Zap1 Regulates Zinc Homeostasis and Modulates Virulence in Cryptococcus gattii
et al. (2012) Zap1 Regulates Zinc Homeostasis and Modulates Virulence in
Cryptococcus gattii. PLoS ONE 7(8): e43773. doi:10.1371/journal.pone.0043773
Zap1 Regulates Zinc Homeostasis and Modulates Virulence in Cryptococcus gattii
Rafael de Oliveira Schneider 0
Natully de Souza Su ffert Fogac a 0
Lvia Kmetzsch 0
Augusto Schrank 0
Marilene Henning Vainstein 0
Charley Christian Staats 0
Kirsten Nielsen, University of Minnesota, United States of America
0 1 Centro de Biotecnologia, Universidade Federal do Rio Grande do Sul, Porto Alegre, Brazil, 2 Departamento de Biologia Molecular e Biotecnologia, Universidade Federal do Rio Grande do Sul , Porto Alegre , Brazil
Zinc homeostasis is essential for fungal growth, as this metal is a critical structural component of several proteins, including transcription factors. The fungal pathogen Cryptococcus gattii obtains zinc from the stringent zinc-limiting milieu of the host during the infection process. To characterize the zinc metabolism in C. gattii and its relationship to fungal virulence, the zinc finger protein Zap1 was functionally characterized. The C. gattii ZAP1 gene is an ortholog of the master regulatory genes zafA and ZAP1 that are found in Aspergillus fumigatus and Saccharomyces cerevisiae, respectively. There is some evidence to support an association between Zap1 and zinc metabolism in C. gattii: (i) ZAP1 expression is highly induced during zinc deprivation, (ii) ZAP1 knockouts demonstrate impaired growth in zinc-limiting conditions, (iii) Zap1 regulates the expression of ZIP zinc transporters and distinct zinc-binding proteins and (iv) Zap1 regulates the labile pool of intracellular zinc. In addition, the deletion of ZAP1 reduces C. gattii virulence in a murine model of cryptococcosis infection. Based on these observations, we postulate that proper zinc metabolism plays a crucial role in cryptococcal virulence.
Funding: This work was supported by grants from the Brazilian agencies Conselho Nacional de Desenvolvimento Cientfico e Tecnolo gico (CNPQ
476029/20104), Financiadora de Estudos e Projetos (FINEP), Coordenacao de Aperfeicoamento de Pessoal de Nvel Superior (CAPES), and Fundacao de Amparo a` Pesquisa do
Estado do Rio Grande do Sul (FAPERGS). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the
Competing Interests: The authors have declared that no competing interests exist.
. These authors contributed equally to this work.
The function of many proteins depends upon the essential role
of Zn, acting as both a catalytic constituent and as a core
component of structural motifs. The zinc metalloproteome consists
of more than 300 proteins in yeast, and the majority of these
proteins are zinc finger transcription factors . The molecular
mechanisms that control zinc homeostasis in cells are best
characterized in Saccharomyces cerevisiae. The critical importance of
zinc in yeast cells is suggested by the fact that approximately 3% of
the proteome requires zinc for proper functioning . Zinc
acquisition from the environment, especially in zinc-limiting
conditions, is mainly performed by the well-characterized ZIP
family of plasma membrane transporters Zrt1p and Zrt2p [3,4]
and by the multi-metal transporter Fet4p . Intracellular
transport of zinc to organelles, a process associated with
highzinc growth conditions, is mediated by the cation diffusion
facilitator families Zrc1p, Cot1p and Msc2p . The expression
of the majority of these transporters is directly regulated by the
master zinc regulator Zap1p , a multi-zinc finger transcription
factor  that specifically binds to zinc-responsive elements in the
promoter regions of over 40 genes in the yeast genome, including
ZRT1 and ZRT2 .
Zinc is an indispensable micronutrient for all organisms.
Pathogenic microorganisms require zinc for successful growth
and development of infectivity . Therefore, mammalians limit
zinc availability as a defensive strategy against invading pathogens
by forming zinc complexes with proteins such as calprotectin
[12,13]. However, in comparison to the well-characterized
microbial iron homeostasis system and its importance in
hostpathogen interactions [14,15], relatively little is known about the
role of zinc in the host-pathogen interplay in the infection milieu.
Zinc metabolism studies in pathogenic fungi have mainly focused
on Aspergillus fumigatus and Candida albicans. The A. fumigatus
transcription factor ZafA is a functional homolog of S. cerevisiae
Zap1p, and its transcript levels are regulated by zinc availability.
Moreover, ZafA is associated with the regulation of the zinc
transporter-encoding genes zrfA and zrfB, further underscoring its
role in zinc homeostasis. Mutant cells lacking ZafA display
attenuated virulence, as assessed in murine models of aspergillosis
. The functional homologues of S. cerevisiae Zap1p in C. albicans
were identified in two independent studies and were named Csr1p
 and Zap1 , respectively. C. albicans mutant cells lacking
Csr1p display a severe growth reduction in low-zinc environments
and defects in filamentous growth, an important
virulenceassociated trait . In addition, this protein positively regulates
the expression of zinc transporters , further reinforcing its role
in zinc homeostasis.
The basidiomycete yeasts Cryptococcus neoformans and C. gattii are
the etiological agents of cryptococcosis, a life-threatening disease
mostly characterized by meningoencephalitis. Cryptococcosis is a
devastating disease in Africa and a major cause of death in
immunosuppressed patients in many countries [19,20]. C.
neoformans var grubii (serotype A) is the most prevalent cause of human
cryptococcosis, accounting for over 95% of cryptococcal cases
worldwide, while C. gattii infections account for less than 1% of
cryptococcosis cases . However, C. gattii is usually associated
with cryptococcosis in immunocompetent individuals . In
addition, cryptococcosis outbreaks caused by a hypervirulent
strain of C. gattii on Vancouver Island  and in the USA [24,25]
reinforce the need for a thorough molecular characterization of
the virulence determinants of this species. At least four
wellcharacterized pathogenic determinants are shared by C. neoformans
and C. gattii: (i) the presence of a polysaccharide capsule, (ii) the
synthesis of a melanin-like pigment, (iii) the ability to proliferate at
human body temperature and (iv) the ability to proliferate inside
macrophages . Nevertheless, only a few genes for other
virulence determinants have been characterized in C. gattii to date
Despite its importance, zinc metabolism is poorly characterized
in both C. neoformans and C. gattii. This is in contrast to both iron
metabolism  and copper metabolism [27,28], which have been
well-studied. Zinc chelation by calprotectin impairs the growth of
C. neoformans, and this mechanism is also likely to be important
during interactions between cryptococcal cells and neutrophils and
other immune cells . Here, we characterized the ZAP1 gene
and its role as a transcriptional regulator of zinc metabolism in C.
gattii. ZAP1 shares structural and functional features with other
fungal zinc regulators, and functional analysis revealed that Zap1
regulates the expression of several genes involved in zinc
metabolism. In addition, the results of this study demonstrate that
Zap1 is necessary for key events in various cryptococcal
Identification of the C2H2 Zn-finger transcription factor
Zap1 in C. gattii
Scrutiny of the C. gattii R265 genome  using the S. cerevisiae
zinc finger metalloregulatory protein Zap1p revealed the presence
of 25 predicted C2H2 zinc finger domain-containing proteins.
