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Table 8.1 Classication of antifungals, their mechanism of action and known genetic mechanism
through which resistance is acquired
Antifungals
Polyenes By binding to ergosterol in the
Azole
Echinocandin
Flucytosine Flucytosine is converted to
Mechanism of action
fungal cell membrane, forming pores
that disrupt cell integrity and lead to
cell death
Inhibits the 14α-demethylase
enzyme, which is essential for the
production of ergosterol, a major
component of fungal cell wall
Inhibiting the β-1,3-glucan synthase
enzyme (Fks1), which is essential for
the synthesis of β-1,3-glucan, a
major component of the fungal cell
wall
5-uorouracil (5-FU) inside the
fungal cell, which with DNA and
RNA synthesis
Mechanism of resistance
• Mutations in genes involved in
ergosterol synthesis or uptake
• Overexpression of drug efux pumps,
which can transport polyene drugs out
of the fungal cell
• Changes in the cell membrane
composition that reduce the binding
afnity of polyene drugs
• Mutations can reduce the binding
afnity of azole drugs to the enzyme or
lead to overexpression of the enzyme,
both of which can make the fungus
resistant to treatment
• Overexpression of drug efux pumps
• Mutations in genes involved in
ergosterol synthesis or uptake
• Mutations in genes involved in the
cell wall stress response
• Mutation in the FKS1 gene, leading
to reduced binding afnity of
echinocandin drugs to the enzyme or
lead to overexpression of the enzyme
• Mutations in genes involved in
β-1,3-glucan synthesis or uptake
• Mutations in genes involved in the
cell wall stress response
• Mutation in gene encoding for
cytosine permease which uptakes
ucytosine into the fungal cell
• Mutations in genes that encode
enzymes, cytosine deaminase, which is
involved in the conversion of
ucytosine to 5-FU
A. Kumar etal.
(c) CDR1: A novel mutation (D709E) was found in the CDR1 gene of C. auris,
which encodes for an ABC efux pump, and contribute to polyene resistance
(Reslan etal. 2022).
(d) MEC3: A mutation in MEC3, a gene mainly known for its role in DNA damage
homeostasis, has been shown to further increase the polyene MIC (Carolus
etal. 2021).
It is to be noted that while these genes have been associated with polyene resistance
in C. auris, the exact mechanisms of resistance are complex and not fully understood. Resistance can also vary between different strains of C. auris.
Flucytosine is another antifungal drug that works by inhibiting the synthesis of
DNA and RNA in fungal cells (Bellmann and Smuszkiewicz 2017). It is taken up
into the cell by the cytosine permease, after which by the action of cytosine
8 Unravelling Drug Resistance in Candida Species: Genetic, Biolm…
deaminase (which is a fungal specic enzyme and not found in mammalian of bacterial cells) (Edlind and Katiyar 2010), the drug is converted to its active form,
5- uorouracil (5-FU). 5-FU gets into a competition with uracil to get incorporated
into the RNA strand during transcription, which results in blocking the RNA synthesis machinery and ultimately impairs protein synthesis inside the fungal cell
(Houst etal. 2020). In addition to this, DNA synthesis can also be halted as 5-FU
gets converted to uoro-deoxyuridylic acid thereby inhibiting thymidylate synthase,
causing DNA damage (Houst etal. 2020). However, some Candida species can
develop resistance to ucytosine, either by reducing the drug uptake or by altering
the target enzymes. This can limit the effectiveness of ucytosine in treating fungal
infections, especially when used alone. This is the prime reason why ucytosine as
a monotherapy is not advised in the case of Candida species (Malani and
Kauffman 2007).
Flucytosine primary resistance is common in Candid aglabrata, Candida krusei,
and Candida guilliermondii and is also found in some strains of Candida albicans,
Candida tropicalis and Candida parapsilosis (Cuenca-Estrella etal. 2001). These
strains may have mutations in the genes encoding cytosine deaminase, uracil phosphoribosyltransferase or uracil permease, which are involved in the activation and
transport of ucytosine into the fungal cell. Flucytosine is usually combined with
other antifungal agents, such as amphotericin B or azoles, as means to enhance its
efcacy and prevent resistance development against ucytosine. However, some
ucytosine-resistant strains of Candida have also shown cross-resistance to azoles,
limiting their use in chronic clinical conditions (Noel etal. 2003).
