Journal of Applied Microbiology, 2023, 134, 1–15

https://doi.org/10.1093/jambio/lxad091
Advance access publication date: 28 April 2023
Research Article

Glucose metabolic reprogramming and modulation in

glycerol biosynthesis regulates drug resistance in clinical
isolates of Candida

Downloaded from https://academic.oup.com/jambio/article/134/5/lxad091/7146826 by AMI - Member Access user on 11 May 2023

Sajad Ahmad Padder1 , Rayees Ahmad Padder2,# , Asiya Ramzan1 , Gulnaz Bashir3 ,
Inayatullah Tahir4 , Reiaz Ul Rehman1,= , Abdul Haseeb Shah 1,*
1
Department of Bioresources, School of Biological Sciences, University of Kashmir, 190006 Hazratbal, Srinagar, India
2
Department of Biotechnology, Jamia Millia Islamia, 110025 New Delhi, India
3
Department of Microbiology, Sheri Kashmir Institute of Medical Sciences, 190011 Soura, Srinagar, India
4
Department of Botany, School of Biological Sciences, University of Kashmir, 190006 Hazratbal, Srinagar, India
∗
Corresponding author. Department of Bioresources, School of Biological Sciences, University of Kashmir, Hazratbal, Srinagar, India.
E-mail: hasb789biotech@gmail.com
#
Present address: Centre for Advanced Biotechnology and Medicine, Rutgers Robert wood Johnson Medical School, New Brunswick, NJ 08854, USA.
=
Co-Correspondence: E-mail: rreiazbiores@gmail.com

Abstract
Aims: The study is aimed at understanding the novel molecular mechanisms governing drug resistance in the opportunistic fungi belonging to
the genus Candida.
Methods and results: This is a multipronged study wherein different assays like drug susceptibility and whole cell proteome analysis, stress
tolerance assay, measurement of total internal glycerol content, western blot analysis, reactive oxygen species (ROS) measurement, glucose
uptake, lactate production, ATP generation, and NADPH measurements were made.
The study reveals an incidence of different species of Candida in the northern most part of India (Kashmir valley). Resistant isolates, mostly
resistant to azoles were reported across all the species. The study revealed a difference in resistance mechanisms between Candida albicans
and C. glabrata clinical isolates. Further, such resistance mechanism (in the case of C. albicans) was mostly mediated by Hexokinase 2 (Hxk2)
and Glucose-6-phosphate dehydrogenase (G6pd). Increased expression of Hxk2 was associated with increased glucose uptake, more lactate
production, and more ATP generation in drug-resistant C. albicans. At the same time, increased G6pd expression was responsible for the
increased production of NADPH, which imparts a better ROS scavenging potential. While in C. glabrata the resistance was linked with glycerol
metabolism, where the drug-resistant isolate tends to accumulate more glycerol as an osmolyte in response to external stresses. This glycerol
accumulation was found to be triggered by the HOG1-MAPK pathway.
Conclusion: The study concludes that, like various human malignant tumors, there is a strong correlation between drug resistance and aberrant
cellular metabolism in the opportunistic fungi belonging to the genus Candida.

Significance and impact of the study

The study is novel in unraveling the role of intermediary metabolism in the acquisition of drug resistance in Candida, which is upon further
evaluation, can lead to the identification of more efficient alternate drug targets.
Keywords: antifungal resistance, Candida, hexokinase 2, 3-bromopyruvate, glucose-6-phosphate dehydrogenase, polydatin

Introduction species (Cryptococcus neoformans), and molds (Aspergillus

Fungi being primitive eukaryotes interact with other life
forms under explicit mutualism, commensalism, or parasitism.
When a fungal species causes mild to severe diseases in its
hosts, it is called a pathogen. Most fungal pathogens in mam-
mals are opportunistic pathogens as they cause diseases only
under compromised host immune response (Sanglard et al.
2009). The Candida genus comprises opportunistic fungi, and
the frequency of these fungal infections has dramatically in-
creased in recent decades, due to the increase in the number
of immunocompromised patients (Sarvikivi et al. 2005, Prasad
et al. 2014). Among the common human fungal pathogens
known, Candida albicans is the most frequent and prevalent
species, followed by other nonalbican (C. glabrata, C. para-
psilosis, C. tropicalis, C. auris, and C. krusei), noncandidal
fumigatus, Micropsorum canis, etc) (Pfaller and Diekema
2007, Chowdhary et al. 2017). The infections caused by these
fungi range from superficial mucosal, like oropharyngeal can-
didiasis, to life-threatening systemic infections such as dissem-
inated candidiasis, cryptococcal meningitis, and invasive as-
pergillosis (Mayer et al. 2013). These fungal infections are
evolving as grave problems of concern in the healthcare sys-
tem (Nami et al. 2019).
With the mounting incidence of opportunistic fungal infec-
tions, the limited availability of antifungal drug classes, or the
decreased response of certain fungi to these antifungals, the
emergence or development of multiple drug resistance by these
pathogens has posed a stern public health concern (Loeffler
and Stevens 2003).

Received: December 20, 2022. Revised: March 31, 2023. Accepted: April 27, 2023

C The Author(s) 2023. Published by Oxford University Press on behalf of Applied Microbiology International. All rights reserved. For permissions, please

