Post-transcriptional Control of Antifungal Resistance in Human Fungal Pathogens

Abstract

Global estimates suggest that over 300 million individuals of all ages are affected by serious fungal infections every year, culminating in about 1.7 million deaths. The societal and economic burden on the public health sector due to opportunistic fungal pathogens is quite significant, especially among immunocompromised patients. Despite the high clinical significance of these infectious agents, treatment options are limited with only three major classes of antifungal drugs approved for use. Clinical management of fungal diseases is further compromised by the emergence of antifungal resistant strains. Transcriptional and genetic mechanisms that control drug resistance in human fungal pathogens are well-studied and include drug target alteration, upregulation of drug efflux pumps as well as changes in drug affinity and abundance of target proteins. In this review we highlight several recently discovered novel post-transcriptional mechanisms that control antifungal resistance, which involve regulation at the translational, post-translational, epigenetic and mRNA stability levels. The discovery of many of these novel mechanisms has opened new avenues for the development of more effective antifungal treatment strategies and new insights, perspectives and future directions that will facilitate this process are discussed.

Introduction

Fungal pathogens affect more than 300 million individuals worldwide, resulting in approximately 1.7 million deaths annually (Global Action Fund for Fungal Infections, https://www.gaffi.org/why/). The most common opportunistic human fungal pathogens include Candida, Aspergillus, Cryptococcus, Pneumocystis and Mucorales species. Candida spp. are the most common cause of healthcare-associated bloodstream infections (BSIs) and the fourth leading cause of hospital-acquired BSIs in the United States with about a 50% attributable mortality rate (Wisplinghoff et al. 2004; Edmond et al. 1999; Denning and Bromley 2015; Toda et al. 2019).

Fungal infections caused by Aspergillus species manifest as varied clinical entities ranging from allergic and chronic to acute invasive diseases (Denning et al. 2011; 2013). Among Aspergillus infections, the global incidence of invasive aspergillosis (IA) ranges from 200,000–400,000 cases annually and specifically includes more than 10% of patients with acute leukemia as well as patients with bone marrow and other transplants (Kosmidis and Denning 2015). Overall, the mortality rate associated with invasive aspergillosis remains high, ranging between 30% and 85% in immunocompromised patient populations, depending upon host immune status, the causative species and drug resistance (Global Action Fund for Fungal Infections, https://www.gaffi.org/why/) (Denning and Bromley 2015).

Cryptococcus neoformans is the principal pathogen of the genus Cryptococcus with worldwide distribution. The most common clinical presentation is cryptococcal meningitis with about 220,000 new cases reported annually worldwide, resulting in 181,000 deaths, mainly in sub-Saharan Africa (Rajasingham et al. 2017).

Mucormycosis is an angio-invasive fungal infection caused by fungi of the order Mucorales (Prakash and Chakrabarti 2021). The highest mortality rates have been observed in patients with disseminated mucormycosis (68%) and the lowest rates have been found in those with cutaneous disease (31%) (Jeong et al. 2019). The emergence of COVID-19–associated mucormycosis has been infrequently reported in the United States but has been described in other parts of the world, particularly in India (Dulski et al. 2021; Pal et al. 2021; Patel et al. 2021; Mejía-Santos et al. 2021). COVID-19 might increase the risk of acquiring mucormycosis due to induced immune dysregulation or associated treatments such as corticosteroids and immunomodulatory drugs (e.g., tocilizumab or baricitinib) that impair host defenses against molds (Narayanan et al. 2021).

The severity of these fungal diseases depends upon the immune status of the patient, antifungal drug efficacy, as well as the virulence and ability of the specific fungal pathogen to evade the host immune system. Early diagnosis, as well as effective antifungal therapy, are necessary for successful clinical outcomes of fungal infections. Currently, there are only three major classes of drugs available to treat these infections: azoles, polyenes, and echinocandins (Perfect 2017). Azole antifungals function by inhibiting the cytochrome P450 lanosterol demethylase Erg11 (Cyp51), which plays an essential role in the ergosterol biosynthesis pathway, leading to either fungistatic growth inhibition or fungicidal killing of the pathogen, dependent on the species (Geißel et al. 2018). Inhibition of lanosterol demethylase results in the replacement of ergosterol by methylated sterols, which exerts severe stress on the plasma membrane (Sanglard 2002). In addition, azole fungicidal effects can occur as a result of the synthesis of cell wall carbohydrate patches that penetrate the cell membrane (Geißel et al. 2018). Polyenes exert a fungicidal effect simply by binding to ergosterol (Gray et al. 2012). The echinocandins are large lipopeptide molecules that act as noncompetitive inhibitors of (1,3)-β-D-glucan synthase, an enzyme involved in fungal cell wall synthesis. The disruption of (1,3)-β-D-glucan synthesis results in the loss of cell wall integrity and severe cell wall stress (Denning 2003). Interestingly, echinocandins are fungicidal against certain pathogenic yeasts, such as Candida spp., but fungistatic against Aspergillus spp. and several other pathogenic filamentous fungi. In the laboratory, treatment with echinocandins leads to hyphal growth arrest with turgescence and blunting of hyperbranched hyphae (Aruanno et al. 2019). Thus, echinocandins are prescribed as an alternative treatment for invasive aspergillosis (Aruanno et al. 2019) and are usually prescribed along with other antifungal compounds, as they are not alone effective in the treatment of molds, including Mucorales, Fusarium, Scedosporium and Trichosporon species. These molds have a low amount of β-(1,3)-D-glucan, as they mainly possess β-(1,6)-D-glucan in their cell walls (Cappelletty and Eiselstein-McKitrick 2007; Aguilar-Zapata et al. 2015).

