Journal of
Fungi
Review
Fungal Drug Response and Antimicrobial Resistance
Paloma Osset-Trénor 1 , Amparo Pascual-Ahuir 1, * and Markus Proft 2, *
Citation: Osset-Trénor, P.;
Pascual-Ahuir, A.; Proft, M. Fungal
Drug Response and Antimicrobial
Resistance. J. Fungi 2023, 9, 565.
https://doi.org/10.3390/jof9050565
Academic Editor: Brian L. Wickes
Received: 23 March 2023
Revised: 27 April 2023
Accepted: 12 May 2023
Published: 12 May 2023
Copyright: © 2023 by the authors.
Licensee MDPI, Basel, Switzerland.
This article is an open access article
distributed under the terms and
conditions of the Creative Commons
Attribution (CC BY) license (https://
creativecommons.org/licenses/by/
4.0/).
J. Fungi 2023, 9, 565. https://doi.org/10.3390/jof9050565
1 Department of Biotechnology, Instituto de Biología Molecular y Celular de Plantas IBMCP,
Universidad Politécnica de Valencia, 46022 Valencia, Spain
2 Department of Molecular and Cellular Pathology and Therapy, Instituto de Biomedicina de Valencia
IBV-CSIC, Consejo Superior de Investigaciones Científicas CSIC, 46010 Valencia, Spain
* Correspondence: apascual@ibmcp.upv.es (A.P.-A.); mproft@ibv.csic.es (M.P.);
Tel.: +34-96-3877770 (A.P.-A.); +34-96-3391769 (M.P.)
Abstract: Antifungal resistance is a growing concern as it poses a significant threat to public health.
Fungal infections are a significant cause of morbidity and mortality, especially in immunocompro-
mised individuals. The limited number of antifungal agents and the emergence of resistance have led
to a critical need to understand the mechanisms of antifungal drug resistance. This review provides
an overview of the importance of antifungal resistance, the classes of antifungal agents, and their
mode of action. It highlights the molecular mechanisms of antifungal drug resistance, including
alterations in drug modification, activation, and availability. In addition, the review discusses the
response to drugs via the regulation of multidrug efflux systems and antifungal drug–target inter-
actions. We emphasize the importance of understanding the molecular mechanisms of antifungal
drug resistance to develop effective strategies to combat the emergence of resistance and highlight
the need for continued research to identify new targets for antifungal drug development and explore
alternative therapeutic options to overcome resistance. Overall, an understanding of antifungal drug
resistance and its mechanisms will be indispensable for the field of antifungal drug development and
clinical management of fungal infections.
Keywords: antifungal resistance; pleiotropic drug response; pathogenic fungi; multidrug efflux;
antifungal drugs
1. Introduction
Fungi can cause a variety of human diseases [1], including (i) local mycoses of the
skin, nails, and hair, (ii) systemic mycoses that spread throughout the body and affect
internal organs, such as histoplasmosis, aspergillosis, and candidiasis, (iii) opportunistic
infections that occur in people with weakened immune systems, such as Pneumocystis
jirovecii and Cryptococcus neoformans, (iv) allergic diseases triggered by an allergic reaction
to fungal spores such as bronchopulmonary aspergillosis, or (v) toxigenic infections caused
by toxic substances produced by fungi, such as aflatoxicosis and ergotism. Every year, over
150 million severe fungal infections occur globally, leading to around 1.7 million deaths
annually. Disturbingly, these numbers are steadily increasing due to social and medical ad-
vancements that have facilitated the spread of these infections. Furthermore, the long-term
use of antifungal drugs in high-risk patients has resulted in the emergence of drug-resistant
fungi, including the highly dangerous Candida auris [2,3]. Very recently, the World Health
Organization (WHO) has raised the alert of threatening fungal infections by ranking a
total of 19 fungal species as priority pathogens for strategic research, development, and
public health actions [4]. The most critical group contains Cryptococcus neoformans, Candida
auris, Aspergillus fumigatus, and Candida albicans. According to this ultimate analysis from
WHO, research into fungal-invasive diseases still receives insufficient funding, with only
1.5% of the total research budget in infectious diseases, and the approved antifungals and
those in clinical trials do not fully solve the challenges posed to healthcare workers due to
https://www.mdpi.com/journal/jof
J. Fungi 2023, 9, 565 2 of 20

treatment limitations and increasing resistance. The widespread use of fungicides (azoles)
in agriculture additionally exacerbates the issue, thus demanding the development of new
types of antifungals with different targets, safer profiles, and mechanisms of action exclu-
sively for human use. Thus, fungal infections are a growing global concern that requires
more effective research to understand and combat fungal antimicrobial resistance [3,5].
This article summarizes and highlights the molecular mechanisms underlying the adaptive
response and acquired resistance to different classes of antifungal compounds.