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2 Padder et al.
Antifungal agents like azoles, polyenes, and echinocandins
are routinely used to combat fungal infections (de Oliveira
Santos et al. 2018). Compared to polyene amphotericin B
(AmpB), azoles and echinocandins are recent antifungals with
enhanced specificity to pathogens. However, their excessive
use has led to the emergence of highly tolerant fungi (Brown et
al. 2012). Among the different resistance mechanisms, azole-
resistant isolates have been found to either change the protein
sequence or its expression (White 2001).
To colonize and cause infection, these opportunistic
pathogens need to withstand various environmental chal-
lenges posed in the host microenvironment like immune de-
fense, limited availability of nutrients, competition with the
inhabitant microbiota, and extreme physiological conditions
(higher/lower pH, oxidative/osmotic stresses) (Calderone and
Fonzi 2001, Marotta et al. 2013, Dong et al. 2015). Microor-
ganisms accumulate compatible solutes like amino acids, ions,
and polyhydroxy compounds to withstand hypertonic condi-
tions (Nevoigt and Stahl 1997). Among these, glycerol is the
most prominent compatible solute accumulated by Saccha-
romyces cerevisiae and some other yeasts (Petelenz-Kurdziel
et al. 2013). Nevertheless, some microorganisms have also re-
ported the accumulation of trehalose, arabitol, mannitol, or
glycine/betaine (Van Eck et al. 1993, Welsh 2000). As an adap-
tation to osmotic stress, cells change the intracellular glyc-
erol content to offset the increasing external osmolarity and
thereby enable survival by buffering the osmotic change and
maintaining normal cellular volume (Reed et al. 1987).
Candida albicans intricately responds to glucose by regu-
lating various genes involved in central carbon metabolism,
whereby a varied glucose concentration significantly affects
the gene regulation and in turn the growth and survival of
the pathogen (Rodaki et al. 2009). Phosphorylation of Hog1
in response to glucose is a very important step that medi-
ates glucose-enhanced resistance to cationic stress (Rodaki et
al. 2009). In the case of human cells, as far the central glu-
cose metabolism is concerned, the rate-limiting enzyme hex-
okinase 2 (Hxk2) holds the key role as it phosphorylates glu-
cose to maintain the magnitude and direction of glucose flux
within cells (Moreno and Herrero 2002, Gancedo and Flores
2008). Normal differentiated human cells primarily rely on
mitochondrial oxidative phosphorylation for energy produc-
tion. However, most cancerous cells preferentially drive their
metabolism away from oxidative phosphorylation to glycoly-
sis irrespective of the oxygen availability for their demands of
energy and biomolecules (Warburg effect) (Hsu and Sabatini
2008, Pedersen 2008). Another important pathway linked to
glucose metabolism is the pentose phosphate pathway (PPP),
a vital source of NADPH owing to its potential for oxidative
stress resistance (Cairns et al. 2011, Kuehne et al. 2015, Chen
et al. 2019).
The present study investigated the intricate relationship be-
tween glucose metabolism and drug resistance in the clinical
isolates of commonly isolated human pathogenic fungi C. al-
bicans and C. glabrata. The focus was to thoroughly under-
mine the basic similarities and differences (if any) in the drug-
resistance mechanisms in clinical isolates belonging to these
two species at the metabolic/molecular level. We report a dif-
ference in drug-resistance mechanisms adapted by C. albicans
and C. glabrata clinical isolates. While C. albicans showed a
Hxk2-dependent resistance mechanism and G6pd-mediated
reactive oxygen species (ROS) scavenging (a feature observed
commonly in human cells), C. glabrata resistant isolate accu-
mulated more intracellular glycerol as an osmolyte to impart
better adaptability to the pathogen in response to stress. The
study provides valuable insights into how deranged cellular
metabolism regulates drug resistance in Candida species, thus
providing unique therapeutic nodules alongside conventional
antifungal therapies to target drug-resistant fungi efficiently.
Materials and Methods
Reagents/materials used
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The reagents/materials used in the study are mentioned in Ta-
ble S1.
Collection of clinical isolates
Clinical isolates of Candida species were procured from the
microbiology division of a tertiary care hospital. Fungal iso-
lates were cultured and maintained on yeast extract peptone
dextrose (YEPD) solid, and liquid media procured from Hi-
Media. Glycerol stocks were made of these freshly revived cul-
tures and stored at −80◦ C for long-term storage and use.
Drug susceptibility assays
Drug susceptibility testing of fluconazole (FLC)-resistant
clinical isolates was done using drugs belonging to dif-
ferent classes, like azoles (ketoconazole, miconazole, and
voriconazole), polyenes (AMB), and echinocandins [caspofun-
gin (CSF)]. The susceptibility testing was done by minimum
inhibitory concentration (MIC) determinations and on solid
agar-drug plates by drug susceptibility spot assays, as dis-
cussed below (Shah et al. 2015).
Broth microdilution assay
The assay was performed to determine the MIC of drugs or
stressors against Candida strains (Pfaller et al. 2011). Briefly,
the cells from freshly streaked YEPD-agar plates were resus-
pended in sterile 0.9% NaCl maintaining OD600 = 0.1. The
cells were further dissolved 100-fold and used for MIC deter-
minations. The results were read using a microplate reader as
well as by visual inspection of the plates.
Serial dilution spot assay
Serial dilution spot assay was done using a previously de-
scribed protocol (Vandeputte et al. 2012, Shah et al. 2014).
Briefly, drug plates were prepared by adding the required con-
centration of the drug to warm YEPD-agar media and pouring
it over plates. The cell suspension (OD600 = 0.1) of each fungal
strain was then serially diluted 5-fold, and 4 μl of the suspen-
sion from all serially diluted strains was spotted on solid drug
plates. Plates were incubated at 30◦ C for 48 h, and growth
inhibition by each drug was calculated.
Checkerboard assay
The assay was performed to check the interaction of Hxk2 in-
hibitor [3-bromopyruvate (3-BP)] and G6pd inhibitor (poly-
datin) in combination with conventional antifungal drug FLC
at their sub-inhibitory concentrations. The assay was per-
formed similar to the broth microdilution assay (explained
above), except those two drugs were used here in combina-
tion at their sub-MIC concentrations in such a way that one
drug was diluted along X-axis and another was diluted both
along X-axis as well as Y-axis. The cells were added, and the
Intermediary metabolism and C andida drug resistance
plates were incubated at 30◦ C for 48 h. The drug interaction
was obtained using the following equation:
FICI = FIC1 + FIC2 = C1 Comb /MIC1 alone + C2 Comb /MIC2 alone ,
where MIC1 alone and MIC2 alone are the MIC of drugs 1 and
2 alone, and C1 Comb and C2 Comb is the concentration of drugs
1 and 2 at the iso-effective combinations (Maurya et al. 2011,
Prasad et al. 2017). The interaction between two drugs is said
to be synergistic if FICI values are ≤0.5. If FICI values fall be-
tween 0.5 and 4, then there is no interaction between the two
drugs and FICI values >4, suggest antagonism between the
two drugs (Septama and Panichayupakaranant 2016, Prasad
et al. 2017).
Whole-cell proteome analysis
A volume of 30 ml cells with a starting OD600 of 0.2 were
grown for up to 6 h and centrifuged at 5000 rpm for 5 min.
Supernatants were discarded, pellet was washed twice in PBS
and snap-frozen in liquid nitrogen. The pellets were thawed
and washed in ice-cold lysis buffer [20 mmol HEPES (pH 7.3),
350 mmol NaCl, 10% glycerol, and 0.1% tween 20] contain-
ing protease inhibitors (1 mmol PMSF 10 mmol Sodium Flu-
oride 1X Yeast/Fungal Protease arrest PhosStop). The cells
were resuspended in ∼200–300 μl lysis buffer and transferred
to a 50 ml falcon tube containing 1 ml chilled glass beads.
The samples were disrupted by bead beating on a vortexer
for 5 × 30 s in spurts and were rested on ice for 1 min each
time. Lysates were recovered by centrifugation and quantified.
A total of 100 μg protein samples were taken and reduced
with 5 mmol TCEP, further alkylated with 50 mmol iodoac-
etamide, and then digested with Trypsin (1:50, Trypsin/lysate
ratio) for 16 h at 37◦ C. Digests were cleaned using a C18
silica cartridge to remove the salt and dried using a speed
vac. The dried pellet was resuspended in buffer A (5% ace-
tonitrile and 0.1% formic acid). This was followed by the
mass spectrometric analysis of peptide mixtures. The exper-
iments were performed using the EASY-nLC 1000 system
(Thermo Fisher Scientific) coupled to Thermo Fisher-Q Ex-
active equipped with a nanoelectrospray ion source. A to-
tal of 1.0 μg of the peptide mixture was resolved using a
25 cm PicoFrit column (360 μm outer diameter, 75 μm inner
diameter, and 10 μm tip) filled with 1.8 μm of C18-resin (Dr.
Maeisch, Germany). The peptides were loaded with buffer A
and eluted with a 0%–40% gradient of buffer B (95% ace-
tonitrile and 0.1% formic acid) at a flow rate of 300 nl min−1
for 100 min. MS data were acquired using a data-dependent
top 10 method, dynamically choosing the most abundant
precursor ions from the survey scan. For data processing,
the samples were processed, and 2 RAW files were gener-
ated and analyzed with Proteome Discoverer (v2.2) against
the Uniprot Candida reference proteome database. For the
Sequest search, the precursor and fragment mass tolerances
were set at 10 ppm and 0.5 Da. The protease used to gen-
erate peptides, i.e. enzyme specificity, was set for trypsin/P
(cleavage at the C terminus of “K/R: unless followed by
P”) along with a maximum missed cleavages value of two.
Carbamidomethyl on cysteine as fixed modification and oxi-
dation of methionine and N-terminal acetylation and phos-
phorylation on-site (S, T, and Y) were considered variable
modifications for database search. Both peptide-spectrum
match and protein false discovery rate (FDR) were set to
0.01.
3
Evaluation of stress resistance
Stress resistance assays were performed by employing the se-
rial dilution spot assay as explained above. Instead of drugs,
different concentrations of stressors were used. Calcofluor
white (CFW) and Congo red (CR) were used to generate cell
wall stress; NaCl was used to generate cationic stress; H2 O2
was used to generate oxidative stress; and tert-butyl hydro-
peroxide was used to generate super oxides. The plates were
incubated at 30◦ C (in dark for oxidative stresses) for 24 to
48 h and growth was visualized. Plates were incubated either
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at higher or lower temperatures to generate thermal stress.
Intracellular glycerol levels
The intracellular glycerol production was measured according
to the previously described protocol (Ene et al. 2012). Briefly,
Candida strains were grown overnight, and an aliquot of 5 ×
107 cells was treated with 1 mol NaCl in YEPD for 30 min. In-
ternal glycerol levels were measured by subtracting extracel-
lular glycerol content from total glycerol content using Free
Glycerol Reagent.
Western blot analysis
A volume of 30 ml of exponentially growing cells with or
without the exposure to indicated stress agent/inhibitor were
harvested by centrifugation (3000 × g for 5 min). For checking
the phosphorylation of Hog1, cells were harvested at three-
time points 0 (untreated), 30, and 60 min post-stress induc-
tion. While checking the inhibition of Hxk2 and G6pd by
the inhibitors, cells were harvested at 0, 2.5, and 5 h post-
treatment. The supernatants were discarded, and the pellets
snapfrozen in liquid nitrogen. The pellets were thawed and
washed in ice-cold lysis buffer [20 mmol HEPES (pH 7.3),
350 mmol NaCl, 10% glycerol, and 0.1% Tween 20] con-
taining protease inhibitors (1 mmol PMSF; 10 mmol sodium
fluoride; 1X Yeast/Fungal Protease arrest; and PhosStop). The
cells were resuspended and disrupted in ∼200–300 μl lysis
buffer containing 1 ml chilled glass beads. Lysates were re-
covered by centrifugation, and an equal amount of protein
(40–50 μg) was subjected to SDS–PAGE on 10% gels. The
protein was transferred onto a preactivated PVDF membrane.
The membranes were blocked for 1 h at room temperature in
5% BSA in 1X TBST and incubated overnight in the primary
antibodies against the indicated proteins at 4◦ C on the rocker.
This was followed by washing (three times, 5 min each) and
1.5 h incubation in specific HRP-conjugated secondary anti-
bodies. The membranes were again washed thrice and devel-
oped using Immobilon Forte Western Substrate. β-Actin was
used as a loading control.
ROS generation
The fluorescent probe dichloro-dihydro-fluorescein diac-
etate (DCFDA) was used for the measurement of ROS gen-
eration (Dhamgaye et al. 2012). Briefly, secondary fungal cul-
tures were set up in 5 ml of YEPD broth and grown up to an
absorbance (OD600 ) of 0.8, followed by adding 0.078 mmol
G6pd inhibitor (polydatin) in one tube, and another tube was
kept untreated (control). Tubes were incubated for 80 min at
30◦ C at 200 rpm. Samples were centrifuged at 5000 rpm for
5 min, washed and resuspended in equal volume (5 ml) of
YNB, and incubated for 1 h at 30◦ C. Cell pellet from here was
taken and resuspended in 1 ml fresh YNB to final OD600 of 1.
4 Padder et al.

