Critical Reviews in Microbiology

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Candida albicans targets that potentially synergize

with fluconazole

Hui Lu, Manjari Shrivastava, Malcolm Whiteway & Yuanying Jiang

To cite this article: Hui Lu, Manjari Shrivastava, Malcolm Whiteway & Yuanying Jiang (2021)
Candida�albicans targets that potentially synergize with fluconazole, Critical Reviews in
Microbiology, 47:3, 323-337, DOI: 10.1080/1040841X.2021.1884641

To link to this article: https://doi.org/10.1080/1040841X.2021.1884641

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CRITICAL REVIEWS IN MICROBIOLOGY
2021, VOL. 47, NO. 3, 323–337
https://doi.org/10.1080/1040841X.2021.1884641

REVIEW ARTICLE

Candida albicans targets that potentially synergize with fluconazole

Hui Lua, Manjari Shrivastavab, Malcolm Whitewayb and Yuanying Jianga
a
Department of Pharmacology, Shanghai Tenth People’s Hospital, Tongji University School of Medicine, Shanghai, China; bDepartment
of Biology, Concordia University, Montreal, QC, Canada

ABSTRACT ARTICLE HISTORY

Fluconazole has characteristics that make it widely used in the clinical treatment of C. albicans Received 15 August 2020
infections. However, fluconazole has only a fungistatic activity in C. albicans, therefore, in the Revised 11 January 2021
long-term treatment of C. albicans infection with fluconazole, C. albicans has the potential to Accepted 29 January 2021
acquire fluconazole resistance. A promising approach to increase fluconazole’s efficacy is identify- Published online 15 February
2021
ing potential targets of drugs that can enhance the antifungal effect of fluconazole, or even
make the drug fungicidal. In this review, we systematically provide a global overview of potential KEYWORDS
targets of drugs synergistic with fluconazole in C. albicans, identify new avenues for research on Fluconazole; synergistic
fluconazole potentiation, and highlight the promise of combinatorial strategies with fluconazole targets; Candida albicans
in combatting C. albicans infections.

Introduction genes that have synthetic lethality with Erg11 (Parsons

Candida albicans is an opportunistic yeast that causes
diseases ranging from superficial and mucosal infec-
tions to severe widely disseminated and bloodstream
infections in immunocompromised individuals that
include AIDS patients, cancer patients, and organ trans-
plant patients (Enoch et al. 2017). Infections caused by
C. albicans are frequently treated with azole drugs that
inhibit ergosterol biosynthesis. As a representative anti-
fungal azole, fluconazole has the useful characteristics
of a wide antifungal spectrum, low toxic effects, and
high bioavailability, and is widely used in the clinical
treatment of C. albicans infections. However, flucon-
azole is a fungistatic drug that inhibits growth but does
not kill the pathogenic fungus, thereby providing the
opportunity for the development of fluconazole resist-
ance (Pristov and Ghannoum 2019; Berman and Krysan
2020). Given the utility of fluconazole in treating fungal
infections, there is considerable interest in preventing
fluconazole resistance, and one promising strategy is to
increase the efficacy of the drug against C. albicans.
A promising strategy to increase the efficacy of flu-
conazole is identifying targets of synergistic drugs that
can enhance the antifungal effect of fluconazole, or
even curb the emergence of fluconazole resistance by
making fluconazole fungicidal. There are more than 400
et al. 2004; Davierwala et al. 2005) and at least 75 genes
have synthetic lethality with fluconazole in S. cerevisiae
(Parsons et al. 2004; Jansen et al. 2009). These provide
useful clues to identify synergistic targets of fluconazole
in C. albicans. For example, the identification of GCS1 as
an S. cerevisiae gene (ortholog in C. albicans is the AGE3
gene) whose inactivation leads to lethality when
coupled with fluconazole treatment allowed the subse-
quent characterization that Brefeldin A, which acts on
the same circuit in C. albicans, could act synergistically
with fluconazole in the pathogen (Epp, Vanier,
et al. 2010).
In this review, we have systematically identified
those genes (total 220 genes, including 96 genes from
our studies) whose inactivation enhanced C. albicans
response to fluconazole (Table S1). We then investi-
gated pathway and process enrichment analysis to link
these genes into several biological processes (Figure 1,
Table S2). Finally, we evaluated the possibility of these
biological processes becoming synergistic fluconazole
targets to potentiate the activity of fluconazole for the
treatment of C. albicans infections. This review provides
global potential targets synergistic with fluconazole,
and highlights the promise of combinatorial strategies
with fluconazole in combatting C. albicans infections