2. Antifungal Drugs: Classes and Modes of Action

Currently, only four classes of systemic antifungal treatments are used in clinical
practice: azoles, echinocandins, polyenes, and pyrimidines [6] (Figure 1). Azole antifungal
drugs, first reported in the late 1960s [7], work by inhibiting the synthesis of ergosterol, an
essential component of fungal cell membranes. This results in increased permeability of the
fungal cell, leading to its destruction. Azoles are used to treat a variety of fungal infections,
including dermatophytosis, candidiasis, and aspergillosis. There are several classes of
azole antifungal drugs [8], including (i) imidazoles, such as clotrimazole, econazole, and
miconazole, which are commonly used to treat skin and nail infections, (ii) triazoles, such
as fluconazole, itraconazole, and voriconazole, which are used to treat systemic fungal
infections and (iii) allylamines, such as terbinafine, which is used to treat dermatophyte
infections. Ergosterol acts as a control mechanism for the fluidity, asymmetry, and overall
stability of the cell membrane in fungal cells [9]. For the cell membrane to maintain its
integrity, the incorporated sterols must not have a C-4 methyl group. Azoles primarily
impact the heme protein Cyp51 (Erg11), which catalyzes the cytochrome P-450-dependent
removal of a methyl group from lanosterol through the process of 14α-demethylation within
the ergosterol biosynthesis pathway [10,11]. Azole drugs inhibit the enzyme reaction by
non-competitive reversible interaction with the heme and prevent accessing of protons
to the active site [12]. When the 14α-demethylase is inhibited, the levels of ergosterol
decrease, and the levels of its precursors, such as lanosterol, 4,14-dimethylzymosterol, and
24-methylenedihydrolanosterol, increase, leading to structural and functional changes in
the plasma membrane [13].
Echinocandins are a class of antifungal agents discovered in the 1980s and 1990s. They
work by targeting a key component of the fungal cell wall, β(1,3)-glucan, which is essential
for its stability and integrity (Figure 1). The unique composition of the fungal cell wall,
which includes compounds such as mannan, chitin, and α- and β-glucans, has made it
a prime target for antifungal agents [14]. These components are exclusive to fungi and
not present in other organisms, making them a desirable target for drugs to selectively
target and kill fungal cells while minimizing damage to other cells [15]. The majority
of our understanding of the cell wall composition of fungi that are medically significant
comes from research conducted on C. albicans [16]. This yeast species has a complex cell
wall structure made up of multiple layers, including chitin, β-glucan, and mannoprotein.
These last two components make up approximately 80% of the overall mass of the cell wall,
thus highlighting the importance of β-glucan for fungal cell stability [17]. Echinocandins
inhibit the synthesis of β(1,3)-glucan by binding to the enzyme β(1,3)-glucan synthase, a
large (210 kDa) integral membrane heterodimeric protein, thereby disrupting the fungal
cell wall and leading to cell death [18,19]. The mechanism of action of echinocandins
makes them effective against a variety of clinically important fungi, including Candida and
Aspergillus species. They are typically used to treat serious invasive fungal infections, often
in combination with other antifungals.
Polyenes are a class of antifungal agents discovered in the 1950s [20]. Similar to
azoles, they target the unique fungal membrane component ergosterol; however, in this
case, by direct binding to the sterol, thereby causing a change in the permeability of the
membrane and disruption of the membrane potential [21,22] (Figure 1). Polyenes have a
broad spectrum of activity against various medically important fungi, as they inactivate
all cells with a significant content of sterols in their outer membranes [23]. The most
J. Fungi 2023, 9, 565 3 of 20

important drawback of the use of polyenes is their intrinsically high host toxicity, which
limits their therapeutic index [24,25]. One of the most well-known polyenes is amphotericin
B, which was initially derived from the soil bacterium Streptomyces nodosus. Amphotericin
B was the first effective antifungal agent and is still widely used today, together with
nystatin and natamycin, especially for the treatment of serious or life-threatening fungal
infections [20,26].

Figure 1. Mechanisms of action of traditional and novel antifungal drugs. Representative compounds
are indicated for each class. FUMP = 5-fluorouridylic acid; Fcy1, Fca1 = cytosine deaminases;
Fur1 = Uracil phosphoribosyltransferase; Ac-Phosphatidyl inositol = Acyl-Phosphatidyl inositol;
GPI = Glycosylphosphatidyl inositol; DHODH = Dihydroorotate dehydrogenase.

Pyrimidine analogs are a different class of antifungal agents that act by inhibiting
DNA and RNA synthesis [27]. Flucytosine (5FC) is the most commonly used pyrimidine
analog for the treatment of fungal infections. The 5FC has been shown to inhibit the
growth of various yeasts, including Candida and Cryptococcus neoformans. However, the
high prevalence of primary resistance in many fungal species has reduced its initial promise
as an antifungal agent [28–30]. Nowadays, 5FC is mostly used in combination with other
antifungals, such as amphotericin B and fluconazole, and is rarely used as a single agent.
The mechanism of action of 5FC involves entering the fungal cell through a permease
enzyme and being converted to 5-fluorouracil (5FU) by the enzyme cytosine deaminase.
This is followed by the conversion of 5FU into 5-fluorouridylic acid (FUMP) by UMP
pyrophosphorylase, which is further phosphorylated and incorporated into RNA, causing
J. Fungi 2023, 9, 565 4 of 20

disruption of protein synthesis [31]. Additionally, 5FU is converted to 5-fluorodeoxyuridine