Table 1. MIC80 (in μgml−1 ) of different classes of antifungal drugs against clinical isolates of (a) C. glabrata and (b) C. albicans.

(a)
Strain FLC KTC MICO CSF AMB
M183 64 (R) 0.5 0.25 0.5 1
M283 128 (R) 0.5 0.25 0.5 1
M198 128 (R) 0.5 0.025 0.05 4
M203 8 0.0625 0.0625 0.25 1
M1037 16 0.5 0.25 0.25 2
M1057 32 0.5 0.25 0.25 2
M349 32 0.25 0.0625 1.6 4
M626 32 0.0312 2 0.8 4

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M49 32 0.25 0.0156 0.0039 8
WT 1 0.00097 0.00097 0.0125 2
(b)
Strain FLC KTC MICO CSF AMB
M1038 64 (R) 0.125 1 0.0125 0.5
M66 32 (R) 2 0.5 0.0078 2
M83 32 (R) 2 0.5 0.0078 2
M2 2 0.00097 0.0039 0.025 1
M3 2 0.00097 0.0039 0.025 2
M6 2 0.00097 0.0039 0.025 2
M169 1 0.0078 0.0625 0.0078 2
M295 2 0.0078 0.0625 0.00156 2
M39 2 0.0039 0.0039 0.00312 2
M100 1 0.00097 0.0625 0.025 1
M35 1 0.00097 0.0625 0.025 2
M738 1 0.00195 0.0625 0.025 1
M194 0.5 0.00195 0.0625 0.025 1
M46 0.5 0.00097 0.0039 0.025 1
M195 0.5 0.00097 0.0039 0.0125 2
M67 0.5 0.00097 0.0625 0.0125 2
M26 0.5 0.00097 0.0039 0.0125 1
M54 0.5 0.00195 0.0625 0.025 2
WT 2 0.00097 0.0039 0.025 2

(R) Represents resistant isolates.