Understanding the molecular pathways and mechanisms involved in fungal
pathogenesis can reveal new drug targets. Investigating specic enzymes, receptors,
or regulatory proteins that play crucial roles in the fungal life cycle may provide
opportunities for targeted drug development. This includes targeting processes
involved in cell wall biosynthesis, signal transduction, and essential metabolic pathways. Research efforts should focus on the discovery and development of novel
antifungal compounds with unique mechanisms of action. Screening natural
sources, chemical libraries, and employing advanced computational methods can
aid in identifying compounds that are effective against drug-resistant fungal strains.
Combining existing antifungal drugs with other compounds or therapies can create
synergistic effects, enhancing overall efcacy and reducing the likelihood of resistance development. This approach may involve combination drug therapies, immunomodulation, or the use of adjuvants that potentiate the antifungal activity of
existing drugs.
211
8.3 Contribution ofLipids inDrug Resistance
inCandida Species
While antifungal agents serve as the primary treatment for Candida infections, their
efcacy is constrained by the rising prevalence of resistance in Candida species.
This resistance can arise from a complex interplay of intrinsic and acquired factors,
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A. Kumar etal.
encompassing variations in target enzymes, heightened expression or activation of
efux pumps, and alterations in membrane composition or permeability.
Signicantly, the role of lipids is pivotal in determining the susceptibility or resistance of Candida species to various types of antifungal treatments (Spampinato and
Leonardi 2013; Turner and Butler 2014). Lipids play a crucial role in biological
membranes by inuencing various aspects of their structure and function. These
include uidity, asymmetry, permeability, and interaction with proteins. Moreover,
lipids are involved in signalling pathways and metabolic processes that regulate cell
growth, differentiation, stress response, and adaptation. Different classes of lipids
exist, such as phospholipids, sterols, sphingolipids, and glycolipids. Each class
comprises distinct subtypes with unique chemical and physical properties. The
diverse lipid composition and metabolism found in Candida species reect their
remarkable ability to adapt to different environmental conditions and host niches
(Arendrup and Patterson 2017).
The main classes of antifungals used to treat Candida infections include poly-
enes, azoles, echinocandins, and ucytosine. Each class operates differently, and
targets are specic within the fungal cell. The susceptibility and resistance of
Candida species to these antifungal classes can be inuenced by various lipidrelated mechanisms. Polyenes have the amazing ability to bind to ergosterol, one of
the main components found in fungal membranes, and effectively create pores that
disrupt the membrane’s integrity and function. However, resistance to its effects can
arise from a decrease in ergosterol levels or alterations in its structure within the
membrane, which can be attributed to mutations or overexpression of genes involved
in ergosterol biosynthesis, like ERG1, ERG3, ERG6, ERG11, or ERG25. On the
other hand, resistance can also stem from heightened membrane uidity or asymmetry that hinders the binding or insertion of polyenes (Fig.8.3). This can be caused
by changes in the distribution or composition of phospholipids or sphingolipids
within the membrane. Azoles act by inhibiting the activity of Erg11p; this results in
the build-up of harmful sterol by-products and a decrease in the amount of ergosterol present in the fungal membrane. Changes in the composition or permeability
Fig. 8.3 ERG3 mutation substitutes the ergosterolin membrane with alternative non-toxic sterols
8 Unravelling Drug Resistance in Candida Species: Genetic, Biolm…
213
of the membrane caused by mutations can also impact the uptake and retention of
azoles within the cell. Furthermore, the key role of ABC transporter proteins such
as CaCdr1p in developing drug resistance is well-established. These transporters
have a preference for lipid raft microdomains in the membrane, where they interact
strongly with components like sphingolipids and ergosterol. When these raft components are altered, this can directly inuence the susceptibility of C. albicans to
antifungal drugs. Specically, changes in these components can impact the localization of these transporter proteins, ultimately affecting the effectiveness of treatment
(Pasrija etal. 2005, 2008; Mukhopadhyay etal. 2004).