monophosphate, a potent inhibitor of thymidylate synthase, an enzyme involved in DNA
synthesis and nuclear division [32]. Thus, 5FC acts by disrupting pyrimidine metabolism,
as well as RNA, DNA, and protein synthesis in the fungal cell (Figure 1).
Although there is a growing need for more antifungal drug options [33,34], no new
classes of antifungal drugs have become available in the past 20 years. However, there
is some good news, as several new antifungal classes are currently in late-stage clinical
development. These include promising drugs currently in clinical trials with previously un-
explored modes of action, such as fosmanogepix (a novel inhibitor of the Gwt1 enzyme) or
olorofim (a new inhibitor of the dihydroorotate dehydrogenase enzyme) [35–37] (Figure 1).
Fosfomanogepix is a N-phosphonooxymethyl prodrug, which after drug adminis-
tration, is converted into the active form of manogepix by systemic phosphatases [38].
Manogepix has a new mode of inhibition that targets the maturation of glycosylphos-
phatidylinositol (GPI)-anchored proteins by inhibiting the fungal enzyme Gwt1 at the
endoplasmic reticulum [39]. This enzyme, which is crucial for the trafficking and anchoring
of mannoproteins to the fungal cell membrane and wall, is an inositol acyltransferase [40].
GPI-anchored mannoproteins act as adhesives, enabling fungi to stick to mucosal and ep-
ithelial surfaces in the host before colonization and infection. Several fungal adhesins and
virulence factors are derived from GPI-anchored proteins and GPI biosynthesis is essential
in fungi [41,42]. The inhibition by manogepix appears to be unique to fungal pathogens
because it does not impede human inositol acylation via the nearest mammalian ortholog,
PIGW [38,43]. Manogepix has broad-spectrum activity against numerous pathogenic fungi
including Candida and Aspergillus spp. [44,45].
Olorofim is a member of a novel class of antifungal drugs named orotomides. Olorofim
impairs fungal growth through inhibition of the fungal dihydroorotate dehydrogenase
(DHODH) enzyme, which is located in mitochondria and involved in pyrimidine synthe-
sis [46,47] (Figure 1). Olorofim does not show any significant cross-reactivity with the
human dihydroorotate dehydrogenase, thus limiting its drug toxicity [48]. While olorofim
may not be effective against a wide range of fungal infections, it possesses remarkable nov-
elty and a spectrum of activity that is anticipated to be significant in the future. Olorofim
does not demonstrate any efficacy against yeast. Nevertheless, it is effective against various
clinically important fungal groups, including dimorphic molds such as Histoplasma and
Coccidioides spp. or hyaline hyphomycetes such as Aspergillus spp. [35,46].

3. Impact of Fungal Antimicrobial Resistance

Infections are a leading cause of mortality and disability worldwide, with drug-
resistant bacterial infections causing 1.27 million direct deaths and contributing to an
estimated 4.95 million deaths annually [49]. This burden is particularly high in resource-
limited settings. Fungal diseases cause over 1.5 million deaths and affect over a billion
individuals [50]. Serious fungal infections often occur as a consequence of underlying
health conditions such as asthma, AIDS, cancer, organ transplantation, and corticosteroid
therapies. The WHO recognizes that the incidence of invasive fungal diseases (IFDs) is
increasing globally, particularly among immunocompromised populations [4]. However,
there are challenges in diagnosing and treating IFDs, including limited access to quality
diagnostics and treatments and the emergence of antifungal resistance. Despite the growing
concern, fungal infections receive limited attention and resources, resulting in a scarcity
of data on their distribution and patterns of antifungal resistance [4,51–53]. Thus, it is not
possible to accurately estimate their burden.
The acquisition and emergence of antifungal drug resistance can be explained as an
evolutionary response to the selective pressure exerted by the drug. The likelihood of
resistance emerging due to genetic changes is influenced by several factors, including the
number and doubling rate of fungal cells exposed to the drug, the number of different
pathways that confer resistance, and the fitness costs associated with each pathway [54].
It is important to note that antifungal drug resistance can originate either in the host or
J. Fungi 2023, 9, 565 5 of 20

in the environment. In vivo resistance develops in individuals during antifungal therapy,

leading to treatment failure for a range of pathogenic fungi [55,56]. This is particularly
relevant for Candida spp., which is a major cause of nosocomial bloodstream infections
and frequently shows the emergence of resistance to antifungals [57]. For example, azole
resistance in C. albicans has been documented in individuals with HIV who were undergoing
prolonged fluconazole therapy [58]. The demographics of patients susceptible to IFDs are
progressively broadening, with a particular emphasis on populations such as the elderly,
individuals with compromised immune systems due to HIV, cancer chemotherapy, or
immune-suppression therapy required for transplantation, as well as those suffering from
severe viral infections such as influenza virus and COVID-19 [59–62]. Environmental
resistance can also emerge due to prior exposure of human pathogenic fungi to fungicides
in nature. Fungicides are commonly used to protect crops and materials against fungal
infections, and the resulting selective pressure drives the evolution of resistance against all
major classes of fungicides, including azoles [63].

4. Mechanisms of Acquired Antifungal Resistance

At a mechanistic level, antifungal resistance is typically acquired through modifi-
cations that impact the interaction between the drug and its target, either directly or
indirectly [64,65]. The development of resistance can be attributed to genetic alterations in
the binding site of the target, such as mutations in the genes encoding lanosterol demethy-
lase for azoles or β-glucan synthase for echinocandins [6]. Resistance may also result from
an increase in the amount of target available [66], or through changes in the effective drug
concentration, as a consequence of elevated drug efflux activity for intracellular drugs such
as azoles [67,68], or through the inhibition of prodrug activation for flucytosine [69]. In
scientific terminology, antifungal resistance is distinct from antifungal tolerance, which
refers to the capacity of drug-sensitive cells to survive in the presence of drug concen-
trations beyond the minimum inhibitory concentration (MIC). This ability is associated
with various general stress responses and/or epigenetic pathways, as comprehensively
reviewed in [70]. Notably, tolerance is most pronounced when using fungistatic drugs and
has been extensively examined in Candida albicans strains treated with fluconazole [71].
Nevertheless, the clinical significance of antifungal tolerance remains uncertain. In this
review, we focus on the molecular mechanisms, which enable fungal cells to respond and
protect from antibiotic drugs and eventually develop drug resistance.