The cells were centrifuged and resuspended in 250 μl YNB,
to which 10 μmol DCFDA was added and incubated at 30◦ C
for another 30 min. Fluorescence was measured with an exci-
tation and emission wavelength at 488 and 540 nm.
Measurement of glucose uptake/lactate production,
ATP, and NADPH
Glucose uptake and lactate production were measured using
the respective kits following the manufacturer’s protocol (TeS-
laa and Teitell 2014). The ATP levels were measured using
ATP Colorimetric/Fluorometric Kit, and NADPH levels were
quantified using Colorimetric NADPH Assay Kit as per man-
ufacturer protocols.
Statistical analyses
All statistical analyses, including analysis of variance
(ANOVA) and significance of data were calculated using
Graph Pad Prism v 8.4. All the data are represented as
mean ± SEM of at least three replicates unless otherwise
stated.
Results:
Drug susceptibility analysis reveals the presence of
FLC resistant clinical isolates
A total of 84 clinical isolates of pathogenic fungi were pro-
cured from the microbiology division of a local tertiary care
hospital. Preliminary identification of clinical isolates by the
Vitek 2 system (bioMe’rieux, inc., France) revealed that 84
clinical isolates were spread across different Candida species
as detailed in Table S2.
Initially, all the procured isolates were tested for their sus-
ceptibilities by the Vitek 2 system and with the commonly
used azole drug FLC using broth microdilution assay. The
interpretive guidelines for drug susceptibility testing for dif-
ferent antifungals against Candida species are mentioned in
Table S3. The MIC80 values obtained using the FLC drug
revealed that out of nine C. glabrata isolates, eight isolates
(M183, M283, M198, M1037, M1057, M349, M49, and
M626) were resistant to FLC, and one isolate (M203) was
susceptible to it (Table 1a). Among the eight resistant iso-
lates, three (M183, M283, and M198) showed a high re-
sistance (MIC80 ≥ 64 μgml−1 ) while the other five (M1037,
M1057, M349, M49, and M626) showed intermediate re-
sistance (MIC80 = 16–32 μgml−1 ) to FLC (Table 1a). Simi-
larly, among the 12 C. parapsilosis isolates, 4 (M276, M742,
M529, and M551) showed resistance toward FLC, and the
rest 8 (M463, M336, M1068, M30, M717, M478, M168, and
M342) were sensitive to FLC (Table S4). Out of the total 18
C. albicans isolates, 3 (M1038, M66, and M83) were resistant
to FLC, whereas the rest 15 were sensitive to it (Table 1b). All
the 39 C. krusei isolates were mostly resistant to FLC, which
generally points to their intrinsic resistance against this azole
drug, while all these isolates were susceptible to polyene AMB
and echinocandin CSF (Table S5). Out of the two C. rugosa
isolates, one (M90) showed resistance to FLC while the other
(M196) was sensitive to it (Table S6). Both the C. tropicalis
Intermediary metabolism and C andida drug resistance
Figure 1. Mass spectrometry-based expression profiles and pathway analysis of proteins belonging to basic metabolic pathways in drug-resistant and
drug-susceptible isolates (a–c) represents expression profiles of proteins belonging to glycolytic pathway, PPP, and nucleotide biosynthetic pathway,
respectively, in drug-resistant C. albicans (M1038) compared to drug-susceptible C. albicans (M2). (d) Represents expression profiles of proteins
belonging to glycerol biosynthetic pathway in drug-resistant C. glabrata (M183) compared to drug-susceptible C. glabrata isolate (M203).
isolates (M666, M729) identified were sensitive to FLC, while
the Meyerozyma guilliermondii (M144) and Yarrowia lipoly-
tica (M328) isolates showed resistance to FLC (Table S6).
Further, we performed extensive drug susceptibility test-
ing of the clinical isolates by MIC80 determinations using
drugs belonging to different classes, which include azoles (ke-
toconazole and miconazole), polyenes (AMB), and echinocan-
dins (CSF). This drug susceptibility testing revealed that FLC-
resistant C. glabrata isolates show cross-resistance to azole
antifungal drugs, while there was only marginal resistance
against echinocandin drug CSF and no resistance was seen
against polyene AMB (Table 1a). Likewise, FLC-resistant C.
albicans isolates show cross-resistance to other azoles (Table
1b). Most of the C. parapsilosis isolates showed slight resis-
tance to polyene AMB (Table S3). While C. krusei isolates
were resistant mostly to FLC only (Table S4).
The clinical isolates of the two most frequently isolated
(globally) fungal species, C. albicans and C. glabrata were fur-
ther studied to get mechanistic insights into drug resistance.
Drug susceptibility spot assays mostly confirmed the results of
broth microdilution assays and thus signify that clinical iso-
lates show resistance mostly against azole drugs compared to
echinocandins and polyenes (Figure S1).
The identity of the procured isolates made by conventional
methods such as colony color on Hi-Chrome Candida agar
and cornmeal agar, was further authenticated (only of resis-
tant ones) by Candida TM identification kits and by PCR-
based RFLP digestion using MspI enzyme for molecular iden-
tification (Mirhendi et al. 2006, Fatima et al. 2017) (Fig. 1).
Growth curve analysis of drug-resistant C. albicans and C.
glabrata isolates was performed to check the inherent dif-
ferences in growth patterns among different isolates. The
growth curve analysis revealed no significant differences in
the inherent growth pattern of drug-resistant clinical isolates
5
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compared to the drug-susceptible clinical isolates and respec-
tive Wild Type (WT) strains (Figure S2).
Whole-cell proteome analysis provides novel
insights into drug resistance in clinical fungal
isolates
Despite the ample exploration of mechanisms governing drug
resistance in pathogenic Candida, be it the involvement of ef-
flux pumps (like CDR1/CDR2), drug target alterations (like
SNPs in ERG11), etc., drug resistance has not been curbed
successfully. The possible reasons for this are the dynamic
adaptability and metabolic flexibility of these opportunistic
pathogens (Padder et al. 2022). With this intent, the focus
of our study relied on the exploration of other mechanisms,
which shall be explored in depth to look for alternate targets
to curb the ever-increasing menace of drug resistance. With
this aim, we performed the mass spectrometry-based whole-
cell proteome analysis with one representative drug-resistant
(M1038 for C. albicans and M183 for C. glabrata) versus
one representative susceptible isolate (M2 for C. albicans and
M203 for C. glabrata) to look for the global picture of pro-
tein expression profile changes in drug-resistant isolates. The
results revealed a significant difference in the expression pat-
tern of proteins involving various pathways (Figure S3a–f).
For C. glabrata, 1892 proteins were picked by mass spectrom-
etry analysis. Of those, 1750 proteins were found common in
both M183 and M203 isolates, while 78 proteins were found
specific to isolate M183, and 61 proteins were found spe-
cific to M203 isolate. In the case of C. albicans, a total of
3070 proteins were picked up by mass spectrometry. Among
those, 2817 proteins were reported to be common in M183
and M269 were found specific to M1038 isolate, and 184
were specific to M2 isolate. A detailed representation of the
pathway analysis and differential expression analysis of drug-
6 Padder et al.

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Figure 2. Identification of drug-resistant and representative drug-susceptible clinical isolates of C. albicans and C. glabrata. (a) Colorimetric kit-based
identification of C. albicans, where M1038, M66, and M83 are the drug-resistant clinical isolates of C. albicans and M2 is the respective drug-susceptible
isolate. For C. glabrata, the change in color of the 12th well of the kit (having trehalose as a carbon source) from red to yellow implies a positive result.
For C. albicans, the change in colors of the 4th, 5th, 6th, 9th, and 12th wells (containing maltose, sucrose, galactose, xylose, and trehalose, respectively)
from red to yellow depicts the positive result. (b) Colorimetric kit-based identification of C. glabrata, where M183, M283, and M198 are the
drug-resistant clinical isolates of C. glabrata, and M203 is the respective drug-susceptible isolate. (c) PCR-RFLP-based molecular identification of C.
albicans, where lane 1 represents 50 bp DNA ladder; lanes 2, 4, 6, and 8 represent PCR products of ITS1-4 amplification products of different C. albicans
isolates, M1038, M66, M83, and M2, respectively); lanes 3, 5, 7, and 9 represent Msp1 digestion products of corresponding PCR products. Digestion
products are approximately the size 338 and 297 bp, as expected for C. albicans. (d) PCR-RFLP-based molecular identification of drug-resistant and
representative drug-susceptible isolates of C. glabrata. Where lanes 1 and 8 represent 50 and 100 bp DNA ladder, lanes 2, 4, 6, and 9 represent PCR
products of ITS1-4 amplification of different C. glabrata isolates M183, M283, M198, and M203, respectively; lanes 3, 5, 7, and 10 represent Msp1
digestion products of corresponding PCR products. Digestion products are approximately the size of 557 and 314 bp as expected for C. glabrata.