The lipid composition of Candida species varies depending on their species,
strain, growth phase, and environmental conditions. Lipids have emerged as one of
the critical contributors in the acquisition of multidrug resistance (MDR) in Candida.
Interestingly, the comparative lipidomics of Candida species revealed that there is
no typical lipid marker which separates them, rather each Candida strain has a typical lipid imprint (Prasad and Singh 2013). A detailed lipidomic study of human
pathogenic fungi C. auris revealed that it contains 5 major lipid groups: phosphoglycerolipids (PGLs), sphingolipids (SLs), sterols, diacylglycerols (DAGs), and
triacylglycerols (TAGs), along with their molecular lipid species, and were able to
identify 582 different lipid species belonging to the ve major lipid group mentioned above (Shahi etal. 2020). Using lipidomics, they were also able to elucidate
the difference in the lipid composition of an azole susceptible and an azole-resistant
C. auris strain, gaining some crucial insights into the role of various lipid species in
azole drug resistance of C. auris. Some of the notable differences were the molecular lipid imprint of PGLs, which shows a stark difference between the azole susceptible and resistant strain, in addition to, azole-resistant strain showing elevated
levels of polar lipids. Also, the azole-resistant clinical strain was reported to have a
higher unsaturation index and free ergosterol (both ergostatetranol and ergosterol)
content, when in comparison with the azole susceptible strain (Shahi etal. 2020).
To sum up lipids play an inevitable role, in determining the susceptibility and resis-
tance of Candida species to types of antifungal treatments. They can inuence aspects
of membrane structure and function ultimately impacting how antifungals work and
where they target. Lipids also are involved in signalling pathways and metabolic processes that regulate the expression or activity of enzymes and transporters related to
resistance. Therefore, gaining an understanding of lipidomics in Candida species and
their interactions with antifungals can offer insights into the mechanisms behind antifungal resistance. This knowledge can pave the way for strategies or approaches to
combat resistance by developing antifungal agents or methods.
8.4 Role ofEfflux Pumps inDrug Resistance
In drug-resistant human fungal pathogens, efux pump upregulation contributes signicantly to their resistance. Nevertheless, various groups of transporters play a pivotal role in mitigating antifungal effects by actively participating in drug efux from
the cellular interior (Shapiro etal. 2011). While diverse transporter groups exist in the
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A. Kumar etal.
cell membrane of Candida species. The efux of drugs by the transporters is inuenced by two primary factors: the upregulation of efux pump expression and heightened transporter activity (Bachmann etal. 2002). Under drug treatment conditions,
these transporters become activated, actively expelling drugs, aiding the fungus in
managing toxicity and adapting to therapeutic challenges (Albertson etal. 1996). In
Candida isolates resistant to azoles, a prevalent trend is the increased expression of
genes related to drug efux pumps, originating primarily from two major superfamilies: ATP-binding cassette (ABC) and major facilitator superfamily (MFS) (Schubert
etal. 2011). These efux pumps play a pivotal role in expelling drugs, reducing the
intracellular concentration of uconazole and thereby contributing to infection persistence (Albertson et al. 1996). Both ABC (ATP-binding cassette) and MFS (major
facilitator superfamily) efux pumps are crucial contributors to multidrug resistance
(MDR) in yeast (Albertson etal. 1996; Holmes etal. 2016). ABC efux pump proteins employ energy from the hydrolysis of ATP to drive the efux of drugs (Schubert
etal. 2011). In the context of azole resistance, the MFS class is also implicated, as it
relies on the proton motive force to expel substrates from the fungal cell membrane.
ABC superfamily transporters are primary active transporters, while MFS is a secondary carrier transporter. The ABC group of transporters, notably CDR1, CDR2, and
SNQ2, actively plays a crucial role in azole drug resistance (Fig.8.4) (Mukherjee etal.
2003; Ramage 2002). Various drug efux pumps, such as PDR5 (pleiotropic drug
resistance), SNQ2 (sensitivity to 4- nitroquinoline N-oxide), and YOR1 (yeast oligomycin resistance), among other Candida species, as well play important role in drug
resistance (Harris etal. 2021; Oliveira etal. 2001).