CONTACT Yuanying Jiang jiangyy@tongji.edu.cn Department of Pharmacology, Shanghai Tenth People’s Hospital, Tongji University School of
Medicine, No.1239 Siping Road, Shanghai 200092, China; Malcolm Whiteway malcolm.whiteway@concordia.ca Department of Biology, Concordia
University, Montreal, QC, Canada
Supplemental data for this article can be accessed here.
ß 2021 Informa UK Limited, trading as Taylor & Francis Group
324 H. LU ET AL.
Figure 1. Terms of GO biological processes of fluconazole synergistic genes. Starting with the 220 genes identified through sys-
tematic searches, GO biological processes enrichment analysis was carried out. All genes in the C. albicans genome have been
used as an enrichment background. Terms with a p-value <0.05 and a minimum count of 2 were collected and grouped into
clusters based on their membership similarities.
Ergosterol biosynthetic process
Ergosterol is a significant component of the cell mem-
brane of C. albicans and plays an important role in
maintaining membrane integrity and fluidity and ensur-
ing proper function of some membrane-bound
enzymes. Fluconazole blocks the ergosterol biosyn-
thetic pathway in both Saccharomyces cerevisiae
(Sagatova et al. 2015) and C. albicans (Warrilow et al.
2010; Hargrove et al. 2017), by directly inhibiting lano-
sterol 14a-demethylase (Erg11), the enzyme that cata-
lyzes the oxidative removal of the 14a-methyl group
from lanosterol. The activity of the Erg11 requires the
formation of a membrane-bound complex with Ncp1
(Lamb et al. 1999), which is an NADPH-dependent cyto-
chrome P450 reductase and acts as an electron donor.
A C. albicans strain losing one copy of the NCP1 gene
shows increased sensitivity to fluconazole, potentially
through reduced activity of Erg11 (Xu et al. 2007).
Squalene epoxidase (Erg1) is another key enzyme in the
ergosterol biosynthetic process and represents the tar-
get of terbinafine. A conditional ERG1 gene mutant
strain (MET3p-ERG1/erg1D) displayed increased sensitiv-
ity to fluconazole under repressing conditions (Pasrija
et al. 2005). Furthermore, the combination of terbina-
fine with fluconazole has shown some promising results
against C. albicans (Barchiesi et al. 1997; Khodavandi
et al. 2014). C. albicans strains deleted for the C-8 sterol
isomerase (Erg2) and the C-4 sterol methyl oxidase
(Erg251) also were reported to show increase sensitivity
to fluconazole (Mukhopadhyay et al. 2004; Chen et al.
2018). The NSG2 gene is proposed to be involved in the
regulation of C14-methylated sterol biosynthesis in C.
albicans. Our recent study showed that a C. albicans
strain lacking the gene NSG2 displayed increased sensi-
tivity to fluconazole (Chen et al. 2018). This was con-
firmed by our independent study showing the
homozygous deletion of the NSG2 gene in C. albicans
enhanced the therapeutic efficacy of fluconazole
in vitro and in vivo (Lv et al. 2018). Taken together, since
strains with decreased levels of Ncp1, Erg1, Erg2,
Erg251, and Nsg2 increase sensitivity to fluconazole,
these enzymes involved in ergosterol biosynthesis may
be potential synergistic targets with fluconazole against
C. albicans (Figure 2). However, reduction of some
enzymes involved in ergosterol biosynthesis, such as
Erg3 (Sanglard, Ischer, Parkinson, et al. 2003; Chen et al.
2018; Luna-Tapia et al. 2018), Erg4 (Chen et al. 2018),
and Erg6 (Xu et al. 2007; Chen et al. 2018) were not
found to increase the efficacy of fluconazole.
Upc2 is a global transcriptional factor of the ERG
genes (Znaidi et al. 2008; Flowers et al. 2012), and Upc2