resistant and drug-susceptible isolates of both species is shown
in Figure S3a–f and Figure S4a and b.
In the case of C. albicans and C. glabrata, we reported
many proteins belonging to different pathways, whose ex-
pression was significantly different between drug-resistant
and drug-susceptible isolates (Table S7a and b). Various pro-
teins involved in carbohydrate metabolism (glycolytic path-
way) were significantly upregulated in C. albicans drug-
resistant isolate M1038 compared to its susceptible isolate M2
(Fig. 2a). These proteins include Hexokinase, Phosphomu-
tase, and Glyceraldehyde-3-phosphate dehydrogenase. Other
glycolytic proteins like glucose-6-phosphate isomerase, phos-
phoglycerate kinase, phosphoglycerate mutase, and phos-
phopyruvate hydratase were also upregulated in M1038
as compared to M2 (Fig. 2a). In contrast, pyruvate ki-
nase and triosephosphate isomerase showed a reduced ex-
pression in M1038 compared to M2 (Fig. 2a). So far, the
expression of the PPP is concerned; there was a signifi-
cantly increased expression of 6-phosphogluconate dehydro-
genase, 6-phosphogluconolactonase and glucose-6-phosphate
dehydrogenase (G6pd) in M1038 as compared to suscepti-
ble isolate M2 (Fig. 2b). A significantly increased expres-
sion of nucleotide biosynthesis proteins like ribose-phosphate
pyrophosphokinase, inosine-5 -monophosphate dehydroge-
nase, GMP synthase, adenylosuccinate lyase, and nucleo-
side diphosphate kinase was also seen in drug-resistant
isolate M1038 as compared to susceptible isolate M2
(Fig. 2c).
Unlike the C. albicans-resistant (M1038) isolate, the pro-
teins belonging to glycolysis, PPP, or nucleotide biosynthesis
pathways did not show significant differential expression pat-
terns in the C. glabrata-resistant isolate (M183). In M183,
there was an increased expression of glycerol biosynthesis-
related proteins like glycerol-3-phosphatase and glycerol-3-
phosphate dehydrogenase. At the same time, the expression
of glycerol catabolism-related protein (glycerol kinase) was
significantly downregulated in M183 compared to M203
(Fig. 2d).
Drug-resistant clinical isolates show enhanced
stress tolerance and elevated glycerol
accumulation
Different types of stresses, which a pathogen usually encoun-
ters in the external environment or the host microenviron-
ment, were given to check the response of drug-resistant and
drug-susceptible Candida isolates. Evaluation of stress resis-
tance was done through serial dilution spot assays with differ-
ent stressors against drug-resistant and drug-susceptible clin-
ical isolates of C. albicans and C. glabrata (Fig. 3). The as-
say was also performed to evaluate the effects of higher (5%)
and lower (0.2%) glucose concentrations. Besides an alternate
carbon source, glycerol (a nonfermentable carbon source) was
also used to examine the difference in growth patterns (if any)
of drug-resistant and susceptible clinical isolates. The resistant
isolates (M1038 and M183) showed more resistance to some
stressors than their respective drug-susceptible isolates (Fig. 3)
albeit to a lesser extent but collectively seem enough to have
a significant impact on drug resistance. However, there was
some difference in resistance pattern between the resistant iso-
lates. For example, at 0.015% SDS, M1038 showed more re-
sistance than M183, while the latter showed more resistance
to CR (300 μgml−1 ), at which no growth was witnessed in
the former. Both the resistant isolates showed resistance to-
ward peroxide stress. However, the resistance was more in
M183 than in M1038, as the former showed resistance at a
very higher concentration of H2 O2 (15 mmol) while the latter
could not grow at this concentration. So far, the thermal stress
is concerned; both the resistant isolates showed resistance to a
higher temperature (37◦ C) compared to the respective suscep-
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Figure 3. Spot assay analysis of drug-resistant and drug-susceptible isolates of C. albicans and C. glabrata against different stressors. M1038 and M2
represent drug-resistant and drug-susceptible isolates of C. albicans, respectively, while M183 and M203 represent drug-resistant and drug-susceptible
isolates of C. glabrata, respectively. CFW, CR, t-BOOH, and H2 O2 are abbreviations for CFW, CR, tert-butyl hydroperoxide, and hydrogen peroxide. The
results are representative of at least three independent experiments.

tible isolates (Fig. 3). However, at a lower temperature (23◦ C),

there was no significant difference in growth in C. glabrata
isolates, while C. albicans-resistant isolate (M1038) showed
increased growth compared to its susceptible isolate (M2) at
this temperature. The resistant isolates (M1038 and M183)
showed increased growth at higher and lower glucose concen-
trations compared to their respective drug-susceptible isolates.
Upon exposure to alternate carbon sources (2% glycerol), the
resistant isolates showed comparatively more growth than the
drug-susceptible isolates (Fig. 3). Overall, the drug-resistant
isolates revealed a better stress resistance than their respective
susceptible isolates.
To withstand the hypertonic conditions, microorganisms
accumulate compatible solutes (Khatibi et al. 2019). More-
over, the whole cell proteome profile insights revealed an in-
creased expression of glycerol biosynthetic genes, particularly
in the drug-resistant C. glabrata isolate (Fig. 2d). To inves-
tigate the correlation between stress resistance and osmotic
balance and confirm the proteomics data, we measured the in-
tracellular glycerol accumulation in drug-resistant and drug-
susceptible clinical isolates on exposure to different stres-
sors for different time periods. Three stressors (1 M NaCl,
1.25 mM t-BOOH, and 200 μg ml−1 CFW) were used, and
glycerol accumulation was measured at 30- and 60-min post-
treatment in both the resistant and susceptible isolates of
C. albicans and C. glabrata (Fig. 4). As can be seen from
Fig. 4, the internal glycerol accumulation was the same in
all the isolates when no stress was given (basal glycerol con-
tent); however, upon exposure to different stresses, the resis-
tant isolates tend to significantly accumulate glycerol com- Figure 4. Intracellular glycerol content in drug-resistant and
pared to the respective susceptible isolates (Fig. 4). Addition- drug-susceptible isolates upon exposure to different stresses. (a–c)
Represents total intracellular glycerol accumulation in drug-resistant
ally, C. glabrata (M183) showed more glycerol biosynthe-
(M183) and susceptible (M203) isolates of C. glabrata under NaCl (1 M),
sis/accumulation compared to C. albicans (M1083), indicat- CFW (200 μgml−1 ), and t-BOOH (1.25 mM) stress, respectively, at
ing a more dependence of the former on the glycerol biosyn- different time points (n = 3, mean ± SEM). (d–f) represents total
thesis pathway (Fig. 4). In drug-susceptible isolates, glycerol intracellular glycerol accumulation in drug-resistant (M1038) and
accumulation either remained unchanged or did not change susceptible (M2) isolates of C. albicans under NaCl (1 mol), CFW
significantly in response to stress (Fig. 4). (200 μg ml−1 ), and t-BOOH (1.25 mmol) stress, respectively, at different
time points (n = 3, mean ± SEM).

Glycerol biosynthesis/accumulation as a stress

response mediated via Hog1 activation in
drug-resistant clinical isolates
Osmotic/oxidative or heavy metal stress adaptation is pri-
marily linked to high osmolarity glycerol 1 (Hog1) mitogen-
activated protein kinase (Hog1-MAPK) signal transduction
pathway (Ren et al. 2022). As the resistant isolates show
an increased glycerol accumulation upon exposure to differ-
ent stress-mimicked environments, we sought to investigate
whether glycerol accumulation is linked to Hog1 activation
(phosphorylated Hog1). It was found that Hog1 was activated
8 Padder et al.