Considering that S. cerevisiae Zap1p is characterized by a
concentration of zinc fingers in its C-terminus , proteins
without this feature were not considered for further analysis. The
best-hit protein matching these criteria is encoded by the gene
CNBG_4460, which contains four C2H2 domains. As previously
demonstrated, S. cerevisiae Zap1p possesses seven zinc finger
binding domains . Thus, a direct comparison of the predicted
CNBG_4460 encoded protein with the orthologs of the C.
neoformans H99 and JEC21 strains was performed. The overall
identity between proteins from the two C. neoformans strains was
very high (.98%). However, when each predicted C. neoformans
protein was compared to the putative C. gattii protein, the
similarity was found to be only 78% (Figure S1), which may be
due to a gap in the sequence corresponding to the C-terminus of
the C. gattii protein. Analysis of the CNBG_4460 locus sequence
relative to proteins from both C. neoformans strains was conducted
employing the Gene Wise algorithm (http://www.ebi.ac.uk/
Tools/Wise2/) to search for possible frameshift or annotation
errors. The resulting sequence of the C. gattii Zap1 protein
displayed increased identity with proteins from both C. neoformans
strains (84%), and the similarity is likely due to the presence of an
additional 70-bp exon (Figure S1). Moreover, RNA-seq confirmed
the presence of an extra exon in the C-terminal coding region of
the C. gattii ZAP1 gene. In this way, the proposed annotation for
the C. gattii ZAP1 gene is quite identical to the C. neoformans
orthologs (Figure S1). Thus, the ZAP1 gene encodes a 699-aa
protein containing six C2H2 zinc finger domains that are
distributed along its sequence, as observed in fungal Zap1
functional homologs (Figure 1A). The domain architecture in C.
gattii Zap1 protein is similar to those observed in C. neoformans
functional homologs. Additionally, phylogenetic analysis
(Figure 1B) including Zap1 sequences from different fungal species
demonstrated that C. gattii Zap1 is highly similar to Zap1 from C.
neoformans and A. fumigatus but less similar to Zap1 from C. albicans.
The expression of Zap1p is regulated by zinc levels in S. cerevisiae
. To further characterize the functional homology of C. gattii
Zap1, the transcript levels from the ZAP1 gene were assessed by
qRT-PCR analysis using RNA isolated from C. gattii that were
cultured in both the presence and the absence of the zinc chelator
TPEN. A significant increase in ZAP1 transcripts (25-fold) was
detected in zinc-limiting conditions compared to control
conditions (Figure 1C), suggesting a possible role for C. gattii Zap1 in
zinc metabolism. An increase in the transcript levels of distinct ZIP
family zinc transporters (ZIP1 CNBG_6066, ZIP2
CNBG_2209, and ZIP3 CNBG_5361) could also be detected
in the same experimental conditions (Figure 1D). This result
indicates the utility of TPEN as a tool to evaluate the zinc
deprivation responsiveness of C. gattii, and confirms that ZAP1
transcript levels can be regulated by zinc availability.
C. gattii Zap1 regulates zinc transport and growth during
To evaluate the function of Zap1 in C. gattii, null mutants and
complemented strains were constructed. Knockout and
complementation of the ZAP1 gene were confirmed by both Southern
blotting and RT-PCR analysis (Figure S2). To evaluate the role of
C. gattii Zap1 in zinc homeostasis, the ability of WT, zap1D mutant
and zap1D::ZAP1 complemented strains to grow in zinc-limiting
conditions (YNB containing TPEN) were assayed. Zinc
deprivation induced decreased growth in all strains analyzed in
comparison to their growth in zinc-rich medium. However, this
growth reduction was much more pronounced in the zap1D
mutant (Figure 2A). To evaluate the possibility that the growth
arrest in zap1D mutants exposed to zinc deprivation conditions
might be related to lower intracellular zinc levels, fluorometric
analyses were performed with the cell permeable zinc probe
Fluozin 1 AM. Relative fluorescence levels of zap1D mutant were
approximately 5 times lower than those of the WT strain and were
comparable to background fluorescence levels obtained from the
treatment of WT and zap1D mutant cells with the membrane
permeable TPEN (Figure 2B). Because fungal Zap1 orthologs
regulate the expression of ZIP family zinc transporters [16,18,31],
qRT-PCR analyses were conducted to evaluate the relative
transcript levels of three genes encoding ZIP domain containing
proteins. The expression of two of these genes (ZIP1
CNBG_6066 and ZIP2 CNBG_2209) was regulated by Zap1
because the relative transcript levels of these genes were drastically
reduced in the zap1D mutant cells compared to WT cells
(Figure 2C). Collectively, these results confirm that C. gattii Zap1
plays a key role in the regulation of zinc homeostasis.
C. gattii cells lacking Zap1 display alterations in oxidative
Zinc deficiency generates a burst of oxidative stress in S. cerevisiae
cells, and the adaptive responses to overcome some of the damage
caused by reactive oxygen species (ROS) can be mediated by
Zap1p . Assays to evaluate the sensitivity of WT, zap1D
mutant and zap1D::ZAP1 complemented strains to hydrogen
peroxide, menadione or T-BOOH revealed no differences in their
relative growth when exposed to these distinct ROS generators
(data not shown). However, assays employing the intracellular
fluorescent ROS probe CM-H2CFDA revealed an accumulation
of ROS in mutant cells, compared to the level in WT and
complemented strains, after cultivation in YNB with TPEN
(Figure 3A). To gain insight into this phenomenon, the relative
transcript levels of three catalase- (CAT1 CNBG_4696, CAT2
CNBG_5786 and CAT3 CNBG_4667) and two superoxide
dismutase (SOD)-encoding genes (SOD1 CNBG_0599 and SOD2
CNBG_2661) were measured by qRT-PCR. Comparison of the
relative transcript levels of the CAT genes from WT and zap1D
mutant cells revealed no statistically significant differences
(Figure 3B). Nevertheless, a slight increase (1.9-fold) was observed
in the levels of the Cu-Zn SOD encoded by CNBG_0599 but not
in the Mn SOD encoded by CNBG_2661 (Figure 3C).