Fig. 8.4 Efux pumps play a crucial role in expelling drugs, leading to a decrease in intracellular
drug concentration and consequently contributing to the persistence of infections. Multidrug resistance (MDR) in Candida is signicantly inuenced by both ATP-binding cassette (ABC) and
major facilitator superfamily (MFS) efux pumps. (a) Elevated expression of drug transporters,
governs resistance to azole drugs in Candida species. (b) ABC transporters (CDR1 and CDR2) and
MFS transporters (MDR1) play key roles in intracellular-drug efux across the cell membrane
8 Unravelling Drug Resistance in Candida Species: Genetic, Biolm…
215
8.4.1 ATP-Binding Cassette (ABC) Transporters
The ATP-binding cassette (ABC) superfamily is renowned for its role in facilitating
the import and export of ions and molecules in bacteria. However, their signicance
as drug transporters is now rmly established, extending even to eukaryotes (Cannon
etal. 2009). The diverse range of xenobiotic compounds that the ABC family can
export contributes signicantly to clinical resistance against Candida species. In
Candida species, the upregulation of the ABC efux pump is a pivotal factor in the
development of drug resistance, particularly evident with the azole class of antifungals (Bauer etal. 1999). These transporters typically consist of two transmembrane
domains (TMD) and two nucleotide-binding domains (NBD), which are essential
features of the ABC group. The NBD domains play a crucial role in facilitating
substrate movement across the membrane through ATP hydrolysis (Decottignies
and Goffeau 1997; Sipos and Kuchler 2006).
The well-characterized ABC proteins in Candida albicans, including Cdr1p,
Cdr2p, Cdr3p, and Cdr4p, have garnered attention due to their roles as drug transporters (Sanglard etal. 1995). Cdr1 and Cdr2 deletion strains of C. albicans exhibited higher accumulation of uconazole compared to the wild-type host, establishing
a direct connection between uconazole accumulation and efux mediated by these
transporters. Overexpression of CaCDR1 and CaCDR2 is commonly observed in
azole-resistant clinical isolates of C. albicans, in oral, systemic, and vaginal contexts alongside, higher expression of CaFLU1 and CaPDR16 has been observed in
azole-resistant isolates (Schubert etal. 2011; Ramage 2002; Cannon etal. 2009).
C. auris exhibits numerous ABC and MFS orthologs of transporter genes, like
Candida albicans (Chowdhary etal. 2017). Phylogenetic studies have conrmed
the presence of homologs for Snq2, Mdr1, Cdr1/Cdr2, and Cdr4 in C. auris
(Morschhäuser etal. 2007). The relationship between efux pump transporters and
azole drug resistance in C. auris is of paramount importance. Azole-resistant clinical isolates indicate an elevated expression of Cdr1 drug pumps in C. auris and its
deletion results in an eightfold increase in susceptibility to azole in C. auris (Adams
etal. 2018; Chowdhary etal. 2017; Horton and Nett 2020).
The CaCDR1 and CaCDR2 are under the regulation of the transcription factor
CaTac1p, while the regulation of CaMDR1 is mediated by the transcription factor
CaMrr1p (Harris etal. 2021; Bhattacharya et al. 2020). Both transcription factors
Tac1 and Mrr1 belong to the zinc-cluster transcription factors (Zn2-Cys6) (Dunkel
et al. 2008a; Liu et al. 2018). Mutant strains in tac1, characterized by gain-offunction mutations, result in the constitutive overexpression of CDR1 and CDR2
(Coste etal. 2006). Similarly, activating mutations in MRR1 leads to an upregulation of MDR1 (Dunkel etal. 2008b). In particular, resistance in clinical isolates has
been associated with various gain-of-function (GOF) mutations in CaTAC1, such as
T225A, V736A, N972D, N977D, G980E, and G980W, and in CaMRR1, including
P683S and P683H (Dunkel etal. 2008a; Coste etal. 2006).
