Involvement of the Aspergillus nidulans protein kinase C with farnesol tolerance is related to the unfolded protein response

Article (PDF Available)inMolecular Microbiology 78(5):1259-79 · December 2010with22 Reads
DOI: 10.1111/j.1365-2958.2010.07403.x · Source: PubMed
Abstract
Previously, we demonstrated that the Aspergillus nidulans calC2 mutation in protein kinase C pkcA was able to confer tolerance to farnesol (FOH), an isoprenoid that has been shown to inhibit proliferation and induce apoptosis. Here, we investigate in more detail the role played by A. nidulans pkcA in FOH tolerance. We demonstrate that pkcA overexpression during FOH exposure causes increased cell death. FOH is also able to activate several markers of endoplasmic reticulum (ER) stress and the unfolded protein response (UPR). Our results suggest an intense cross-talk between PkcA and the UPR during FOH-induced cell death. Furthermore, the overexpression of pkcA increases both mRNA accumulation and metacaspases activity, and there is a genetic interaction between PkcA and the caspase-like protein CasA. Mutant analyses imply that MAP kinases are involved in the signal transduction in response to the effects caused by FOH.
Involvement of the
Aspergillus nidulans
protein kinase C
with farnesol tolerance is related to the unfolded
protein response
mmi_7403 1259..1279
Ana Cristina Colabardini,
1
Patrícia Alves de Castro,
1
Paula Fagundes de Gouvêa,
1
Marcela Savoldi,
1
Iran Malavazi,
3
Maria Helena S. Goldman
2
and
Gustavo Henrique Goldman
1,4
*
1
Faculdade de Ciências Farmacêuticas de Ribeirão
Preto and
2
Faculdade de Filosofia, Ciências e Letras de
Ribeirão Preto, Universidade de São Paulo, São Paulo,
Brazil.
3
Departamento de Genética e Evolução, Centro de
Ciências Biológicas e da Saúde (CCBS), Universidade
Federal de São Carlos, Brazil.
4
Laboratório Nacional de Ciência e Tecnologia do
Bioetanol CTBE, Caixa Postal 6170, 13083-970
Campinas, São Paulo, Brazil.
Summary
Previously, we demonstrated that the Aspergillus
nidulans calC2 mutation in protein kinase C pkcA was
able to confer tolerance to farnesol (FOH), an iso-
prenoid that has been shown to inhibit proliferation
and induce apoptosis. Here, we investigate in more
detail the role played by A. nidulans pkcA in FOH
tolerance. We demonstrate that pkcA overexpression
during FOH exposure causes increased cell death.
FOH is also able to activate several markers of endo-
plasmic reticulum (ER) stress and the unfolded
protein response (UPR). Our results suggest an
intense cross-talk between PkcA and the UPR during
FOH-induced cell death. Furthermore, the overex-
pression of pkcA increases both mRNA accumulation
and metacaspases activity, and there is a genetic
interaction between PkcA and the caspase-like
protein CasA. Mutant analyses imply that MAP
kinases are involved in the signal transduction in
response to the effects caused by FOH.
Introduction
Protein kinase C (PKC) is a conserved enzyme among
eukaryotes. Although mammalian cells have at least 10
isoforms, fungi only have one or two PKCs (Levin, 2005;
Shea and Poeta, 2006; Rhome and Poeta, 2009). Sac-
charomyces cerevisiae has only one PKC (PKC1) that
has a serine/threonine domain, regulatory domains (C1,
C2 and HR1), and a pseudosubstrate sequence. The
PKC1 C1 and HR1 domains interact with the small
GTPase Rho1 (Nonaka et al., 1995; Schmitz et al., 2002).
PKC1 is involved in the regulation of cell construction via
the activation of a MAP kinase signalling cascade in
response to cell wall damage (Kamada et al., 1995).
Bck1p, the first component in the cell wall integrity (CWI)
MAPK signalling cascade, activates Slt2p that phospho-
rylates and activates the transcription factor Rlm1p, which
regulates the expression of genes whose products are
important in cell wall biosynthesis (Jung and Levin, 1999).
