Glutathione biosynthesis in the yeast pathogens
Candida glabrata and Candida albicans: essential
in C. glabrata, and essential for virulence in
Amit Kumar Yadav,1Prashant Ramesh Desai,1Maruti Nandan Rai,2
Rupinder Kaur,2Kaliannan Ganesan1and Anand Kumar Bachhawat13
Anand Kumar Bachhawat
Received 31 August 2010
Accepted 10 October 2010
1Institute of Microbial Technology (CSIR), Sector 39-A, Chandigarh 160 036, India
2Centre for DNA Fingerprinting and Diagnostics, Building 7, Gruhakalpa 5-4-399/B, Nampally,
Redox pathways play a key role in pathogenesis. Glutathione, a central molecule in redox
homeostasis in yeasts, is an essential metabolite, but its requirements can be met either from
endogenous biosynthesis or from the extracellular milieu. In this report we have examined the
importance of glutathione biosynthesis in two major human opportunistic fungal pathogens,
Candida albicans and Candida glabrata. As the genome sequence of C. glabrata had suggested
the absence of glutathione transporters, we initially investigated exogenous glutathione utilization
in C. glabrata by disruption of the MET15 gene, involved in methionine biosynthesis. We observed
an organic sulphur auxotrophy in a C. glabrata met15D strain; however, unlike its Saccharomyces
cerevisiae counterpart, the C. glabrata met15D strain was unable to grow on exogenous
glutathione. This inability to grow on exogenous glutathione was demonstrated to be due to the
lack of a functional glutathione transporter, despite the presence of a functional glutathione
degradation machinery (the Dug pathway). In the absence of the ability to obtain glutathione from
the extracellular medium, we examined and could demonstrate that c-glutamyl cysteine synthase,
the first enzyme of glutathione biosynthesis, was essential in C. glabrata. Further, although c-
glutamyl cysteine synthase has been reported to be non-essential in C. albicans, we report here
for what is believed to be the first time that the enzyme is required for survival in human
macrophages in vitro, as well as for virulence in a murine model of disseminated candidiasis. The
essentiality of c-glutamyl cysteine synthase in C. glabrata, and its essentiality for virulence in C.
albicans, make the enzyme a strong candidate for antifungal development.
Glutathione, c-Glu-Cys-Gly, is an essential metabolite in
almost all eukaryotic organisms (Fahey & Sundquist, 1991;
Meister & Anderson, 1983), and plays a key role in redox
homeostasis and in the cellular response to oxidative stress
(Meister & Anderson, 1983; Penninckx, 2002; Sipos et al.,
2002). The importance of oxidative stress responses in the
virulence and survival of pathogens in their natural
environment has been suggested by many studies, but the
relative importance of the glutathione-dependent redox
pathway, as opposed to other pathways for redox
homeostasis and oxidative stress, has not been rigorously
evaluated. In parasitic protozoans such as Leishmania
infantum and Trypansoma brucei, however, glutathione
biosynthesis (which leads to trypanothione synthesis), is
essential (Huynh et al., 2003; Mukherjee et al., 2009), while
in the malarial parasite Plasmodium berghei, glutathione
biosynthesis has been shown to be essential for the survival
of the protozoan during its passage through insects (Vega-
Rodrı ´guez et al., 2009). Further, in L. infantum, even the
deletion of one of the two copies of the GSH1 gene is not
tolerated by the organism. These findings describing the
essentiality of glutathione biosynthesis in these pathogens
prompted us to investigate the importance, if any, of
Abbreviations: FOA, 5-fluoroorotic acid; ROS, reactive oxygen species;
UTR, untranslated region.
3Present address: Indian Institute of Science Education and Research
(IISER), Mohali Transit Campus: MGSIPAP Complex, Sector 26,
Chandigarh 160 019, India.
A supplementary figure, showing the growth of C. glabrata wild-type
(ABG2367) and met15D (ABG2370) strains on inorganic sulphate and
methionine, and a supplementary table, listing the primers used in this
study, are available with the online version of this paper.
Microbiology (2011), 157, 484–495
484045054G2011 SGM Printed in Great Britain
glutathione biosynthesis in the survival of yeast pathogens
in their mammalian host. Candida albicans and Candida
glabrata are the two most important yeast pathogens that
cause bloodstream infections (Fidel et al., 1999; Pfaller
et al., 2010; Wingard, 1995). Although C. glabrata is
phylogenetically much closer to Saccharomyces cerevisiae
than to C. albicans, both are human commensals and are
rarely seen in the environment outside the human host,
where their ecological niche is the vaginal mucosa, skin and
blood (Fidel et al., 1999; Kaur et al., 2005).
Work with the yeasts S. cerevisiae and Schizosaccharomyces
pombe have shown that yeasts have the ability to obtain
their glutathione requirements from both endogenous
biosynthesis and the extracellular medium (Bourbouloux
et al., 2000; Thakur et al., 2008). Disruption of glutathione
biosynthesis in the yeasts Schizosaccharomyces pombe and
S. cerevisiae, carried out by knocking out the first enzyme
of glutathione biosynthesis, c-glutamyl cysteine synthase
(GSH1 or GCS1), leads to glutathione auxotrophy, in
which the cells become dependent for growth on
exogenous glutathione, the uptake of which is mediated
by high-affinity glutathione transporters. Furthermore, in
S. cerevisiae, the exogenous glutathione can also be utilized
as a sulphur source, and this utilization depends on the
presence of the glutathione transporter as well as on an
alternative pathway of glutathione degradation that
involves the ‘Dug pathway complex’, comprising the
three proteins Dug1p, Dug2p and Dug3p (Ganguli et al.,
In C. albicans, disruption of glutathione biosynthesis has
been shown to cause glutathione auxotrophy similar to that
observed in S. cerevisiae and Schizosaccharomyces pombe,
and the disruptant displays higher reactive oxygen species
(ROS) levels and undergoes apoptosis (Baek et al., 2004).