The impact of gain-of-function mutations extends to the homologs of Tac1b and
Mrr1a in C. auris, providing uconazole resistance. For example, an extensive
study involving 304 clinical isolates of C. auris worldwide, each representing one
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A. Kumar etal.
of the four major genetic clades, identied three common TAC1b mutations: A640V
in clade Ic, A657V in clade Ib, and F862_N866del substitutions in clade IV (Rybak
etal. 2019). These mutations in TAC1b were found to signicantly contribute to
azole resistance across various clades, as demonstrated by another TAC1b mutation
(encoding S611P) associated with improved uconazole resistance in clade-IV clinical isolates (Li etal. 2021). This evidence underscores the role of TAC1 mutations
in fostering resistance to azoles across diverse clinical strains.
In C. glabrata, the primary mechanism driving resistance to azole involves the
upregulation of ABC transporters, specically CDR1, CDR2, SNQ2, and PDH1
(Tsai etal. 2006). Additionally, gain-of-function (GOF) mutations in the transcription factor CgPdr1 (pleiotropic drug resistance) induce the expression of CgCDR1,
CgCDR2, Cg SNQ2, and Cg PDH1 (Ferrari etal. 2009). These GOFs encompassing
D261G, L280F, R293I, S316I, L328F, LS343F, R376G, G583S, Y584C, T588A,
R592S, T607S, N691D, D876Y, D1082G, and E1083Q (Ferrari etal. 2009). These
GOFs are crucial in elevating resistance to uconazole (Ferrari et al. 2009).
Moreover, the increased expression of the efux pump also exhibits a correlation
with heightened azole resistance in C. parapsilosis and C. dubliniensis (Silva
etal. 2012).
Fluconazole tolerance is also linked to the overexpression of MDR1in C. albi-
cans, where activating single nucleotide polymorphisms (SNPs) in Mrr1 results in
azole resistance (Liu and Myers 2017). While Flr1 shares homology with C. albi-
cans CDR1, it has not demonstrated a signicant role in azole resistance (Chen etal.
2007). These ndings underscore the diverse molecular mechanisms at play in the
regulation of drug resistance, shedding light on the intricate interplay of transcriptional activators, genetic mutations, and transporter expression.
Efux transporters have also been studied in C. auris biolms, revealing their
contribution to biolm-mediated resistance (Horton and Nett 2020). Several transcriptional factors, including Tac1, and Mrr1, and their activation and constitutive
expression networks, play a crucial role in regulating the efux mechanism under
different drug treatment conditions (Dunkel etal. 2008b). These mechanisms contribute to drug resistance in clinical isolates of C. auris strains (Adams etal. 2018).
The role of mitochondria in efux pump-mediated azole resistance in C. gla-
brata is noteworthy. There are many mitochondrial membrane-localized proteins
such as ABC and Atm1 protein in iron homeostasis as well (Kispal etal. 1997). The
mitochondrial system indirectly regulates the calcium signalling and pleiotropic
drug resistance pathway thus, results in azole resistance, aligning with the upregulation of genes such as CgCDR1, CgPDH1, and others (Brown et al. 2014; Sun
etal. 2019).
Notably, even petite yeast strains, characterized by mitochondrial functional
abnormalities, exhibit upregulated ABC transporters. This adaptive response
enhances their tness and amplies resistance to azoles in C. glabrata (Ferrari
etal. 2009).
ABC transporters in fungus possess distinct domains dedicated to interacting
with the membrane as well as ATP binding and hydrolysis (Higgins 2001). These
transporters exhibit remarkable promiscuity, binding to various substrates within
8 Unravelling Drug Resistance in Candida Species: Genetic, Biolm…
217
the expansive binding pocket, albeit with different binding residues (Prasad etal.
2015). This study has suggested that the ABC pumps have the presence of at least
three drug-binding sites in these transporters (Golin etal. 2003). The constitutive
expression of these transporters also plays a crucial role in shaping the drug resistance pattern (Prasad etal. 2006). Overall, these ndings highlight the importance
of understanding the molecular mechanisms involving ABC transporters in Candida
species to address and combat antifungal resistance effectively.
Previously, there was limited understanding regarding the expression of efux
pumps in the context of echinocandins and polyenes. The uncertainty stemmed
from the fact that these drugs primarily act on the outer surface of the plasma membrane, and it was unclear whether efux transporters regulated them, given their
large size and hydrophobic nature (Cannon etal. 2009; Perfect 2017; Robbins etal.
