Broad antifungal resistance mediated by RNAi-dependent epimutation in the basal human fungal pathogen Mucor circinelloides

Mapping Intimacies ◽

10.1101/526459 ◽

2019 ◽

Author(s):

Zanetta Chang ◽

R. Blake Billmyre ◽

Soo Chan Lee ◽

Joseph Heitman

Keyword(s):

Drug Resistance ◽

Target Gene ◽

Drug Exposure ◽

Antifungal Agents ◽

Mucor Circinelloides ◽

Antifungal Resistance ◽

Rnai Pathway ◽

Srna Library ◽

Human Fungal Pathogen

Mucormycosis - an emergent, deadly fungal infection - is difficult to treat, in part because the causative species demonstrate broad clinical antifungal resistance. However, the mechanisms underlying drug resistance in these infections remain poorly understood. Our previous work demonstrated that one major agent of mucormycosis, Mucor circinelloides, can develop resistance to the antifungal agents FK506 and rapamycin through a novel, transient RNA interference-dependent mechanism known as epimutation. Epimutations silence the drug target gene and are selected by drug exposure; the target gene is re-expressed and sensitivity is restored following passage without drug. This silencing process involves generation of small RNA (sRNA) against the target gene via core RNAi pathway proteins. To further elucidate the role of epimutation in the broad antifungal resistance of Mucor, epimutants were isolated that confer resistance to another antifungal agent, 5-fluoroorotic acid (5-FOA). We identified epimutant strains that exhibit resistance to 5-FOA without mutations in PyrF or PyrG, enzymes which convert 5-FOA into the active toxic form. Using sRNA hybridization as well as sRNA library analysis, we demonstrate that these epimutants harbor sRNA against either pyrF or pyrG, and further show that this sRNA is lost after reversion to drug sensitivity. We conclude that epimutation is a mechanism capable of targeting multiple genes, enabling Mucor to develop resistance to a variety of antifungal agents. Elucidation of the role of RNAi in epimutation affords a fuller understanding of mucormycosis. Furthermore, it improves our understanding of fungal pathogenesis and adaptation to stresses, including the evolution of drug resistance.

Download Full-text

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

10.1101/636050 ◽

2019 ◽

Author(s):

Zanetta Chang ◽

Joseph Heitman

Keyword(s):

Drug Resistance ◽

Murine Model ◽

Fungal Pathogen ◽

Fungal Pathogens ◽

Antifungal Agents ◽

Mucor Circinelloides ◽

Antifungal Resistance ◽

Morbidity And Mortality ◽

The Brain

ABSTRACTThe 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.

Download Full-text

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

mBio ◽

10.1128/mbio.02579-19 ◽

2019 ◽

Vol 10 (6) ◽

Cited By ~ 51

Author(s):

Zanetta Chang ◽

Joseph Heitman

Keyword(s):

Drug Resistance ◽

Murine Model ◽

Fungal Pathogen ◽

Fungal Pathogens ◽

Antifungal Agents ◽

Mucor Circinelloides ◽

Antifungal Resistance ◽

Content Type ◽

The Brain

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.

Download Full-text

Mutations in efflux pump Rv1258c (Tap) cause resistance to pyrazinamide and other drugs inM. tuberculosis

10.1101/249102 ◽

2018 ◽

Author(s):

Jiayun Liu ◽

Wanliang Shi ◽

Shuo Zhang ◽

Gail Cassell ◽

Dmitry A. Maslov ◽

...

Keyword(s):