In the filamentous fungus Aspergillus nidulans, there are
two putative PKC-encoding genes, pkcA (AN0106.3) and
pkcB (AN5973.3) (Herrmann et al., 2006; Ichinomiya
et al., 2007; Ronen et al., 2007; Teepe et al., 2007).
Although sequence similarity of PkcB with other PKCs is
limited to the catalytic region in the carboxy-terminus,
PkcA has all the characteristic features of fungal PKCs,
including an extended N-terminal regulatory domain con-
taining C1, C2 and HR1 domains (Herrmann et al., 2006).
Knockdown of pkcA expression leads to reduced growth,
conidiation and penicillin production (Herrmann et al.,
2006).
Recently, Teepe et al. (2007) shown that an A. nidulans
calC2 mutation that confers hypersensitivity to calcofluor
white along with other drug sensitivities (indicating a
defect in CWI and polar growth) was complemented by
pkcA. The pkcA allele of the calC2 strain contains a muta-
tion predicted to introduce a charged arginine residue in
place of neutral glycine at a conserved site located imme-
diately beside the C1B regulatory domain. Ronen et al.
(2007) and Ichinomiya et al. (2007) generated a condi-
tional promoter mutant of pkcA and observe that its
repression resulted in increased conidial swelling,
decreased rates of hyphal growth, changes in the ultra-
structure of the cell wall and increased sensitivity to anti-
fungal agents.
Farnesol (FOH), a non-sterol isoprenoid produced by
dephosphorylation of farnesyl pyrophosphate, has been
Accepted 15 September, 2010. *For correspondence. E-mail
ggoldman@usp.br; Tel./Fax (+55) 16 36024280/4281/4311.
Molecular Microbiology (2010) 78(5), 1259–1279 doi:10.1111/j.1365-2958.2010.07403.x
First published online 15 October 2010
© 2010 Blackwell Publishing Ltd
shown to inhibit proliferation and induce apoptosis in neo-
plastic cell lines and also to be effective in chemopreven-
tion and chemotherapy in several in vivo cancer models
(Adany et al., 1994; Ohizumi et al., 1995; Miquel et al.,
1996; He et al., 1997; Hudes et al., 2000; Burke et al.,
1997; Wiseman et al., 2007; Joo and Jetten, 2010). In
fungi, FOH plays a role as an extracellular quorum-
sensing molecule in the dimorphic fungus Candida albi-
cans (Nickerson et al., 2006; Langford et al., 2009). FOH
inhibits the yeast to mycelium dimorphic transition and
induces C. albicans to grow as actively budding yeasts
(Hornby et al., 2001). In S. cerevisiae, FOH blocks growth
by raising the concentration of mitochondrial reactive
oxygen species (ROS) (Machida et al., 1998; 1999). FOH
has no effect on A. nidulans hyphal morphogenesis, but it
triggers morphological features characteristic of apoptosis
through ROS accumulation (Semighini et al., 2006;
Savoldi et al., 2008). In mammalian cells, FOH-mediated
cell death can be reduced by the addition of phorbol
esters (Voziyan et al., 1995; Taylor et al., 2005). Phorbol
esters are non-metabolizable structural mimics of diacylg-
lycerol that bind to and activate proteins containing C1
domains, such as PKCs (Yang and Kazanietz, 2003).
There is an increase of FOH sensitivity when PKC inhibi-
tors are added, suggesting a role for PKCs as the C1
domain-containing proteins that regulate FOH sensitivity
(Voziyan et al., 1995; Taylor et al., 2005; Fairn et al.,
2007). Phosphatidylcholine (PC), phosphatidic acid (PA)
and diacylglycerol (DAG) were able to prevent induction
of apoptosis by FOH in mammalian cells (Taylor et al.,
2005). S. cerevisiae FOH-induced growth inhibition could
be effectively prevented by the coaddition of a DAG ana-
logue (Machida et al., 1999). In C. albicans, FOH-growth
defect was also relieved by addition of a DAG analogue,
implicating phophatidylinositol signalling in the delay
(Uppuluri et al., 2007). However, a mutant strain deleted
for PKC1 responded to FOH and DAG analogue similar to
wild-type, suggesting the PKC is not the target of the DAG
analogue. Fairn et al. (2007) provided a more substantial
evidence of the involvement of the PKC1 pathway in FOH
sensitivity. These authors showed that Pkc1 relocalized to
the mitochondria upon FOH addition and inactivation of
the non-essential and non-redundant member of the Pkc1
signalling pathway, BCK1, resulted in FOH sensitivity.