is frequently modified in fluconazole-resistant clinical
isolates. Increased fluconazole susceptibility was
reported in UPC2 deletion strains (Silver et al. 2004;
MacPherson et al. 2005), and gain of function UPC2
alleles trigger overproduction of Erg11 and ultimately
lead to fluconazole resistance (Flowers et al. 2012). Efg1
is directly involved in the ergosterol biosynthetic pro-
cess through negative transcriptional regulation of the
ERG3 gene (Lo et al. 2005). A strain containing a homo-
zygous deletion of the EFG1 gene shows an enhanced
susceptibility to fluconazole that may be caused by
overexpression of the ERG3 gene and consequent accu-
mulation of toxic sterols (Prasad et al. 2010). A third
ergosterol biosynthesis-related transcription factor,
Ndt80, also plays a role in fluconazole resistance in C.
albicans (Sellam, Tebbji, et al. 2009). A C. albicans strain
lacking the NDT80 gene showed increased sensitivity to
fluconazole (Chen et al. 2004; Homann et al. 2009;
Sellam, Tebbji, et al. 2009; Vandeputte et al. 2012).
These results suggest that Upc2, Efg1, and Ndt80, as
transcriptional regulators of ergosterol biosynthesis-
related genes, might be potential synergistic flucon-
azole targets in C. albicans (Figure 2).
Biofilm formation and filamentous growth
One of the main characteristics of C. albicans biofilms is
that they display innate resistance to currently available
antifungal drug classes, except for the echinocandins
(Hawser and Douglas 1995; Tournu and Van Dijck 2012;
Figure 2. Genes from the pathway of ergosterol synthesis might be potential synergistic targets of fluconazole against C. albicans
infection.
CRITICAL REVIEWS IN MICROBIOLOGY 325
Mathe and Van Dijck 2013; Zarnowski et al. 2018; de
Barros et al. 2020). Candida albicans biofilm formation
can be divided into four major phases: adhesion, prolif-
eration, maturation, and dispersal (Lohse et al. 2018;
Wall et al. 2019). In the adhesion phase, yeast cells
adhere to host tissues or material surfaces and form a
basal layer that will anchor the C. albicans biofilm to
the surface. Deletion strains of regulators Bcr1, Cas5,
Dal81, Def1, Mrr2, and Snf5, are all defective for adher-
ence during biofilm formation (Finkel et al. 2012), and
these mutations lead to increased sensitivity to flucon-
azole in C. albicans (Homann et al. 2009; Vandeputte
et al. 2012; Desai et al. 2013; Chen et al. 2018). This sug-
gests that an impaired adhesion process is beneficial
for fluconazole potentiation.
Adherence is followed by a proliferation phase,
which is characterized by the initiation of hyphae for-
mation. Ergosterol synthesis plays an important role in
hyphae formation because disruption of genes involved
in ergosterol synthesis resulted in hyphal growth
defects, and azoles inhibited hyphal growth (Odds et al.
1985; Umebayashi and Nakano 2003; O’Meara et al.
2015). It appears that hyphal defects may enhance the
efficacy of fluconazole, for filamentous-growth-related
deletion mutant strains for genes such as HDA1, GPI19,
MNN2, and SPT6 show increased sensitivity to flucon-
azole (Victoria et al. 2010; Li, Cai, et al. 2015; Chen et al.
2018; Caldara and Marmiroli 2020). Ace2 is a transcrip-
tion factor involved in the regulation of morphogenesis
(Kelly et al. 2004) and is required for normal biofilm
326 H. LU ET AL.
formation in normoxia (Stichternoth and Ernst, 2009). It
is reasonable that deletions of genes involved in regula-
tion of Ace2 and morphogenesis (RAM) network, such
as the CBK1, KIC1, CAS4, MOB2, and SOG2 genes, caused
increased sensitivity to fluconazole (Song et al. 2008;
Chen et al. 2018). However, although Nrg1 and Tup1
repress expression of hypha-specific genes and fila-
mentous growth of C. albicans, deletions of the NRG1
and TUP1 genes caused predominantly hyphal growth
(Braun et al. 2001; Murad et al. 2001) and fluconazole
hypersensitivity in C. albicans (Homann et al. 2009). This
suggested that deletions of the NRG1 and TUP1 genes
enhanced sensitivity to fluconazole by influencing other
biological processes as discussed later, and not by
affecting the filamentous growth process. Genes
involved in hyphal growth in suspension cultures are
also required for proper biofilm formation. In the bio-
film phase, this process is mainly mediated by the Bcr1,
Efg1, and Ndt80 transcription factors involved in both