The accumulation of intracellular ROS in C. gattii zap1D mutant
cells suggested that other mechanisms might also be involved in
the regulation of ROS homeostasis in such cells. The S. cerevisiae
Zap1p is also known for its role in the regulation of the expression
of distinct genes involved in sulfur and glutathione metabolism
[33,34]. To test this possibility, the sensitivity of zap1D mutant cells
to the glutathione depletion agent diethyl malate (DEM) was
examined. While the addition of DEM to WT or zap1D::ZAP1
complemented strains reduces their growth to approximately 60%
of the level of non-treated cultures, it caused a more intense
growth inhibition in the zap1D mutant strain to approximately
40% of the level of non-treated cultures (Figure 4A). This result
suggests that the lack of ZAP1 leads to a reduction in glutathione
levels in these cells. In an attempt to correlate the proposed
diminished glutathione levels with the elevated intracellular ROS
levels, the relative transcript levels of two glutathione peroxidase
(GPx)-encoding genes (GPX1 CNBG4202 and GPX2
CNBG5153) were evaluated. GPxs catalyze the reduction of
hydrogen peroxide with the consumption of reduced glutathione,
yielding oxidized glutathione . The relative GPX2 gene
transcript levels were 4 times higher in the zap1D mutant
compared to WT cells (Figure 4B), confirming an imbalance in
glutathione and ROS metabolism in the absence of ZAP1. In
addition, analysis of the sensitivity of WT cells, zap1D mutants and
zap1D::ZAP1 complemented strains to the reactive nitrogen species
(RNS) generator DETA-NONOate revealed that the absence of
ZAP1 gene activity leads to a marked decrease in the viability of
the zap1D mutant compared to the WT or complemented strains
Effects of ZAP1 deletion on C. gattii virulence
The zap1D strain was evaluated for its ability to synthesize
melanin and capsule and for its ability to grow at 37uC, as these
are the most well studied virulence factors of C. gattii . The lack
of ZAP1 does not interfere with any of these traits (Figure S3), but
a decrease in the ability to cause experimental cryptococcosis was
observed in zap1D cells. Using an intranasal model of murine
infection, we found that mice infected with the ZAP1 null mutant
obtained for the actin gene. Data are shown as the mean 6 SD from
three experimental replicates of three biological replicates. ** P,0.01. ***
P,0.001. NS, not significant.
survived longer (median survival 11.5 days) than those infected
with the WT (P = 0.0078) and complemented strains (P = 0.0253)
(median survival 6 and 6.5 days, respectively; Figure 6A). In this
context, interactions between yeast cells and macrophages play a
pivotal role in the pathobiology of C. neoformans . To evaluate
whether the lower virulence of the zap1D mutant is associated with
changes in the interplay between macrophages and C. gattii,
phagocytosis assays were performed employing the macrophage
cell-like RAW264.7 line. Assays with the WT, zap1D mutant and
zap1D::ZAP1 complemented strains revealed that cells lacking
ZAP1 have increased CFU counts after 18h of interaction with
macrophages (Figure 6B). In conjunction, these results confirm the
role of ZAP1 in C. gattii virulence.
ZAP1 regulates the expression of zinc transporters,
oxidative stress-related proteins and zinc-binding
To evaluate the response of the ZAP1 regulon to zinc
deprivation, RNA-seq analyses were conducted using RNA
isolated from WT or zap1D mutant cells after a 2-h exposure to
TPEN, as this condition promotes the intense expression of ZAP1
and ZIP zinc transporters (Figure 1C and 1D). Analysis of the two
transcriptomes revealed a total of 183 and 328 genes that were
significantly (p,0.05) up-regulated and down-regulated,
respectively, in WT cells compared to zap1D mutant cells. As expected,
two ZIP zinc transporters were found among the up-regulated
genes, providing a positive control for the analysis. In addition,
one gene related to nitrosative stress, the flavohemoglobin
encoding gene, was also up-regulated in WT cells (Table 1). The
full list of ZAP1-regulated genes and the corresponding
foldchanges are shown in Table S1. To further characterize the genes
regulated by ZAP1 in C. gattii, a screen for genes encoding putative
zinc-binding proteins was conducted, employing gene ontology
(GO) classification after analysis on the UFO server . Among
the 183 WT up-regulated genes analyzed, four zinc-binding
proteins were detected, and all are transcription factors containing
distinct zinc finger domains (Table 1). However, when the 328
WT down-regulated genes were subjected to the same analysis,
thirteen different genes encoding putative zinc-binding proteins
were identified. The majority of these genes encode proteins with
dehydrogenase activity, and only two encode zinc finger
transcription factors (Table 1).
Zinc is a fundamental micronutrient in cell physiology, as it is a
key component of the cores of several proteins . Fungal cells
have evolved a regulatory mechanism to acquire and distribute
zinc inside cells . In all fungal systems characterized to date,
including S. cerevisiae, C. albicans and A. fumigatus, the Zap1p
transcription factor and its functional homologs control zinc
homeostasis through the modulation of zinc transporter and
zincbinding protein expression [4,1618]. In the present study, the
functional homolog of S. cerevisiae Zap1p was characterized in C.
gattii, allowing us to analyze the influence of zinc in cryptococcal
virulence. Four lines of evidence support the assumption that Zap1
is a zinc-responsive transcriptional regulator that is responsible for
zinc homeostasis in C. gattii. First, ZAP1 transcript levels increase in
response to zinc deprivation. Second, in silico analysis of the
predicted Zap1 protein sequence identified several C2H2 zinc
finger domains that are common to all fungal Zap1 functional
homologs described to date [4,16,17]. Third, Zap1 is associated
with the regulation of zinc transporter expression because in C.
gattii ZAP1 null mutants, the relative transcript levels of genes
encoding the ZIP family transporters are drastically reduced
compared to WT cells. Fourth, the growth of the C. gattii zap1D
strain is reduced in zinc-limiting conditions. Additionally, C. gattii
null mutants for the ZAP1 gene display attenuated virulence in the
intranasal murine model of cryptococcosis, and these mutants
associate more with macrophages than do WT and complemented
strains in phagocytosis assays. The modulation of virulence was
also demonstrated for mutants of the ZAP1 homolog in A. fumigatus
(ZafA gene) , further indicating the importance of zinc
homeostasis in the virulence of fungal pathogens.
At least three transcription factors that regulate metal
homeostasis were described in the phylogenetically related yeast C.
neoformans. The CIR1 and HAPx genes are involved in the
regulation of iron homeostasis [39,40], and CUF1 regulates copper
metabolism in C. neoformans . Lack of CIR1 in C. neoformans
yields cells with enhanced melanization, decreased capsule size,
and temperature-sensitive growth , while C. neoformans CUF1
null mutants are hypomelanized . The CUF1 and CIR1
mutant phenotypes contribute to their reduced virulence, as
assayed by murine models of cryptococossis [28,40]. The analyses
presented here demonstrate that C. gattii ZAP1 null mutants also
display reduced virulence in the same animal models and that this
phenotype could not be associated with any detectable alteration
in the classical virulence traits, such as melanin and capsule
formation and host temperature growth. The results observed with
C. gattii ZAP1 are in agreement with those described for C.
neoformans, in which the lack of ZAP1 leads to a severe defect in
murine infectivity and reduced melanization . A recent report
evaluated the phenotypes associated with the knockout of distinct
genes encoding functional homologs belonging to distinct
functional classed in both C. neoformans and C. gattii. Despite
conservation in phenotypes concerning the predicted activity of
the encoded protein, subtle differences could be found when
compared to the classical virulence traits among the species . It
is therefore reasonable to assume that the decrease in virulence of
C. gattii ZAP1 mutants can be attributed to defects in their ability to
grow in low-zinc conditions and to take up zinc from the
environment. Indeed, the reduction in zinc availability in the
infection milieu is a host strategy designed to hamper pathogen
replication . For instance, a decrease in cytoplasmic zinc
concentration is observed in murine macrophages infected with
Histoplasma capsulatum or treated with cytokines that induce
antimicrobial activity . It has also been reported that abscesses
resulting from infection with the bacterium Staphylococcus aureus are
rich in the SB100 zinc-binding protein calprotectin, leading to zinc
chelation and thereby reducing zinc availability to the pathogen
. Furthermore, the exposure of C. neoformans cells to
calprotectin leads to growth inhibition and cell death .