Furthermore, expression of activated alleles of PKC1,
BCK1 and MKK1 increased tolerance to FOH and hydro-
gen peroxide. Savoldi et al. (2008) reported that A. nidu-
lans PkcA calC2
mutation was able to confer FOH
tolerance. In contrast to S. cerevisiae, A. nidulans
PkcA::GFP did not relocalize to the mitochondria upon
exposure of germlings to FOH, but instead the fluores-
cence was completely relocalized to the entire cytoplasm.
Here, we investigate in more detail the role played by A.
nidulans pkcA in FOH tolerance. We demonstrate that
pkcA overexpression during FOH exposure causes
increased cell death. FOH is also able to activate several
markers of endoplasmic reticulum (ER) stress and the
unfolded protein response (UPR). Our results suggest an
intense cross-talk between PkcA, CWI and UPR during
FOH-induced cell death.
Results
Overexpression of pkcA increases FOH sensitivity
We have previously shown that a point mutation (calC2)in
pkcA was able to confer FOH tolerance (Savoldi et al.,
2008). We considered the possibility that PkcA overex-
pression would increase the FOH sensitivity. Thus, we
constructed an alcA::pkcA mutant by replacing the endog-
enous pkcA promoter by the alcA promoter and verified its
survival to FOH when PkcA is overexpressed. The alcA
promoter is repressed by glucose, derepressed by glyc-
erol and induced to high levels by ethanol or
L-threonine
(Flipphi et al., 2002). We were able to select a transfor-
mant that when grown in the presence of glycerol 2% as
single carbon source for 16 h and transferred to glycerol
2% + ethanol 2% for 6 h at 37°C, the pkcA mRNA accu-
mulates about 10-fold (Fig. 1A). When the alcA::pkcA
mutant strain was grown in MM + glycerol 2% + ethanol
2% for 16 h at 37°C, the PkcA activity was about three
times higher than in the wild-type strain (Fig. 1B). As
previously reported by other groups (Ichinomiya et al.,
2007; Ronen et al., 2007), the alcA repression by growing
the alcA::pkcA mutant strain in the presence of 4%
glucose, decreased dramatically the colony diameter
(Fig. 1C), indicating once more that pkcA is an essential
gene. The pkcA overexpression increased the suscepti-
bility to FOH (Table 1 and Fig. 5D). When PkcA was over-
expressed and the alcA::pkcA mutant strain was exposed
to FOH, the PkcA activity increased only about 20% (com-
pared with the alcA::pkcA mutant not exposed to FOH;
Fig. 1B). There are no differences in the survival of both
strains in the absence of FOH.
Along this manuscript, we evaluated A. nidulans cell
death by using three different methods: nuclear conden-
sation, germling viability and 10-fold dilutions of a starting
suspension of conidia. Thus, to verify if the alcA::pkcA
mutant is more sensitive to cell death caused by other
stressing agents, we overexpressed PkcA and exposed
conidia of this strain to several concentrations/dosages of
amphotericin, itraconazole, caspofungin, camptothecin,
hydroxyurea, 4-nitroquinoline oxide, sirolimus, calcium,
menadione, hydrogen peroxid and paraquat. Overexpres-
sion of pkcA increased the survival to caspofungin and
posoconazole but not for Congo red (A.C. Colabardini,
unpublished results). The wild-type and the alcA::pkcA
mutant strains showed comparable levels of susceptibility
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© 2010 Blackwell Publishing Ltd, Molecular Microbiology, 78, 1259–1279
to all these agents (data not shown). The calC2 mutant
that displays increased FOH tolerance also did not show
any growth difference with the wild-type strain, except
when exposed to FOH (see Table 1 and Fig. 5D). We also
observed that both mRNA accumulation and PKC activity
is increased by FOH (Fig. 1B and D).