4.1. Fungal Multidrug Efflux Systems: Components, Regulation, and Impact on Drug Resistance
A notable proportion of observed clinical resistance to antifungal agents can be at-
tributed to the action of ATP-binding cassette (ABC) and major facilitator superfamily
(MFS) transport pumps located at the fungal plasma membrane [72,73]. The majority
of studies in this field have been conducted with azole drugs [74]; however, multidrug
efflux affects resistance to other classes of antifungals [75]. The ABC and MFS transporters
are the two most extensively researched transporter families implicated in efflux [73].
Fungi allocate a significant portion of their genomic resources to encoding transporters,
with fungal genomes containing approximately 10 to 30 transporter-encoding genes per
megabase of genomic DNA [76]. The ABC transporter superfamily comprises primary
efflux transporters, which use ATP hydrolysis to export substrates [77,78]. This superfamily
can be further divided into five families of transporters (ABCA, ABCB, ABCC, ABCD,
and ABCG), with three families being involved in the efflux of toxic compounds. These
three families, known as multi-drug resistance (MDR), multi-drug resistance-associated pro-
tein (MRP), and pleiotropic drug resistance (PDR), respectively, have been extensively inves-
tigated in Saccharomyces cerevisiae, offering insights into their potential roles in pathogenic
fungi [79,80]. The PDR family exhibits the lowest level of phylogenetic conservation among
the ABC transporter families, with evidence of gene loss and duplication observed in yeasts
and filamentous fungi [81]. This implies that the members of the PDR family evolve rapidly
in response to external selective pressures.
J. Fungi 2023, 9, 565 6 of 20

The conserved structure of ABC transporters comprises a nucleotide-binding domain

(NBD) located either preceding or following a transmembrane domain (TMD), which is
formed by six transmembrane-spanning helices. This NBD-TMD6 configuration is observed
in half ABC transporters, which dimerize to produce a complete functional protein. In
contrast, most fungal ABC transporters have undergone evolutionary changes that have
led to the fusion of two NBD-TMD6 half transporters, resulting in the formation of a single
functional protein instead of a dimeric structure. ABC transporters are a highly complex
and fascinating class of proteins. Although some members of this superfamily exhibit
high substrate specificity, ABC multidrug transporters are highly promiscuous in their
substrate recognition. These proteins harness the energy generated by ATP binding and
hydrolysis at the NBDs to induce conformational changes that enable the transporters to
bind and release drug substrates [82]. The binding pocket is constructed from the TMDs
and can accommodate large substrates. The transporter adopts a high-affinity inward-
facing (IF) conformation during the binding phase, while the substrate is released from
the outward-facing (OF) structure with lower affinity. As a result, these proteins exhibit
polytopic characteristics. The nature of the drug-binding and transport sites is relevant
to understanding resistance mechanisms mediated by these transporters. For example,
the large drug-binding pockets of P-glycoprotein and Pdr5 contain multiple overlapping
sites [83,84]. Notably, it has been observed that a single drug can bind to multiple locations
within these sites [85] and that different drugs can simultaneously bind to different locations
within the binding pocket [86].
The clinical relevance of efflux pumps extends beyond ABC multidrug transporters, as
some members of the MFS family also mediate hyper resistance to drugs [87]. MFS proteins
are typically composed of 12 or 14 transmembrane helices, with each transmembrane
domain consisting of 6 or 7 helix bundles that pack together to create a cavity lined
with amino acids that bind transport substrates. These transporters are generally smaller
than ABC transporters and, similar to their counterparts, exhibit considerable substrate
promiscuity. However, MFS transporters utilize the energy derived from a proton motive
force rather than ATP hydrolysis to facilitate efflux.
Extensive research has been conducted on fungal multidrug transporters, with a
particular focus on those found in Saccharomyces cerevisiae, Candida albicans, and Candida
glabrata [77,88–90]. A group of S. cerevisiae strains was discovered that exhibited resistance
to a wide range of drugs and natural toxins, known as pleiotropic drug resistance (PDR) [91].
Subsequent studies identified the primary genes involved in PDR of budding yeast to be
ABC and MFS transporters and, importantly, zinc cluster transcription factors (TFs) [92–95]
(Figure 2). The best-characterized PDR ABC transporters, Pdr5, Snq2, and Yor1, each play a
role in resistance against hundreds of functionally and structurally unrelated drugs [96].
The most frequently mutated loci in PDR strains were found to encode two paralogous zinc
cluster TFs, Pdr1 and Pdr3, which share 36% identity and are an essential part of the PDR
network [97]. Recently, it has been shown in yeast that multiple PDR TFs contribute to the
overall multidrug response with distinguishable sensitivities towards different xenobiotic
molecules [98]. Pdr TFs were found to bind to pleiotropic drug resistance elements (PDREs)
within the promoters of different multidrug transporter encoding genes, which is essential
for PDR inducibility [99–102]. Pdr1 and Pdr3 are constitutively bound to DNA, regardless
of the presence of drugs, suggesting that their activation is not dependent on changes in
nuclear occupancy or DNA-binding [103]. Thakur et al. [104] showed that the regulatory
domains XBD (xenobiotic binding domains) of both Pdr1 and Pdr3 bind the antifungal
ketoconazole at low micromolar affinity, suggesting that ligand binding may increase their
transcriptional activity. Similar to other known zinc cluster proteins, partial deletions or
mutations within the regulatory domains of Pdr1 and/or Pdr3 result in constitutively active
TFs that induce strong expression of ABC transporters [105–107], leading to an instance of
up to 10-fold higher minimum inhibitory concentrations of ketoconazole. The pathogenic
fungi Candida glabrata and Candida albicans have different mechanisms for controlling the
expression of their drug efflux ABC transporters, with Pdr1 regulating Cdr1, Pdh1, and
 DNA-binding [103]. Thakur et al. [104] showed that the regulatory domains XBD (xeno-
biotic binding domains) of both Pdr1 and Pdr3 bind the antifungal ketoconazole at low
micromolar affinity, suggesting that ligand binding may increase their transcriptional
activity. Similar to other known zinc cluster proteins, partial deletions or mutations
within the regulatory domains of Pdr1 and/or Pdr3 result in constitutively active TFs that
J. Fungi 2023, 9, 565 7 of 20
induce strong expression of ABC transporters [105–107], leading to an instance of up to
10-fold higher minimum inhibitory concentrations of ketoconazole. The pathogenic fungi
Candida glabrata and Candida albicans have different mechanisms for controlling the ex-
Snq2 transporters
pression of their C. glabrata
in drug efflux[108,109] and Tac1 and
ABC transporters, Mrr1
with controlling
Pdr1 regulatingtheCdr1,
expression
Pdh1,ofand
Cdr1,
Snq2Cdr2 or Mdr1 transporters
transporters C. albicans
in C. glabratain[108,109] Tac1 andAspergillus
[110–113].
and fumigatus
Mrr1 controlling thelacks a Pdr1 of
expression
homolog but has intrinsic drug resistance through AbcG1 and its zinc cluster TF
Cdr1, Cdr2 or Mdr1 transporters in C. albicans [110–113]. Aspergillus fumigatus lacks aregulator
AtrR [114,115].
Pdr1 homolog but has intrinsic drug resistance through AbcG1 and its zinc cluster TF
regulator AtrR [114,115].