Microbial drug resistance includes both primary resistance, which is associated with inherited reduced susceptibility to a drug class, as well as secondary resistance, which is acquired in an otherwise susceptible strain following the drug exposure (Cowen et al. 2015). In the clinic antifungal resistant strains are defined as those that display a minimum inhibitory concentration (MIC) greater than or equal to the clinical breakpoint (CBP) when tested according to the Clinical and Laboratory Standards Institute (CLSI) or European Committee on Antimicrobial Susceptibility testing (EUCAST) methods (CLSI 2020; Arendrup et al. 2020). Clinical breakpoints take into account the in vivo aspects of drug efficacy and help in providing guidance regarding the likelihood of whether a strain will respond to the therapy (CLSI 2020; Wiederhold, 2021). In situations where the clinical breakpoints have not yet been established, epidemiological cut-off values help in the identification of isolates with acquired resistance by using MICs (Pfaller et al. 2012; Lockhart et al. 2017). In research laboratory settings, strains with in vitro antifungal resistance are defined as those showing reduced susceptibility to a drug and high MICs as compared to the control or reference strain (Berman and Krysan 2020). Resistance generally occurs as a result of genetic changes that directly affect the drug target and/or intracellular drug concentration of the strain, resulting in a heritable effect on the entire cell population of a given isolate (Berman and Krysan 2020).

It is pertinent to consider here that in spite of low levels of antifungal resistance, for most human fungal pathogens, such as Candida albicans, clinical outcomes remain poor and mortality levels are high, as existing therapies often fail to treat infections caused by these species (Pfaller et al. 2019). Host factors, immune status of the patient and pharmacologic issues (drug-fungus interactions) can explain these outcomes, which directly or indirectly affect therapeutic responses (Lepak and Andes 2014). This discordant relationship between low levels of clinical resistance and overall clinical outcomes/therapeutic failures can be attributed to antifungal tolerance. Tolerance/trailing growth is usually characteristic of susceptible strains that tend to grow slowly at inhibitory drug concentrations and is less dependent on the drug concentration (Berman and Krysan 2020). Tolerant cell growth is slower, more time-dependent and sensitive to environmental stresses such as changes in pH, temperature, and nutrients (Rosenberg et al. 2018). The degree of tolerance varies depending on intrinsic allele diversity of each isolate, phenotypic heterogeneity, the size of the sub-population of cells that can grow above the MIC, and the rate at which colonies become visible at supra-MIC concentrations (Rosenberg et al. 2018). Importantly, the terms “antifungal drug resistance” and “tolerance” are frequently used interchangeably in the literature (Berman and Krysan 2020) and thus many studies investigating the responses of fungal pathogen strains to antifungals are difficult to interpret.

Increased prescription of antifungals for prophylaxis, empiric or pre-emptive therapies has led to the emergence of antifungal resistance worldwide, limiting the overall availability of treatment options (Perlin et al. 2017). Also, the widespread use of medically related antifungals in agriculture has resulted in environmental reservoirs for some drug-resistant pathogens, eventually leading to increased antifungal resistance in the clinic (Toda et al. 2021). A steady increase in worldwide reports of azole resistance in clinically relevant fungal pathogens, resulting in therapeutic failures, has been a matter of clinical concern (Beardsley et al. 2018; Arastehfar et al. 2020). Most previous studies on azole resistance in these species (e.g. Candida, Aspergillus) have focused on transcriptional mechanisms and genetic mutations that alter the expression or function of ERG11 (the azole target) or drug efflux pumps and components of sterol biosynthesis pathways (Nishimoto et al. 2020; Chowdhary et al. 2017; Arastehfar et al. 2020). Gain or loss of chromosomes has also been known to contribute to azole resistance and attenuated virulence in C. albicans (Perepnikhatka et al. 1999; Chen et al. 2004).

Echinocandin resistance is usually associated with mutations in hotspot (HS) regions of FKS genes, which encode the catalytic subunits of β−1,3-D-glucan synthase (Arendrup and Perlin 2014; Jiménez-Ortigosa et al. 2017). Echinocandin resistance in clinical strains of C. albicans is relatively rare (<1–2%) (Grossman et al. 2014). However, significant echinocandin resistance (2–10%) has been reported for Candida glabrata, Candida tropicalis and Candida auris (Arastehfar et al. 2020; Pfaller et al. 2019; Chow et al. 2020; Nishimoto et al. 2020). In certain cases, mutations outside of the HS regions can cause echinocandin resistance and occasionally echinocandin-susceptible strains also harbor weak mutations in the HS region resulting in therapeutic failure (Arastehfar et al. 2020; Hou et al. 2019). In a recent study by Satish, et al., a novel, fks1-independent mechanism of echinocandin resistance in A. fumigatus was identified (Satish et al. 2019). This mechanism involves caspofungin-induced reactive oxygen species (ROS)-mediated changes in the lipid composition of the microenvironment of β−1,3-D-glucan synthase to alter the drug-target interaction. The prominent lipid changes, mainly in dihydrosphingosine and phytosphingosine, have been shown to reduce sensitivity of A. fumigatus to echinocandins (Satish et al. 2019).