A Poly-Caspases FLICA apoptosis detection kit showed
the presence of green fluorescence in 80% of the FOH-
exposed wild-type germlings (n = 100), indicating the acti-
vation of intracellular caspases, whereas no fluorescence
was observed in the unexposed germlings (Fig. 1E, upper
panel). Consistently, when the levels of pkcA mRNA are
increased, there is green fluorescence even in the
absence of FOH (Fig. 1E, lower panel). We examined a
possible necrotic role in the FOH-cell death by staining
protoplasts with propidium iodide (PI). When the wild-type
was grown in the presence of glycerol + 100 mM threo-
nine and not exposed to FOH, approximately 10% of the
germlings were labelled with PI (Fig. 1E); when the wild-
type was exposed to FOH 50 mM for 30 min, 30% of the
germlings were labelled with PI (Fig. 1E). The alcA::pkcA
mutant grown in the presence of glycerol + 100 mM threo-
nine and not exposed to FOH showed 15% of the germ-
lings labelled with PI (Fig. 1E); when the alcA::pkcA
mutant was exposed to FOH 50 mM for 30 min, 30% of the
germlings were labelled with PI (Fig. 1E). All the germlings
that showed positive labelling for FLICA also were positive
for PI, and vice versa. We were not able to perform
TUNEL and Annexin assays for the alcA::pkcA mutant
because it is not possible to produce protoplasts from this
mutant even when it is grown in the presence of glycerol
(data not shown). These results indicate that FOH induces
Fig. 1. Overexpression of pkcA increases the susceptibility to FOH.
A. Wild-type and alcA::pkcA strains were grown for 16 h in MM + glycerol 2% at 37°C and transferred for MM + glycerol 2% + ethanol 2% and
grown for further 6 h. The relative quantification of pkcA and tubulin gene expression was determined by a standard curve (i.e. C
T
values
plotted against logarithm of the DNA copy number). The results are the means standard deviation of four sets of experiments.
B. Protein kinase C activity of the wild-type and alcA::pkcA strains exposed or not to FOH 50 mM. The results are the means standard
deviation of three sets of experiments. Statistical differences were determined by one-way analysis of variance (
ANOVA) followed, when
significant, by Newman–Keuls multiple comparison test, using GraphPad Prism statistical software (GraphPad Software, version 3, 2003).
C. Wild-type and alcA::pkcA strains were grown for 72 h at 37°C in either MM + glycerol 2% or MM + glucose 4%.
D. Wild-type strain was grown for 16 h in YG at 37°C and transferred for fresh YG with either 0, 10, 50 or 100 mM FOH and grown for further
2 h. The relative quantification of pkcA and tubulin gene expression was determined by a standard curve (i.e. C
T
values plotted against
logarithm of the DNA copy number). The results are the means standard deviation of four sets of experiments.
E. Activation of intracellular caspases and PI labelling in germlings grown for 16 h in MM + 2% glycerol + 100 mM threonine exposed or not to
FOH, indicated by green or red fluorescence respectively. Bars, 5 mm. This figure is available in colour online at wileyonlinelibrary.com.
Aspergillus nidulans
, farnesol and protein kinase C
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Fig. 1. cont.
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et al
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© 2010 Blackwell Publishing Ltd, Molecular Microbiology, 78, 1259–1279
caspase-like proteases and necrosis in A. nidulans;
however, increased PkcA expression does not enhance
the necrotic process but activates caspase-like proteases.
In addition, increased levels of PkcA expression enhance
the A. nidulans FOH-induced cell death and influence of
PkcA on cell death seems to be specifically related to
FOH.
FOH promotes the UPR
Recent studies have revealed that induction of ER stress
and the subsequent activation of the UPR play a critical
role in the induction of apoptosis by FOH in lung carci-
noma cells (for a review, see Joo and Jetten, 2010).