Altogether, these data reinforce the dependence of C. neoformans
and C. gattii on zinc availability for development within the host
We observed that C. gattii ZAP1 mutant cells display several
defects in their ability to handle oxidative stress. In addition to the
accumulation of intracellular ROS, such cells display alterations in
glutathione metabolism. For instance, C. gattii ZAP1 mutant cells
display increased GPX2 transcript levels and are more sensitive to
diethyl malate compared to WT cells. This suggests a decreased
concentration of intracellular glutathione in C. gattii ZAP1 mutant
cells. In light of these results, we hypothesize that as an adaptive
response to the zinc deprivation-induced accumulation of
intracellular ROS levels, C. gattii ZAP1 mutant cells modulate the ROS
balance through glutathione metabolism. Via the activity of GPx,
cells can detoxify ROS with the concomitant consumption of
glutathione . Accordingly, GPX null mutants of C. neoformans
are hypersensitive to oxidative stress . It is well documented
Figure 4. Lack of ZAP1 leads to alterations in glutathione metabolism. (A) The WT, zap1D mutant and zap1D::ZAP1 complemented strains
were incubated in YNB or YNB +0.5 mM DEM. After 24 h of incubation, the cell density was spectrophotometrically determined. The ratio between
growth in DEM and control conditions is shown as the mean 6 SD from three biological replicates. (B) Quantitative real time RT-PCR of GPX gene
transcripts after growth of C. gattii WT or zap1D mutant cells in YNB + TPEN. The measured quantity of the mRNA in each of the samples was
normalized using the Ct values obtained for the actin gene. Data are shown as the mean 6 SD from three experimental replicates of three biological
replicates. *P,0.05. **P,0.01. ***P,0.001. NS, not significant.
that zinc depletion results in enhanced ROS levels inside S.
cerevisiae cells [32,44]. As an adaptive response to elevated ROS
levels caused by zinc deprivation, S. cerevisiae cells activate the
expression of the TSA1 gene, which encodes a peroxiredoxin, to
degrade hydroperoxides . The ZAP1 gene from S. cerevisiae is
also involved in the regulation of the catalase encoding gene CTT1
in low-zinc conditions, suggesting that this enzyme plays a role in
ROS detoxification under conditions of zinc deprivation . The
uptake and metabolism of sulfate is largely dependent upon ZAP1
activity in S. cerevisiae and is repressed in zinc-limiting conditions
. Methionine, cysteine, and (most likely) other metabolites
from sulfur metabolism, including glutathione, are found at lower
concentrations in zinc-limited S. cerevisiae cells, suggesting that the
detoxification of ROS in zinc-limiting conditions also relies on
proper glutathione metabolism . Altogether, these findings
suggest that C. gattii likely evolved a Zap1-independent strategy to
cope with the elevated ROS levels caused by zinc-limiting
conditions, unlike those observed in S. cerevisiae.
The generation of RNS is a common strategy used by immune
cells to hamper the development of C. neoformans and other fungi
. The successful growth and virulence of C. neoformans in
nitrosative conditions, both in vitro and in vivo, depends on the
activity of flavohemoglobin denitrosylase and S-nitrosoglutathione
reductase, which are encoded by the FHB1 and GNO1 genes,
respectively [46,47]. C. gattii ZAP1 null mutants are more sensitive
to the RNS generator DETA-NONOate, possibly as a
consequence of reduced transcript levels of the FBH1 ortholog in C.
gattii, as observed in our transcriptome analysis. Therefore, in
addition to defects in zinc transport and metabolism, the
attenuated virulence observed in the C. gattii ZAP1 null mutants
may also be associated with defects in dealing with nitrosative
The comparison of the C. gattii ZAP1 regulon with its
counterparts from S. cerevisiae and C. albicans revealed some
overlapping circuits. The ZIP family of zinc transporters is
positively regulated by ZAP1 in yeasts [18,31]. However, Zap1
regulates several other genes that are not necessarily associated
with zinc homeostasis. When specifically analyzing the
zincbinding proteins, as inferred from their GO annotations, we
observed that Zap1 is a negative regulator of several zinc-binding
proteins. The results presented here show that some alcohol
dehydrogenases are downregulated in WT cells compared to the
ZAP1 null mutants. The same expression pattern is also observed
in S. cerevisiae and C. albicans [18,31]. This strategy could represent
an adaptation to low zinc availability named zinc conservation
, in which other metalloproteins necessary to survive in such
conditions are preferentially associated with zinc. Alcohol
dehydrogenases are among the most abundant zinc-binding proteins in
the cell, representing a significant proportion of the total cellular
zinc . As a result, the reduced expression of some zinc-binding
proteins would make zinc available for other proteins, such as Cu/
Figure 6. Zap1 is required for full C. gattii virulence in mice and influences phagocytosis by macrophages. (A) Virulence assay of WT,
zap1D mutant and zap1D::ZAP1 complemented strains in an intranasal inhalation infection model with BALB/c mice. (B) CFU counts after macrophage
infection with WT, zap1D mutant and zap1D::ZAP1 complemented strains. *** P,0.001.
Zn SODs, as a strategy to cope with the harsh conditions of a
In conclusion, this report describes the identification and
characterization of Zap1, a general regulator of zinc homeostasis
in C. gattii. Two key events are responsible for the observed
reduced virulence of C. gattii ZAP1 null mutants in the intranasal
murine model of infection: the reduced zinc load in cells and the
corresponding increase in intracellular ROS. The C. gattii Zap1
regulon includes zinc transporters and several zinc-binding
proteins, and there is some conservation between it and the
regulatory circuits of other yeast Zap1 regulons.
Materials and Methods
The use of animals in this work were performed with approval
of The Universidade Federal do Rio Grande do Sul Ethics
Committee for Use of Animals (CEUA protocol number 19801).
Mice were housed in groups of four in kept in filtered top
ventilated cages, maintained on 12 h dark/light cycle, with food
and water ad libitum. The animals were cared according to the
Brazilian National Council for Animal Experimentation Control
(CONSEA) and Brazilian College of Animal Experimentation
(COBEA) guidelines. All efforts to minimize animal suffering were
made. Before mortality analysis, mice were intraperitoneally
anesthetized with 100 mg/kg Ketamine and 16 mg/kg Xylazine.
Mice were analyzed twice daily for any signals of suffering, defined
by weight loss, weakness or inability to obtain feed or water. In the
first signals of suffering, mice were humanely sacrificed.