2016). The speculation arose due to the unsuitability of these drugs to bind in the
transporter’s binding pocket. However, recent reports have shed light on the potential involvement of efux pumps in the case of Amphotericin B (Cannon etal.
2009). It has been conrmed that there are few non-synonymous mutations in the
putative membrane transporters of C. auris clinical isolates. Nevertheless, a comprehensive study is required to elucidate how these mutations contribute to clinical
isolates gaining drug resistance through efux pump mechanisms (Escandón
etal. 2019).
8.4.2 Major Facilitator Superfamily (MFS) Transporter
The major facilitator superfamily (MFS) represents one of the two most extensive
groups of membrane transporters found across various domains of life, encompassing bacteria, archaea, and eukaryotes (Pao etal. 1998). MFS transporters, classied
as secondary active transporters, are recognized for their role in exporting a diverse
range of substrates (Decottignies and Goffeau 1997). These include organic and
inorganic ions, drugs, metabolites, neurotransmitters, nucleosides, amino acids, and
various intermediates, functioning as uniporters, symporters, or antiporters (Pao
etal. 1998). The MFS superfamily stands out as the largest family of secondary
active membrane carriers, and its transporters exhibit a remarkable diversity in their
capabilities to transport various substrates (Decottignies and Goffeau 1997; Pao
etal. 1998). These substrates range from small molecules, encompassing organic
and inorganic ions, to more complex biomolecules like peptides and lipid components. Within the MFS superfamily, certain members of the drug/H+ antiporter family function as multidrug transporters, utilizing proton-driven downhill transport to
expel their respective substrates (Calabrese etal. 2000).
Typically, MFS transporter proteins consist of 12 or 14 putative transmembrane
segments (TMS), with lengths varying between 400 and 600 amino acids (Saier
1998, 1999). These single-polypeptide secondary carriers transport small solutes by
responding to chemiosmotic ion gradients (Cannon etal. 2009). Members of the
MFS superfamily are ubiquitous in all domains of life, holding direct therapeutic
and pharmacological signicance. They exhibit specicity for a wide array of
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A. Kumar etal.
substances, including sugars, polyols, drugs, neurotransmitters, metabolites, phosphorylated glycolytic intermediates, and amino acids.
While the MFS superfamily is widespread, only a limited subset of MFS trans-
porters, such as MDR1, FLU1, NAG3, NAG4, JEN1, ARN1, and NGT1, have been
identied and studied in C. albicans thus far (Pasrija etal. 2007; Sengupta and
Datta 2003).
The MFS family transporters MDR1 and FLU1 are recognized for their role in
exporting antifungal compounds. Notably, MDR1, belonging to the DHA1 MFS
transporter group, has been implicated in conferring resistance to uconazole and
ketoconazole (Pasrija etal. 2007). Upregulation of MDR1 in C. albicans has also
been associated with resistance to brefeldin A and cerulenin. Structural and functional analyses of MDR1 have highlighted a critical region, TMS5, responsible for
drug/H+ trafcking (Calabrese etal. 2000).
Among the C. albicans MFS, MDR1 and its alleles are identied as the sole drug
efux pump transporters.MDR1 was initially found to confer resistance against the
tubulin-binding drug benomyl and the tetrahydrofolate reductase inhibitor methotrexate (Becker et al. 1995; Gupta et al. 1998). MDR1 overexpression has been
linked to azole resistance in C. albicans. Expression of MDR1 in S. cerevisiae
imparts resistance to various unrelated compounds. Notably, methotrexate, benomyl, and several unrelated drugs induce increased expression of MDR1 in C. albi-
cans cells, with some azole-resistant clinical isolates demonstrating heightened
MDR1 expression (Ben-Yaacov etal. 1994; Fling etal. 1991).
FLU1, another DHA1 MFS gene in C. albicans, functions as a transporter, as
evidenced by increased susceptibility to mycophenolic acid upon FLU1 deletion. In
a study generating 21 itraconazole-resistant mutants through mutagenesis, approximately 50% of these mutants exhibited resistance due to the overexpression of
MDR3, a DHA2 family MFS-type transporter (Anon etal. 2018).
The Drug: H+ Antiporter-1 (DHA1) family comprises members that include
MDR and drug-specic efux pumps, widely distributed across various organisms.