Figure 2. The fungal pleiotropic drug resistance (PDR). Upper panel: General features of PDR reg-
ulation.
Figure Different
2. The xenobioticdrug
fungal pleiotropic molecules, including
resistance antibiotic
(PDR). Upper drugs,
panel: are features
General recognized by regula-
of PDR transcrip-
tional activators (Pdr-TFs) via their xenobiotic binding domains (XBD). Pdr-TFs bound at plei-
tion. Different xenobiotic molecules, including antibiotic drugs, are recognized by transcriptional
otropic drug resistance promoter elements (PDRE) stimulate via their transactivation domains
activators (Pdr-TFs) via their xenobiotic binding domains (XBD). Pdr-TFs bound at pleiotropic drug
(AD) the expression of PDR, MDR, or MFS genes encoding plasma membrane transporters of the
resistance promoter
pleiotropic drugelements (PDRE)
resistance, stimulate
multidrug via theirortransactivation
resistance, domains
major facilitator (AD) the expression
superfamilies. Lower panel:
of PDR, MDR, or MFS genes encoding plasma membrane transporters of the
molecular mechanisms leading to antifungal drug resistance via the PDR system. pleiotropic drug resis-
tance, multidrug resistance, or major facilitator superfamilies. Lower panel: molecular mechanisms
leading to antifungal
The drug resistance
major mechanism of via the PDRantifungal
acquired system. resistance via drug efflux is the rein-
forcement of the expression of one or several efflux transporters (Figure 2). This can be
The major mechanism of acquired antifungal resistance via drug efflux is the reinforce-
achieved by an increase of genomic copies of the relevant PDR genes (gene amplifica-
ment of the expression of one or several efflux transporters (Figure 2). This can be achieved
by an increase of genomic copies of the relevant PDR genes (gene amplification), the gain of
function mutations in PDR TFs, or modification of PDR TFs mRNA stability. Amplification
of genes, including ERG11 and TAC1, plays a significant role in the multidrug resistance
of Candida species [116–118], leading to a linear relationship between gene copy number
and the minimum inhibitory concentration of fluconazole. Aneuploidy leading to drug
resistance has been observed in C. albicans, C. neoformans, and C. auris [119–121]. These
findings show that increasing the copy number of genes involved in multidrug efflux, even
modestly, creates enough hyper-resistance to have clinical implications. More recently, the
J. Fungi 2023, 9, 565 8 of 20

