Aspergillus fumigatus calcineurin interacts with a nucleoside
Taı ´sa Magnani Dinamarcoc,1, Neil Andrew Browna,1, Ricardo Sergio Couto de Almeidab,
Patrı ´cia Alves de Castroa, Marcela Savoldia, Maria Helena de Souza Goldmanc,
Gustavo Henrique Goldmana,d,*
aFaculdade de Cie ˆncias Farmace ˆuticas de Ribeira ˜o Preto, Universidade de Sa ˜o Paulo, Sa ˜o Paulo, Brazil
bDepartment of Microbiology, University of Londrina, Londrina, Parana ´, Brazil
cFaculdade de Filosofia, Cie ˆncias e Letras de Ribeira ˜o Preto, Universidade de Sa ˜o Paulo, Sa ˜o Paulo, Brazil
dLaborato ´rio Nacional de Cie ˆncia e Tecnologia do Bioetanol, CTBE, Caixa Postal 6170, 13083-970 Campinas, Sa ˜o Paulo, Brazil
Received 20 March 2012; accepted 10 May 2012
Available online 22 May 2012
The Ca2þ-calcineurin pathway affects virulence and morphogenesis in filamentous fungi. Here, we identified 37 CalA-interacting proteins
that interact with the catalytic subunit of calcineurin (CalA) in Aspergillus fumigatus, including the nucleoside diphosphate kinase (SwoH). The
in vivo interaction between CalA and SwoH was validated by bimolecular fluorescence complementation. A. fumigatus swoH is an essential
gene. Therefore, a temperature-sensitive conditional mutant strain with a point mutation in the active site, SwoHV83F, was constructed, which
demonstrated reduced growth and increased sensitivity to elevated temperatures. The SwoHV83Fmutation did not cause a loss in virulence in the
Galleria mellonella infection model. Taken together these results imply that CalA interacts with SwoH.
? 2012 Institut Pasteur. Published by Elsevier Masson SAS. All rights reserved.
Keywords: Aspergillus fumigatus; Calcineurin; Nucleoside diphosphate kinase; Galleria mellonella
Calcium ions are essential for signal transduction. In
eukaryotic cells important calcium mediators are calmodulin
and the phosphatase calcineurin [1,2]. Calcineurin is a heter-
odimeric protein composed of a catalytic subunit A and
a regulatory subunit B . In fungi, calcineurin plays an
important role in the control of cell morphology and virulence
[1e3]. The main mode of action of calcineurin is through the
dephosphorylation of the transcription factor Crz1p [2,3].
Calcineurin dephosphorylates Crz1p in response to an increase
in cytosolic calcium, promoting its nuclear translocation [2,3].
Besides Crz1 homologs, few direct targets of calcineurin are
known in fungi [4e6]. We and others have been characterizing
the Ca2þ-calcineurin pathway in the human pathogenic fungus
Aspergillus fumigatus [2,7]. Previously, we have shown that
the calcineurin catalytic subunit, calA, was not an essential
gene, but it is required for hyphal extension, branching and
conidial architecture . Furthermore, the A. fumigatus DcalA
mutant strain demonstrated decreased fitness in a low dose
murine infection and could not grow in fetal bovine serum
(FBS) probably due to a defect in phosphate transport.
We were interested in identifying the proteins that interact
with calcineurin within A. fumigatus. It has been suggested
that the concomitant usage of antifungal drugs and specific
fungal calcineurin inhibitors could be an important advance-
ment in antifungal therapy. The understanding of the targets
and pathways that are associated with Ca2þ-calcineurin in this
* Corresponding author. Faculdade de Cie ˆncias Farmace ˆuticas de Ribeira ˜o
Preto, Universidade de Sa ˜o Paulo, Sa ˜o Paulo, Brazil. Tel./fax: þ55 16
E-mail address: firstname.lastname@example.org (G.H. Goldman).
1These authors contributed equally to this work.
Microbes and Infection 14 (2012) 922e929
1286-4579/$ - see front matter ? 2012 Institut Pasteur. Published by Elsevier Masson SAS. All rights reserved.
organism would facilitate a greater understanding of fungal
virulence and assist the discovery of new antifungal agents.
Here, we investigated which proteins interact with the cata-
lytic subunit of calcineurin within A. fumigatus.