Based on this evidence, we raised two hypotheses: (i)
FOH is affecting ER stress and activating the UPR in A.
nidulans and (ii) the specific effect of PkcA on FOH-
induced cell death could be due to a synergistic combi-
nation of PkcA activity and FOH on ER stress and
activation of UPR. To start addressing these hypotheses,
we overexpressed PkcA in the presence of agents that
disrupt endoplasmic reticulum homeostasis (ER), such as
2-deoxy-D-glucose (2-DG) and dithiothreitol (DTT). These
two agents induce ER stress by impairing the N-linked
glycosylation that promotes folding of nascent polypeptide
chains in the ER and unfolding proteins directly by reduc-
ing disulphide bonds respectively (Helenius and Aebi,
2004; Back et al., 2005). The overexpression of PkcA
increases the susceptibility of alcA::pkcA mutant strain to
these agents (Fig. 2A and B).
The second hypothesis suggests that A. nidulans PkcA
is influencing the UPR. The accumulation of aberrant
folded proteins in the ER activates the bifunctional trans-
membrane kinase/endoribonuclease Ire1 (Korennykh
et al., 2009). Ire1 excises an intron from yeast HAC1
cytoplasmic precursor mRNA HAC1u (uninduced), remov-
ing it to generate the induced form of the HAC1 mRNA,
HAC1i (induced) (Cox and Walter, 1996). This creates a
frame-shift in the mRNA, allowing for the translation of a
transcription factor that moves to the nucleus and regu-
lates the expression of UPR target genes (Travers et al.,
2000; Kimata et al., 2006). Recently, a homologue of
HAC1 was identified in A. fumigatus (Richie et al., 2009).
The A. fumigatus DhacA mutant was unable to activate
the UPR in response to ER stress and was hypersensitive
to agents that disrupt ER homeostasis or the cell wall.
We identified an A. nidulans HAC1 homologue, hacA
(AN9397.4), that displays about 75% identity and 83%
similarity with A. fumigatus hacA (7e-155) and about 40%
identity and 56% similarity with S. cerevisiae HAC1 (2e-
11). In S. cerevisiae, the Ire1p-mediated splicing of a
252-nucleotide (nt) unconventional intron from HAC1
mRNA relieves the transcript from a translational block
(Kawahara et al., 1997; Ruegsegger et al., 2001). In con-
trast, in mammalian cells, the HAC1 homologue XBP1 is
activated in a similar way by the splicing of a 26 nt uncon-
ventional intron (Lee et al., 2002), whereas Trichoderma
reesei hac1 and A. nidulans, A. niger and A. fumigatus
hacA are activated by the splicing of a 20 nt unconven-
tional intron (Saloheimo et al., 2003; Mulder et al., 2004;
Richie et al., 2009). We developed an RT-PCR assay to
distinguish the increased accumulation of A. nidulans
hacA
i
(Fig. 2C and D). First, we exposed A. nidulans to
1 mM DDT for 60 min and verified a 6.0 and 3.5 times
increase of the mRNA accumulation of hacA
i
in the wild-
type and calC2 mutant strains respectively (Fig. 2D).
Table 1. Non-condensed nuclei (%) and viability (%) of wild-type and mutant strains exposed to different FOH concentrations.