Polyene resistance is rarely reported even though these drugs have been used in clinics for decades. This could be due to poor solubility of the drug and severe host renal toxicity, which prohibit long-term use of polyenes. However, certain Candida species, such as Candida lusitaniae, Candida guillermondii, Candida haemulonii, as well as Aspergillus terreus, display intrinsic resistance to amphotericin B (AMB) (Ramos et al. 2015; Kathuria et al. 2015). Missense or nonsense mutations in ERG6, encoding C-24 methyltransferase, which converts zymosterol to fecosterol in the ergosterol biosynthesis pathway, was found to be responsible for AMB resistance in a clinical strain of C. glabrata (Vandeputte et al. 2007; 2008). ERG11 deletion or mutations in several other components of the ergosterol biosynthesis pathway were also found to be associated with reduced AMB susceptibility in C. glabrata strains (Geber et al. 1995; Hull et al. 2012a; b). Loss of ERG6- and ERG2 (C-8 sterol isomerase)-encoded enzyme activities specifically leads to the accumulation of zymosterol and fecosterol, respectively, resulting in the absence of ergosterol in Candida cell membranes, ultimately reducing AMB susceptibility (Vandeputte et al. 2007; 2008; Geber et al. 1995; Hull et al. 2012a; 2012b).

While the roles of transcriptional mechanisms, genetic mutations and chromosomal alterations in antifungal resistance have been well-characterized, many recent studies in the field are beginning to highlight novel gene expression mechanisms associated with this process. More specifically, a number of significant advances have been made to improve our understanding of how antifungal resistance mechanisms are mediated at the post-transcriptional level, particularly through translational, post-translational, epigenetic and mRNA stability mechanisms. This review article will serve to highlight many of these recently discovered novel mechanisms. New insights and perspectives, as well as possible future directions, are discussed, which may assist in the identification of novel therapeutic drug targets and, eventually, the development of more effective antifungal treatment strategies.

Regulation of antifungal resistance at the level of mRNA stability

Considerably little is known about how mRNA stability mechanisms control antifungal resistance in human fungal pathogens. Azole resistance in C. albicans is often associated with increased expression of genes encoding multidrug efflux pumps, such as CDR1 and CDR2, the ATP-binding cassette transporter genes, and/or MDR1, encoding a major facilitator superfamily transporter (Prasad et al. 1995; Riggle and Kumamoto, 2006; Sanglard et al. 1997; White, 1997; Wirsching et al. 2000). However, Manoharlal, et al., demonstrated that in addition to increased transcription, enhanced mRNA stability results in elevated CDR1 transcript levels in azole-resistant C. albicans strains (Manoharlal et al. 2008). Interestingly, a subsequent study demonstrated that the CDR1 3’ untranslated region (UTR) was hyperadenylated in these strains (Manoharlal et al. 2010). The nuclear poly(A) polymerase (PAP1), responsible for mRNA adenylation and poly(A) tail synthesis in C. albicans, is located on chromosome 5 at the mating type-like (MTL) locus. The MTL loci of C. albicans contain unique genes, including OBP (an oxysterol-binding protein), PAP1 (a poly(A) polymerase 1) and PIK1 (a phosphoinositol kinase) at both MTL-a and the MTL-α alleles (Hull & Johnson, 1999). In most cases wild-type C. albicans strains are diploid and have both the MTL-a and MTL-α loci (Magee & Magee 2000). However, on analyzing PAP1 allelic status, Manoharlal, et al., found that PAP1 heterozygosity (PAP1-a/PAP1-α) and homozygosity (PAP1-α/PAP1-α) were associated with azole susceptible and resistant strains, respectively (Manoharlal et al. 2010). Also, a heterozygous deletion of PAP1-a (Δpap1-a/PAP1-α) led to enhanced resistance to fluconazole, terbinafine and cycloheximide, polyadenylation and increased transcript stability of CDR1 in an azole-susceptible isolate (Manoharlal et al. 2010).

The Ccr4-NOT-Pop2 mRNA deadenylase complex possesses exonuclease activity that functions to shorten mRNA poly(A) tails and regulate mRNA decay and translation. In Saccharomyces cerevisiae, ccr4/pop2 mutants exhibit sensitivity to cell wall perturbing agents (Hata et al. 1998; Markovich et al. 2004). While the molecular mechanism(s) responsible for these phenotypes are unknown, it was subsequently demonstrated that ccr4/pop2 mutants displayed reduced cell wall glucan levels and sensitivity to the echinocandin drug caspofungin (Dagley et al. 2011). Furthermore, cell wall defects in these mutants were linked to dysfunctional mitochondria and defective phospholipid homeostasis (Figure 1). Screening of a collection of mitochondrial mutants in S. cerevisiae identified several mitochondrial proteins necessary for caspofungin tolerance. Interestingly, these proteins assist in the maintenance of phospholipid homeostasis, thereby contributing to cell wall integrity and echinocandin resistance (Figure 1). Caspofungin susceptible mitochondrial protein mutants had impaired mitochondrial biosynthesis of phospholipids, including phosphatidyl glycerol and cardiolipin (Dagley et al. 2011).

Figure 1.

Diagrammatic representation of the role of the Pop2-Ccr4-NOT deadenylase complex in controlling antifungal resistance via a mRNA stability mechanism in S. cerevisiae. Under normal cellular conditions this complex controls the stability of mRNAs via poly(A) tail shortening, or deadenylation, thereby maintaining the integrity of the cell wall. Cell wall biogenesis occurs by the coordinated role of the Pop2-Ccr4-NOT complex, endoplasmic reticulum and mitochondria which results in activation of phospholipid biosynthesis and incorporation of phospholipids in the plasma membrane (left panel). Absence of the Pop2-Ccr4-NOT deadenylase complex affects stability of target mRNAs, leading to mitochondrial dysfunction which results in a defect in phospholipid homeostasis. This defect leads to a decrease in cell wall glucans, thereby rendering the cells more sensitive to echinocandins (right panel). PE, phosphatidylethanolamine; PG, phosphatidylglycerol; CL, cardiolipin; PS, phosphatidylserine; PI, phosphatidylinositol; PC, phosphatidylcholine. Orange oval, cell wall. Blue oval, cell membrane.