2. Materials and methods
2.1. Strains and culture conditions
A. fumigatus strains used in this study were CEA17
(pyrG?), CEA17-80 (as the wild-type strain in all experi-
ments), Af293 (wild-type), DcalA, calA::S-tag, and the
SwoHV83Fmutant. The media used were of two basic types,
i.e. complete and minimal. The complete media comprised the
following three variants: YAG (2% w/v glucose, 0.5% w/v
yeast extract, 2% w/v agar, trace elements), YUU [YAG sup-
plemented with 1.2 g l-1 (each) of uracil and uridine], and
liquid YG or YG þ UU medium with the same composition
(but without agar). A modified minimal medium (MM: 1%
glucose, original high nitrate salts, trace elements, 2% agar,
pH 6.5) was also used. Trace elements, vitamins, and nitrate
salts were included as described . Strains were grown at
37?C unless indicated otherwise.
2.2. Construction of A. fumigatus mutants
A gene replacement cassette was constructed by “in vivo”
recombination in Saccharomyces cerevisiae as previously
described by . Briefly, approximate 2.0-kb regions on either
side of each ORF were selected for primer design. For
construction, the primers 5F and 5R, were used to amplify the
50-UTR flanking region of the targeted ORF. Likewise, the
primers 3F and 3R were used to amplify the 30-UTR ORF
flanking region. The primers 5F and 3R also contained a short
homologs sequence to the Multiple Cloning Site (MCS) of the
plasmid pRS426. Both fragments, 50- and 30-UTR, were PCR-
amplified from A. fumigatus genomic DNA. The pyrG gene
used in the A. fumigatus cassette was used as marker for
transformation prototrophy. Deletion cassettes were achieved
by transforming each fragment together with the plasmid
pRS426 previously digested with BamHI and EcoRI into the S.
cerevisiae strain SC94721. The DNA of the yeast trans-
formants was extracted, dialyzed and transformed by electro-
poration into Escherichia coli strain DH10B to rescue the
pRS426 plasmid harboring the cassette. The cassette was
PCR-amplified from these plasmids and used for A. fumigatus
transformation. The primers used are listed in Supplementary
The calcineurin gene was fused to the S-tag tail in the C-
terminal region, according to . The amplification of the
cassette GA5:S-tag:pyrG was performed by PCR using the
pHL81 vector as a template and the primers pHL81 GA50S-
Tag and pHL81 30pyrG (The primers used are listed in
Supplementary Table 1). Similarly, the regions 50- and 30-UTR
flanking the calcineurin gene were amplified by PCR. The
cassette calcineurin:S-tag wasconstructed byin vivo
recombination in S. cerevisiae as described above. Southern
blot analysis is shown in Supplementary Fig. S1.
2.3. Extraction and purification of S-tagged proteins
Five-liter cultures containing 2 ? 106conidia/ml of A.
fumigatus, containing the calcineurin:S-tag:pyrG, were sub-
jected to different growth conditions such as, incubation at
37?C for 16 h and subsequent treatment with 200 mM CaCl2
for 5, 10 and 30 min. After this period, the cultures were
filtered and washed with 100 ml of stop buffer (0.9% NaCl,
1 mM NaN3, 10 mM EDTA, 50 mM NaF). Cells were then
immediately frozen in liquid nitrogen, freeze dried overnight
and macerated in liquid nitrogen. For every 0.1 g dry weight
1.3 ml HK solution (300 mM NaCl; 1ug/ml Pepstatin A,
10 ug/ml Leupeptin; 10 ug/ml Trypsin ChymoT I, 10 ug/ml
Aprotinin, 10 ug/ml TPCK, 2 mM TAME, 5 mM Benzami-
dine, 5 mM EDTA (pH 8.0), 25 mM TriseHCl (pH 7.5),
15 mM EGTA (pH 8.0), 500 uM Na Vanadate 0.5% NP40,
15 mM PNPP, 1 mM DTT, 50 ug/ml PMSF) was added. The
suspension was centrifuged at 21.000 rpm at 4?C for 30 min.