Strains
Non-condensed nuclei (%) Viability (%)
20 mM FOH 50 mM FOH 20 mM FOH 50 mM FOH
Wild-type (A89) 97.5 0.2 31.0 5.6 73.0 2.1 31 3.6
DmpkC 20.5 6.0 10.5 4.9 36.0 3.4 12.0 3.2
DhogA 95.0 0.0 2.5 0.7 11.0 1.2 2.0 0.8
DmpkA 100.0 0.0 79.0 1.7 94.0 6.9 68.5 2.6
Wild-type (BPU1) 99.3 0.1 53.0 3.5 85.0 4.5 53.5 5.4
DatfA 100 0.0 40.3 2.7 71.0 6.8 40.0 4.5
DnapA 99.7 0.1 54.3 4.2 79.0 5.8 54.2 2.9
Wild-type (CLK3) 100.0 0.0 29.3 1.1 71.0 4.9 31.0 2.5
DsrrA 100.0 0.0 34.6 4.5 72.0
5.1 36.2 3.6
DsskA 98.0 2.0 34.0 6.0 64.0 3.0 34.5 4.1
DsrrA DsskA 98.7 0.2 43.7 4.8 81.0 1.5 42.5 6.7
Wild-type (GR5) 98.7 0.2 22.0 5.3 93.0 1.9 61.0 2.9
calC2 99.0 0.0 99.0 0.0 98.0 0.0 97.0 1.0
alcA::pkcA 23.0 0.8 11.7 5.1 65.0 3.7 49.0 4.1
alcA::pkcA DcasA 83.0 0.9 50.0 2.9 90.0 7.3 67.0 1.9
DcasA 99.0 0.1 78.0 1.9 95.0 1.7 71.0 2.5
DcasB 69.0 2.8 7.0 4.0 56.0 4.9 23.0 1.5
DcasA DcasB 92.0 0.1 72.0 1.2 84.0 3.5 58.0
1.6
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© 2010 Blackwell Publishing Ltd, Molecular Microbiology, 78, 1259–1279
When the wild-type strain was exposed to increasing con-
centrations of FOH, there was a hacA
i
mRNA accumula-
tion of about 0.5, 2.5 and 3.5 times at 10, 50 and 100 mM
FOH respectively. In contrast, the calC2 mutant strain did
not show any difference in the hacA
i
mRNA accumulation
when exposed to FOH (Fig. 2E). By overexpressing pkcA
in the absence of FOH, we were able to see a three-fold
increase in the hacA
i
mRNA accumulation (Fig. 2E). The
hacA levels of alcA::pkcA and its corresponding wild-type
are lower than the wild-type exposed or not to FOH
because in the former the strains were grown in MM
supplemented with 2% glycerol plus threonine 100 mM
while the later was grown in complete medium. We also
investigated if FOH led to induction of the UPR as judged
by the transcriptional induction of A. nidulans homologues
of A. niger genes known to be affected by ER stress, bipA,
pdiA and pdiB (Guillemette et al., 2007). These genes
showed diverse degrees of increased mRNA accumula-
tion when A. nidulans was exposed to different FOH
concentrations (A. nidulans homologues AN2062.4,
Fig. 2. hacA
i
mRNA expression is increased
in the presence of FOH.
A. Wild-type and alcA::pkcA strains were
grown for 96 h at 37°C in liquid MM + glycerol
2% + 100 mM threonine in the presence of
DTT 0.5 or 1.0 mM.
B. Wild-type and alcA::pkcA strains were
grown in MM + 2% glycerol + threonine
100 mM in the presence of increasing
concentrations of 2-DG.
C. DNA sequences that border the intron
processing of the A. nidulans hacA gene.
Arrows indicate the processing of the 20 bp
intron from hacA
u
(uninduced) to the hacA
i
(induced) mRNA. PCR amplification of the
hacA and tubC (encoding b-tubulin) cDNAs
when A. nidulans wild-type and calC2 mutant
strains were exposed to DTT (D) and FOH
(E). This figure is available in colour online at
wileyonlinelibrary.com.
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© 2010 Blackwell Publishing Ltd, Molecular Microbiology, 78, 1259–1279
AN7436.4 and AN0248.4; Fig. 3A). FOH also enhances
the mRNA accumulation of other genes involved in the
UPR, such as the transcription factor Hsf1, and the
chaperones Hsp90 and Hsp104 (A. nidulans homologues
AN8035.4, AN8269.4 and AN0858.4 respectively;
Fig. 3B).
Taken together, these data suggest FOH is activating
the UPR and that PkcA plays a role in the activation of this
process in A. nidulans.