A previous study provided evidence that a pumilio-domain and fem-3 binding factor (PUF) RNA-binding protein, Puf3, regulates a post-transcriptional mRNA network important for C. albicans biofilm formation (Verma-Gaur et al. 2015), which is known to contribute to antifungal resistance. Puf3 recruits Ccr4 to mRNAs to control transcript stability. The deletion of CCR4 resulted in differential expression of mitochondria-related genes in C. albicans biofilms. Also, biofilm structure was altered in the ccr4Δ/Δ mutant, as observed by scanning electron microscopy, with an increase in the ratio of yeast to filamentous cells as well as increased production of extracellular matrix material. The authors proposed that changes to mitochondrial activity and biogenesis in the biofilm environment, possibly due to hypoxia, establish a signal that leads to activation of protective mechanisms. These events ultimately result in extracellular matrix accumulation, as a consequence of crosstalk between mitochondrial proteins and pathways involved in cell wall biogenesis as well as overall cell stability. Ccr4 thus senses the hypoxic environment and orchestrates biofilm maturation by regulating the expression of genes associated with the cell wall as well as mitochondrial biogenesis and activity. Although a direct role for the C. albicans Puf3-Ccr4 mechanism of biofilm maturation in promoting antifungal resistance has not yet been demonstrated, this remains a fruitful line of research.

mRNA stability mechanisms are also likely to play a role in intrinsic echinocandin resistance of the major human fungal pathogen Cryptococcus neoformans. More specifically, because C. neoformans caspofungin resistance is calcineurin-dependent, post-transcriptional regulation of RNA processing targets of calcineurin could play a role in this process (Kalem et al. 2021). The PUF domain-containing RNA-binding protein Puf4 is one such target and binds to mRNAs encoding nucleolar ribosomal RNA-processing factors (Kalem et al. 2021). C. neoformans puf4 deletion mutants showed increased caspofungin resistance, whereas PUF4 overexpression led to caspofungin sensitivity. The 5’ UTR of the transcript encoding the FKS1 caspofungin target contains three Puf4 binding elements (PBEs) and Puf4 has been shown to bind specifically to this region (Kalem et al. 2021). puf4Δ mutants demonstrated reduced levels of FKS1 mRNA, suggesting that Puf4 functions as a positive regulator of FKS1 mRNA stability. In addition, the puf4Δ mutant had increased chitin content, suggesting that its cell wall composition is less reliant on β−1,3-glucan than that of a wild-type strain. Thus, Puf4 controls cell wall biogenesis in C. neoformans by a mRNA stability mechanism, thereby regulating caspofungin resistance (Kalem et al. 2021).

The studies discussed above (Manoharlal et al. 2008; Manoharlal et al. 2010; Dagley et al. 2011; Verma-Gaur et al. 2015; Kalem et al. 2021) are important as they suggest that novel mechanisms of post-transcriptional regulation involving control of mRNA stability result in antifungal resistance. Future studies on antifungal resistant fungal species, which examine linkages among mitochondrial biogenesis, phospholipid homeostasis and cell wall biogenesis, might help in designing new antifungal strategies to combat drug resistance. Further elucidation of mRNA stability regulatory mechanisms may also open new avenues of research to enhance antifungal susceptibility in a variety of pathogenic fungal species through adjunctive therapy.

Translational control of antifungal resistance

While translational regulation of antifungal resistance in human fungal pathogens has not been well-studied, several reports have begun to shed light on this area. Adaptive mistranslation (regulated erroneous protein translation) results in proteome plasticity and phenotypic diversification in C. albicans (Weil et al. 2017). C. albicans has a CUG codon usage system in which environmental cues modulate translation fidelity. Recognition and aminoacylation of a mutant serine tRNA by both leucyl- and seryl-tRNA synthetases (Santos et al. 1997), results in a charged tRNA that can incorporate either leucine (Leu) or serine (Ser), respectively, at CUG codons. This codon ambiguity, combined with variation in the basal levels of Ser and Leu in different ecological niches and environmental conditions, results in increased proteome diversity and generates advantageous phenotypic variations. Bezerra, et al., demonstrated that an increase in Leu misincorporation at CUG sites to 22% resulted in a strain that is more tolerant to fluconazole, although the mechanism was not well-understood (Bezerra et al. 2013). A subsequent study by Weil, et al., investigated the contribution of CUG mistranslation towards acquisition of fluconazole resistance by evolving both wild-type and hypermistranslating (HM) strains in the presence and absence of fluconazole (Weil et al. 2017). Acquisition of fluconazole resistance was accelerated in HM versus wild-type strains. In addition, the wild-type strain exhibited increased drug efflux via the Mdr1 multidrug efflux pump, whereas the HM strain overexpressed both CDR1 and CDR2, which encode ABC superfamily transporters (Weil et al. 2017). It is pertinent to mention that the increased accumulation of loss-of-heterozygosity events at ChrR and Chr5, aneuploidy, translational and cell surface modifications in HM strains, appear to mediate more rapid acquisition of drug resistance. Mistranslation in HM strains also led to upregulation of amino acid metabolism and mutations in tRNA synthetases. In addition, a large number of single nucleotide polymorphisms (SNPs) and codon changes in cell wall proteins were observed in HM strains, suggesting a mechanism by which CUG codon ambiguity could lead to antifungal resistance. Future studies may help to identify and characterize genes necessary for evolving antifungal resistance in HM strains and determine whether mistranslation plays a major role in this process.