The supernatant was removed and centrifuged again for
10 min under the same conditions. After determining protein
concentration, S-tag fused proteins were specifically purified
with S-protein Agarose (Novagen). Approximately 150 ul
packed volume of resin was added for every 100 mg of
protein. The mixture was kept stirring at 4?C for 2 h. The
suspension was then centrifuged and the resin washed 6 times
with an equal volume of HK solution. The resin was subse-
quently transferred to an Eppendorf tube and washed at least
two times at 4?C with 1 ml HK. After washes, sample buffer
was added and the mixture boiled for 5 min. The proteins were
resolved on a NuPAGE 4e12% Bis-Tris gel (Invitrogen),
excised and sent for identification by mass spectrometry at the
Institute Pasteur de Montevideo.
2.4. Bimolecular fluorescence complementation (BiFC)
The interaction between CalA and SwoH in A. fumigatus
was confirmed by BiFC analyses. The N-terminal half of the
yellow fluorescent protein (YFPN) or the C-terminal half of
the YFP (YFPC) was fused to the N-terminus of the protein
of interest). The YFPN(154 amino acids of YFP and 5-amino
acids linker) and YFPC(86 amino acids of YFP and 17-
amino acids linker) were cloned into vectors pDV7 and
pDV8. To create an N-terminal YFPCfusion construct of
SwoH, the AscI-PacI fragment was amplified using primers
AscI swoH fw and PacI swoH rev and after digestion, cloned
into the corresponding sites of pDV8, yielding pDV8:swoH.
Using the same approach, to create N-terminal YFPNfusion
constructs, CalA was amplified using AscI calA Fwd and
PacI calA Rev, and cloned into the corresponding sites of
pDV8:swoH andpDV7:calA were
923 T. Magnani Dinamarco et al. / Microbes and Infection 14 (2012) 922e929
Identification of proteins that interact with A. fumigatus S-tag::CalA.
enolase/allergen Asp F 22
ATP synthase F1,
Mitoc. F1 ATPase sub.alpha
ATP carrier protein
to protein synthesis
60S ribosomal protein L27e
Ribosomal protein S5
60S ribosomal protein L10
40S ribosomal protein S15
Elongation factor 3
Eukaryotic translation initiation factor 4
Proteins related to heat-shock
Heat shock protein 70 from Aspergillus sps
Molecular chaperone Hsp70
Heat shock protein Hsp90
Hsp70 chaperone (HscA)
Proteins related to glucose metabolism
pyruvate decarboxylase PdcA
1 gij710011645.53 61,91210 1315
2 gij1591294085.8671,932 13 876
8.31 32,356 201172
10 gij7098507010.96 40,47924 1725
10.21 28,8606 851
16 gij709962329.35 25,712 10 852
24gij709995905.53 61,912 101315
T. Magnani Dinamarco et al. / Microbes and Infection 14 (2012) 922e929
2.5. Analysis of NDK protein and enzymatic activities
For protein extraction, 1 ? 106conidia/ml of wild-type and
DcalA strains were grown in YG medium for 16 h at 37?C in
a reciprocal shaker (250 rpm). After this period, 200 mM of
CaCl2 was added to the medium and the germlings were
collected after 10 and 30 min exposure. Mycelia were freeze
dried and crude protein extracts were prepared as described
To analyze NDK autophosphorylation, the crude extracts
were adjusted to the same protein concentration and incubated
with 200 nM [g-32P] ATP (6000 Ci/mmol. PerkinElmer) for
15 min at 25
TriseHCl, pH 8.0, 50 mM NaCl and 1 mM MgCl2. The
reaction was stopped by adding equal volume of 2% SDS-
PAGE sample buffer. The reacted protein samples were
separated by SDS-PAGE (12.5%) electrophoresis, dried and
The thin layer chromatography (TLC) assay for NDK
activity was performed as previously described . The TLC
plates were autoradiographed and the amount of dGTP was
determined via a densitometry analysis using the Image J
?C in reaction buffer containing 20 mM
2.6. Staining and microscopy
For nuclear DAPI (40,6-diamino-2-phenylindole) staining
of the germlings, conidia were inoculated on coverslips. After
incubation at 30?C, 37?and 44?C, coverslips with adherent
germlings were formaldehyde fixed, stained with DAPI (100
ng/ml, Sigma) and then visualized on a epifluorescence Carl
Zeiss (Jena, Germany) microscope using a 100X magnification
oil immersion objective lens (EC Plan-Neofluar, NA 1.3) as
previously described .