regulation of the morphological transition (Nobile et al.
2012) and in fluconazole response (Chen et al. 2004; Lo
et al. 2005; Homann et al. 2009; Sellam, Tebbji, et al.
2009; Prasad et al. 2010; Desai et al. 2013; Chen et al.
2018). Although the effect of hyphal defects on ergos-
terol synthesis remains elusive, targeting the filament-
ous growth process may aid in fluconazole
potentiation.
The maturation phase occurs when the hyphal yeast
scaffold produces exo-polymeric substances that essen-
tially act as an adhesive to hold the entire biofilm archi-
tecture. A well-known matrix component, b-1,3 glucan,
is known to provide resistance against fluconazole by
sequestering the drug (Nett et al. 2010b). Smi1 acts
through a positive regulator of matrix production,
Rlm1, to govern the expression of the GSC1 gene
encoding glucan synthase (Nett et al. 2011). As well, the
chaperone Hsp90 may affect the expression or activity
of the GSC1 gene, perhaps through the Smi1-Rlm1
pathway (Robbins et al. 2011). The BGL2 gene, encoding
a glucan transferase, the PHR1 gene, encoding a cell
surface glycosidase, and the XOG1 gene encoding exo-
1,3-b-glucanase are responsible for the delivery and
arrangement of b-1,3 glucan in the matrix (Nett et al.
2010a; Taff et al. 2012). Deletion strains of the BGL2,
HSP90, GSC1, PHR1, XOG1, RLM1, and SMI1 genes dis-
played increased sensitivity to fluconazole (Sarthy et al.
1997; Canton et al. 2005; Cowen et al. 2009; Nett et al.
2011; Robbins et al. 2011; Taff et al. 2012). This suggests
that targeting C. albicans biofilm matrix-associated
genes/proteins might lead to increased efficacy of
fluconazole.
The dispersal phase is the last phase of biofilm for-
mation and is characterized by the dispersal of yeast
cells and/or pieces of the biofilm from the body of the
biofilm. Nrg1 is a key regulator that is involved in the
dispersion of cells from the biofilm (Uppuluri et al.
2010; Uppuluri et al. 2018; Wall et al. 2019). Hsp90 has
also been implicated in C. albicans biofilm dispersal, as
depletion of Hsp90 leads to hyper-filamentation, and
markedly reduces the number of dispersed cells from a
biofilm (Robbins et al. 2011) As discussed above, strains
lacking the NRG1 and HSP90 genes showed increased
sensitivity to fluconazole (Cowen et al. 2009; Homann
et al. 2009; Robbins et al. 2011). This suggests that tar-
geting C. albicans biofilm dispersal genes might also
lead to increased efficacy of fluconazole.
Ion homeostasis
Dysfunction of ion homeostasis may increase sensitivity
to fluconazole based on our GO analysis, as ions partici-
pate in membrane potential maintenance in C. albicans.
Therefore, strategies that target ion homeostasis regula-
tion may serve to uncover new antifungal combinations
interacting with fluconazole. We identified genes
involved in the regulation of hydrogen (Hþ), calcium
(Ca2þ), iron (Fe3þ), and zinc (Zn2þ) ions that influenced
response to fluconazole in C. albicans (Figure 3).
Adjustment of pH of the medium can eliminate flu-
conazole tolerance of C. albicans (Marr et al. 1999;
Rosenberg et al. 2018), suggesting that Hþ homeostasis
links to fluconazole sensitivity. Environmental pH is
sensed by three plasma membrane receptor proteins:
Rim9, Dfg16, and Rim21. Under alkaline pH conditions,
Rim8 is hyperphosphorylated, leading to endocytosis of
the membrane complex Rim9, Dfg16, and Rim21 and
recruitment of the endosomal sorting complexes
required for transport (ESCRT) I, II, and III (Wolf et al.
2010). Rim20 and Rim13 are then recruited, leading to
cleavage of the C-terminal inhibitory domain of Rim101
and generating a Rim101 active form (Garnaud et al.
2018). Mutant strains with defects in genes encoding
proteins that compose the ESCRT I, II, and III complexes
increased the sensitivity of C. albicans to fluconazole
(Cornet et al. 2006; Zarnowski et al. 2018). Mutants
missing the SNF7 and VPS20 genes, which play roles in
the proteolytic activation of Rim101, also showed
increased sensitivity to fluconazole (Cornet et al. 2006;
Zarnowski et al. 2018). While a strain lacking the RIM13
gene showed increased resistance to fluconazole (Chen
et al. 2018), a strain lacking the RIM101 gene exhibited
hypersensitivity to fluconazole (Davis, 2003; Cornet
et al. 2006; Baek et al. 2008; Homann et al. 2009;