In C. glabrata, the genome sequence has revealed that a
large number of ORFs have been lost in comparison with
its close relative S. cerevisiae (De Hertogh et al., 2006;
Dujon et al., 2004), and among the ORFs that appear to be
absent are homologues of members of the oligopeptide
transporter family, to which the yeast high-affinity
glutathione transporters (HGT1 of S. cerevisiae and PGT1
of Schizosaccharomyces pombe and others) belong. It was
relevant, therefore, first to investigate the importance of
glutathione biosynthesis and assimilation in C. glabrata.
We demonstrate in this study that C. glabrata lacks the
ability to utilize exogenously provided glutathione, owing
to the lack of a glutathione transporter, despite possessing a
functional Dug complex for the degradation of glutathione.
We also demonstrate that in C. glabrata, c-glutamyl
cysteine synthase (GSH1) is an essential enzyme, and that
in C. albicans, despite the ability of Cagcs1D strains to grow
in the presence of exogenous glutathione, CaGCS1 is
essential for virulence. The demonstration of the essential-
ity of c-glutamyl cysteine synthase for the virulence of these
yeast pathogens makes this enzyme an attractive target for
Materials. All chemicals and reagents were of analytical reagent
grade and were procured from different commercial sources.
Oligonucleotide primers were synthesized by Sigma-Genosys, India.
Medium components were purchased from BD (Difco). Restriction
enzymes, DNA polymerases and other DNA-modifying enzymes were
obtained from New England Biolabs. Gel extraction kits and plasmid
miniprep columns were obtained from Qiagen.
Strains, media and growth conditions. The Escherichia coli strain
DH5a was used as a cloning host. S. cerevisiae, C. glabrata and C.
albicans strains used in the study are described in Table 1. S. cerevisiae
and C. glabrata strains were regularly maintained on yeast extract
peptone dextrose (YPD) medium. Synthetic defined minimal
medium contained yeast nitrogen base, ammonium sulphate and
glucose, supplemented with methionine, cysteine, glutathione,
homocysteine, histidine, leucine, lysine (as per requirement) and
uracil at 50 mg l21. Yeast transformations were carried out using the
modified lithium acetate method, as described for C. glabrata (Gietz
et al., 1992), and for C. albicans using electroporation (Reuss et al.,
Cloning of CgMET15. The CgMET15 (CAGL0D06402g) ORF along
with its 59 untranslated region (59 UTR) (600 bp) was PCR-amplified
from the genomic DNA of C. glabrata using primers CgMET15-600F
and CgMET15R, yielding a 2 kb product with an SmaI site. This PCR
product was then digested with SmaI and cloned into pGRB2.2 to
yield plasmid pGRB2.2-CgMET15.
Subcloning of ScGSH1 and HGT1 from S. cerevisiae into C.
glabrata expression vector pGRB2.2. Plasmid p416TEF-ScGSH1
(Sharma et al., 2000) was digested with BspDI and HindIII to release
the 2 kb insert from vector backbone of p416TEF (5.5 kb).
Subsequently, this insert of 2 kb was blunted and then ligated at
the SmaI site of pGRB2.2 to yield plasmid pGRB2.2-ScGSH1. Plasmid
p416TEF-ScHGT1 (Kaur et al., 2009) was digested with BamHI and
EcoR1 to release the 2.4 kb insert from the vector backbone of
p416TEF (5.5 kb). Subsequently, this insert was cloned into pGRB2.2
at the same sites (BamHI and EcoRI) to yield plasmid pGRB2.2-
Construction of strains
Construction of the Cgmet15D strain. The CgMET15 gene was
recombination. Briefly, 850 bp of the 59 UTR of MET15 was PCR-
amplified from wild-type genomic DNA using primer pair OgRK17
and OgRK18, and digested and cloned upstream of the hygromycin-
resistance gene (hph) (under the PGK1 promoter) in the KpnI and
HindIII sites of a plasmid containing the URA3 gene as a selection
marker (plasmid pRK9). Next, the 39 UTR of MET15 (about 750 bp)
was amplified with primers OgRK19 and OgRK20 from wild-type
genomic DNA, digested with SpeI and SacI, and cloned downstream
of the hph gene in the SpeI/SacI sites of pRK9, so that the hph gene
was flanked by the 59 and 39 UTRs of MET15 (plasmid pRK13). For a
one-step replacement strategy, plasmid pRK13 was digested with Bcg1
(sites engineered during primer design), the linear fragment carrying
the HYG cassette (hph gene flanked by the 59 and 39 UTRs of MET15)
was transformed into wild-type strains, and transformants were selected
for hygromycin resistance. Hygromycin-resistant transformants were
then screened for methionine auxotrophy. Replacement of the
MET15 ORF with the hph gene was verified by PCR using a primer
external to the cloned fragment [at both the 59 (OgRK21) and 39
(OgRK22) ends] and a primer that annealed within the plasmid
(OgRK45 and OgRK46). In addition, the lack of amplification with
primers (OgRK23 and OgRK24) that annealed to the region within
Glutathione is essential for virulence in Candida spp.
the ORF of MET15 was taken as final evidence of the disruption of
MET15 with the hph gene.