2.7. Galleria mellonella infection model
The infection assay was performed as described . The
conidial suspension was adjusted to two different concentra-
tions: 1 ? 106/5 ml for 37?C infections and 5 ? 106/5 ml for
1 ? 106/5 ml does not caused larval death at 30?C (data not
shown, 12) and it is therefore necessary to increase the inoc-
ulum for the incubation at this temperature.
?C infections. This was done because an inoculum of
3. Results and discussion
3.1. Identification of CalA interacting proteins
We identified a variety of proteins that were able to interact
with the CalA:S-tag protein when the modified A. fumigatus
strain was exposed to 200 mM CaCl2for 5, 10 and 30 min
(Supplementary Fig. S2). In contrast, only a negligible number
of proteins could be isolated from the lysates of the wild-type
control (Supplementary Fig. S2). All the protein bands were
excised and selected bands were identified by peptide mass
fingerprinting. We were able to identify 37 CalA-interacting
enolase/allergen Asp F 22
malate dehydrogenase, NAD-dependent
ATP citrate lyase, subunit 1.
Other cell metabolism proteins
Fatty acid synthase beta subunit
conserved hypothetical protein;
Calcineurin catalytic subunit CnaA
Peptidyl-prolyl cis-trans isomerase/cyclophilin
Kunitz-type trypsin inhibitor KTI1
nucleoside diphosphate kinase
cobalamin-independent methionine synthase MetH/D
tubulin alpha-1 subunit
925T. Magnani Dinamarco et al. / Microbes and Infection 14 (2012) 922e929
proteins that were involved in a variety of cellular processes;
they were classified into FunCat functional categories (http://
frame.html) and divided into five functional categories: (i)
proteins related to mitochondria, (ii) proteins related to protein
synthesis, (iii) proteins related to heat-shock, (iv) proteins
related to glucose metabolism, and (v) other cell metabolism
proteins (Table 1). Some of these proteins like Hsp90, ATP
synthase, 40S ribosomal protein, and cyclophilin have already
been identified as interacting with the S. cerevisiae, A. fumi-
gatus, and Cryptococcus neoformans calcineurin catalytic
subunit [2,6]. As an initial step to investigate the protein
interactions and considering the important role played by
calcineurin in polar growth, we decided to validate the inter-
action between the nucleoside diphosphate kinase (NDK)
identified in the screen and CalA. NDKs are present in
bacteria, archaea and eukaryotes and are responsible to
maintain the pool of nucleoside triphosphates (NTPs) by
a reaction that catalyzes the transfer of 50-triphosphates to
nucleoside 50-diphosphates [13,14]. NDKs are not only
involved in the regulation of the NTPs pool but they are
involved in cellular proliferation, differentiation, tumor
metastasis, stress, and photosignalling [13,14]. In Neurospora
crassa NDK regulates hyphal development, catalase activity
and perithecial polarity [11,15e17]. The Aspergillus nidulans
NKD (named SwoH) was previously identified by comple-
mentation of the temperature-sensitive swoH1 mutant, previ-
ously identified in a screen for defects in polar growth .
3.2. CalA and SwoH interaction is modulated by calcium
The interaction between CalA and SwoH in A. fumigatus
was confirmed by bimolecular fluorescence complementation
(BiFC) analyses. In the strain expressing only YFPN-CalA or
YFPC-SwoH, no YFP fluorescence was detected either in the
absence or in the presence of 200 mM CaCl2for 10 min
(Fig. 1A). In contrast, in the strain expressing both the YFPN-
CalA and YFPC-SwoH, the YFP signal was detected along the
gemlings when exposed to CaCl2(Fig. 1A). Aiming to confirm
SwoH sub-cellular localization,
a SwoH:GFP mutant strain. The SwoH:GFP mutant strain had
a phenotypic behavior identical to the wild-type strain (data
not shown). Visualization of SwoH:GFP along 4, 6, 8, and 16-
h growth demonstrated a disperse distribution along the
germlings with more intense fluorescence at 6- and 8-h growth
(Supplementary Fig. S3). The localization pattern of the YFP
signal was similar to that of SwoH:GFP (Supplementary
3.3. SwoH is essential for A. fumigatus growth
We were not able to delete A. fumigatus swoH gene (data
not shown), which is known to be essential in A. nidulans .