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Figure 5. Immunoblot analysis of pHog1 under different stress conditions. Stressors used include 1 mol NaCl, 200 μg ml−1 CFW, and 1.25 mmol t-BOOH
in drug-resistant and drug-susceptible isolates of C. glabrata and C. albicans. (a) Representative western blots, where 0, 30, and 60 represent time in
minutes post-stress treatment, respectively, and (b) represents the corresponding densitometry of immunoblots, where 1, 2, and 3 represent 0, 30, and
60 min post-stress treatment, respectively. The results are representative of at least three independent experiments.

upon stress induction (1 mol NaCl, 1.25 mmol t-BOOH, and
200μgml−1 CFW) in a time-dependent manner. Such activa-
tion was specific to resistant isolates only (Fig. 5). Of note,
in the absence of external stress, there was no significant dif-
ference in the expression of pHog1 between the susceptible
and resistant isolates, further indicating Hog1 activation as
an adaptive response to adverse environmental exposures in
resistant fungi (Fig. 5).
Additionally, Hog1 activation was more pronounced in C.
glabrata-resistant isolate (M183) compared to C. albicans-
resistant isolate (M1038) (Fig. 5). Overall, these findings indi-
cate that elevated glycerol accumulation is used as an adaptive
measure to withstand environmental stresses brought about
by various classes of antifungal drugs and the host immune
response. More importantly, such adaptation seems to be reg-
ulated via Hog1 activation.
Enhanced glucose fermentation accounts for
reduced drug response in resistant fungi
Hxk2 was one of the many proteins with elevated expression
levels in the whole-cell proteomic data. The enzyme being the
first-rate limiting enzyme in glucose energy metabolism, we
were interested in seeing if this enzyme is related to reduced
drug response in these fungi. To this end, we studied the ex-
pression levels of Hxk2 in these fungi employing western blots
from whole-cell protein lysates, and it was found that drug-
resistant (M1038) C. albicans had significantly higher expres-
sion levels of Hxk2 compared to the corresponding suscepti-
ble isolate (M2), (Fig. 6a and c). However, the trend was not
similar in C. glabrata, where the drug-resistant (M183) and
susceptible (M203) isolates did not show any considerable dif-
ference in the expression of Hxk2 (Fig. 6b and d). To confirm
the role of Hxk2 in mediating drug resistance, we knocked
down its expression by treating these fungal isolates with 3-BP,
which selectively inhibits the expression of the human Hxk2
enzyme (Sun et al. 2015, Zou et al. 2015, Rai et al. 2019).
Interestingly, low-dose treatments at 0.39 mM (sub MIC80 )
were found to significantly inhibit the expression of Hxk2
(Fig. 6a–d). Of note, 3-BP treatment inhibited Hxk2 expres-
sion in both C. albicans and C. glabrata species irrespective
of the resistant or susceptible nature of the isolates (Fig. 6a–
d), further confirming the specificity of the inhibitor to inhibit
Hxk2 in fungi as well. After confirming the inhibitory effects
of 3-BP on Hxk2 expression, we sought to evaluate whether
3-BP was able to rescue drug resistance in these fungi, and it
was found that 3-BP not only increased FLC drug sensitiv-
ity but also reduced its MIC80 values in C. albicans but not
C. glabrata (Table S8a). Interestingly, such effects were more
pronounced in the resistant (M1038, with a 16-fold reduc-
tion in MIC of FLC) compared to the susceptible (M2, with
an approximately of 4-fold reduction in FLC MIC) isolates
(Table S8a), indicating a specific role of Hxk2 in mediating
drug resistance in C. albicans. Also, as expected, 3-BP could
not rescue drug resistance in C. glabrata to significant levels
(Table S8a), further confirming little or no role of Hxk2 or
related compensatory mechanisms in mediating resistance in
C. glabrata.
To further ascertain the metabolic relevance of Hxk2 ex-
pression and drug resistance, we tried to determine whether
Hxk2-mediated drug resistance is associated with increased
glucose fermentation, a phenomenon associated with cancer
progression and chemotherapeutic resistance in humans (Li et
al. 2020). It was observed that resistant C. albicans (M1038)
fermented glucose at a faster rate compared to the susceptible
(M2) isolates, besides producing more ATP (Fig. 7a–c), indi-
cating the importance of an acidic microenvironment to with-
stand various stress signals. Expectedly, no significant differ-
ence could be observed in glucose fermentation rate between
resistant (M183) and susceptible (M203) C. glabrata isolates
(Fig. 7d–f). To rule out whether such glucose fermentation was
Intermediary metabolism and C andida drug resistance 9

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Figure 6. Immunoblot analysis of Hxk2 in drug-resistant and drug-susceptible isolates of C. albicans and C. glabrata with and without 3-BP treatment. (a)
Represents western blots showing varied expression of Hxk2 in drug-resistant (M1038) and drug-susceptible (M2) C. albicans isolates with and without
3-BP treatment (b) Represents western blots showing varied expression of Hxk2 in drug-resistant (M183) and drug-susceptible (M203) C. glabrata
isolates with and without 3-BP treatment. In a and b, 0, 2.5, and 5 represents time in hours post stress application (c and d) represent the densitometry
of immunoblots A and B, respectively. In c and d, 0, 1, and 2 represent 0, 2.5, and 5 h post-stress application, respectively.

Figure 7. Glucose consumption rate, lactate production, and ATP generation of drug-resistant and drug-susceptible isolates of C. albicans and C.
glabrata. (a–c) Represents relative glucose consumption rates, relative lactate production, and relative ATP generation of drug-resistant and
drug-susceptible clinical isolates of C. albicans with and without 3-BP treatment (n = 3, mean ± SEM). (d–f) Represents relative glucose consumption
rates, relative lactate production, and relative ATP generation of drug-resistant and drug-susceptible clinical isolates of C. glabrata with and without 3-BP
treatment (n = 3, mean ± SEM).
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Figure 8. Immunoblot analysis of G6pd in drug-resistant and drug-susceptible isolates of C. albicans and C. glabrata with and without polydatin
treatment. (a) Represents western blots showing varied expression of G6pd in drug-resistant (M1038) and drug-susceptible (M2) C. albicans isolates
with and without polydatin treatment. (b) Represents western blots showing varied expression of G6pd in drug-resistant (M183) and drug-susceptible
(M203) C. glabrata isolates with and without polydatin treatment. In (a and b) 0, 2.5, and 5 represent time in hours post stress application. (c and d)
Represent the densitometry of immunoblots A and B, respectively. In (c and d) 0, 1, and 2 represent 0, 2.5, and 5 h post-stress application, respectively.

Hxk2 dependent, we knocked down its expression using its
pharmacological inhibitor 3-BP. It was found that 3-BP was
able to rescue glucose fermentation and ATP production rates
in the resistant C. albicans isolates (M1038) (Fig. 7a–c). To
the best of our knowledge, we, for the first time, were able
to show a phenomenon related to the Warburg effect (glucose
fermentation) operative in C albicans. Overall, these results in-
dicate a pivotal role of glucose fermentation brought about by
Hxk2 in mediating drug resistance in C. albicans. The results
further confirmed the difference in resistance mechanisms op-
erative in C. albicans and C. glabrata drug-resistant clinical
isolates.
effects of polydatin on G6pd expression, we sought to eval-
uate whether polydatin was able to rescue drug resistance in
these fungi, and it was found that the latter not only increased
drug (FLC) sensitivity but also reduced its MIC80 values in C.
albicans but not C. glabrata (Table S8b). Interestingly, such ef-
fects were more pronounced in the resistant (M1038, with an
8-fold reduction in MIC of FLC) compared to the susceptible
(M2, with an ∼4-fold reduction in FLC MIC) isolates (Table
S8b), indicating a specific role of G6pd in mediating drug re-
sistance in C. albicans. Also, expectedly polydatin could not
rescue drug resistance in C. glabrata to significant levels (Table
S8b).