Strains and culture conditions
The C. gattii strain R265 was used in this work. It was routinely
cultured in YPD (2% glucose, 2% peptone, and 1% yeast extract).
Agar was added to a final concentration of 1.5% when solid media
was used. YPD plates supplemented with 100 mg/ml
nourseothricin were used to select for the C. gattii zap1D mutant strain.
YPD +200 mg/ml hygromycin was used to select for the C. gattii
zap1D::ZAP1 complemented strain. Phenotypic assays were
conducted in Yeast Nitrogen Base (YNB, without amino acids
and ammonium sulfate) with the addition of asparagine to a final
concentration of 40 mM. The murine macrophage-like cell line
was used for the evaluation of phagocytosis-related phenotypes. It
was routinely cultured in Dulbeccos Modified Eagle Medium
(DMEM) with 10% heat-inactivated fetal bovine serum (FBS) in a
humidified incubator at 37uC and 5% CO2.
In silico identification and characterization of C. gattii
The putative C. gattii ZAP1 gene was identified by BLAST
analysis of the Broad Institute C. gattii R265 database using the S.
cerevisiae Zap1p sequence as a query (NCBI accession number
NP_012479.1). Amino acid sequences of Zap1 orthologs from S.
cerevisiae, C. albicans, A. fumigatus, C. gattii and C. neoformans were
aligned using ClustalX2 . Mega5 was utilized for phylogenetic
analysis applying the Neighbor-Joining method; tree architecture
was inferred using 1000 bootstraps . Searches for zinc-binding
domains were conducted using the ScanProsite tool .
Construction of knockout and complemented C. gattii
The Delsgate methodology was used to construct the ZAP1 gene
inactivation allele by employing the vector pDONR-NAT, as
previously described for C. neoformans [51,52]. The nourseothricin
resistance cassette from pAI4 was subcloned into the EcoRV site of
pDONR201 (Gateway donor vector, Invitrogen) to construct
plasmid pDONR-NAT. The 59 and 39 ZAP1 flanking sequences
(953 and 801 bp, respectively) were PCR-amplified and gel
purified (Illustra GFX PCR DNA and Gel Band Purification kit,
GE Healthcare). The plasmid pDONR-NAT and each PCR
product were mixed at equimolar ratios in a BP clonase reaction
according to manufacturers instructions (Invitrogen). This
reaction was then transformed into Escherichia coli OmniMAX 2-T1
cells. The inactivation construct plasmid was linearized by I-SceI
digestion prior to C. gattii biolistic transformation . For
complementation, a 5-kb fragment spanning the ZAP1 gene was
amplified from C. gattii R265 DNA and subcloned into the EcoRV
sodium: inorganic phosphate symporter
RNA polymerase III smallest subunit
DNA-directed RNA polymerase I polypeptide
conserved hypothetical protein
conserved hypothetical protein
conserved hypothetical protein
conserved hypothetical protein
conserved hypothetical protein
extracellular elastinolytic metalloproteinase
ZIP zinc/iron transport family
ZIP zinc/iron transport family
Phosphate transporter family
Oxidoreductase NAD-binding domain
Pyridoxal-phosphate dependent enzyme
TFIIB zinc-binding; Brf1-like TBP-binding domain
Transcription factor S-II (TFIIS)
Transcription factor S-II (TFIIS)
Putative GTPase activating protein for Arf
Alcohol dehydrogenase GroES-like domain
Alcohol dehydrogenase GroES-like domain
Alcohol dehydrogenase GroES-like domain
Alcohol dehydrogenase GroES-like domain
Fungal Zn(2)-Cys(6) binuclear cluster domain
Alcohol dehydrogenase GroES-like domain;
Fungalysin metallopeptidase (M36);
Phosphomannose isomerase type I
A: Gene accessions, descriptions and PFAM descriptions were obtained from the Broad Institute Database.
B: Data are presented as the ratio of FPKM (fragments per kilobase of exon per million fragments mapped) of genes in WT cells compared to their expression in ZAP1
mutant cells during growth in YNB + TPEN using a log scale.
C: Zinc-binding proteins were selected based on UFO analysis  using the predicted protein sequences of significantly differentially expressed genes in the WT or
zap1D strains as input. The sequences that contained the GO term zinc ion binding (GO:0008270) were selected for further functional classification.
site of pJAF15, and the plasmid was transformed into the C. gattii
zap1D strain. Correct integration of the inactivation cassette into
the WT ZAP1 locus was evaluated by Southern blot and RT-PCR
analysis. The primers used in these constructions are listed in
10 % FBS that was prepared with India ink. Relative capsule sizes
were defined as the ratio between the capsule thickness and cell
diameter. ImageJ software was utilized to determine the capsule
measurements of 100 cells of each strain. Niger seed medium
plates were used for melanin synthesis evaluation.
The viability of mutant cells in zinc-limiting media was assessed
by pre-growing the WT, zap1D and zap1D::ZAP1 strains in YPD
overnight (30uC). The cells were then washed with water and
16106 cells were inoculated in YNB containing 10 mM TPEN or
YNB containing 10 mM TPEN and 10 mM ZnCl2. After 24 h of
incubation at 30uC, the OD600 was determined. To evaluate the
viability of cells when exposed to other chemicals, the cells were
washed after the YPD incubation, and 16106 cells were inoculated
in YNB with various concentrations of diethyl malate or
DETANONOate. After 24 h of incubation at 30uC, the OD600 was
determined. Capsule formation was examined by microscopy after
incubation for 24 h (37uC and 5 % CO2) in DMEM media with
WT and zap1D cells were pre-grown in YPD media (30uC for
24 h) and then inoculated in YNB media for a further period of
growth (30uC for 18 h). YNB + 10 mM TPEN was inoculated with
16107 cells/mL and incubated for additional 4 h at 30uC. Cells
were collected by centrifugation (10,000 g for 10 min), and RNA
was isolated by Trizol (Invitrogen) after cellular lysis via mortar
and pestle. RNA integrity and concentration were assessed by
electrophoresis on a 1% agarose gel and by fluorometry analysis
using a Qubit fluorometer and a Quant-iT RNA assay kit
according to the manufacturers instructions (Invitrogen). mRNA
was purified from total RNA samples, processed and sequenced
using Solexa technology on an Illumina Genome Analyzer GAII
(Fasteris Life Sciences SA, Plan-les-Ouates, Switzerland). The
resulting fastq files were aligned to the C. gattii R265 reference
sequence  with the help of Tophat . Aligned transcripts
were quantified using cufflinks  with the current annotation of
the C. gattii R265 genome provided by the Broad Institute.
Differential expression was evaluated by the cuddiff module of
cufflinks with a False Discovery Rate (FDR) set at 5%. Genes with
an FDR corrected p-value ,0.05 were considered to be
statistically significant. Functional classification against PFAM
and GO of differentially expressed genes was performed using the
web server UFO .