Sequencing of the temperature-sensitive swoH1 mutant of A.
nidulans predicted a V83F change . Structural modeling
suggested that the swoH1 mutation would lead to the
perturbation of the NDK active site. A. fumigatus SwoH has
82% identity and 91% similarity with A. nidulans SwoH (e-
value 2.2e-67) and the Valine at position 83 is conserved.
Therefore, we decided to construct a conditional mutant for
the A. fumigatus SwoH by introducing the V83F change. At
showed the same radial growth (Fig. 1B). However, the
SwoHV83Fmutant strain was temperature sensitive and
demonstrated reduced radial growth at 37 and 44?C when
compared to the wild-type strain (Fig. 1B). Germlings of the
SwoHV83Fmutant had comparable length and number of
nuclei to the wild-type strain at 30 and 37?C, but swollen
morphology and a reduced number of nuclei at 44
(Fig. 1C). These results strongly indicate A. fumigatus swoH is
an essential gene.
?C, both the wild-type and SwoHV83Fmutant strains
3.4. NDK levels reduced in DcalA mutant
The interaction between CalA and SwoH could reflect the
fact that CalA is controlling SwoH activity. Thus, to start
addressing this hypothesis we decided to measure SwoH
activity. The wild-type and the DcalA mutant strains showed
comparable growth rates in complete medium up to 16 h
growth (Fig. 2A). After 48 h, the DcalA mutant strain had
approximately 50% the dry weight of the wild-type strain
(Fig. 2A). Equal amounts of proteins were used for the NDK
enzymatic assay (Fig. 2B). When the wild-type strain was
exposed to CaCl2there was no change in the NDK activity
levels (Fig. 2B). In contrast, the levels of NDK activity in the
DcalA strain before and after CaCl2exposure were approxi-
mately two fold lower than the wild-type strain (Fig. 2B)
suggesting that CalA influences NDK activity in A. fumigatus.
3.5. SwoHV83Fmutation does not affect virulence
Next, we evaluated if the V83F mutation could affect A.
fumigatus virulence in a Galleria mellonella model. These
assays were performed at 30 and 37?C to evaluate the ther-
mosensibility of DswoH strain during infection. At 30?C there
was no difference in virulence among the three strains
(Fig. 2C). However, there was a significant difference between
strain, which killed about 20% of the
G. mellonella larvae, and both the wild-type and the com-
plemented SwoHV83F:SwoHþstrains, which were able to kill
all the larvae at 37?C (Fig. 2C and C). The obvious attenu-
ation at 37?C can be explained by the poor growth of the
mutant at this temperature.
3.6. Possible role for CalAeSwoH interaction in
temperature-sensitive polarized growth
To start to understand the nature of the interaction between
A. fumigatus CalA and SwoH, we investigated the possibility
that CalA is able to bind to SwoH by using yeast two-hybrid
analysis. Our results did not reveal any interaction between
these two proteins (data not shown), suggesting that there are
other proteins or conditions that mediate this interactions that
926 T. Magnani Dinamarco et al. / Microbes and Infection 14 (2012) 922e929
could not be completely recapitulated by using yeast two-
hybrid assays. Polarized growth is an essential feature of
filamentous fungi. Therefore, is SwoH interacting with CalA
in a larger complex that is involved in the control of polarized
growth? In N. crassa the NDK is involved in perithecial
polarity and morphogenesis, while in A. nidulans, and now
also A. fumigatus, swoH that encodes the homologous NDK
appears to be involved in polarized growth. Establishment and
maintenance of cell polarity are coordinated by signaling
pathways such as the calcium and NDR (nuclear Dbf2-related)
protein-kinase pathways [19,20]. The CotA kinase, a member
of the NDR protein-kinase family, forms a complex with
MobB to regulate cell polarized growth in A. nidulans [19,20].