Construction of Cgdug3D. CAGL0J11484g, the orthologue of
ScDUG3, was PCR-amplified from the genomic DNA of C. glabrata
using primers CgDug3F and CgDug3R, yielding a 2.7 kb fragment
that was then digested with EcoRV and KpnI, and ligated to an
EcoRV- and KpnI-digested pBSK vector to generate plasmid pBSK-
CgDug3. pBSK-CgDug3 was digested with SnaBI and EcoNI, blunted,
and ligated with the hisG-URA3-hisG cassette [obtained from plasmid
PHUKH3 (Earley & Crouse, 1996) by PvuII digestion], to yield
plasmid pBSK-CgDug3D:HisG-URA3-HisG. The disruption cassette
met15Dura3D background, and transformantswereselectedonminimal
medium lacking uracil and containing methionine as the sulphur
source. Screening was done using diagnostic PCR. To remove the URA
marker from the disruptant ABG2703 (Cgmet15Dura3Ddug3D::hisG-
URA3-hisG), 5-fluoroorotic acid (FOA) selection was performed by
Table 1. List of strains and plasmids used in this study
Strain or plasmidDerived from
Genotype or descriptionSource or reference
S. cerevisiae MATa his3D1 leu2D0 met15D0 ura3D0
C. glabrata wild-type strain
C. glabrata met15D::hph HygRura3D::Tn903 G418R
C. glabrata met15D::hph HygRura3D::Tn903 G418R
C. glabrata met15D::hph HygRura3D::Tn903 G418Rdug3D::hisG
C. glabrata met15D::hph HygRura3D::Tn903 G418R
C. albicans wild-type strain
Laboratory of R. Kaur
Laboratory of R. Kaur
ABA2240 (Sc 5314)Laboratory of K.
Dr S. O. Kang
ABA2772 (YB204) C. albicans ura3-iro1D::imm434/ura3-iro1D::imm434
C. albicans ura3-iro1D::URA3-IRO1/ura3-iro1D::imm434 Dgcs1::hisG/
C. albicans ura3-iro1D::URA3-IRO1/ura3-iro1D::imm434
p416TEF CEN-vector bearing a URA3 marker and TEF-promoter-MCS-terminator
for S. cerevisiae expression and Amprmarker for selection in E. coli
CEN-vector bearing URA3 marker and PGK1-promoter-MCS-terminator
for C. glabrata expression and Amprmarker for selection in E. coli
E. coli expression vector (Stratagene)
S. cerevisiae GSH1 gene cloned in BspDI and HindIII sites of p416TEF
S. cerevisiae HGT1 gene cloned in BamHI and EcoRI sites of p416TEF
S. cerevisiae HGT1 gene cloned in BamHI and EcoRI sites of pGRB2.2
S. cerevisiae GSH1 gene cloned in SmaI site of pGRB2.2
C. glabrata MET15 gene along with its promoter (600 bp) cloned in
SmaI site of pGRB2.2
Plasmid containing hph expression cassette for hygromycin resistance
(HygR) under the PGK1 promoter flanked by FRT sites followed by
the MCS region and URA3 as a selection marker
59 UTR of MET15 (850 bp) cloned in KpnI and HindIII sites of plasmid
39 UTR of MET15 (750 bp) cloned in SpeI and SacI sites of plasmid
pRK9 so that the hph gene is flanked by the 59 and 39 UTRs of MET15
CgDUG3 along with its 59 UTR (593 bp) and 39 UTR (1017 bp) cloned
in EcoRV and KpnI sites of pBSK
Plasmid pBSK-CgDug3 digested with SnaBI and EcoNI followed by
blunting and ligation with the hisG-URA3-hisG cassette
Plasmid containing hisG-URA3-hisG cassette.
Mumberg et al.
Laboratory of R. Kaur
Sharma et al. (2000)
Kaur et al. (2009)
Laboratory of R. Kaur
pBSK-CgDug3 This study
Earley & Crouse
This study pBSK-CgGSH1 CgGSH1 along with its 59 UTR (629p) cloned in XbaI and XhoI
sites of pBSK
Plasmid pBSK-CgGSH1 digested with SmaI, followed by ligation with
CgMET15 (released from plasmid pGRB2.2-CgMET15 using SmaI)
A. K. Yadav and others
using 0.1% FOA and selecting transformants on minimal medium
containing uracil (1.2 mg 100 ml21) and methionine to yield strain
Construction of Cggsh1D:CgMET15. CgGSH1 (CAGL0L03630g)
was PCR-amplified using primers CgGSH1DF and CgGSH1DR to
yield a 3 kb fragment that was digested with XbaI and XhoI and
cloned into pBSK to yield plasmid pBSK-CgGSH1. pBSK-GSH1 was
then digested with SmaI, and the CgMET15 ORF with its own
promoter was inserted into this plasmid to generate plasmid
pBSKCggsh1D:CgMET15. Before disrupting CgGSH1 in a C.
glabrata met15D background, S. cerevisiae GSH1 on plasmid
pGRB2.2 (pGRB2.2-ScGSH1) was transformed in a C. glabrata
met15D background, and transformants were selected on minimal
medium lacking uracil and containing methionine. A Cgmet15D
strain carrying the pGRB2.2-ScGSH1 plasmid was then used as the
host strainto createa CgGSH1
Cggsh1D:CgMET15 disruption cassette that was excised from
pBSK-Cggsh1D:CgMET15 using SnaBI and XhoI. Transformants
were selected on minimal medium lacking uracil and any organic
sulphur source except ammonium sulphate for the selection of the
marker CgMET15. Screening for disruptants was done using
Reintegration of the URA3 and IRO1 genes in the ABA2772
(ura3D iro1D gcs1D) strain of C. albicans. In order to study the
virulence of the C. albicans gcs1D strain it was essential to reintegrate
the URA3 and IRO1 genes in their native locus. Primers CaURA1134F
and CaURA3792R were used to obtain a PCR product of 2.6 kb,
which was then transformed in strain ABA2772 (ura3D iro1D gcs1D)
by electroporation (Reuss et al., 2004). Transformants were selected
on minimal medium supplemented with 1 mM glutathione and
lacking uracil. Diagnostic PCR was carried out with the transformants
to check the locus of the reintegrated URA3 and IRO1 genes using a
primer upstream of the reintegration cassette (CaURA1041F) and one
specific to the reintegration cassette (CaURA3792R) to obtain strain
Reintegration of CaGCS1. Strain ABA2878 was used for the
reintegration of CaGCS1. The CaGCS1 ORF (orf19.5059) was PCR-
amplified from the genomic DNA of C. albicans using primers
CaGSH1F and CaGSH1R, and transformed into strain ABA2878, and
subsequently the transformants were selected on medium lacking
glutathione to obtain strain ABA2993 (CaURA3+IRO+GCS1+).