Elevated expression of G6pd mediates drug
resistance in C. Albicans
The proteomic data also revealed that another closely related
protein, G6pd, was also observed as one among the elevated
proteins in mass spectrometry data of C. albicans. We em-
ployed the immunoblot approach to confirm the expression
levels of G6pd between resistant versus susceptible C. albi-
cans, which revealed a significantly increased expression of
G6pd in the resistant isolate (M1038) compared to the suscep-
tible isolate (M2) (Fig. 8a and c). However, no such difference
in the expression of G6pd was seen between C. glabrata resis-
tant (M183) and susceptible (M203) isolates (Fig. 8b and d).
Next, we attempted to seek the role of G6pd in mediating drug
response in these fungi. To this end, we knocked down G6pd
by using its pharmacological inhibitor polydatin (Reservatrol-
3-β-mono-D-glucoside). Low dose (0.312 mM and sub-MIC)
polydatin treatment led to a significant inhibition in G6pd ex-
pression in both the resistant and susceptible fungi (Fig. 8a–
d), further confirming the specificity of the inhibitor to in-
hibit G6pd in fungi as well. After confirming the inhibitory
G6pd generates reducing equivalents to scavenge
ROS in resistant isolates
G6pd being the critical rate-limiting enzyme in the PPP gen-
erates the reduced equivalents (NADPH) needed to maintain
the antioxidant (GSH) levels within the cell (Xiao et al. 2018).
This prompted us to seek out whether elevated G6pd levels
in these resistant strains (M1038) have any role in antioxi-
dant defense. Interestingly, we found a significantly increased
NADPH production in drug-resistant (M1038) C. albicans
compared to susceptible isolate (M2) (Fig. 9a). In contrast, no
such difference in NADPH production was seen in C. glabrata
isolate (Fig. 9b). In line, ROS levels were significantly lower
in the resistant (M1038) compared to the susceptible (M2)
C. albicans isolate (Fig. 9c and e). We also did rescue experi-
ments, in which we knocked down G6pd using its pharmaco-
logical inhibitor and polydatin (0.078 mM). It was found that
post-drug treatment, cellular NADPH levels declined signifi-
cantly in both the resistant and susceptible C. albicans isolates
(Fig. 9a). Interestingly, these rescue experiments were able to
increase ROS generation in otherwise ROS scavenging resis-
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Figure 9. ROS generation and relative NADPH production in drug-resistant and drug-susceptible clinical isolates of C. albicans and C. glabrata. (a)
Represents ROS generation in drug-resistant (M1038) and drug-susceptible (M2) isolates of C. albicans under normal cellular state and after the addition
of polydatin (M1038 + P and M2 + P). (b) Represents relative fluorescence intensities of drug-resistant (M1038) and drug-susceptible (M2) isolates of C.
albicans under normal cellular state and after the addition of polydatin (M1038 + P and M2 + P). (c) Relative NADPH production in drug-resistant (M1038)
and drug-susceptible (M2) isolates of C. albicans under normal cellular state and after the addition of polydatin (M1038 + P and M2 + P). (d) Represents
ROS generation in drug-resistant (M183) and drug-susceptible (M203) isolates of C. glabrata under normal cellular state and after the addition of
polydatin (M183 + P and M203 + P). (e) Represents relative fluorescence intensities of drug-resistant (M183) and drug-susceptible (M203) isolates of C.
glabrata under normal cellular state and after the addition of polydatin (M183 + P and M203 + P). (f) Relative NADPH production in drug-resistant (M183)
and drug-susceptible (M203) isolates of C. glabrata under normal cellular state and after the addition of polydatin (M183 + P and M203 + P).