Real time RT-PCR analysis
RNA samples were prepared as above. cDNAs were prepared
from DNAse (Promega)-treated total RNA samples (500 ng) with
ImProm-II Reverse transcriptase (Promega) using oligo-dT.
qRTPCR was performed on a Real-time PCR StepOne Real-Time
PCR System (Applied Biosystems) with thermal cycling conditions
set with an initial step at 95uC for 5 min followed by 40 cycles at
95uC for 15 s, 55uC for 15 s and 60uC for 60 s. Platinum SYBR
green qPCR Supermix (Invitrogen) was used as a reaction mix,
supplemented with 5 pmol of each primer and 1 ml of the cDNA
template in a final volume of 25 ml. All experiments were
performed using three independent cultures, and each cDNA
sample was analyzed in triplicate with each primer pair. Melting
curve analysis was performed at the end of the reaction to confirm
the presence of a single PCR product. Data were normalized to
actin cDNAs amplified in each set of PCR experiments. Relative
expression was determined by the 22DCT method . Statistical
analyses were conducted via a two-tailed Students t-test. The
primers used in these analyses are listed in Table S2.
Cells were grown overnight in YPD medium at 30uC, washed in
YNB and incubated for an additional 24 h in YNB. Cells were
washed and inoculated at a cell density of 16107 cells/mL in fresh
YNB +10 mM TPEN. The acetoxymethyl ester of
dichlorodihydrofluorescein diacetate (CM-H2DCFDA) (Molecular Probes) was
added to a final concentration of 10 mM and incubated for an
additional 2 h to load the dye into cells. After 2 h, the cells were
washed with PBS and analyzed for fluorescence determination
using a SpectraMax M4 plate reader fluorometer (Molecular
Devices) with the emission and excitation wavelengths set at 488
and 520 nm, respectively. Fluorescence values were normalized to
cell count, based on the OD600 determination. Statistical analyses
were conducted with a two-tailed Students t-test.
Intracellular zinc level determinations
Cells were cultivated for ROS assessment. After a 24-h
incubation in YNB, cells were washed and inoculated at a density
of 16107 cells/mL in fresh YNB. The acetoxymethyl ester of
Fluozin-1 (Fluozin-1 AM Molecular Probes) was added to a final
concentration of 10 mM and incubated for an additional 2 h to
load the dye into the cells. A control experiment was conducted in
which TPEN was included to evaluate the fluorescence
background. After 2 h, the cells were washed with PBS and analyzed
for fluorescence determination in a SpectraMax M4 plate reader
fluorometer (Molecular Devices) with its emission and excitation
wavelengths set to 495 and 517 nm, respectively. Fluorescence
values were normalized to the cell count, as determine by OD600
measurement. Statistical analyses were conducted using a
twotailed Students t-test.
Phagocytosis assays were conducted to evaluate the
susceptibility of WT, zap1D and zap1D::ZAP1 cells to the antifungal action of
phagocytes. Macrophage-like RAW264.7 cells were seeded at a
density of 16105 cells/100 ml of DMEM supplemented with 10%
FBS in each well of the 96-well culture plates (TPP). After 24 h of
incubation (37uC and 5% CO2), the medium was replaced with
fresh medium containing 16106 cells of each fungal strain,
obtained after a 24-h incubation in YPD and extensive washing in
PBS. The plates were further incubated (18 h, 37uC, and 5%
CO2), and yeast cells that were not associated with the
macrophages were removed by PBS washes. Fungal survival was
evaluated after macrophage lysis with sterile ice-cold Milli-Q water
and subsequent plating on YPD for CFU determination. This
assay was performed in triplicate for each strain. A Students t-test
was used to determine the statistical significance of the observed
differences in fungal survival.
Virulence studies were conducted as previously described .
Briefly, fungal cells were cultured in 50 ml YPD medium at 30uC
overnight with shaking, washed twice and re-suspended in PBS.
Groups of eight female BALB/c mice (approximately 5 weeks old)
intraperitoneally anesthetized with 100 mg/kg Ketamine and
16 mg/kg Xylazine were infected with 16107 yeast cells
suspended in 50 ml PBS and monitored twice daily for moribund
signals. We used a high C. gattii inoculum since BALB/c mouse is
moderately resistant to cryptococcal infection . The median
survival values were calculated by KaplanMeier survival analysis.
Animal studies were approved by the Federal University of Rio
Grande do Sul Ethics Committee.
Figure S1 In silico characterization of C. gattii Zap1.
Multiple sequence alignment of the Zap1 ortholog sequences of C.
neoformans serotype D Zap1 (CryneoD_Zap1 Genbank
XP_572252) and C. neoformans serotype A Zap1 (CryneoA_Zap1
Broad Institute CNAG_05392) with the C. gattii Zap1 Broad
Institute (A) and the proposed sequences (B). The predicted zinc
fingers are shown as shaded black boxes. (C) Comparison of the
CNBG_4460 locus (Supercontig 11: 588643591042) automatic
annotation and the proposed annotation with the annotated
sequences of ZAP1 from C. neoformans serotype D (CryneoD_ZAP1
Genbank NC_006693) and C. neoformans serotype A
(CryneoA_ZAP1 Broad Institute CNAG_05392). Exons are depicted with
arrows and arrowheads.
Figure S2 Construction of the C. gattii ZAP1 gene
knockout and complemented strains. A. ZAP1 deletion
scheme. TV represents the targeting vector constructed by
Delsgate methodology. 5 ZAP1 and 3 ZAP1 represent the 59
and 39 flanking regions of the ZAP1 gene, respectively. 5F and 5R:
primers utilized to amplify the 59 flanking region of ZAP1. 3F and
3R: primers utilized to amplify the 39 flanking region of ZAP1.
Nat: cassette that confers nourseothricin resistance. WT represents
the wild type locus of the ZAP1 gene in the R265 strain. D
represents the ZAP1 locus in the zap1 mutant strain. The cleavage
sites of the BglII restriction enzyme are indicated. B. Confirmation
by Southern blotting. Genomic DNA (10 mg) from WT (lane 1),
zap1D::ZAP1 complemented (lane 2) and zap1D mutant (lane 3)
strains was digested with BglII. The 39 flanking region was used as
the probe for Southern hybridization. Numbers on the left indicate
the hybridization signal sizes based upon the position of the
molecular size marker. C. Semi-quantitative RT-PCR using
cDNA from WT, zap1D mutant and zap1D::ZAP1 complemented
strains as the template. RNA samples were used as templates for
reactions employing (+) reverse transcriptase. Control reactions
without reverse transcriptase addition (2) were used to confirm the
absence of genomic DNA. The upper panel shows the ZAP1
amplicons, while the lower panel shows the ACT1 amplicons
Figure S3 Analysis of virulence-related phenotypes of
the C. gattii ZAP1 null mutant. (A) Melanin production was
assessed by plating ten-fold serial dilutions of WT, zap1D mutant
and zap1D::ZAP1 complemented strains in niger seed agar and
incubating for 48 h. (B) Ability to replicate at body temperature
was assessed by plating ten-fold serial dilutions of WT, zap1D
mutant and zap1D::ZAP1 complemented strains in YNB agar and
incubating at 30 or 37uC for 24 h. (C) Capsule production was
evaluated by analysis of the capsule/cell ratio of 100 distinct cells
from WT, zap1D mutant and zap1D::ZAP1 cells cultured in
List of primers used in this work.