Figure 3. Schematic diagram depicting the regulation of different ion systems, as well as the potential synergistic targets of flu-
conazole based on ion signalling pathways in C. albicans.
Garnaud et al. 2018). Because the Rim pathway is fungal
specific, it could provide an interesting target to syner-
gize with fluconazole in the treatment of C. albi-
cans infections.
While the environmental pH is mainly regulated by
the Rim signalling pathway, the intracellular pH is
mainly regulated by the V-ATPase, a master pH regula-
tor that participates in stress response and morphology
transitioning in C. albicans (Li et al. 2018). This multi-
subunit enzyme is made up of the V1 complex (the per-
ipheral membrane subunits responsible for hydrolyzing
ATP) and the V0 complex (the integral membrane pro-
teins acting as a proton transporter). The V1 complex
consists of eight subunits from V1A toV1H (Olsen, 2014),
while the V0 domain contains the six subunits V0a, V0c,
V0c0 , V0c", V0d, and V0e (Kane, 2016). Deletion strains of
the V1A, V1E, V1G, and V1H subunits showed increased
sensitivity to fluconazole (Epp, Vanier, et al. 2010; Jia
et al. 2015; Kim et al. 2019), as did a strain lacking the
VMA11 gene, which encodes the V0c0 subunit (Chen
et al. 2018; Weissman et al. 2008). The V0c’ subunit is a
fungal-specific subunit, which could make it a desirable
target for fluconazole adjuvants. These results sug-
gested that dampening the activity of V-ATPase could
serve as a strategy for enhancing the efficacy of flucon-
azole against C. albicans.
Synergistic effects of fluconazole and Ca2þchannel
blockers or Ca2þ chelator agents against C. albicans fur-
ther suggest that Ca2þ homeostasis plays an important
role in fluconazole sensitivity (Yu et al. 2013; Liu, Yue,
et al. 2016; Casalinuovo et al. 2017). When C. albicans is
targeted by fluconazole, Ca2þ flows into cells through
CRITICAL REVIEWS IN MICROBIOLOGY 327
the Cch1-Mid1 channel increasing intracellular Ca2þ
concentration and activating the calcium-calcineurin
signalling pathway, which is an important mechanism
for C. albicans to regulate fluconazole tolerance.
Deletion of the CCH1 gene or the MID1 gene individu-
ally or in combination causes hypersensitivity to flucon-
azole (LaFayette et al. 2010; Chen et al. 2018). Pmc1 is a
vacuolar calcium P-type ATPase and is crucial for regu-
lating intracellular Ca2þ concentration. A deletion strain
of the PMC1 gene exhibited increased resistance to flu-
conazole; this may be caused by Ca2þ released from
the vacuole resulting in an increased intracellular Ca2þ
concentration (Sanglard, Ischer, Marchetti, et al. 2003;
Luna-Tapia et al. 2019). Calcineurin is a protein phos-
phatase consisting of two subunits, a catalytic subunit
encoded by the CMP1 gene and a regulatory subunit
encoded by the CNB1 gene. Homozygous deletion of
the CMP1 gene (Sanglard, Ischer, Marchetti, et al. 2003;
Bader et al. 2006; LaFayette et al. 2010) and the CNB1
gene (Onyewu et al. 2004) in C. albicans caused hyper-
sensitivity to fluconazole. In fact, calcineurin inhibitors
have already been identified to synergize with flucon-
azole (Sanglard, Ischer, Marchetti, et al. 2003; Uppuluri
et al. 2008; Lee et al. 2018; Rosenberg et al. 2018). Crz1
is transported into the cell nucleus and generates toler-
ance to fluconazole, which can eventually lead to flu-
conazole resistance (Thewes, 2014). Similarly,
homozygous deletion of the CRZ1 gene mutant caused
increased sensitivity to fluconazole (Onyewu et al. 2004;
Bruno and Mitchell, 2005; Homann et al. 2009; Jia et al.
2009). Deletion of other calcium-calcineurin signalling
pathway-related genes, such as the RTA2 gene (Jia et al.
328 H. LU ET AL.
2009) and the RCN1 gene (Reedy et al. 2010), were also
reported to cause increased sensitivity to fluconazole.
Taken together, it appears designing new agents based
on dysregulation of Ca2þ homeostasis is a promising
direction for enhanced efficacy of fluconazole against
C. albicans.
Iron acquisition plays a crucial role in the process of
the transition from commensal behaviour to pathogen-
icity in C. albicans (Mamouei et al. 2017; Fourie et al.
2018). Fe3þ transport into the intracellular space occurs
through the high-affinity permeases encoded by the
FTR1, FTR2, FTH1, and FTH2 genes (Bairwa et al. 2017).
Deletions of FTR1 and FTR2 resulted in increased sensi-
tivity to fluconazole in C. albicans (Prasad et al. 2006),
while deletion of the FTH2 gene generated increased