Drug Resistance ◽

Drug Targets ◽

Drug Exposure ◽

Efflux Pump ◽

Active Form ◽

Point Mutations ◽

Drug Susceptibility ◽

Clinical Isolates

AbstractAlthough drug resistance inM. tuberculosisis mainly caused by mutations in drug activating enzymes or drug targets, there is increasing interest in possible role of efflux in causing drug resistance. Previously, efflux genes are shown upregulated upon drug exposure or implicated in drug resistance in overexpression studies, but the role of mutations in efflux pumps identified in clinical isolates in causing drug resistance is unknown. Here we investigated the role of mutations in efflux pump Rv1258c (Tap) from clinical isolates in causing drug resistance inM. tuberculosisby constructing point mutations V219A, S292L in Rv1258c in the chromosome ofM. tuberculosisand assessed drug susceptibility of the constructed mutants. Interestingly, V219A, S292L point mutations caused clinically relevant drug resistance to pyrazinamide (PZA), isoniazid (INH), and streptomycin (SM), but not to other drugs inM. tuberculosis. While V219A point mutation conferred a low level resistance, the S292L mutation caused a higher level of resistance. Efflux inhibitor piperine inhibited INH and PZA resistance in the S292L mutant but not in the V219A mutant. S292L mutant had higher efflux activity for pyrazinoic acid (the active form of PZA) than the parent strain. We conclude that point mutations in the efflux pump Rv1258c in clinical isolates can confer clinically relevant drug resistance including PZA and could explain some previously unaccounted drug resistance in clinical strains. Future studies need to take efflux mutations into consideration for improved detection of drug resistance inM. tuberculosisand address their role in affecting treatment outcome in vivo.

Download Full-text

A non-canonical RNAi pathway controls virulence and genome stability in Mucorales

10.1101/2020.01.14.906289 ◽

2020 ◽

Cited By ~ 2

Author(s):

C. Pérez-Arques ◽

M.I. Navarro-Mendoza ◽

L. Murcia ◽

E. Navarro ◽

V. Garre ◽

...

Keyword(s):

Genome Stability ◽

Mucor Circinelloides ◽

Antifungal Drugs ◽

Regular Growth ◽

Rnai Pathway ◽

Ribonuclease Iii ◽

Transcriptomic Response ◽

Stressful Environment ◽

Macrophage Phagocytosis

AbstractEpimutations in fungal pathogens are emerging as novel phenomena that could explain the fast-developing resistance to antifungal drugs and other stresses. These epimutations are generated by RNA interference (RNAi) mechanisms that transiently silence specific genes to overcome stressful stimuli. The early-diverging fungus Mucor circinelloides exercises a fine control over two interacting RNAi pathways to produce epimutants: the canonical RNAi pathway and a new RNAi degradative pathway. The latter is considered a non-canonical RNAi pathway (NCRIP) because it relies on RNA-dependent RNA polymerases (RdRPs) and a novel ribonuclease III-like named R3B2 to degrade target transcripts. Here in this work, we uncovered the role of NCRIP in regulating virulence processes and transposon movements through key components of the pathway, RdRP1, and R3B2. Mutants in these genes are unable to launch a proper virulence response to macrophage phagocytosis, resulting in a decreased virulence potential. The transcriptomic profile of rdrp1Δ and r3b2Δ mutants revealed a pre-exposure adaptation to the stressful phagosomal environment even when the strains are not confronted by macrophages. These results suggest that NCRIP represses key targets during regular growth and release its control when the fungus is challenged by a stressful environment. NCRIP interacts with the RNAi canonical core to protect genome stability by controlling the expression of centromeric retrotransposable elements. In the absence of NCRIP, these retrotransposons are robustly repressed by the canonical RNAi machinery; thus, supporting the antagonistic role of NCRIP in containing the epimutational pathway. Both interacting RNAi pathways might be essential to govern host-pathogen interactions through transient adaptations, contributing to the unique traits of the emerging infection mucormycosis.Author summaryMucormycosis is an emergent and lethal infectious disease caused by Mucorales, a fungal group resistant to most antifungal drugs. Mucor circinelloides, a genetic model to characterize this infection, can develop drug resistance via RNAi epimutations. This epimutational RNAi mechanism interacts with a novel non-canonical RNAi pathway (NCRIP), where the ribonuclease III-like R3B2 and the RNA-dependent RNA polymerase RdRP1 are essential. The analysis of the transcriptomic response to phagocytosis by macrophage in rdrp1Δ and r3b2Δ mutants revealed that NCRIP might control virulence in M. circinelloides. These mutants showed constitutive activation of the response to phagocytosis and a reduction in virulence in a mouse model, probably caused by a disorganized execution of the genetic program to overcome host defense mechanisms. The antagonistic role of the NCRIP and the RNAi canonical core is evident during post-transcriptional regulation of centromeric retrotransposons. These retrotransposons are silenced by the canonical RNAi pathway, but this regulation is restrained by NCRIP, proven by an overproduction of small RNAs targeting these loci in NCRIP mutants. These new insights into the initial phase of mucormycosis and transposable element regulation point to NCRIP as a crucial genetic regulator of pathogenesis-related molecular processes that could serve as pharmacological targets.