fast and reversible amplification of PDR genes leading to drug resistance in C. albicans has
been investigated mechanistically, revealing the exchange between long, inverted repeats
flanking the amplified region on multiple chromosomes. It is evident from these and other
results that hyper resistance by gene duplication is frequent in many fungal species and
can be achieved by multiple mechanisms, including aneuploidy, nonhomologous extension
after double-strand breaks, and chromosomal exchange between indirect and direct re-
peats [122]. Because aneuploidities generally reduce fitness, they seem to arise only during
severe changes in environment such as drug exposure, and do not persist once the selective
pressure is removed [123,124]. Remarkably, aneuploidities were found in a great portion of
clinical S. cerevisiae strains [123].
Pleiotropic drug resistance by gain of function (GOF) mutations in TFs is dominated
in budding yeast by the zinc cluster factors Pdr1 and Pdr3. It is important to note that,
although not understood in detail on many occasions, these GOF mutations generally
do not lead to overexpression of the TFs; instead, they create hyperactive TFs, which
constitutively overexpress their transporter target genes. Amino acid substitutions in
Pdr1, such as M308I in PDR1-2, F815S in PDR1-3, K302Q in PDR1-6, P298A in PDR1-7
and L1036W in PDR1-8 mutants, all result in multidrug-resistant phenotypes [105]. Three
regions within the Pdr1 regulator with implications for drug resistance were identified,
and mutant alleles PDR1-3, PDR1-6, and PDR1-8 were constructed to investigate their
phenotypes, with PDR1-3 being the most effective in activating target promoters [105].
Similar to Pdr1, multiple amino acid substitutions in Pdr3, which cluster in short regions of
the TF, can cause drug resistance [106]. According to [125], the presence of GOF mutations
isolated from patients suggests that the Candida glabrata homolog Pdr1 plays an important
role in the development of clinical antifungal resistance. In this case, Pdr1 expression
itself is heavily regulated; thus, even a modest up-regulation of the wild-type Pdr1 copy
contributes to drug resistance in this pathogenic fungus [126]. Extensive studies on CgPdr1
have shown an extraordinarily high number of GOF substitutions, which lead to enhanced
drug resistance [125,127–129]. Recent advances point to the existence of an extensive reg-
ulatory domain within CgPdr1, which contributes to the highly flexible transactivation
capacity via contacts with other co-activator proteins [126,130]. In Candida albicans, Tac1
is the main contributor to drug resistance via GOF mutations. Many points and small
deletion mutations have been correlated in this TF with antifungal resistance by enhanc-
ing its transactivation capacity in the context with additional transcriptional co-activator
complexes [112,131–133]. Additionally, multidrug resistance in C. albicans is caused by the
Mrr1 regulator, which resembles Tac1. Mutations in Mrr1 can confer hyper resistance to
benomyl, with many GOF mutations found in distinct locations exhibiting a dominant
phenotype [113,134]. In the emerging high-risk pathogen Candida auris, two genes similar
to C. albicans’ TAC1 were identified, with a majority of GOF mutations reported in one of
them, TAC1B, that map to regions analogous to those in the C. albicans homolog [135,136].
This suggests a potential link between the regulatory mechanisms of the two proteins.
Rahman et al. [137] identified a novel mechanism of hyper resistance in S. cerevisiae in-
volving a set of serine residues in the Pdr5 linker region. Their study showed that alanine
mutations in six different serines exhibited significant hyper resistance, and changing S837
to aspartate produced the same phenotype, which was the result of a 2–3x increase of Pdr5
that was due to an increased Pdr5 mRNA half-life. Mixed effects on both PDR transporter
expression and mRNA stabilization had been previously characterized in azole-resistant
C. albicans isolates [138]. In any case, it is important to note that overexpression of multidrug
efflux transporters interferes with general cell homeostasis, most likely by interfering with
proper intracellular metabolites [139], thus only emerges upon a strong selection in the
presence of antifungal agents.
A novel mechanism of antifungal resistance has emerged recently by investigating
the A666G mutation in the yeast Pdr5 transporter, which was found in several screens for
hyper-resistant mutants [140,141]. It conferred 2–5x increased substrate-specific resistance
but did not alter susceptibility to clotrimazole. Importantly, the mutant had no difference
J. Fungi 2023, 9, 565 9 of 20

in the quantity of Pdr5 or the level of ATPase activity compared to the wild type, but
it had enhanced cooperativity, which led to higher transport of xenobiotic compounds
per molecule of ATP hydrolyzed [82,141].