Diagnostic PCR was also done to confirm the reintegration of GCS1.
Mouse virulence assay. All animal experiments were approved by
the institutional animal ethics committee of the Institute of Microbial
Technology (IMTECH). Male BALB/c mice 6–8 weeks of age were
used for the survival experiment and were obtained from the
IMTECH animal house. To investigate the virulence of the Cagcs1D
strain in comparison with the C. albicans wild-type strain, both
strains were grown in minimal medium supplemented with 1 mM
glutathione for 16 h, followed by washing both strains with 16 PBS
and finally resuspending them in the same buffer. To correlate the
number of cells to 1 OD600unit, haemocytometer cell counting was
done. Mice were individually injected intravenously with 56105cells
of C. albicans wild-type and gcs1D and then observed for survival. All
mouse experiments were carried out twice with a set of seven to 10
mice per strain in each experiment.
Growth assays by dilution spotting. For dilution spotting assays,
the different strains were grown overnight in minimal medium with
nutrient supplements added as required, and were reinoculated into
fresh medium to OD6000.1 and grown for 6 h. The exponential phase
cells were harvested, washed with water and resuspended in water to
OD600 0.2. They were then serially diluted to 1:10, 1:100 and
1:1000. Of these cell suspensions, 10 ml of each dilution was spotted
onto the appropriate plates. Plates were incubated for 2 days and
End-point dilution survival assay. THP1 (human monocyte) cells
were treated with 16 nM phorbol 12-myristate 13-acetate (PMA) to
differentiate them to macrophages and seeded at 26105cells per well
in 96-well flat-bottomed tissue culture plates. Cells were incubated at
37 uC under 5% CO2, and RPMI medium was replaced with fresh
medium after 12 h of PMA treatment. Overnight cultures of C.
albicans wild-type and gcs1D mutant, grown in either YPD or
YPD+1 mM glutathione, were collected, washed with PBS and
vortexed to disrupt any cell clumps. The OD600of C. albicans cell
suspensions was taken, followed by diluting the cell suspension to
26106cells ml21in PBS. A 200 ml volume of the suspension was
added to two wells of a 96-well plate and serial fourfold dilutions were
made in PBS. To infect THP1 cells at an m.o.i. of 1:4, 25 ml of the
original cell suspension (26106cells ml21) was added to wells
containing differentiated THP1 cells (26105cells) in 150 ml RPMI
medium. As a control, an equal number of cells was incubated in
RPMI medium containing either no glutathione or 1 mM glu-
tathione. A total of five different m.o.i. values, 1:4, 1:16, 1:64, 1:256
and 1:1024, were used for the end-point dilution survival assay,
which involved counting C. albicans colonies from the THP1 seeded
wells where they could be visualized and comparing that number with
the number of colonies that appeared in the wells of the same dilution
in the absence of macrophages. After 90 min co-incubation with
macrophage cells, extracellular yeasts were removed by three PBS
washes followed by the addition of either RPMI medium or RPMI
medium supplemented with 1 mM glutathione. The co-culturing of
macrophages and C. albicans cells was continued for an additional
24 h, and the total number of C. albicans colonies at different m.o.i.
values that appeared in wells seeded with or without THP1 cells was
counted under the microscope. C. albicans survival was quantified by
dividing the number of colonies formed in the presence of THP1 cells
by the number of colonies formed in RPMI medium, and was
expressed as percentage growth inhibition. The end-point survival
assays were carried out twice, and each experiment was done in
Macrophage viability assay. Macrophage viability was determined
after 24 h by the trypan blue staining method. Briefly, at various time
points post-infection of THP1 cells, supernatant was aspirated from
the wells and 40 ml trypan blue (0.2%) was added to wells. After 3–
5 min incubation at room temperature, trypan blue was removed
from the wells, and cells were washed twice with PBS and observed
under the microscope. At 8 and 24 h post-infection, a minimum of
100 macrophages was counted to determine the presence of live
(unstained) and dead (blue) cells.
The C. glabrata met15D strain is a strict organic
sulphur auxotroph that can utilize cysteine and
methionine but not glutathione
To gain insights into glutathione utilization by C. glabrata,
we created a disruption of the CgMET15 (CAGL0D06402g)
gene. The MET15 gene encodes O-acetyl homoserine
thiolase (OAHSH), and disruption of this enzyme in S.
cerevisiae leads to strict organic sulphur auxotrophy, which
has greatly facilitated the study of organic sulphur
utilization in this yeast. A similar disruption made in C.
albicans, however, revealed that in C. albicans, the
Glutathione is essential for virulence in Candida spp.
disruptants have a severe (but not total) growth defect on
inorganic sulphate (Viaene et al., 2000), suggesting that a
secondary pathway of sulphate assimilation through
cysteine synthase (OASSH) might be present in this yeast.
On examination of the C. glabrata met15D strain, it was
observed that it was unable to grow on inorganic sulphate,
and like its S. cerevisiae counterpart, was a strict organic
sulphur auxotroph (Supplementary Fig. S1). Furthermore,
C. glabrata met15D could utilize either cysteine or
methionine as a sulphur source (Fig. 2), suggesting the
presence of both forward and reverse transulphuration
pathways in this yeast (Fig. 1). However, when we tested the
growth of the C. glabrata met15D strain on glutathione as a
sulphur source, it was found that the strain could not utilize
glutathione as a sulphur source (Fig. 2). This was quite
distinct from the behaviour of S. cerevisiae met15D strains,
which can efficiently utilize glutathione as a sulphur source.