Although the translational machinery in yeast and mammalian systems is quite similar, the above studies suggest that several components of this machinery may still hold potential for exploitation as antifungal drug targets. In support of this notion, Iyer, et al., have shown that rocaglate compounds, which specifically inhibit translation initiation in mammalian cells and in S. cerevisiae through interaction with eIF4A (Sadlish et al. 2013; Iwasaki et al. 2016; 2019; Shen et al. 2020), cause hyperacidification and fragmentation of the vacuolar compartment, mitochondrial depolarization, and increased caspase-like activity in C. auris (Iyer et al. 2020). Translational inhibition mediated by rocaglates induces a noncanonical form of programmed cell death in C. auris, which has autophagy- and apoptosis-related attributes (Iyer et al. 2020). Sordarin is another such natural compound that specifically binds to the elongation factor-2 (EF-2)-ribosome complex and inhibits the release of EF-2 from post-translocational ribosomes in S. cerevisiae; EF-2 encodes a protein essential for catalyzing ribosomal translocation during protein synthesis (Justice et al. 1998). Sordarin and its analog were shown to direct antifungal effects by specifically inhibiting the protein synthesis elongation cycle in C. albicans, C. glabrata, and C. neoformans and had no effect in mammalian systems (Domínguez et al., 1998). Another novel drug that inhibits fungal translation, icofungipen, is a beta amino acid and has a novel mechanism-of-action compared to drugs in other antifungal classes (Hasenoehrl et al. 2006). The active accumulation of icofungipen in yeast cells leads to competitive inhibition of isoleucyl-tRNA synthetase, consequently disrupting protein synthesis (Ziegelbauer 1998a; Ziegelbauer et al. 1998b). In the future, high-throughput compound library screening, focusing on compounds that target protein synthesis specifically in fungi, could lead to the discovery of potential new drug targets. Studies that focus on determining the in vivo efficacy of these compounds for potential treatment of fungal infections should be prioritized.

Post-translational modifications and epigenetic mechanisms that regulate antifungal resistance

Through biochemical reactions, post-translational modifications (PTMs) reversibly or irreversibly alter the structure and properties of proteins which, in turn, allows cells to dynamically regulate their signal integration and physiological states. Li, et al., recently demonstrated that mitochondrial dysfunction in A. fumigatus induces calcium signalling, which further leads to overexpression of multidrug transporters, thereby promoting drug resistance (Li et al. 2020). In their study, multidrug-resistant transporters, chitin synthases, and calcium signalling-related genes were significantly up-regulated, while mitochondrial genes responsible for scavenging reactive oxygen species (ROS) such as cytochrome c oxidase, the terminal enzyme of the electron transport chain, were significantly down-regulated in drug-resistant cox10 mutants (Figure 2). Promoters of the genes with increased expression share a consensus calcium-dependent serine threonine phosphatase-dependent response element (CDRE) motif which is the binding site of the calcium-signaling transcription factor CrzA. It has been previously reported that upon perceiving external stress stimuli, the Ca²⁺ sensor protein calmodulin normally binds cytosolic Ca²⁺, which is increased (Juvvadi et al. 2014; 2017). These events lead to activation of the phosphatase calcineurin, which dephosphorylates CrzA. In the dephosphorylated form, CrzA is translocated to the nucleus and promotes the expression of downstream target genes important for drug resistance (Figure 2). Interestingly, cox10 drug-resistant mutants demonstrated increased cytosolic Ca²⁺ transients and persistent CrzA nuclear localization in both the presence and absence of calcium stimuli, leading to overexpression of multidrug transporters and chitin synthases (Figure 2). Calcium chelators were able to restore drug susceptibility and increase the efficacy of itraconazole treatment in both laboratory-generated and clinical A. fumigatus cox10 mutant strains (Li et al. 2020).

Figure 2.

A schematic representation of calcium-signaling activation induced by mitochondrial dysfunction in A. fumigatus cox10 mutants contributing to antifungal resistance. Upon stimulation by various external stresses, including drugs, the Ca²⁺ sensor protein calmodulin (CaM) binds the increased cytosolic Ca²⁺, subsequently activating the phosphatase calcineurin. Activated calcineurin dephosphorylates the transcription factor CrzA, leading to its nuclear translocation, which causes induction of downstream genes important for drug resistance. In the cox10 mutant when calcium signaling is increased, the CrzA transcription factor is persistently localized to the nucleus by an unknown mechanism and further activates genes encoding multidrug transporters, therefore leading to enhanced drug resistance (note that in this case it is uncertain whether CrzA is dephosphorylated in the nucleus). Red diamonds, calcium, star denotes cox10 defective mutant. Orange oval, cell wall. Blue oval, cell membrane (adapted from Li et al. 2020).

In addition to the calcineirin-calmodulin pathway, phosphorelay pathways involving PKC and MAPK signaling have been found to be important for regulation of antifungal resistance. Pkc1 partially regulates the response to ergosterol biosynthesis inhibitors via a MAPK cascade in C. albicans and S. cerevisiae (LaFayette et al. 2010). In C. albicans, Pkc1 and calcineurin independently regulate resistance via a common target. Pkc1 inhibition in C. albicans phenocopies inhibition of calcineurin or Hsp90, a molecular chaperone, thereby reducing drug resistance in a clinical isolate. Reduced levels of Hsp90 result in destabilization of the terminal MAPK pathway component Mkc1, thereby blocking PKC signaling. This finding suggests that Hsp90 regulates basal tolerance and resistance to ergosterol biosynthesis inhibitors through Mkc1 in addition to the established connection with calcineurin. Additionally, abrogation of C. albicans Pkc1 renders fungistatic drugs fungicidal and attenuates virulence of C. albicans, thus having significant therapeutic potential (LaFayette et al. 2010).