The authors thank Dr. Joseph Heitman and Dr. Alex Idnurm for providing
the pJAF15 and pAI4 plasmids. The authors also thank Dr. Marcio L.
Rodrigues for his critical reading of the manuscript. Automated DNA
sequencing was performed at the facilities of the Brazilian Genome
Network at the Center of Biotechnology, CBiot-UFRGS-RS. The authors
declare no conflict of interest.
Conceived and designed the experiments: ROS NSSF LK AS MHV CCS.
Performed the experiments: ROS NSSF LK. Analyzed the data: ROS
NSSF LK CCS. Contributed reagents/materials/analysis tools: AS MHV
CCS. Wrote the paper: ROS CCS.
1. Ehrensberger KM , Bird AJ ( 2011 ) Hammering out details: regulating metal levels in eukaryotes . Trends Biochem Sci 36 : 524 - 531 .
2. Eide DJ ( 1998 ) The molecular biology of metal ion transport in Saccharomyces cerevisiae . Annu Rev Nutr 18 : 441 - 469 .
3. Zhao H , Eide D ( 1996 ) The yeast ZRT1 gene encodes the zinc transporter protein of a high-affinity uptake system induced by zinc limitation . Proc Natl Acad Sci U S A 93 : 2454 - 2458 .
4. Zhao H , Eide DJ ( 1997 ) Zap1p, a metalloregulatory protein involved in zincresponsive transcriptional regulation in Saccharomyces cerevisiae . Mol Cell Biol 17 : 5044 - 5052 .
5. Waters BM , Eide DJ ( 2002 ) Combinatorial control of yeast FET4 gene expression by iron, zinc, and oxygen . J Biol Chem 277 : 33749 - 33757 .
6. Kamizono A , Nishizawa M , Teranishi Y , Murata K , Kimura A ( 1989 ) Identification of a gene conferring resistance to zinc and cadmium ions in the yeast Saccharomyces cerevisiae . Mol Gen Genet 219 : 161 - 167 .
7. MacDiarmid CW , Gaither LA , Eide D ( 2000 ) Zinc transporters that regulate vacuolar zinc storage in Saccharomyces cerevisiae . EMBO J 19 : 2845 - 2855 .
8. Li L , Kaplan J ( 2001 ) The yeast gene MSC2, a member of the cation diffusion facilitator family, affects the cellular distribution of zinc . J Biol Chem 276 : 5036 - 5043 .
9. Eide DJ ( 2003 ) Multiple regulatory mechanisms maintain zinc homeostasis in Saccharomyces cerevisiae . J Nutr 133 : 1532S - 1535S .
10. Lyons TJ , Gasch AP , Gaither LA , Botstein D , Brown PO , et al. ( 2000 ) Genomewide characterization of the Zap1p zinc-responsive regulon in yeast . Proc Natl Acad Sci U S A 97 : 7957 - 7962 .
11. Kehl-Fie TE , Skaar EP ( 2010 ) Nutritional immunity beyond iron: a role for manganese and zinc . Current Opinion in Chemical Biology 14 : 218 - 224 .
12. Corbin BD , Seeley EH , Raab A , Feldmann J , Miller MR , et al. ( 2008 ) Metal chelation and inhibition of bacterial growth in tissue abscesses . Science 319 : 962 - 965 .
13. Kehl-Fie TE , Chitayat S , Hood MI , Damo S , Restrepo N , et al. ( 2011 ) Nutrient metal sequestration by calprotectin inhibits bacterial superoxide defense, enhancing neutrophil killing of Staphylococcus aureus . Cell Host Microbe 10 : 158 - 164 .
14. Howard DH ( 1999 ) Acquisition, transport, and storage of iron by pathogenic fungi . Clin Microbiol Rev 12 : 394 - 404 .
15. Jung WH , Kronstad JW ( 2008 ) Iron and fungal pathogenesis: a case study with Cryptococcus neoformans . Cell Microbiol 10 : 277 - 284 .
16. Moreno MA , Ibrahim-Granet O , Vicentefranqueira R , Amich J , Ave P , et al. ( 2007 ) The regulation of zinc homeostasis by the ZafA transcriptional activator is essential for Aspergillus fumigatus virulence . Mol Microbiol 64 : 1182 - 1197 .
17. Kim MJ , Kil M , Jung JH , Kim J ( 2008 ) Roles of Zinc-responsive transcription factor Csr1 in filamentous growth of the pathogenic Yeast Candida albicans . J Microbiol Biotechnol 18 : 242 - 247 .
18. Nobile CJ , Nett JE , Hernday AD , Homann OR , Deneault JS , et al. ( 2009 ) Biofilm matrix regulation by Candida albicans Zap1 . PLoS Biol 7 : e1000133 .
19. Prado M , Silva MB , Laurenti R , Travassos LR , Taborda CP ( 2009 ) Mortality due to systemic mycoses as a primary cause of death or in association with AIDS in Brazil: a review from 1996 to 2006 . Mem Inst Oswaldo Cruz 104 : 513 - 521 .
20. Park BJ , Wannemuehler KA , Marston BJ , Govender N , Pappas PG , et al. ( 2009 ) Estimation of the current global burden of cryptococcal meningitis among persons living with HIV/AIDS . AIDS 23 : 525 - 530 .
21. Lin X ( 2009 ) Cryptococcus neoformans: morphogenesis, infection, and evolution . Infect Genet Evol 9 : 401 - 416 .
22. Chaturvedi V , Chaturvedi S ( 2011 ) Cryptococcus gattii: a resurgent fungal pathogen . Trends Microbiol 19 : 564 - 571 .
23. Kidd SE , Hagen F , Tscharke RL , Huynh M , Bartlett KH , et al. ( 2004 ) A rare genotype of Cryptococcus gattii caused the cryptococcosis outbreak on Vancouver Island (British Columbia, Canada) . Proc Natl Acad Sci U S A 101 : 17258 - 17263 .
24. Byrnes EJ , Heitman J ( 2009 ) Cryptococcus gattii outbreak expands into the Northwestern United States with fatal consequences . F1000 Biol Rep 1.
25. Byrnes EJ , 3rd , Li W , Lewit Y , Perfect JR , Carter DA , et al. ( 2009 ) First reported case of Cryptococcus gattii in the Southeastern USA: implications for travelassociated acquisition of an emerging pathogen . Plos One 4 : e5851 .
26. Ma H , May RC ( 2009 ) Virulence in Cryptococcus species . Adv Appl Microbiol 67 : 131 - 190 .
27. Ding C , Yin J , Tovar EM , Fitzpatrick DA , Higgins DG , et al. ( 2011 ) The copper regulon of the human fungal pathogen Cryptococcus neoformans H99 . Mol Microbiol 81 : 1560 - 1576 .
28. Waterman SR , Hacham M , Hu G , Zhu X , Park YD , et al. ( 2007 ) Role of a CUF1/CTR4 copper regulatory axis in the virulence of Cryptococcus neoformans . J Clin Invest 117 : 794 - 802 .