C. glabrata lacks a glutathione transporter but
retains a functional Dug pathway required for
Glutathione utilization in S. cerevisiae has been shown to
require transport through a high-affinity glutathione
transporter (Hgt1p), followed by its degradation through
the Dug complex pathway for glutathione degradation
(involving Dug1p, Dug2p and Dug3p) (Bourbouloux et al.,
2000; Ganguli et al., 2007). Putative orthologues of the Dug
complex proteins (Dug1p, Dug2p and Dug3p) can be
identified in C. glabrata by in silico analysis. Earlier
comparative studies of transporters from different yeasts
had already indicated that C. glabrata lacks any member of
the oligopeptide transporter family to which Hgt1p belongs
(Dujon et al., 2004). Our analysis also confirmed that even
remote homologues of the glutathione transporter Hgt1p
are not found in this yeast. This is in contrast to other
yeasts, which contain at least two to three members of this
family. The possible absence of a glutathione transporter
correlated well with the inability of a Cgmet15D strain to
grow on glutathione; however, the presence of the
glutathione degradation machinery was intriguing, con-
sidering the fact that this complex has been presumed to
have evolved for the utilization of exogenous glutathione.
These observations needed further investigation. To
examine therefore whether the inability of the C. glabrata
met15D strain to utilize glutathione as a sulphur source was
a consequence of glutathione not being transported inside
the cell in the absence of an Hgt1p orthologue, we
expressed the S. cerevisiae glutathione transporter Hgt1p in
C. glabrata met15D strains and examined the transformants
for their ability to grow on glutathione. Interestingly, the
presence of Hgt1p enabled C. glabrata to utilize glutathione
as a sulphur source (Fig. 3). This indicates that C. glabrata
contains a functional glutathione utilization pathway,
except for the glutathione transporter. To see whether this
utilization of glutathione occurred through the Dug
complex, which we have previously shown to have evolved
for the utilization of exogenous glutathione, we disrupted
the CgDUG3 gene (CAGL0J11484g), which encodes one of
the proteins of the Dug complex. On deleting CgDUG3 it
was found that C. glabrata failed to utilize exogenous
glutathione, implying that the Dug complex is indeed
functional in C. glabrata (Fig. 3). The presence of a
functional Dug complex in C. glabrata suggests that apart
Fig. 1. Schematic showing the sulphur assim-
ilatory pathways in C. glabrata.
A. K. Yadav and others
from exogenous glutathione utilization, the Dug complex
is also involved in intracellular glutathione homeostasis.
GSH1, c-glutamyl cysteine synthase, is an
essential enzyme in C. glabrata
As glutathione is an essential metabolite for the growth of
eukaryotic cells, the absence of a glutathione transporter in
C. glabrata suggested that the enzyme c-glutamyl cysteine
synthase (GSHI) might be essential in this yeast. However,
the possibility also existed that glutathione was not an
essential metabolite in this yeast, as is the case in E. coli
(Murata & Kimura, 1982). To examine these possibilities,
we created a knockout of the C. glabrata GSH1 gene using
CgMET15 as a marker in cells harbouring a copy of the S.
cerevisiae GSH1 gene (CAGL0L03630g) on a URA3 plasmid.
The essentiality of the CgGSH1 gene was then examined by
removing the ScGSH1-containing plasmid using FOA (Fig.
4). Even in the presence of 100 mM glutathione, the cells
lacking ScGSH1 could not grow, indicating that CgGSH1 is
essential in C. glabrata (data not shown). At significantly
higher concentrations of glutathione, however, we did
observe some growth upon prolonged incubation of C.
glabrata strains carrying a knockout of the CgGSH1 gene,
indicating that at high concentrations of glutathione alone,
small amounts are taken up by C. glabrata cells, allowing
their growth (data not shown).
The C. albicans gcs1D strain shows decreased
growth rates and eventual growth stasis upon
The essentiality of glutathione biosynthesis in C. glabrata
prompted us to re-examine the importance of glutathione
Fig. 3. Expression of a glutathione transporter
from S. cerevisiae permits growth on glu-
tathione in a C. glabrata met15D (ABG2370)
strain but not in a C. glabrata dug3D met15D
(ABG2704) strain. C. glabrata met15D and
met15D dug3D strains were transformed with
plasmid pGRB2.2 (vector, lanes 3 and 1,
HGT1 (ScHGT1, lanes 4 and 2, respectively).
The transformants were grown in minimal
medium (containing methionine), and cells
were harvested, washed and resuspended to
OD6000.2. Serial dilutions of OD6000.2, 0.02,
0.002 and 0.0002 were then spotted onto
tathione, methionine or ammonium sulphate
(Sulphate) (having no organic sulphur source).
200 mM glu-
Fig. 2. Growth of C. glabrata met15D
(ABC733) strains on different sulphur sources.
The met15D strains were grown in minimal
medium (containing leucine, histidine, uracil
and methionine), and cells were harvested,
washed and resuspended to OD6000.2. Serial
dilutions to OD600 0.2, 0.02, 0.002 and
0.0002 were made, and 10 ml of each dilution
was spotted onto minimal medium containing
200 mM methionine,
(Sulphate), cysteine, homocysteine or glu-
tathione as the sulphur source. Plates were
photographed after 2 days.