In C. auris, the genetic removal of SSK1, encoding a response regulator in the mitogen-associated protein kinase Hog1 pathway, results in restoration of amphotericin B and caspofungin susceptibility (Shivarathri et al. 2020). Loss of both SSK1 and HOG1 causes alterations in membrane lipid permeability, cell wall mannan content, and hyperresistance to cell wall-perturbing agents, suggesting a distinct change in plasticity that affects cell wall function, stress adaptation, and multidrug resistance. Therefore two-component signal transduction systems could prove to be suitable targets for restoring C. auris susceptibility to antifungals.

In addition to posttranslational mechanisms, epigenetic modifications have also recently been shown to play an important role in antifungal resistance. These modifications can be categorized as either RNA-based or chromatin-based. RNA-based epigenetic modifications include those mediated by RNA interference (RNAi) and non-coding RNAs. RNAi mechanisms involve repression of gene expression by small non-coding RNAs (sRNAs) produced by RNA-dependent RNA polymerases (mainly RNA polymerase III) and the endonuclease Dicer (Villalobos-Escobedo et al. 2016). These processed sRNAs are then captured by Argonaute proteins (components of the RNA-induced silencing complex (RISC)), which further induce degradation or inhibit translation of the target RNA. On the other hand, long non-coding RNAs (lncRNAs) are typically >200 nt RNA molecules that are transcribed by RNA polymerase II (Carninci et al. 2005). Most lncRNAs are capped, spliced and polyadenylated in a similar manner to that of mRNA. They are predominantly found in the nucleus, are rapidly degraded by exosomes and were thus originally regarded as transcriptional noise (Struhl 2007). However, recent studies suggest that lncRNAs play important roles in the epigenetic regulation of drug susceptibility in fungi, which will be discussed here (Dimond and Fraser 2013; Mikkelsen et al. 2007; Moran et al. 2012).

Epimutation is a “heritable change in gene activity that is not associated with a DNA mutation but rather with gain or loss of DNA methylation or other heritable modifications of chromatin” (Oey and Whitelaw 2014). Epimutation also involves the intrinsic RNAi silencing pathway, which transiently suppresses expression of fungal drug target genes. Epimutants generate antisense sRNAs specific to the target gene that trigger mRNA degradation and thereby transiently prevent production of the drug target. This mechanism was first discovered in Mucor circinelloides, a dimorphic fungal pathogen, and is associated with the ability to develop transient resistance to the antifungal agents FK506 and rapamycin (Calo et al. 2014). These drugs bind to the peptidylprolyl isomerase FKBP12, encoded by the fkbA gene, and inhibit calcineurin and TOR (target of rapamycin), respectively (Figure 3A). The endogenous expression of sRNAs against fkbA causes mRNA degradation, resulting in M. circinelloides strains becoming resistant to FK506. sRNA sequencing of the M. circinelloides epimutants showed the presence of sense and antisense sRNAs mapping to the fkbA locus and the epimutation in these strains was transient and reversible. A subsequent study demonstrated that epimutation in M. circinelloides can confer transient resistance to 5-fluoroorotic acid (5-FOA) by the accumulation of sense and antisense sRNAs against either pyrF or pyrG (Chang et al. 2019). Both pyrF and pyrG encode enzymes in the pyrimidine biosynthetic pathway important for conversion of 5-FOA from an inactive prodrug into a toxic nucleotide analog (Boeke et al. 1984). Recently, Torres-Garcia, et al., have also demonstrated that antifungal resistance in Schizosaccharomyces pombe can arise through heterochromatin-dependent epimutations (Torres-Garcia et al. 2020). Heterochromatin heritability may allow cells to grow under certain stress conditions by acquiring epimutations that could result in unstable gene silencing, rather than DNA modifications, thereby influencing phenotypes (Oey and Whitelaw 2014). Torres-Garcia, et al., identified caffeine resistant heterochromatin-dependent S. pombe epimutants (Torres-Garcia et al. 2020); caffeine is an analogue of purine bases that has been involved in a variety of cellular processes in eukaryotic cells, including mammals, plants and fungi (Calvo et al., 2009) and is known to demonstrate a wide array of pharmacological and biological effects that interfere with DNA repair and recombination pathways, delay cell cycle progression and modulate intracellular calcium homeostasis (Gentner and Werner, 1975; Osman and McCready, 1998; Loprieno et al., 1974). Some of these epimutants showed unstable caffeine resistance and demonstrated heterochromatin islands with reduced expression of genes including hba1, ncRNA.394, ppr4, grt1, fio1 and mbx2. The authors also demonstrated that resistance can also arise as a result of forced heterochromatin formation at defined loci via heterochromatin-mediated silencing. Notably, caffeine resistant strains with forced heterochromatin silencing at either hba1 or ncRNA.394 exhibited resistance to the widely-used antifungals fluconazole, clotrimazole and tebuconazole. The unstable caffeine resistant strains with heterochromatin islands over hba1 or ncRNA.394 also showed antifungal resistance and produced small interfering RNAs (siRNAs) homologous to surrounding genes. Deletion of the RNAi components dcr1 or ago1 from unstable caffeine resistant strains abolished caffeine resistance, suggesting a role for RNAi in this process. These results indicate that epimutations targeting specific loci not only drive resistance to antifungals but can also generate phenotypic plasticity, leading to rapid adaptation to environmental stresses (Torres-Garcia et al. 2020). Thus reengineering of existing drugs that target histone-modifying enzymes, as well as identification of novel agents that specifically inhibit fungal heterochromatin with no impact on the host, could serve as fruitful avenues for anifungal drug development. Future studies may provide new insights into the role of epimutations in controlling drug resistance and stress responses of a variety of human fungal pathogens, including C. neoformans, A. fumigatus and C. auris.