Subsequent research uncovered its role in resistance to benzotriazoles, cycloheximide, and sulfometuron methyl (Ben-Yaacov et al. 1994; Goldway et al. 1995).
Disruption of the MDR1 gene decreased the pathogenicity of C. albicans
(Morschhäuser etal. 2007). The Drug: H+ Antiporter-2 (DHA2) family of drug: H+
antiporters in C. albicans consists of nine members sharing signicant sequence
similarity with other transport proteins. These DHA2 proteins, particularly ATR1,
have been associated with conferring resistance to structurally dissimilar chemicals
like aminotriazole and 4-nitroquinolone-N-oxide (Gömpel-Klein and Brendel 1990;
Kanazawa etal. 1988). Additionally, SGE1 is connected to resistance against ethidium bromide and crystal violet (Amakasu etal. 1993; Goffeau etal. 1997). While
no member of the DHA2 family has been identied in C. albicans, insights from the
study of SGE1 and ATR1in S. cerevisiae provide valuable information about poten-
tial functions and roles within this family (Kanazawa et al. 1988; Amakasu
etal. 1993).
Additionally, other members of the DHA1 family in C. albicans, such as NAG4,
NAG3, and, FLU1, have been identied. The disruption of FLU1 leads to
8 Unravelling Drug Resistance in Candida Species: Genetic, Biolm…
219
mycophenolic acid hypersensitivity, indicating that this substance may be the suitable substrate for the drug transporter (Calabrese et al. 2000). Whereas, NAG3
(TMP1) and NAG4 (TMP2) exhibit sensitivity to various compounds, including
cycloheximide and show upregulation specically (Sengupta and Datta 2003).
Furthermore, their expression increases in response to these drugs, indicating their
potential role as diverse drug efux pumps. While FLU1, NAG3, and NAG4 are
members of the DHA1 family, none, except MDR1, has been specically linked to
MDR in C. albicans, making MDR1 a clinically signicant efux pump protein
(Silva etal. 2009). Among the MFS transporters, Mdr1 is extensively identied and
stands out as a signicant multidrug transporter, particularly playing a crucial role
in clinical azole resistance within the DHA1 family (Kohli etal. 2001). The overexpression of MDR1 is frequently observed in azole-resistant clinical isolates, and it is
closely linked with the development of drug resistance (Pasrija etal. 2007). Among
all DHA1 family members, MDR1 is notably the most signicant in terms of its
impact on clinical azole resistance (Gaur etal. 2008).
The upregulation of MDR1 is controlled by various transcription factors interact-
ing with the gene’s promoters. Transcription factors such as Cph1, Mrr1, Upc2, and
Mcm1 play a crucial role in this process, contributing to the upregulation of Mdr1
proteins in clinically resistant Candida strains (Sasse et al. 2011). These factors
exert both positive and negative regulation, with Cph1 and Cap1 demonstrating
negative regulation, while Mrr1, Upc2, and Mcm1 exert positive regulatory effects
(Schubert etal. 2011). MRR1 (multidrug resistance regulator 1) plays a signicant
role in regulating the expression of MDR1. In C. albicans, it exhibits resistance to
uconazole (Dunkel etal. 2008a). Notably, a gain-of-function (GOF) mutation in
Mrr1 is associated with increased MDR1 expression (Dunkel et al. 2008a;
Morschhäuser etal. 2007). These mutant variants can activate MDR1 expression
even in drug-sensitive clinical isolates. Additionally, Upc2 mutant variants are also
capable of triggering MDR1 upregulation (Schubert etal. 2011).
The targeting drug efux pumps in Candida species present a promising avenue
for combatting fungal infections and overcoming drug resistance challenges.
Exploring and harnessing drug efux pump mechanism for therapeutic interventions can lead to novel strategies with potential clinical impact. The development of
specic and potent inhibitors targeting the drug efux pumps in Candida by designing compounds that selectively block or modulate the activity of these pumps could
enhance the efcacy of existing antifungal drugs. This approach would not only
overcome resistance mediated by efux pumps but also potentiate the action of
conventional antifungal agents, offering a dual benet in the treatment of Candida
infections.