tant strains (M1308), and the low ROS scavenging susceptible
isolates (Fig. 9c and e). Nevertheless, polydatin treatment to
C. glabrata also increased ROS production, likely due to over-
all NADPH depletion post G6pd inhibition (Fig. 9d and f).
Additionally, if we compare resistant C. albicans (M1038) to
the corresponding C. glabrata (M183) isolate, the latter had
significantly higher ROS levels (Fig. 9e and f), suggesting al-
ternate mechanism/s governing drug resistance in this specie
(glycerol metabolism regulated via Hog1, Fig. 5). These re-
sults indicate the role of G6pd and/or PPP in maintaining the
cell’s redox state to withstand drug insults. Therefore, the re-
sults report and validate for the first time the G6pd mediated,
NADPH-driven ROS scavenging, leading to enhanced drug re-
sistance in C. albicans.
Discussion
The incidence of fungal infections has been on the rise due
to several factors and the limited arsenal of antifungal drugs
being under continuous use, which has led to the develop-
ment of drug-resistant fungi, thereby posing a growing clini-
cal threat (Ksiezopolska and Gabaldón 2018). Approximately
20%–30% of candidemia cases are due to intrinsically resis-
tant Candida isolates (to FLC or echinocandins) (Arendrup
2014). Of late, these fungal infections were frequently asso-
ciated with COVID-19 infection in India, thereby augment-
ing the healthcare burden (Kuehn 2021, Dubey et al. 2022).
To this end, the present study aimed to evaluate the molec-
ular mechanism of antifungal resistance in the clinical iso-
lates of pathogenic fungi. A total of 84 clinical isolates be-
longing to different Candida species were procured from a
tertiary care hospital in the Northern part of India (Kash-
mir valley). Identification of the isolates revealed that they
belonged to multiple species of Candida. The MIC80 results
using FLC drug revealed that many clinical isolates showed
resistance against this azole. The drug-resistant isolates of two
frequently encountered (globally) Candida species (C. albi-
cans and C. glabrata) were evaluated further to understand
the novel molecular mechanisms governing antifungal drug
resistance.
Mass spectrometry-based whole-cell proteome analysis car-
ried out for one representative drug-resistant isolate of C. al-
bicans and C. glabrata revealed differential protein expres-
sion in drug-resistant Candida isolates, compared to drug-
susceptible isolates. Among them, the proteins involved in
carbohydrate metabolism were significantly upregulated in
C. albicans drug-resistant (M1038) isolate. While we could
observe an increased expression of glycerol biosynthesis-
related proteins like glycerol-3-phosphatase and glycerol-3-
phosphate dehydrogenase in C. glabrata drug-resistant iso-
late but unlike C. albicans resistant isolate, the proteins in-
volved in carbohydrate metabolism did not show signifi-
cant differential expression. On the other hand, the expres-
sion of glycerol catabolism-related protein (glycerol kinase)
was significantly downregulated in C. glabrata drug-resistant
isolate.
12
In line with the proteomic profile of different Candida
isolates, we could also observe a difference in drug resis-
tance mechanisms between C. albicans and C. glabrata drug-
resistant isolates. The former showed a Hxk2-mediated anti-
fungal resistance, wherein an increased expression of Hxk2
was a key factor governing drug resistance via increased glu-
cose uptake, more ATP generation, and enhanced lactate pro-
duction (Warburg effect, as seen in human cells). Metaboliz-
ing glucose at an increased rate is a strategic hallmark of most
if not all, aggressive cancers (Mathupala et al. 2006). Most
cancerous cells preferentially drive their metabolism away
from oxidative phosphorylation to glycolysis irrespective of
the oxygen availability to fulfill their growing anabolic needs
(Hsu and Sabatini 2008, Pedersen 2008). Such metabolic aber-
ration is regulated by the enzyme Hxk2, which maintains a
higher glycolytic flux by catalyzing the initial phosphorylation
step of glucose to glucose-6-phosphate to support more glu-
cose fermentation than normal cells (Hsu and Sabatini 2008,
Pedersen 2008). However, contrary to cancerous cells, Hxk2
in S. cerevisiae, besides its catalytic role, harbors a vital regula-
tory function in the glucose catabolite repression mechanism
(Moreno and Herrero 2002, Gancedo and Flores 2008).
Most importantly, thus far, to the best of our knowledge,
there are scarce reports describing the role of Hxk2 in an-
tifungal resistance, and we have for the first time elucidated
the close role of Hxk2 in mediating antifungal resistance (Wi-
jnants et al. 2020, Laurian et al. 2021). The present study
suggests that like in cancer (Gatenby and Gillies 2004, Lunt
and Vander Heiden 2011), modulating glycolytic flux could
be an efficient strategy to target glucose metabolism in drug-
resistant fungi. The results from our study are in line with
many clinical studies, which have documented a strong cor-
relation between increased Hxk2 expression with the disease
progression (including resistance) in various types of cancers
(Li et al. 2020). We selected a pharmacological inhibitor, 3-BP,
which has been reported to selectively inhibit Hxk2 in human
cells (Zou et al. 2015, Rai et al. 2019) since the in-silico molec-
ular docking approach also revealed a very good binding affin-
ity of 3-BP with C. albicans Hxk2 (Figure S5, Table S9a). For
the first time, we evaluated the Hxk2 inhibitory effect of 3-BP
in this group of human pathogenic fungi, which interestingly
rescued glucose uptake, ATP production, and lactate produc-
tion in these Candida isolates (Fig. 7a–f). Besides, such gly-
colytic phenotype was more pronounced in C. albicans com-
pared to C. glabrata isolates. Importantly, such inhibition also
rescued FLC resistance in the clinical isolate (M1038) of C.
albicans.
Drug-resistant C. albicans also revealed a better G6pd me-
diated ROS scavenging as the resistant isolate showed a signif-
icant upregulation of G6pd that was directly associated with
increased production of NADPH, and hence better ROS scav-
enging. In contrast, there was no such difference in NADPH
production and ROS scavenging in C. glabrata isolates. Of
note, the data related to G6pd and Candida pathogenicity
is scarce. We report G6pd as a strong possible therapeu-
tic target in otherwise drug-resistant fungi. We, for the first
time, used a selective pharmacological inhibitor (polydatin) of
G6pd (Mele et al. 2018, Doustimotlagh and Eftekhari 2021),
and showed that it inhibited the metabolism and pathogenic-
ity in this group of pathogenic fungi. Polydatin treatment was
able to inhibit G6pd expression and rescue ROS scavenging
via NADPH depletion, as reported previously (Aurora et al.
2022). An in-silico molecular docking approach also revealed
Padder et al.
a very good binding affinity of polydatin with G6pd (Figure
S5, Table S9b). It is pertinent to mention that between the
resistant isolates of both species, more ROS production and
less ROS scavenging was observed in M183 than in M1038,
pointing toward different resistance mechanisms seen in the C.
glabrata (glycerol metabolism-related Hog1 activation) clini-
cal isolate.
Nevertheless, unlike C. albicans, resistant isolates from C.
glabrata did not show any difference in the expression of
Hxk2 and G6pd. Rather a glycerol biosynthesis-mediated
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drug resistance was seen in C. glabrata, wherein the resistant
isolate was reported to accumulate more intracellular glyc-
erol as an osmolyte that imparts better adaptability to the
pathogen in response to osmotic stress (Fig. 10). Glycerol is
synthesized by the reduction of dihydroxyacetone phosphate
to glycerol 3-phosphate (G3P), followed by its dephospho-
rylation to glycerol, and the steps are catalyzed by NAD-
dependent cytosolic glycerol 3-phosphate dehydrogenase and
glycerol-3-phosphatase (Van Eck et al. 1993, Eriksson et al.
1995, Nevoigt and Stahl 1997, Welsh 2000, Petelenz-Kurdziel
et al. 2013). In drug-resistant C. glabrata (M183), there
was an increased expression of these glycerol biosynthesis-
related proteins like glycerol-3-phosphatase and glycerol-3-
phosphate dehydrogenase (Fig. 10). At the same time, the ex-
pression of glycerol catabolism related protein (glycerol ki-
nase) was significantly downregulated in M183 compared to
M203 (Fig. 10). Such glycerol accumulation was found to be
driven by the Hog1-MAPK pathway, wherein the phosphory-
lation of Hog1 (and hence its activation) led to the increased
accumulation of intracellular glycerol (Fig. 10) in line with the
previous reports (Wang et al. 2020). Phosphorylated Hog1,
in turn, controls the accumulation of glycerol by stimulat-
ing the transcription of glycerol-producing enzymes (Gpd1,
Gpd2) and glycerol-proton symporter (Slt1), rapid closure of
glycerol facilitator (Fps1) besides activating 6-phosphofructo-
2-kinase (Pfk26/27) (Dihazi et al. 2004, Petelenz-Kurdziel et
al. 2013).
Acknowledgments
We acknowledge the initial support, laboratory space, and
useful suggestions provided by Prof. Rajendra Prasad, Amity
University, Gurugram, Haryana during the course of this
work. S.A.P acknowledges the financial support received in
the form of JRF and SRF from SERB-GOI, Early Career Re-
search Award (ECR/2016/000463) project and SRF received
from ICMR-GOI (AMR/Fellowship/1/2020-ECD-II). We also
acknowledge the help provided with fluorescence microscopy
by Dr. Khalid Z. Masoodi, Transcriptomics Laboratory, Di-
vision of Plant Biotechnology, SKUAST-K, India. We want to
thank VProteomics, New Delhi, India, where Mass Spectrom-
etry data generation and preliminary analysis were performed.
Ethics approval
The study was approved under protocol No. IEC/SKIMS #
Protocol 82/2016 by the institutional ethical committee for
the use of anonymous clinical isolates of fungi.
Supplementary data
Supplementary data is available at JAMBIO Journal online.
Intermediary metabolism and C andida drug resistance 13

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Figure 10. Schematic illustration of drug resistance mechanisms adopted by clinical isolates of C. albicans and C. glabrata. Upon stress exposure, Hog1
is extensively phosphorylated (activated) in resistant isolates of both species compared to the respective drug-susceptible clinical isolates, which impart
better stress adaptability to them by increasing the intracellular glycerol levels (osmolyte). Upregulation of glycerol biosynthesis-related proteins like
glycerol-3-phosphate and glycerol-3-phosphate dehydrogenase and downregulation of glycerol catabolism-related protein-glycerol kinase was seen in C.
glabrata resistant isolate leading to higher intracellular glycerol accumulation in C. glabrata resistant isolate. Contrary to this, drug-resistant clinical isolate
of C. albicans showed an elevated expression of Hxk2 and G6pd, which was associated with increased glucose fermentation (increased glucose uptake,
more lactate production, and more ATP generation) and NADPH mediated ROS scavenging in these resistant isolates, respectively. Upon inhibiting Hxk2
and G6pd with the pharmacological inhibitors 3-BP and polydatin, respectively, drug resistance was rescued in the drug-resistant C. albicans clinical
isolate. Up and down arrows represent the upregulation and downregulation of corresponding genes or metabolites.

Conflict of interest
None declared.
Funding
This work was supported in part by grants to AHS by the Sci-
ence and Engineering Research Board (SERB)-GoI under the
Early Career Research Award (ECR/2016/000 463) and the
Department of Science and Technology (DST)-GoI under IN-
SPIRE Faculty Award (DST/INSPIRE/04/2015/001 575). Fun-
ders have no role in “study design, data collection, and anal-
ysis, the decision to publish, or manuscript preparation.”
Authors contribution
S. A. Padder (Conceptualization, Investigation, Methodology,
Writing – original draft), R. A. Padder (Investigation, Method-
ology, Writing – original draft), A. Ramzan (Investigation), G.
Bashir (Investigation, Funding acquisition, Writing – original
draft), I. Tahir (Visualization Writing – Review & Editing), R.
U. Rehman (Supervision, Writing – Review & Editing) and A.
H. Shah (Conceptualization, Funding acquisition, Investiga-
tion, Supervision, Writing – original draft; Writing – Review
and editing).
Data availability
The mass spectrometry proteomics data have been deposited
to the ProteomeXchange Consortium via the PRIDE partner
repository with the dataset identifier PXD034153.
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