4.2. Mutational Inhibition of Antifungal Drug–Target Interaction

Several antifungal drugs (azoles, echinocandins, manogepix, or orotomides) directly
target specific essential enzymes in the pathogen, such as Erg11/Cyp51, Fks1, Gwt1, or
DHODH. Therefore, conformational changes in the target enzyme caused by mutations
leading to decreased drug recognition while maintaining sufficient enzyme activity are
possible sources of acquired resistance.
Mutations in the ERG11(CYP51) gene are a common azole resistance mechanism in
Candida albicans, altering the inhibition of the lanosterol demethylase enzyme [142–144]. The
effect of these mutations, more than 140 described to date, is not uniform and likely a result
of overall reduced azole-binding affinity to the mutant enzyme [145–147]. Documentation
of new ERG11 mutations in azole-resistant clinical isolates continues to appear, but not all
documented mutations were definitively shown to be tied to azole resistance [148–153].
Homozygous replacement of the native ERG11 alleles with a mutant ERG11 ORF encoding
either a single or double amino acid substitution in the Erg11 protein showed that some
substitutions directly influence or interfere with azole binding, while others indirectly affect
resistance through, for example, the interaction with cytochrome P450 reductase [154]. The
crystal structure of the Erg11 protein of C. albicans has been resolved, leading to further
insight into the interactions between azole drugs and their target and the development
of 3D models useful for future antifungal discovery [155,156]. Aspergillus spp. has two
Cyp51 isoenzymes, Cyp51A, and Cyp51B, that can both act as sterol 14α-demethylase
in vivo, resulting in no phenotypic difference in A. fumigatus strains with gene knock-outs
of either enzyme [157–159]. However, a conditional mutant study showed that growth was
abolished in the absence of both CYP51A and CYP51B [160]. The main azole resistance
mechanism in A. fumigatus is the development of point mutations in CYP51A, which has less
affinity for azole drugs than CYP51B, indicating a molecular basis for this resistance [161].
Lockhart et al. [162] reported that 60% of clinical A. fumigatus isolates had triazole resistance
mutations within CYP51A, with the five most frequent mutations being G54, L98, G138,
M220, and G448. These mutations have been shown to confer azole resistance in A. fumigatus
through gene replacement experiments. Over 40 different CYP51A point mutations have
been reported in clinical A. fumigatus isolates, but not all of them confer an azole resistance
phenotype, and some can occur alone or in combination with other mutations [158,163–169].
Azole resistance is an increasing problem in Cryptococcus spp. due to prolonged treatment
regimens, with CYP51 mutations identified as one of the main mechanisms [170,171]. Point
mutations in CYP51, such as G468S and Y145F, have been shown to confer resistance to
fluconazole and voriconazole in C. neoformans [172,173].
The target of echinocandins, β(1,3)-glucan synthase, is encoded by the FKS1 and FKS2
genes in Candida spp. Limited amino acid substitutions in the Fks subunits of glucan
synthase are responsible for echinocandin resistance leading to clinical failures [174]. These
mutations occur in two highly conserved hot spot regions of FKS1 in C. albicans [175]
and in homologous regions of FKS2 in C. glabrata [176], resulting in elevated minimum
inhibitory concentration (MIC) values. The most frequent and pronounced resistance
phenotype in C. albicans is caused by amino acid changes at S641 and S645, while in
C. glabrata, modifications at S663 in Fks2, S629 in Fks1, and F659 in Fks2 are the most
prominent substitutions [176,177]. Additionally, in Aspergillus spp., although much less
explored, echinocandin resistance due to point-mutated Fks subunits has been reported
recently [178].
Point mutations leading to amino acid substitutions within Gwt1, V163A in C. glabrata,
and V162A in C. albicans can cause resistance to manogepix but do not affect the activity of
other antifungals, including azoles and echinocandins [179,180]. The reduced susceptibility
to manogepix of V162A and V168A Gwt1 mutants expressed in C. albicans and Saccha-
J. Fungi 2023, 9, 565 10 of 20

romyces cerevisiae, respectively, indicates the significance of this particular valine residue in
manogepix binding across various species.
The olorofim target, dihydroorotate dehydrogenase (DHODH), encoded by the pyrE
gene in Aspergillus, also appears to provide a limited resource for mutations leading to
olorofim resistance. A recent study applying long-term olorofim treatment in A. fumigatus
revealed several amino acid substitutions within PyrE with a hotspot at G119 producing
olorofim resistance [181].
Polyene antifungals target an essential membrane compound, ergosterol, instead of
an essential cellular enzyme. This different mechanism of action might explain the rare
occurrence of polyene resistance among fungi. Nevertheless, the most prominent way
to achieve polyene resistance is the alteration of the sterol composition of the plasma
membrane [182,183], which comes with a strong fitness trade-off [184]. Several mutations
in genes involved in the biosynthesis of ergosterol (ERG genes) have been linked to this
mechanism. In C. albicans, when the ERG11 and ERG3 genes, which encode for lanosterol
14α-demethylase and C-5 sterol desaturase, respectively, are dysfunctional, the membrane’s
ergosterol is replaced with alternate sterols such as lanosterol, eburicol, and 4,14-dimethyl-
zymosterol [183,184]. According to other studies, the substitution in ERG11 and the loss of
function of ERG5 (C-22 sterol desaturase) in C. albicans are associated with an alternative
membrane sterol composition and amphotericin B resistance [150,184]. In other Candida
species, the inactivation of ERG6 (C-24 sterol methyl-transferase) and ERG2 (C-8 sterol iso-
merase) has a similar effect [185,186]. A mutation in ERG2, which results in its inactivation,
is one of the few described mechanisms of polyene resistance in C. neoformans [187].

4.3. Resistance by Overexpression of Antifungal Drug Targets

An important strategy of fungal cells to lower the efficient drug concentration inside
the cell is based on titrating the antifungal compound through an increase in the copies of
the target enzyme. A recent high-density mutagenesis screen in S. cerevisiae with several
antimicrobial agents revealed that the gain of function promoter mutants is enriched for the
respective drug target protein on many occasions, pointing to a general strategy to adapt to
antifungal treatments [188]. Resistance by overexpression of the drug target is especially
relevant for azoles. ERG11 overexpression is a frequently observed mutational adaptation
in clinically relevant azole-resistant Candida isolates [189–192]. One prominent mechanism
of constitutive ERG11 expression acts via the gain of function point mutations in the Upc2
TF, which acts as a direct transcriptional activator of ERG11 in Candida spp. [193–195]
In Aspergillus, different promoter rearrangements in the cyp51A gene leading to robust
overexpression have been linked to azole resistance [196].