Glutathione is essential for virulence in Candida spp.
biosynthesis in C. albicans more rigorously. Earlier
researchers have been able to create a gcs1D knockout in
C. albicans (Baek et al., 2004). The Cagcs1D strain was a
glutathione auxotroph on plates, similar to its counterparts
in the yeasts S. cerevisiae and Schizosaccharomyces pombe,
which indicates that C. albicans can acquire its glutathione
requirements from the extracellular medium in the absence
Schizosaccharomyces pombe gcs1D strains show growth
stasis immediately upon glutathione depletion, S. cerevisiae
gsh1D cells show a delayed growth stasis upon glutathione
depletion and manage to grow for up to seven or eight
generations comparably with the wild-type without
glutathione (Sharma et al., 2000). We therefore decided
to investigate the behaviour of the C. albicans strain in
terms of its growth profiles in liquid broth. These growth
studies with Cagcs1D were carried out in strains that were
and IRO1+. (The previously created
Cagcs1D strain was in the C. albicans CAI4 background,
which lacks the URA3 gene and also a part of the adjacent
IRO1 gene, involved in iron metabolism.) As both the
URA3 and the IRO1 gene of C. albicans have been shown to
have a role in pathogenesis (Chibana et al., 2005; Lay et al.,
1998), we reintegrated these genes in their native locus to
make the strains URA3+and IRO1+, as described in
Methods, before carrying out all subsequent experiments.
Upon examination of the growth characteristics of the
Cagcs1D strain, we observed that in medium lacking
glutathione, the strain grew more slowly than the wild-type,
but despite that it did succeed in growing for five to six
generations before entering into growth stasis (Fig. 5a).
Upon reinoculation of the Cagcs1D strain (grown for 24 h in
glutathione-free medium) into fresh glutathione-free med-
ium, complete growth stasis was observed (Fig. 5b). The
substantial viability of the cells even after 24 h growth in
glutathione-free medium (up to 78% viability) indicated
that the cells were not undergoing cell death and were
primarily experiencing growth stasis (data not shown).
When the growth was carried out in medium that was
supplemented with 1 mM glutathione, the Cagcs1D cells
in the presence of 1 mM glutathione, growth was still slower
than that of the corresponding wild-type. Increasing the
glutathione concentration in the medium to 5 mM did not
further increase the growth of the strain (data not shown).
The C. albicans gcs1D strain shows reduced
survival in human macrophages and also displays
attenuated killing of differentiated THP1 cells
The decreased growth seen in Cagcs1D strains even when
grown in medium supplemented with high levels of
glutathione highlighted the importance of glutathione
biosynthesis even in C. albicans, despite its ability to
obtain glutathione from the external medium. Professional
phagocytes (macrophages and neutrophils) constitute the
first line of defence against yeast pathogens, so to examine
the importance of endogenous glutathione biosynthesis as
a response to the initial immune response of the host, we
assessed the survival of C. albicans wild-type and Cagcs1D
cells in differentiated human monocytic THP1 cells by an
end-point dilution survival assay (Rocha et al., 2001).
Fig. 4. CgGSH1 is an essential gene in
strains of C. glabrata were transformed with
S. cerevisiae GSH1 on plasmid pGRB2.2
under the PGK1 promoter (lanes 1 and 2,
respectively). The disruptant and transformant
were then grown in minimal medium without or
with methionine, respectively, and cells were
harvested, washed and resuspended to OD600
0.2. Serial dilutions of OD6000.2, 0.02, 0.002
and 0.0002 were then spotted onto a different
minimal medium (containing ammonium sul-
phate). Methionine, uracil and FOA (the plates
containing FOA had uracil at a concentration
of 1.2 mg 100 ml”1) were added as indicated.
A. K. Yadav and others
490 Microbiology 157
A wide range of m.o.i. values (from 1:4 to 1:1024) was
used to study the interaction of C. albicans with
macrophages. No significant differences were seen in the
internalization rates (about 75%) for C. albicans wild-type
and Cagcs1D cells (data not shown). Co-incubation with
macrophages impaired the growth of both C. albicans wild-
type and Cagcs1D cells. Growth inhibition of 55–70% was
seen for the wild-type C. albicans cells as compared with
growth in RPMI medium at m.o.i. values of 1:256 and
1:1024 (Fig. 6a). Growth of the Cagcs1D strain, in contrast,
was more severely inhibited upon co-incubation with
macrophages, with Cagcs1D cells displaying 3–5% survival
under similar conditions (Fig. 6a). This reduced colony
formation phenotype of the Cagcs1D strain was partially
rescued by supplementing the RPMI medium with 1 mM
glutathione (7–15% survival) (Fig. 6a). Although colonies
formed by the Cagcs1D strain in the presence of macro-
phages were smaller than those of the wild-type (which
could be due to the slow growth rate of the strain),
importantly, no increase in the number of colonies was
observed with prolonged incubation (data not shown).
Interestingly, there was no difference in the rate of germ-
tube formation in wild-type and Cagcs1D cells, as both
strains exhibited morphology switching within the first 2 h
of co-incubation with THP1 cells (Fig. 6b and data not
shown). Taken together, these results suggest that Cagcs1D
cells are unable to survive and escape from the macrophage
To investigate whether Cagcs1D cells would also be
defective in lysing and killing macrophages, macrophage
viability was assessed by trypan blue staining 24 h post-
infection; wells with lower m.o.i. values were used due to
the invisibility of THP1 cells in higher m.o.i. wells. We
observed that THP1 cells infected with the Cagcs1D strain
were able to retain their viability, and only 1–13% of the
macrophages stained blue even after 24 h of co-culture at
different m.o.i. values (Fig. 6b and data not shown). In
contrast, when infected with the C. albicans wild-type
strain under similar conditions, 64–81% of THP1 cells
were unable to efflux trypan blue, indicating significant cell
death (Fig. 6b and data not shown). The reduced killing of
THP1 cells by the Cagcs1D strain even at a higher m.o.i. (or
when co-cultured with THP1 in medium lacking glu-
tathione) suggests that C. albicans Cagcs1D cells are
attenuated for virulence.