resistance to the drug (Chen et al. 2018). Copper is an
essential component of the multicopper oxidase
responsible for iron uptake. It is interesting that dele-
tion mutants of the CCC2 gene encoding the copper
transporter also showed enhanced sensitivity to flucon-
azole (Prasad et al. 2006). Fe3þ deprivation could
increase fluconazole sensitivity, perhaps through a
decreased intracellular Fe3þ concentration increasing
membrane fluidity and permeability through decreased
ergosterol levels via repressed expression of the ERG1,
ERG2, ERG11 and ERG251 genes (Prasad et al. 2006;
Hameed et al. 2011). Zinc is another key ion for the
activity of enzymes such as superoxide dismutase and
metalloproteases which are important for C. albicans
virulence and survival. In C. albicans the uptake of Zn2þ
from the environment is mainly through the two trans-
porters Zrt1 and Zrt2 (Kim et al. 2008). Our recent drug
sensitivity screen showed that mutant strain missing
the ZRT2 gene showed increased sensitivity to flucon-
azole (Chen et al. 2018). Overall, mechanisms impacting
on ion homeostasis may provide insight into synergistic
fluconazole targets.
Cell wall organization and biogenesis
The combination of echinocandins with fluconazole has
shown some promising results against C. albicans
(Karlowsky et al. 2006; Pesee et al. 2016), suggesting
that disruption cell wall integrity may aid fluconazole
potentiation. The cell wall functions in the maintenance
of cell integrity and shape, protection of the plasma
membrane, tolerance to osmotic stress, and morpho-
genesis, all of which play critical roles in fluconazole
sensitivity as discussed in this review. The MAPK Pkc1
signalling pathway plays a key role in the cell wall
integrity process. This pathway is initiated by a family
of cell surface sensors that are coupled to the small G
protein Rho that activates Pkc1. Consequently, a MAPK
cascade, which comprises a linear series of protein kin-
ases including the MAPKKK Bck1, the MAPKK Mkk2, and
the MAPK Mkc1 are activated, and this relays signals to
the terminal transcription factors Rlm1, Cas5 and Swi4/
Swi6 (Dichtl et al. 2016). Deletion strains for the PKC1
gene, the BCK1 gene, and the MKC1 gene all exhibited
increased fluconazole sensitivity (Epp, Vanier, et al.
2010; LaFayette et al. 2010). Deletion of the down-
stream components of the MAPK Pkc1 signalling path-
way, the CAS5 gene, the SWI4 gene, the SWI6 gene, or
both the SWI4 gene and the SWI6 gene rendered strains
hypersensitive to fluconazole (Homann et al. 2009;
LaFayette et al. 2010; Vasicek et al. 2014) (Figure 4).
Another important cell wall integrity control path-
way involves the regulation of Ace2 and morphogen-
esis (RAM) network. The RAM network is comprised of
two serine/threonine protein kinases, Cbk1 and Kic1,
with four associated proteins, Cas4, Hym1, Mob2, and
Sog2, and the terminal transcription factor of the path-
way Ace2 (Saputo et al. 2012). Strains lacking the CBK1,
KIC1, CAS4, MOB2, or the SOG2 gene all showed
increased sensitivity to fluconazole (Song et al. 2008;
Chen et al. 2018) (Figure 4). Deletion strains of other
cell wall-related genes, such as the ARP2 gene (Epp,
Walther, et al. 2010), the BST1 gene (Liu, Zou, et al.
2016), the CHS2 gene (Chen et al. 2018), the MNN10
gene (Zhang et al. 2016), the SUR7 gene (Alvarez et al.
2008), and the RVS161 gene (Douglas et al. 2009), were
also reported to have increased sensitivity to flucon-
azole. Disruption of the synthesis or function of cell
wall components caused by various gene deletions may
lead to cell wall stress and consequently increased effi-
cacy of fluconazole.
Cell cycle and DNA double-strand break repair
Fluconazole can induce growth arrest in C. albicans by
inhibiting ergosterol synthesized; this may be an
important reason for fluconazole tolerance in C. albi-
cans. The cell cycle, coordinated and irreversible periods
of cell division, plays a major role in regulating cellular
morphogenesis in C. albicans (Bachewich and
Whiteway, 2005; Correia et al. 2010). The deletion of the
PCL2 gene, which encodes the G1 cyclin homolog Pcl2
required for bud morphogenesis (Bachewich and
Whiteway, 2005), leads to increased sensitivity to flu-
conazole (Chen et al. 2018). Deletions of the HCM1 and
FKH2 genes, which encode two members of the fork-
head family of transcription factors (Hcm1, Fkh1, Fkh2,
and Fhl1) and play central roles in S phase (Haase and
Wittenberg 2014), resulted in increased sensitivity to