29. Mambula SS , Simons ER , Hastey R , Selsted ME , Levitz SM ( 2000 ) Human neutrophil-mediated nonoxidative antifungal activity against Cryptococcus neoformans . Infect Immun 68 : 6257 - 6264 .
30. Cryptococcus gattii Sequencing Project, Broad Institute of Harvard and MIT - Broad Institute website . Available: http://www.broadinstitute.org/annotation/ genome/cryptococcus_neoformans_b/MultiHome.html. Accessed 2012 Jul 28 .
31. Wu CY , Bird AJ , Chung LM , Newton MA , Winge DR , et al. ( 2008 ) Differential control of Zap1-regulated genes in response to zinc deficiency in Saccharomyces cerevisiae . BMC Genomics 9 : 370 .
32. Eide DJ ( 2009 ) Homeostatic and adaptive responses to zinc deficiency in Saccharomyces cerevisiae . J Biol Chem 284 : 18565 - 18569 .
33. Wu CY , Roje S , Sandoval FJ , Bird AJ , Winge DR , et al. ( 2009 ) Repression of Sulfate Assimilation Is an Adaptive Response of Yeast to the Oxidative Stress of Zinc Deficiency . Journal of Biological Chemistry 284 : 27544 - 27556 .
34. Perrone GG , Grant CM , Dawes IW ( 2005 ) Genetic and environmental factors influencing glutathione homeostasis in Saccharomyces cerevisiae . Mol Biol Cell 16 : 218 - 230 .
35. Flohe L , Toppo S , Cozza G , Ursini F ( 2011 ) A comparison of thiol peroxidase mechanisms . Antioxid Redox Signal 15 : 763 - 780 .
36. Kronstad JW , Attarian R , Cadieux B , Choi J, D'Souza CA , et al. ( 2011 ) Expanding fungal pathogenesis: Cryptococcus breaks out of the opportunistic box . Nat Rev Microbiol 9 : 193 - 203 .
37. Meinicke P ( 2009 ) UFO: a web server for ultra-fast functional profiling of whole genome protein sequences . BMC Genomics 10 : 409 .
38. Maret W ( 2010 ) Metalloproteomics, metalloproteomes, and the annotation of metalloproteins . Metallomics 2 : 117 - 125 .
39. Jung WH , Saikia S , Hu G , Wang J , Fung CK , et al. ( 2010 ) HapX positively and negatively regulates the transcriptional response to iron deprivation in Cryptococcus neoformans . PLoS Pathog 6 : e1001209 .
40. Jung WH , Sham A , White R , Kronstad JW ( 2006 ) Iron regulation of the major virulence factors in the AIDS-associated pathogen Cryptococcus neoformans . PLoS Biol 4 : e410 .
41. Liu OW , Chun CD , Chow ED , Chen C , Madhani HD , et al. ( 2008 ) Systematic genetic analysis of virulence in the human fungal pathogen Cryptococcus neoformans . Cell 135 : 174 - 188 .
42. Winters MS , Chan Q , Caruso JA , Deepe GS , Jr. ( 2010 ) Metallomic analysis of macrophages infected with Histoplasma capsulatum reveals a fundamental role for zinc in host defenses . J Infect Dis 202 : 1136 - 1145 .
43. Missall TA , Cherry-Harris JF , Lodge JK ( 2005 ) Two glutathione peroxidases in the fungal pathogen Cryptococcus neoformans are expressed in the presence of specific substrates . Microbiology 151 : 2573 - 2581 .
44. Wu CY , Bird AJ , Winge DR , Eide DJ ( 2007 ) Regulation of the yeast TSA1 peroxiredoxin by ZAP1 is an adaptive response to the oxidative stress of zinc deficiency . J Biol Chem 282 : 2184 - 2195 .
45. Gross NT , Nessa K , Camner P , Jarstrand C ( 1999 ) Production of nitric oxide by rat alveolar macrophages stimulated by Cryptococcus neoformans or Aspergillus fumigatus . Med Mycol 37 : 151 - 157 .
46. de Jesus-Berrios M , Liu L , Nussbaum JC , Cox GM , Stamler JS , et al. ( 2003 ) Enzymes that counteract nitrosative stress promote fungal virulence . Curr Biol 13 : 1963 - 1968 .
47. Idnurm A , Reedy JL , Nussbaum JC , Heitman J ( 2004 ) Cryptococcus neoformans virulence gene discovery through insertional mutagenesis . Eukaryot Cell 3 : 420 - 429 .
48. Larkin MA , Blackshields G , Brown NP , Chenna R , McGettigan PA , et al. ( 2007 ) Clustal W and Clustal X version 2.0. Bioinformatics 23 : 2947 - 2948 .
49. Tamura K , Peterson D , Peterson N , Stecher G , Nei M , et al. ( 2011 ) MEGA5: molecular evolutionary genetics analysis using maximum likelihood, evolutionary distance, and maximum parsimony methods . Mol Biol Evol 28 : 2731 - 2739 .
50. de Castro E , Sigrist CJ , Gattiker A , Bulliard V , Langendijk-Genevaux PS , et al. ( 2006 ) ScanProsite: detection of PROSITE signature matches and ProRuleassociated functional and structural residues in proteins . Nucleic Acids Res 34 : W362 - 365 .
51. Kmetzsch L , Staats CC , Simon E , Fonseca FL , de Oliveira DL , et al. ( 2010 ) The vacuolar Ca(2)(+) exchanger Vcx1 is involved in calcineurin-dependent Ca(2)(+) tolerance and virulence in Cryptococcus neoformans . Eukaryot Cell 9 : 1798 - 1805 .
52. Kmetzsch L , Staats CC , Simon E , Fonseca FL , Oliveira DL , et al. ( 2011 ) The GATA-type transcriptional activator Gat1 regulates nitrogen uptake and metabolism in the human pathogen Cryptococcus neoformans . Fungal Genet Biol 48 : 192 - 199 .
53. Toffaletti DL , Rude TH , Johnston SA , Durack DT , Perfect JR ( 1993 ) Gene transfer in Cryptococcus neoformans by use of biolistic delivery of DNA . J Bacteriol 175 : 1405 - 1411 .
54. Trapnell C , Pachter L , Salzberg SL ( 2009 ) TopHat: discovering splice junctions with RNA-Seq . Bioinformatics 25 : 1105 - 1111 .
55. Roberts A , Pimentel H , Trapnell C , Pachter L ( 2011 ) Identification of novel transcripts in annotated genomes using RNA-Seq . Bioinformatics 27 : 2325 - 2329 .
56. Livak KJ , Schmittgen TD ( 2001 ) Analysis of relative gene expression data using real-time quantitative PCR and the 2(- Delta Delta C(T)) Method. Methods 25 : 402 - 408 .
57. Huffnagle GB , Boyd MB , Street NE , Lipscomb MF ( 1998 ) IL-5 is required for eosinophil recruitment, crystal deposition, and mononuclear cell recruitment during a pulmonary Cryptococcus neoformans infection in genetically susceptible mice (C57BL/6) . J Immunol 160 : 2393 - 2400 .