Figure 3.

Epigenetic-mediated mechanisms of antifungal drug resistance in human pathogenic fungi. A) FKBP12 is produced normally in a drug-sensitive strain of M. circinelloides and targeted by FK506 and rapamycin, which leads to reduced calcium and TOR signaling (left). However, in a drug-resistant strain, epimutations cause degradation of the FkbA mRNA, which encodes the FKBP12 drug target, via a RNA interference (RNAi)-mediated pathway. The degradation of mRNAs may involve anti-sense transcripts generated by RNA-dependent RNA polymerases (RdRP). B) Deacetylation of Hsp90 by lysine deacteylases (KDACs) allows interaction with the calcineurin client protein, which promotes antifungal resistance (left). The protein kinase CK2 controls Hsp90 function by phosphorylation (star), thereby stabilizing downstream interactors such as Hog1, which are important for promoting antifungal resistance (right). KATs, lysine acetyltransferases; KDACs, lysine deacetylases; Cn, calcineurin; Ac, acetyl group. Orange oval, cell wall. Blue oval, cell membrane.

Ard, et al., also recently showed that a lncRNA regulates drug tolerance in S. pombe (Ard et al. 2014). The lncRNA.1343 controls expression of an adjacent gene encoding the phosphate-responsive permease for glycerophosphodiester 1 (Tgp1) by increasing local nucleosome density. Interestingly, deletion of lncRNA.1343 results in hypersensitivity to a broad spectrum of drugs, including the microtubule-depolymerizing agent thiabendazole, the DNA synthesis inhibitor hydroxyurea, and the inhibitor of cyclic AMP phosphodiesterase caffeine (Ard et al. 2014). Thus, S. pombe lncRNA.1343 controls drug tolerance by transcriptional interference. While lncRNA-mediated RNA interference appears to be generally conserved over evolution, at this point it is unclear whether this mechanism is specifically conserved in other fungi and, if so, whether it can affect the acquisition of antifungal drug resistance in multi-drug-resistant human fungal pathogens such as C. auris.

Epigenetic mechanisms can also alter chromatin either chemically, by histone post-translational modifications, DNA alkylation, phosphorylation, ubiquitination, and sumoylation, or structurally, by chromatin remodeling and DNA-DNA interactions (Allis and Jenuwein 2016; Chen et al. 2017). These modifications alter chromatin organization, allowing transcription factors to selectively access specific genomic regions, thereby controlling gene expression. Histone acetylation has been specifically shown to regulate antifungal resistance in C. albicans (Garnaud et al., 2016; Li et al. 2015; Robbins et al. 2012). The Hsp90 chaperone is known to be regulated by PTMs, including acetylation, in mammals. However, very little is known about conserved regulation of Hsp90 functions by PTMs in fungi. Hsp90 has been shown to govern antifungal resistance by stabilizing signal transducers. In C. albicans, the acetylation of Hsp90 is important for function, suggesting a role for lysine deacetylases (KDACs) in antifungal resistance (Singh et al. 2009). Interestingly, there is a high level of functional redundancy among KDACs, with Hos2, Hda1, Rpd3, and Rpd31 mediating azole resistance and morphogenesis in C. albicans (Li et al. 2017). Hsp90 acetylation at the K27 residue is also required for azole and echinocandin resistance in A. fumigatus (Lamoth et al. 2014; 2015). It is important to note here that the deacetylation of numerous lysine residues by Hda1 and Rpd3 allows Hsp90 to physically interact with calcineurin, leading to the activation of calcineurin-mediated stress responses upon azole-induced stress (Figure 3B). Another mechanism of regulation of Hsp90 includes phosphorylation by regulatory subunits of the CK2 kinase. Hsp90 phosphorylation impacts the stability and function of target proteins such as Hog1, which is necessary for stress responses, including antifungal resistance (Diezmann et al. 2012) (Figure 3B). PkcA is an important kinase in the cell wall integrity (CWI) pathway that is necessary for survival during antifungal exposure. In A. fumigatus, mutations in the C1B domain of PkcA prevented its ability to interact with Hsp90, resulting in a shutdown of the CWI pathway response. These findings suggest that CWI pathway components are substrates for Hsp90, which in turn stabilizes these proteins, regulating the response to temperature and drug stress (Rocha et al. 2021).

HDA1 and RPD3 histone deacetylases (HDACs) are highly expressed in azole-resistant C. albicans strains (Li et al. 2015). However, their level of expression is reduced after resistance is established, suggesting that these genes play a transient, but crucial, role in drug resistance. Interestingly, this effect was attributed to the stability and function of Hsp90 in the absence of deacetylating enzymes (Cowen and Lindquist 2005; Garnaud et al. 2016; Robbins et al. 2012). Singh, et al., demonstrated a role for Hsp90 in regulating the cellular circuitry required for resistance to the echinocandins (Singh et al. 2009). They identified calcineurin as a Hsp90 client protein in C. albicans by reciprocal co-immunoprecipitation validated physical interaction. In their study, the pharmacological or genetic impairment of Hsp90 function reduced tolerance of C. albicans laboratory strains and resistance of clinical isolates to echinocandins, creating a fungicidal combination. Interestingly, genetic impairment of Hsp90 expression enhanced the therapeutic efficacy of an echinocandin (Singh et al. 2009).