Figure 4. Models of the cell wall integrity signalling cascades in C. albicans, as well as the potential synergistic targets of flucon-
azole in these signalling pathways.
fluconazole (Homann et al. 2009; Vandeputte et al.
2012). In addition to membrane damage, fluconazole
may also directly cause DNA damage (Harrison et al.
2014; Robinson 2014). Within the cell cycle, the G1, S,
and G2 phases have checkpoints that monitor DNA dam-
age and control activation of DNA repair mechanisms
(Barnum and O’Connell 2014). DNA double-strand break
repair plays a key role in protecting C. albicans from DNA
damage and maintaining the normal cell cycle. Mre11
and Rad50 are required for both homologous recombin-
ation and nonhomologous end-joining, while Rad52
plays a crucial role specifically in homologous recombin-
ation (Pannunzio et al. 2018; Wright et al. 2018). Sgs1
plays a role in maintaining genome stability by regulat-
ing stalled replication forks (Legrand et al. 2011).
Deletions of the MRE11, RAD50 RAD52, and SGS1 genes
generated increased susceptibility to fluconazole
(Legrand et al. 2007, 2011). This suggest that perturbing
functions of DNA damage response and cell cycle may
improve the efficacy of fluconazole.
Response to stress
The success of C. albicans as a pathogen partly resides
in its ability to adapt to to protect itself from various
stress conditions. The MAPK Hog1 signalling pathway,
CRITICAL REVIEWS IN MICROBIOLOGY 329
one of the most significant signalling pathways respon-
sible for C. albicans’ resistance to different environmen-
tal stresses, is activated by oxidative, osmotic, and
heavy metal stress. The MAPK Hog1 signalling pathway
is composed of MAPKKK Ssk2, the MAPKK Pbs2, and the
MAPK Hog1. Deletion of C. albicans the SSK1 gene
(Chauhan et al. 2007), the PBS2 gene (Blankenship et al.
2010), and the HOG1 gene (Blankenship et al. 2010) ren-
dered normally fungistatic fluconazole fungicidal. These
studies establish a new role for the MAPK Hog1 signal-
ling pathway in drug resistance and suggest that tar-
geting osmatic stress response signalling provides a
promising strategy for treating life-threatening C. albi-
cans infections (Figure 4).
In addition, Hsp90 is a conserved and essential chap-
erone that regulates cellular signalling by stabilizing a
myriad of client proteins (O’Meara et al. 2017). Our
STRING analysis emphasized that Hsp90 is a central glo-
bal cellular regulator that governs stress responses cru-
cial for fluconazole resistance (Figure S1, Table S3). The
heterozygous deletion of the HSP90 gene in C. albicans
enhanced the therapeutic efficacy of fluconazole both
in vitro and in vivo (Cowen et al. 2009; Robbins et al.
2011). Furthermore, Hsp90 inhibitors also converted the
fungistatic activity of fluconazole to fungicidal (Li, An,
et al. 2015; Huang et al. 2020).
330 H. LU ET AL.
Other potential synergistic targets
Apart from these higher-level processes which we dis-
cussed above, there are a variety of specific genes that
can be useful as targets of fluconazole adjuvants. The
Spt-Ada-Gcn5-acetyltransferase (SAGA) coactivator
complex, which regulates numerous cellular processes
through coordination of histone posttranslational modi-
fications (Baker and Grant 2007), has roles in flucon-
azole sensitivity in C. albicans. Deletion of the ADA2
gene, which encodes the transcription coactivator Ada2
for nucleosomal acetylation, enhanced fluconazole sen-
sitivity of C. albicans (Sellam, Askew, et al. 2009; Epp,
Vanier, et al. 2010). Loss of the SPT20 gene, a member
of the SAGA complex that may function in SAGA coacti-
vator complex stability, results in hypersensitivity to flu-
conazole (Epp, Vanier, et al. 2010; Vandeputte et al.
2012; Tan et al. 2014). Deletion strains of the ENO1
gene, which encodes enolase, the MSI3 gene, which
encodes an essential Hsp70 family protein, the HSP60
gene which encodes a heat shock protein, and the RBF1
gene which encodes a transcription factor with roles in
filamentous growth, all of which are downstream tar-
gets of the SAGA coactivator complex (Sellam, Askew,
et al. 2009), show increased susceptibility to fluconazole
(Nagao et al. 2012; Ko et al. 2013; Khamooshi et al.
2014; Chen et al. 2018). Thus, disruption of the stability
of the SAGA coactivator complex, or repressing its activ-
ity, may be beneficial in enhancing the efficacy of flu-
conazole against C. albicans.
It is not surprising that the drug efflux pumps Mdr1,
Cdr1 and Cdr2 play important roles in fluconazole
resistance as they efflux fluconazole out of C. albicans
cells and bringing about decreased intracellular flucon-
azole concentration (White et al. 1997; Franz et al. 1998;
Perea et al. 2001). Several studies have shown that each
of the CDR1 (Sanglard et al. 1996; Umeyama et al. 2002;
Xu et al. 2007; Tsao et al. 2009; Epp, Vanier, et al. 2010),
CDR2 (Tsao et al. 2009), and MDR1 genes deletion
mutants (Wirsching et al. 2001) are hypersensitive to
fluconazole. Tac1 is a Zn (2)-Cys (6) transcriptional acti-
vator of CDR1 and CDR2, while Mrr2 is another Zn (2)-
Cys (6) transcriptional activator that controls the CDR1
gene expression. The TAC1 gene deletion mutant
(Coste et al. 2004; Coste et al. 2006), and the MRR2
gene deletion mutant (Homann et al. 2009) are more
susceptible to fluconazole. The NCB2 gene encodes a b
subunit of the heterodimeric transcription regulator
Nc2 which activates the expression of the CDR1 gene,
and the NCB2 gene deletion mutant also showed hyper-
sensitivity to fluconazole (Chen et al. 2018). Ipt1 is an
inositol phosphoryl transferase required for membrane
localization of the Cdr1. Two previous studies showed
that the deletion of the IPT1 gene led to increased sen-
sitivity to fluconazole (Pasrija et al. 2005; Prasad et al.
2005). At present, no research directly tests the relation-
ship between the deletion of the MRR1 gene encoding
a transcriptional regulator of the Mdr1 and fluconazole
sensitivity in C. albicans, but gain-of-function mutations
of the Mrr1 cause upregulation of the MDR1 gene and
multidrug resistance (Dunkel et al. 2008). Therefore, tar-
geting the genes of drug efflux pumps or their regula-
tors might be an approach for enhancing the efficacy
of fluconazole (Monk and Goffeau 2008).
It is also worth noting that GTPase related genes are
involved in fluconazole sensitivity in C. albicans. Our
previous study showed that the deletion of the AGE3
gene, which encodes an ADP-ribosylation factor GTPase
activating protein, made fluconazole fungicidal (Epp,
Vanier, et al. 2010). Our recent drug sensitivity screen-
ing study showed mutants of three other GTPase
related genes (the BUB2 gene, the BUD7 gene, and the
MTG2 gene) showed increased sensitivity to fluconazole
(Chen et al. 2018). A previous study demonstrated that
constructed a conditional VPS1 gene mutant (tetR-
VPS1), encodes a dynamin-like GTPase, showed signifi-
cant growth defect when exposed to fluconazole
(Bernardo et al. 2008). Taken together, it appears mem-
bers of GTPase superfamily may be potential flucon-
azole synergistic targets against C. albicans.
Farnesol is able to inhibit both biofilm formation and
the yeast-to-hyphal morphological transition in C. albi-
cans; these are important virulence and drug-resistance
traits of this pathogenic fungus (Wongsuk et al. 2016),
and may be reasons for farnesol treatment to show pro-
tection against mucosal candidiasis (Hisajima et al.
2008). Farnesol also exerts a synergistic function with
fluconazole against C. albicans in vitro (Yu et al. 2012;
Cordeiro et al. 2013; Katragkou et al. 2015), perhaps
because it inhibits the activities of Cdr1 and Cdr2
(Sharma and Prasad 2011). A null mutant of the DPP1
gene involved in farnesol production increased resist-
ance to fluconazole (Chen et al. 2018), which might
result from a reduction in farnesol levels. By contrast,
increased levels of farnesol production via deletions of
the NRG1 and TUP1 genes (Kadosh and Johnson 2005;
Kebaara et al. 2008), brought about fluconazole hyper-
sensitivity (Homann et al. 2009). Furthermore, farnesol
down-regulates intracellular cAMP levels via direct
binding to the cyclase domain of the adenylyl cyclase
Cyr1, repressing its activity (Davis-Hanna et al. 2008;
Hall et al. 2011) or through indirectly affecting the small
GTPase Ras1, promoting the cleavage of Ras1 into a sol-
uble form that has a reduced ability to activate Cyr1,
consequently, leading to inhibition of the Ras1-cAMP-