The MobB/CotA complex is a component of the conserved
RAM (Regulation of Ace2 and Morphogenesis) pathway and
serves an important role in cell morphogenesis . An
insertional mutagenesis screen in the human pathogenic
fungus C. neoformans identified homologs of components of
the RAM pathway (e.g., KIC1, CBK1, SOG2, and TAO3)
involved in establishing normal colony morphology . A
targeted deletion of MOB2 showed identical phenotypes to the
other insertion mutants, such as a constitutive hyperpolariza-
tion. These authors demonstrated that the RAM pathway acts
in parallel to the protein phosphatase calcineurin in C. neo-
formans. Thus, it is possible that CalA is participating in
a complex that is involved in polarized growth.
Crude cell extracts from the A. nidulans swoH1 mutant
grown at a permissive temperature had approximately an 80%
decrease in NDK activity compared to the wild-type strain and
did not show any decrease in activity when assayed at higher
Figure 1. A. fumigatus calcineurin interacts with nucleoside diphosphate kinase. (A) Bimolecular fluorescence complementation (BiFC) analyses. The N-terminal
half of yellow protein (YFPN) or the C-terminal half of YFP (YFPC) was fused to the N-terminus of the protein of interest. Bars, 5 mm. (B) Growth phenotypes for
the wild-type and the swoHV83Fmutant grown on YAG medium at 30, 37 and 44?C for 72 h (C) DAPI staining for the wild-type and the SwoH83Fmutant grown for
16 h at 30, 37, and 44?C. Bars, 10 mm, except for the SwoH83Fmutant grown at 44?C where the bars are 5 mm.
927 T. Magnani Dinamarco et al. / Microbes and Infection 14 (2012) 922e929
temperatures . Based on this evidence, the authors
hypothesized that the lack of temperature-sensitive NDK
activity, in the swoH1 mutant, suggests that SwoH NDK is
required for growth at elevated temperatures, rather than for
the maintenance of cell polarity. In C. neoformans calcineurin
has been associated with growth at elevated temperatures such
as 37?C. However, in neither A. nidulans, A. fumigatus nor C.
albicans has calcineurin been linked to growth at elevated
temperatures , arguing against a role for the NDK during
growth at high temperatures.
Fig. 2. The A. fumigatus DcalA has decreased NDK activity. (A) Growth curve of the wild-type and DcalA mutant strains grown in YG liquid medium. All dry
weight measurements were done in triplicate, the results are expressed as mean ? standard deviation and were considered statistically different with a P val-
ue < 0.05, determined by Student’s t-test using GraphPad Prism software version 5 (GraphPad software, Inc., USA). (B) Coomassie staining of a polyacrylamide
gel. One mg of each crude extract from wild-type and DcalA mutant grown for 16 h at 37?C and after incubation with 200 mM CaCl2for 10 and 30 min were
applied to polyacrylamide gel. NDK activity in the crude extracts from wild-type and DcalA mutant, after incubation with 200 mM CaCl2for 10 and 30 min. Crude
extracts containing 1 mg of protein were assayed for NDK activity [the same amount of proteins applied to the left panel]. The amount of nucleoside triphosphate
(dGTP) was determined by densitometric analysis made by using the Image J 240 program (available at http://rsbweb.nih.gov/ij/download.html). (C)
KaplaneMeier plots of survival after infection with A. fumigatus. G. mellonella were infected with 5 ? 106conidia/larvae with 10 larvae per group and incubated
at 30?C (left graph). G. mellonella were infected with 106conidia/larvae with 10 larvae per group and incubated at 37?C (right graph). Larvae injected with PBS
were used as control in both assays.
928 T. Magnani Dinamarco et al. / Microbes and Infection 14 (2012) 922e929
4. Conclusion Download full-text
In summary, we have identified proteins that interact with
the catalytic subunit of calcineurin. However, the role played
by these associated proteins and the function of NDKs during
polarized fungal growth remains to be determined. How this
mechanism is influenced by calcineurin and calcium metabo-
lism requires further investigation.
This research was supported by the Fundac ¸a ˜o de Amparo a `
Pesquisa do Estado de Sa ˜o Paulo (FAPESP) and Conselho
Nacional de Desenvolvimento Cientı ´fico e Tecnolo ´gico
(CNPq), Brazil. We would like to thank the anonymous
reviewers for their comments and suggestions, Dr. Andre ´a
Carla Quiapim and the LBMP- FFCLRP/USP for DNA
sequencing, and Dr. Reinhard Fischer for sending the BiFC
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