance mechanism through which Mucor can also become resistant to calcineurin inhibitors.…”

Section: Discussionmentioning

confidence: 99%

“…In our previous studies, calcineurin inhibitor resistant Mucor strains were found to exhibit hyphal growth instead of yeast growth were found to have mutations in the FKBP12 gene or the calcineurin catalytic A or regulatory B subunit genes (27, 43, 51, 52). In addition, Calo et al, found that Mucor can also silence the FKBP12 gene to become transiently resistant to FK506 and rapamycin via an RNAi-dependent epimutation pathway (51, 52). In this study, we isolated mutants that do not follow the known calcineurin inhibitor resistance mechanisms.…”

Section: Introductionmentioning

confidence: 97%

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A novel resistance pathway for calcineurin inhibitors in the human pathogenic MucoralesMucor circinelloides

Vellanki

Billmyre

Lorenzen

et al. 2019

Preprint

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24Mucormycosis is an emerging lethal fungal infection in immunocompromised 25 patients. Mucor circinelloides (Mucor) is a causal agent of mucormycosis and serves as 26 a model system to understand genetics in Mucorales. Calcineurin is a conserved 27 virulence factor in many pathogenic fungi and calcineurin inhibition or deletion of the 28 calcineurin regulatory subunit (CnbR) in Mucor results in a shift from hyphal to yeast 29 growth. We analyzed thirty-six calcineurin inhibitor resistant or bypass mutants that 30 exhibited hyphal growth in the presence of calcineurin inhibitors or in the yeast-locked 31 cnbR mutant background without carrying any mutations in known calcineurin 32 components. We found that a majority of the mutants had altered sequence in a gene, 33 named here bycA (bypass of calcineurin A). bycA encodes an amino acid permease. 34 We verified that both bycA, and the bycA cnbR double mutant are resistant to the 35 calcineurin inhibitor FK506, thereby demonstrating a novel resistance mechanism 36 against calcineurin inhibitors. We also found that the expression of bycA was 37 significantly higher in the wild type with FK506 and in the cnbR mutants, but 38 significantly lower in the wild type without FK506. These findings suggest that bycA is a 39 negative regulator of hyphal growth and/or a positive regulator of yeast growth in Mucor 40 and calcineurin suppresses the bycA gene at the mRNA level to promote hyphal growth. 41BycA is involved in the Mucor hyphal-yeast transition as our data demonstrates a 42 positive correlation between bycA expression, protein kinase A activity, and Mucor 43 yeast-growth. Also, calcineurin activity rather than hyphal morphology primarily 44 contributes to virulence traits including phagosome maturation blockade, host cell 45 damages, and pro-angiogenic growth factor induction during interactions with hosts. 46 3 Importance 47 Mucor is intrinsically resistant to most known antifungals, which makes 48 mucormycosis treatment challenging. Calcineurin is a serine/threonine phosphatase 49 widely conserved across eukaryotes. When calcineurin function is inhibited in Mucor, 50 growth shifts to a less-virulent yeast growth form which makes calcineurin an attractive 51 target for development of new antifungal drugs. Previously we identified two distinct 52 mechanisms through which Mucor can become resistant to calcineurin inhibitors 53 involving Mendelian mutations in the gene for FKBP12, calcineurin A or B subunits and 54 epimutations silencing the FKBP12 gene. Here, we identified a third novel mechanism 55 where loss of function mutations in the amino acid permease encoding the bycA gene 56 contribute to resistance against calcineurin inhibitors. When calcineurin activity is 57 absent, BycA can activate PKA to promote yeast growth via a cAMP-independent 58 pathway. Our data also shows that calcineurin activity, not morphology, primarily 59 contributes to host -pathogen interactions in the pathogenesis of Mucor. 60 61 62

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Section: Resultsmentioning

confidence: 95%

Section: Discussionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 97%

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A novel resistance pathway for calcineurin inhibitors in the human pathogenic MucoralesMucor circinelloides

Vellanki

Billmyre

Lorenzen

et al. 2019

Preprint

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“…The phenomenon of epimutation was recently characterized as a novel mechanism of RNAi-dependent drug resistance in Mucor (17-19). Here, we report the first analysis of the impact of epimutation during infection.…”

Section: Discussionmentioning

confidence: 99%

“…In epimutation, Mucor endogenous RNAi pathways are activated and induce silencing of various drug target genes to result in antifungal resistance (17, 18). The mechanism of epimutation requires the core RNAi pathway and competes with an alternative RNA degradation pathway in Mucor (19).…”

Section: Introductionmentioning

confidence: 99%

Drug-resistant epimutants exhibit organ-specific stability and induction during murine infections caused by the human fungal pathogenMucor circinelloides

Chang

Heitman

2019

Preprint

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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, 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, as compared to those 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.IMPORTANCEThe 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 described in 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 be both induced and reverted after in vivo passage through a mouse model; 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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Understanding Different Mechanisms Involved in Cancer Drug Resistance

Malviya¹,

Singh²,

Yadav³

2023

Multi‐Drug Resistance in Cancer

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A non-canonical RNA degradation pathway suppresses RNAi-dependent epimutations in the human fungal pathogen Mucor circinelloides

Cited by 57 publications

References 38 publications

A novel resistance pathway for calcineurin inhibitors in the human pathogenic MucoralesMucor circinelloides

A novel resistance pathway for calcineurin inhibitors in the human pathogenic MucoralesMucor circinelloides

Drug-resistant epimutants exhibit organ-specific stability and induction during murine infections caused by the human fungal pathogenMucor circinelloides

Understanding Different Mechanisms Involved in Cancer Drug Resistance

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