PKA signalling pathway (Piispanen et al. 2013). As well,
direct loss of the CYR1 gene caused increased sensitivity
to fluconazole (Jain et al. 2003), while the loss of
phosphodiesterase Pde2, which results in increases in
intracellular cAMP levels (Bahn et al. 2003), increased
resistance to fluconazole (Jung et al. 2005). Thus, farne-
sol treatment, as well as other processes that directly or
indirectly modulate cAMP levels, can influence the sen-
sitivity of C. albicans to fluconazole. (Figure 3).
Mitochondria, as power houses, are needed for flu-
conazole resistance in C. albicans (Sun et al. 2013),
which may be caused by mitochondrial dysfunction
resulting in downregulation of transporter genes, the
ergosterol synthesis genes, and iron homeostasis
(Thomas et al. 2013), all of which are related to flucon-
azole susceptibility as discussed above. Electron trans-
port chain complex I related null mutants showed
increased sensitivity to fluconazole. Furthermore, inhibi-
tors of mitochondrial complex I can improve the out-
come of fluconazole treatment in patients or lab
isolates (Sun et al. 2013). These results suggested that
dysfunction of mitochondria may improve fluconazole
potentiation.
Conclusions and perspectives
In the current review, we summarize the consequences
of deletions of 220 genes that generated hypersensitiv-
ity to fluconazole and suggest that targeting the bio-
logical processes these genes are involved with may
support fluconazole potentiation. Here, we propose
that there are at least four potential pathways for flu-
conazole potentiation: (1) fluconazole may be syner-
gized if a drug’s action helps fluconazole availability in
the C. albicans cell by increasing intracellular flucon-
azole concentration. For instance, drugs targeting bio-
film formation or drug efflux pumps related genes and/
or proteins may help to increase the efficacy of flucon-
azole against C. albicans; (2) fluconazole may be syner-
gized if a drug targets another protein involved in the
ergosterol biosynthetic process. This combination
allows greater effects and/or reduced toxicities due to a
lower fluconazole dose; (3) fluconazole may be syner-
gized if a drug targets a protein on a parallel pathway
that converges on an essential process. For example,
drugs targeting cell wall organization or DNA double-
strand break repair-related genes and/or proteins may
enhance the efficacy of fluconazole against C. albicans;
(4) fluconazole may be synergized if a drug helps flu-
conazole do more damage by inhibiting functions con-
ferring resistance to fluconazole in C. albicans. For
instance, drugs targeting the calcium-calcineurin
CRITICAL REVIEWS IN MICROBIOLOGY 331
signalling pathway may help to increase the efficacy of
fluconazole against C. albicans.
Many studies have tried to identify synergistic tar-
gets of fluconazole against C. albicans. The calcineurin
and the Hsp90 circuits represent promising synergistic
targets for fluconazole potentiation (Uppuluri et al.
2008; Li, An, et al. 2015; Huang et al. 2020), but, so far,
there are no fluconazole synergistic drugs available for
clinical treatment of C. albicans. This situation is mainly
caused by the following four reasons: (1) C. albicans as
a eukaryote, so potential synergistic targets such as cal-
cineurin and Hsp90 may be conserved in the human
host. Such conservation results in compounds having
good synergy with fluconazole in vitro, but working
poorly in vivo because the compounds have toxic side
effects. Future studies could develop new antifungal
drugs that target fungal-specific proteins or fungal-spe-
cific parts of proteins; (2) the mechanism of the fungi-
static activity of fluconazole remains elusive. At present,
we know that fluconazole induces altered sterol com-
position resulting in growth arrest (Kelly et al. 1997).
However, details of this growth arrest remain to be
firmly established: is it cell cycle arrest at a specific
point, or poisoning that may be expected to generate a
random population of non-dividing cells? Future stud-
ies could focus on uncovering the mechanisms of flu-
conazole-induced growth arrest; (3) crystal structures of
C. albicans proteins are limited. This prevents efficient
protein targeted virtual drug screening and computer-
aided drug design. More crystal structures of synergistic
targets of fluconazole will be needed in the future; (4)
Because C. albicans is diploid and has no normal mating
cycle, efficient large-scale gene interaction studies are
difficult in C. albicans. Unlike S. cerevisiae, the whole
genome fluconazole collaborative network has not
been constructed. In the future, more potential flucon-
azole synergistic targets may be found using
CRISPR–Cas9-based gene drive platforms (Shapiro et al.
2018). This could accelerate the development of anti-
fungal drugs with synergistic interactions with
fluconazole.
Disclosure statement
No potential conflict of interest was reported by
the author(s).
Funding
This work was supported by a scientific research project
funded by Shanghai Science and Technology Commission
[Grant No. 19431902800].
332 H. LU ET AL.
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