4.4. Alterations of Drug Modification, Activation, and Availability

Flucytosine (5-FC) stands out among antifungal agents due to its distinctive mode of
action that involves inhibiting DNA, RNA, and protein synthesis and the fact that it acts
as a prodrug, which means that it requires metabolic conversion through the pyrimidine
salvage pathway to become activated [197]. The majority of research on 5-FC resistance
mechanisms has concentrated on S. cerevisiae and C. albicans, with particular attention
given to identifying mutations in crucial enzymes involved in the pyrimidine salvage
pathways. These studies revealed that 5-FC resistance may occur due to the absence or
alteration of any of the enzymes responsible for the activity of cytosine permease (FCY2),
cytosine deaminase (FCA1, FCY1), or uracil phosphoribosyltransferase (FUR1). In S. cere-
visiae, several studies have indicated that mutations in FCY1, FCY2, and FUR1 can result
in resistance to 5-FC [198,199]. Disruption of cytosine deaminase is linked to high lev-
els of dose-independent resistance, whereas loss of cytosine permease results in a lower,
dose-dependent level of resistance, suggesting the involvement of two additional cyto-
sine permeases, FCY21 and FCY22. Additionally, alternative entry routes of 5-FC exist
via other cytosine adenine permease homologs, including TPN1, FUR4, and YOR071c,
and can contribute significantly to 5-FC toxicity [200]. Kern et al. [201] have associated
J. Fungi 2023, 9, 565 11 of 20

phenotypic 5-FC resistance with a single point mutation, R134S, in the FUR1 gene. For
C. albicans, acquired 5-FC resistance has been functionally linked to a single R101C amino
acid substitution in the Fur1 enzyme [202,203], and several point mutations in the FUR1
gene have been described in clinical 5-FC resistant isolates of C. glabrata [69]. In the emerg-
ing multiresistant C. auris, an I211F substitution in the FUR1 gene has been documented in a
5-FC-resistant isolate [204]. Bhattacharya et al. [119] have further exhibited the potential of
transient gene duplication in C. auris during replicative aging, leading to the development
of tolerance to 5-FC.
Another related mechanism of resistance is the decrease of the effective drug con-
centration by the selective impairment of azole influx into the fungal cell. This has been
recently reported to be relevant for PDR- and ERG11-independent azole-resistant C. glabrata
isolates. Here, the molecular key to resistance is mutations in the CgHXT4/6/7 hexose
importer, which contributes to azole import across the fungal cell membrane [205].

5. Concluding Remarks
The indiscriminate use of antifungal drugs for prophylactic and empirical treatment
of suspected invasive fungal diseases in high-risk individuals such as patients with cys-
tic fibrosis, critically ill, or haemato–oncology patients is a worrying trend. Antifungal
stewardship is essential to ensure the appropriate use of these drugs and preserve the
limited antifungal options available, particularly in cases of highly transmissible fungal
infections such as Candida spp. and Trichophyton spp. [206]. In recent years, systems biology
approaches utilizing laboratory evolution techniques have demonstrated the impressive
ability of Saccharomyces and Candida species to acquire resistance to existing antifungal
agents [207–209]. Despite the ongoing threat of drug-resistant fungal infections to human
health, there is renewed optimism in academia and industry to enhance our knowledge of
the mechanisms underlying antifungal resistance and to develop new therapeutic strategies
to combat these pathogens. Combination antimicrobial treatment is an effective strategy
to prevent secondary antimicrobial resistance and is established for various bacterial and
viral infections. For example, combining antifungal agents such as amphotericin B and
flucytosine or fluconazole and flucytosine can prevent the selection of resistant fungal
populations and limit the development of clinical antifungal resistance [210,211]. Further
investigation is required to unravel the full potential of combinatorial antifungal treatment
in the battle against secondary resistance. Another promising approach is to target the
resistance mechanisms themselves, with the aim of restoring the efficacy of current antifun-
gals [212,213]. Alternative methods are currently being developed to protect antifungals
from resistance, including host-directed approaches such as immunotherapy [214], fungal
vaccines [215,216], and improved antifungal targeting [217]. In addition, cell-based thera-
pies, such as dendritic cell transfer and CAR T-cell therapy [218–221], have demonstrated
promising outcomes in vitro but still need to be investigated in clinical trials. These ap-
proaches provide potential alternative options for managing antifungal resistance. As such,
ongoing and future investigations into the mechanisms of antifungal drug resistance are
expected to yield important insights and pave the way for improved clinical outcomes for
diverse populations affected by drug-resistant fungal infections worldwide.

Author Contributions: P.O.-T., A.P.-A. and M.P. wrote, revised the manuscript, and agreed on the
final version of the manuscript. All authors have read and agreed to the published version of
the manuscript.
Funding: Research by the authors was funded by grants from Ministerio de Ciencia, Innovación
y Universidades PID2019-104214RB-I00 (A.P.-A., M.P.) and Agencia Valenciana de la Innovación
INNEST/2021/266 (A.P.-A.).
Institutional Review Board Statement: Not applicable.
Informed Consent Statement: Not applicable.
Data Availability Statement: Not applicable.
J. Fungi 2023, 9, 565 12 of 20

Acknowledgments: We apologize to all researchers whose work on related topics was not cited here
due to space limitations.
Conflicts of Interest: The authors declare no conflict of interest.

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