Download Full-text

Transposon mobilization in the human fungal pathogen Cryptococcus is mutagenic during infection and promotes drug resistance in vitro

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.2001451117 ◽

2020 ◽

Vol 117 (18) ◽

pp. 9973-9980 ◽

Cited By ~ 6

Author(s):

Asiya Gusa ◽

Jonathan D. Williams ◽

Jang-Eun Cho ◽

Anna Floyd Averette ◽

Sheng Sun ◽

...

Keyword(s):

Drug Resistance ◽

Target Genes ◽

Opportunistic Infections ◽

Temperature Shift ◽

Antifungal Drugs ◽

Rnai Pathway ◽

Human Pathogens ◽

Human Fungal Pathogen

When transitioning from the environment, pathogenic microorganisms must adapt rapidly to survive in hostile host conditions. This is especially true for environmental fungi that cause opportunistic infections in immunocompromised patients since these microbes are not well adapted human pathogens. Cryptococcus species are yeastlike fungi that cause lethal infections, especially in HIV-infected patients. Using Cryptococcus deneoformans in a murine model of infection, we examined contributors to drug resistance and demonstrated that transposon mutagenesis drives the development of 5-fluoroorotic acid (5FOA) resistance. Inactivation of target genes URA3 or URA5 primarily reflected the insertion of two transposable elements (TEs): the T1 DNA transposon and the TCN12 retrotransposon. Consistent with in vivo results, increased rates of mutagenesis and resistance to 5FOA and the antifungal drugs rapamycin/FK506 (rap/FK506) and 5-fluorocytosine (5FC) were found when Cryptococcus was incubated at 37° compared to 30° in vitro, a condition that mimics the temperature shift that occurs during the environment-to-host transition. Inactivation of the RNA interference (RNAi) pathway, which suppresses TE movement in many organisms, was not sufficient to elevate TE movement at 30° to the level observed at 37°. We propose that temperature-dependent TE mobilization in Cryptococcus is an important mechanism that enhances microbial adaptation and promotes pathogenesis and drug resistance in the human host.

Download Full-text

Molecular Mechanisms of Resistance to Antifungals in Candida albicans

10.5772/intechopen.96346 ◽

2021 ◽

Author(s):

Estela Ruiz-Baca ◽

Rosa Isela Arredondo-Sánchez ◽

Karina Corral-Pérez ◽

Angélica López-Rodríguez ◽

Iván Meneses-Morales ◽

...

Keyword(s):

Drug Resistance ◽

Candida Albicans ◽

Molecular Mechanisms ◽

Antifungal Agents ◽

Point Mutations ◽

Antifungal Resistance ◽

Antifungal Drug ◽

Antifungal Drug Resistance ◽

Etiological Agents ◽

Nosocomial Disease

Invasive Candidiasis (IC) presents a global mortality rate greater than 40%, occupying the fourth place worldwide as the most frequent opportunistic nosocomial disease. Although the genus Candida consists of around 200 species, only 20 are reported as etiological agents of IC, being Candida albicans the most frequent causal agent. Even when there is a broad range of antifungals drugs for Candida infections, azoles, polyenes, and echinocandins are considered among the most effective treatment. However, there is some incidence for antifungal resistance among some Candida strains, limiting treatment options. Several molecular mechanisms with antifungal agents have been reported for C. albicans where insertions, deletions, and point mutations in genes codifying target proteins are frequently related to the antifungal drug resistance. Furthermore, gene overexpression is also frequently associated to antifungal resistance as well as an increase in the activity of proteins that reduce oxidative damage. This chapter summarizes the main molecular mechanisms to C. albicans antifungal drug resistance, besides offering an overview of new antifungal agents and new antifungal targets to combat fungal infections.

Download Full-text