GCS1 is essential for virulence of C. albicans in
The reduced survival of the C. albicans gcs1D strain in a
macrophage cell line prompted us to examine whether
glutathione biosynthesis might also play a role in the
virulence of C. albicans in a systemic murine model of
candidiasis. BALB/c mice were infected intravenously with
an inoculum of 56105yeast cells and mice survival was
monitored as a function of time.
As shown in Fig. 7(a), mice infected with wild-type C.
albicans could not survive beyond 9 days, with death onset
seen as early as day 4. In contrast, all mice infected with the
C. albicans gcs1D strain remained healthy and were still
alive 15 days post-infection. To examine whether the
decreased mouse mortality seen with the C. albicans
gcs1D strain was linked to reduced fungal dissemination
within the host and colonization of different organs, we
Fig. 5. A C. albicans Cagcs1D strain (ABA2878) shows delayed
growth stasis upon glutathione depletion. (a) Growth of C.
CaGCS1 reintegrated strains (ABA2993) on minimal medium
with 1 mM glutathione or without glutathione. Cells were grown
overnight in minimal medium containing glutathione, washed twice
with minimal medium and reinoculated to OD6000.1. OD600was
measured on a Shimadzu UV-1800 spectrophotometer. C.
albicans wild-type (&), Cagcs1D ($), Cagcs1D+1 mM glu-
tathione (m), CaGCS1 (.), CaGCS1+1 mM glutathione (X). (b)
Cagcs1D (ABA2878) cells were grown in glutathione-free medium
for 24 h and freshly reinoculated into minimal medium with ($) or
without glutathione (m); C. albicans wild-type (&) was the control.
Glutathione is essential for virulence in Candida spp.
quantified the fungal load in different tissues (kidney, liver
and spleen) at various time points (day 2, day 3, day 4 and
day 6) post-tail vein injection. Although no significant
c.f.u. were recovered for C. albicans wild-type and Cagcs1D
strains from the liver and spleen at any time point, renal
loads were vastly different for the two strains throughout
the course of infection. While about 106yeasts could be
recovered from the kidneys of mice infected with wild-type
C. albicans cells at day 6, the kidneys harvested from mice
injected with Cagcs1D cells were almost sterile (Fig. 8).
Taken together, these results suggest that CaGCS1 is
required for virulence in C. albicans.
Fig. 6. Survival of C. albicans wild-type (ABA2240) and gcs1D (ABA2878) cells in differentiated THP1 cells, as determined by
an end-point dilution survival assay. (a) THP1 cells were infected with C. albicans wild-type and gcs1D cells as described in
Methods, and yeast colonies observed in wells with macrophages were counted and expressed as the ratio with respect to
colonies seen in wells containing RPMI medium alone 24 h post-infection. One representative experiment (out of two) is shown.
Experiments were performed in triplicate (means±SD). (b) THP1 cells were infected with yeast cells at an m.o.i. of 1:16, and
images were taken 8 h post-infection with an upright microscope (?40 magnification). The viability of THP1 cells was measured
by trypan blue staining 24 h post-infection. One representative experiment (out of two) is shown. Experiments were performed
in triplicate (means±SD).
A. K. Yadav and others
To confirm that the avirulent phenotype seen in Cagcs1D
strains was a consequence of the deletion of the GCS1 gene,
we integrated a copy of the CaGCS1 gene in the double
disruptant strain. These strains regained the ability to grow
in glutathione-free medium, although the total growth seen
was still a little lower than that of the wild-type (Fig. 5a).
Next, to examine whether the C. albicans strain carrying a
reintegrated copy of CaGCS1 would be virulent in the
murine model of systemic candidiasis, we injected the mice
intravenously either with the wild-type strain or with the
single-copy CaGCS1 reconstituted strain. We observed that
the strain carrying a reintegrated copy of CaGCS1 behaved
like the wild-type, and a parallel mice survival pattern was
seen in the two strains (Fig. 7b). This suggests that the
capability to synthesize glutathione, even with a single copy
of the Gcs1p enzyme, is sufficient for Cagcs1D cells to
regain their virulence attributes in mice. The results clearly
demonstrate that even a single copy of the gene allows the
Cagcs1D strain to regain not only its ability to synthesize
glutathione but also simultaneously its virulence.
In thisstudy wehave examined the essentiality of glutathione
biosynthesis in two fungal pathogens, C. albicans and C.
glabrata, and have uncovered a link between glutathione
biosynthesis and their virulence, raising the possibility of
exploiting metabolic strategies to attenuate Candida infec-
tion. In C. glabrata, the absence of a glutathione transporter
showed that glutathione biosynthesis is an essential process
in this yeast. In C. albicans, although a Cagcs1D strain could
grow on plates when supplied with exogenous glutathione,
depleted for glutathione, the strain was completely defective
in its ability to cause infection in mice.
The complete avirulence of Cagcs1D strains in mice
experiments was surprising, considering the ability of these
strains otherwise to grow on exogenous glutathione. With
abundant glutathione in the human host, one would have
expected C. albicans to have exploited the host glutathione
for its survival. In a recent study on inositol, also an essential
metabolite that can be both endogenously synthesized and
transported by specific transporters, it was observed that
merely disrupting the biosynthesis gene (INO1) does not
have any effect on the virulence capabilities unless the
transporters (ITR1/ITR2) are disrupted as well (Chen et al.,
Fig. 7. Comparison of virulence of a C. albicans gcs1D strain (ABA2878) and a Cagcs1D strain containing a single copy of
GCS1 (ABA2993) with that of a C. albicans wild-type strain (ABA2240) in mice. (a) Percentage survival of BALB/c mice
following intravenous challenge with 5?105cells of C. albicans wild-type (ABA2240, &) and Cagcs1D mutant (ABA2878, $).
(b) Percentage survival of BALB/c mice following intravenous challenge with 5?105cells of C. albicans wild-type (ABA2240,
&) and Cagcs1D::GCS1 strains (ABA2993, $).