Wurtele, et al., reported that the depletion of H3K56 acetylation (critical for proper packaging of DNA into chromatin during DNA replication and DNA damage repair) results in enhanced sensitivity of C. albicans to echinocandins but not fluconazole (Wurtele et al. 2010). Also, reduced C. albicans virulence was noted in murine models of infection arising due to genetic or pharmacological alterations in H3K56 acetylation levels. The core Set3 histone deacetylase complex, composed of Set3, Hos2, Snt1, and Sif2, was shown to mediate C. albicans drug resistance by promoting biofilm formation (Nobile et al. 2014). In addition, reduced pathogenicity, as well as increased sensitivity to various stresses, was also observed in C. neoformans upon deletion of genes encoding HDACs. Hda1 was specifically found to be important for regulating the expression of C. neoformans genes controlling mating, virulence and stress adaptation (Brandao et al. 2015; 2018).

Histone acetyltransferases (HATs) and chromatin remodelling complexes also play important roles in antifungal drug resistance and biofilm formation. Tscherner, et al., demonstrated a role for the chromatin assembly-associated histone acetyltransferase complex NuB4 in regulating oxidative stress resistance, antifungal drug tolerance and virulence in C. albicans (Tscherner et al. 2015). Interestingly, deletion of the regulatory subunit of the NuB4 complex resulted in a marked increase in antifungal drug tolerance. The Swi/Snf chromatin remodelling complex has been recently described as a coactivator for Mrr1, a zinc cluster transcription factor that enhances the expression of Mdr1, which encodes a drug efflux pump (Liu and Myers 2017). Deletion of SNF2, which encodes the catalytic subunit of the Swi/Snf complex in C. albicans, significantly reduces Mdr1 expression, thereby resulting in enhanced susceptibility to fluconazole (Liu and Myers 2017). Altogether, these recent discoveries suggest that post-translational and epigenetic modifications play a significant role in mediating antifungal resistance. Ultimately, several of these mechanisms could serve as targets for novel classes of antifungal therapies.

Conclusions

Antifungal resistance can evolve during patient therapy but has been well-documented to occur in agriculture (Ballard et al. 2018; Hare et al. 2019). Azoles used in the agricultural fields and modern medicine are structurally similar and mechanistically indistinguishable (Berger et al. 2017). The triazoles are currently the most widely-utilized antifungal compound in agriculture due to their high efficiency and broad spectrum of target pathogens (Morton and Staub 2008; Maertens 2004). Currently, about 32 azoles are available for plant protection and five are available for use in patient clinics (FRAC Code List 2019; Fisher et al. 2018). The widespread use of antifungals, and particularly azoles, in agriculture is likely to have contributed, in part, to increased antifungal resistance in the clinic (Toda et al. 2021). Given the rise in antifungal resistance and limited treatment options for patients suffering from fungal diseases, there is a pressing need to employ novel strategies to abrogate and halt the development of drug resistance. It is also important to develop a better understanding of molecular and genetic mechanisms that drive antifungal resistance and tolerance, which should help to improve our knowledge of how fungal pathogens respond to drugs. Although we have some understanding of the transcriptional control of antifungal resistance, very little is known about mRNA stability, translational, post-translational and epigenetic mechanisms that control this process. One caveat to studying these post-transcriptional mechanisms is that they are expected to more likely result in antifungal drug tolerance, rather than true resistance. It is also unclear at this point which of these post-transcriptional mechanisms function to drive antifungal resistance and/or tolerance in the host environment. However, gaining a better understanding of post-transcriptional mechanisms is still likely to lead to the identification of regulatory components and protein targets that could eventually be used in the development of new therapeutic strategies.

The currently available antifungal drugs in the azole and echinocandin classes affect ergosterol biosynthesis and β-glucan synthesis, respectively, thereby targeting cell membranes and cell wall integrity. Thus, components of post-transcriptional mechanisms, such as Ccr4-Pop2 mRNA stability mechanism, which are important for cell wall integrity, antifungal resistance, and maturation of pre-formed biofilms, may serve as promising targets. However, conservation of the Ccr4–Pop2 deadenylase complex in higher eukaryotes is a matter of concern as it could present toxicity problems in relation to drug development. The largest challenge in selecting antifungal targets is their evolutionary conservation in the human host, thereby making it more important to develop fungal-specific inhibitors. Therefore, fungal-specific components of mRNA stability, translational, post-translational and epigenetic mechanisms that govern antifungal resistance and/or are important for cell viability would represent the most promising potential targets for antifungal drug discovery. It is pertinent to mention the study by Iyer, et al., which found that rocaglates inhibit translation initiation in C. auris, leading to activation of a cell death program (Iyer et al. 2020). In contrast, C. albicans, due to an amino acid variant in the drug-binding domain of translation initiation factor 1 (Tif1), showed inherent resistance to the rocaglates (Iyer et al. 2020). Thus, it is also important to take into consideration the divergence that might occur in related fungal pathogens in pathways that direct cellular responses to translational inhibition. In addition, fungal-specific regions and/or structural moieties of conserved proteins in the translational machinery of pathogenic fungi may also represent promising targets. Several highly effective antibiotics are known to target components of the bacterial translation machinery. For example, macrolides, oxazolidinones, and pleuromutilins bind the large 50S ribosomal subunit, while aminoglycosides and tetracyclines interfere with the smaller 30S ribosomal subunit (Bhattacharjee 2016; Chellat et al. 2016). However, targeting of translational regulators and components of the translational machinery remains a largely unexplored and unexploited avenue for the development of novel antifungal strategies. Thus, the development of novel deep sequencing approaches, such as ribosome profiling, as well as large-scale mutant library collections, should also facilitate the study of post-transcriptional mechanisms that control antifungal resistance in certain pathogenic fungi. Additional studies in this area are likely to open up a variety of new and fruitful avenues of research, leading to the development of novel classes of antifungal therapies.