Fig. 8. Organ load experiments. BALB/c mice were infected by the
intravenous route with 5?105cells of the Cagcs1D (ABA2878,
white bars) and wild-type (ABA2240, grey bars) strains. Mice were
sacrificed at specified time intervals, and organs were isolated and
crushed in a tissue homogenizer and plated on YPD+1 mM
glutathione medium. Results for kidney tissue are shown.
Glutathione is essential for virulence in Candida spp.
2008).Oneexplanationforthe striking difference seen in the
case of glutathione may be the inefficient uptake of
glutathione by this yeast. C. albicans contains eight members
of the oligopeptide transporter family, CaOPT1–CaOPT8,
many of which have been shown to transport oligopeptides
(Reuss & Morschha ¨user, 2006). Among these, CaOPT1 has
been considered to be a possible orthologue of the S.
cerevisiae glutathione transporter HGT1, owing to the high
sequence similarity. However, we have recently shown that
the CaOPT1 protein is in fact a very weak glutathione
transporter, although glutathione is unlikely to be its
primary substrate (Thakur & Bachhawat, 2010). This is
especially important when one considers the low concentra-
tions of glutathione in blood plasma [reported to be about
1–3 mM (Guttormsen et al., 2004)]. Within the host,
pathogens experience a variety of stress conditions that
include exposure to ROS and hypoxia, in which glutathione
plays very vital roles (Ernst & Tielker, 2009). The increased
requirement for and inadequate supply of glutathione in the
absence of endogenous biosynthesis is likely to affect the
ability of the pathogen to survive the harsh in vivo
conditions. Glutathione limitation would thus be expected
to significantly affect the virulence of the organism, and the
results described here, in which Cagcs1D strains were
completely avirulent, are in complete agreement with these
expected increased requirements for glutathione in vivo. It is
also possible that glutathione, which plays a central role
under stress conditions, is also important for other processes
specifically required for virulence, although one such
process, the ability to form hyphae, did not appear to be
affected. Specific depletion of the fungal glutathione would
thus clearly impinge on fungal survival, and in this context,
it is interesting to note that an antifungal compound from
garlic, diallyl sulphide, and the protein kinase inhibitor
staurosporine, have both been shown to cause their
antifungal activity by depletion of glutathione in C. albicans
(Castro et al., 2010; Lemar et al., 2007).
C. glabrata appears to be the only yeast that lacks any
member of the glutathione transporter family (the oligopep-
tide transporter family; OPT) and the reason for this absence
is not clear. Comparative evolutionary genomics analysis has
revealed that whole-genome duplication in C. glabrata was
followed by extensive gene loss (Dujon et al., 2004).
Favouring the reductive gene evolution theory, this yeast
(Kaur et al., 2005). This gene loss may reflect the diverse
selective pressures encountered inside the human host and
the successful adaptation of C. glabrata to its new ecological
niche. The lack of the nicotinic acid biosynthetic pathway in
C. glabrata has recently been shown to be linked with the
expression of the genes required for efficient colonization of
the host (Domergue et al., 2005). In light of these findings,
the loss of HGT1 in C. glabrata might stem from the fact that
despite the presence of the transporter, biosynthesis would
still be essential, since particularly in blood plasma,
glutathione levels are exceedingly low to meet the much
higher glutathione requirement in vivo. What is even more
interesting, however, is that in spite of ‘losing’ the
glutathione transporter, C. glabrata has retained a func-
tional glutathione utilization machinery (Dug complex).
Prior to this observation, the Dug pathway was presumed
to have evolved along with the high-affinity glutathione
transporter for the utilization of exogenous glutathione. Its
functional presence in C. glabrata thus suggests that the
Dug pathway has not evolved solely for the utilization of
exogenous glutathione, and also plays a role in intracellular
The essentiality of Gsh1p in C. glabrata and its requirement
for virulence in C. albicans make c-glutamyl cysteine
synthase (Gcs1p) a possible candidate for antifungal drug
development in addition to being previously considered as
an anti-protozoal target. The c-glutamyl cysteine synthases
have evolved into three different lineages. Yeasts share the
same lineage as their protozoan and mammalian counter-
parts, but even so, significant differences exist between the
human and the yeast enzymes. The mammalian enzyme is
heterodimeric, with catalytic and regulatory subunits, while
C. glabrata and C. albicans, like S. cerevisiae, seem to lack
the regulatory subunit (Biterova & Barycki, 2009). C.
albicans Gcs1p has a length of 772 amino acids and appears
to contain a 90 amino acid insertion when compared with
the sequences of C. glabrata and S. cerevisiae. To determine
whether this is a genuine insertion, we examined the Gcs1p
sequences of other Candida species, such as Candida
dubliniensis and Candida tropicalis. A similar insertion was
found in these species, thus suggesting that the insertion in
C. albicans is in fact a genuine insertion rather than a
sequence artefact. Although approximately 40% identity
exists between the CaGcs1p/CgGsh1p enzyme and the
catalytic subunit of human GCS, the differences in subunit
structure (absence of a regulatory subunit) and the extra
portion of about 90 amino acids in C. albicans raise
possibilities for further study and exploitation.
We thank Dr S. O. Kang, Washington University Medicine School, St.
Louis, MO, USA, for generously providing us with the C. albicans
gcs1D strains, and Dr G. F. Crouse, Emory University, Atlanta, GA,
USA, for the pKHUH3 plasmid. We thank Mr Rajkumar and Mr
Hariom for assistance with some of the experiments, and Dr S. Paul
and Dr D. Ganguli for helpful advice. A.K.Y. is a recipient of a
Research Fellowship from the Council of Scientific and Industrial
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Edited by: K. Kuchler
Glutathione is essential for virulence in Candida spp.