Metacaspases are induced by FOH
The caspases are members of a family, or structurally
related group, known as cysteine proteases (for a review
see Mazzoni and Falcone, 2008). A cell death stimulus
activates a cascade of caspases that ultimately cleaves
substrates essential for cell death (Donepudi and Grutter,
2002; Degterev et al., 2003; Lavrik et al., 2005). Richie
et al. (2007) showed that A. fumigatus metacaspases
CasA and CasB play a role under conditions of ER stress.
Thus, we decide to investigate a possible connection
among A. nidulans PkcA, FOH-induced cell death and
metacaspases. In A. nidulans, we have identified two
putative caspase-like proteins, fitting into the type I cat-
egory of metacaspases, and named them casA
(AN2503.3) and casB (AN5712.3). The A. nidulans CasA
and CasB have about 64% identity and 76% similarity
(e-value: 2e-30), and S. cerevisiae Yca1p metacaspase
Fig. 2. cont.
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, farnesol and protein kinase C
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© 2010 Blackwell Publishing Ltd, Molecular Microbiology, 78, 1259–1279
has great similarity to both CasA (57% identity, 70% simi-
larity, e-value: 8e-21) and CasB (58% identity, 71% simi-
larity, e-value: 1e-101). When the wild-type strain is
exposed to FOH, there is an increased accumulation of
casA mRNA of 27.0 and 137.5 times, and casB mRNA of
2.2, 8.0 and times at 10 and 50 mm FOH respectively
(Fig. 4A). PkcA overexpression increases the level of
mRNA accumulation of both casA and casB mRNA by 2,
and 4.5 times, respectively, even in the absence of FOH
(Fig. 4B). To gain more insight on A. nidulans meta-
caspase function, we constructed deletion strains for both
casA and casB genes; we also constructed a double
mutant strain by crossing both DcasA and DcasB deletion
mutants. None of these mutants displayed either growth
reduction or any other apparent phenotype when com-
pared with the corresponding wild-type strain.
We also investigated if necrosis and other caspase-like
proteases would be present upon FOH-cell death of the
metacaspase mutants by staining with PI and FLICA.
When the wild-type and DcasA mutant were grown in
complete medium and not exposed to FOH, approxi-
mately 5% and 0% of the germlings showed PI and FLICA
labelling respectively (Fig. S1). Germlings of the DcasB
and DcasA DcasB mutants not exposed to FOH showed
about 13–15% PI and 15% FLICA labelling (Fig. S1).
Upon exposure to FOH 50 mM for 30 min, the wild-type
showed germlings with 15% PI and 15% FLICA labelling
(Fig. S1). The DcasB and DcasA DcasB mutants exposed
to FOH displayed about 70% PI and 100% FLICA labelling
(Fig. S1). Interestingly, when the DcasA mutant was
exposed to 50 mM FOH for 30 min, it showed germlings
with 50% PI and 60% FLICA labelling (Fig. S1). We have
not observed any PI minus FLICA positive cells. Curiously,
these results suggest that the activation of necrosis and
caspase-like proteases by FOH in A. nidulans is depen-
dent to metacaspases since the absence of at least one of
the metascaspase genes increases PI and FLICA
labelling. However, casB seems to play a more important
role in this activation because in its absence the levels of
activation are even higher.
We investigated in the detail the viability of germlings of
A. nidulans DcasA, DcasB and DcasA DcasB mutants
comparatively to the wild-type strain. To verify if these
mutants are more sensitive to cell death caused by other
oxidative stressing agents, we exposed conidia of these
strains to several concentrations/dosages of the agents
mentioned earlier in the section Overexpression of pkcA
increases FOH sensitivity. The wild-type and the DcasA-B
mutant strains showed comparable levels of susceptibility
to all these agents (data not shown). However, when the
mutant strains were exposed to ER disrupting agents,
such as 2-DG and DTT, they showed differential suscep-
tibility (Fig. 5A and B). The DcasA and DcasA D
casB
strains were as resistant to DTT as the wild-type strain
Fig. 3. Several genes involved in the unfolded protein response
have increased mRNA accumulation when A. nidulans is exposed
to FOH. Wild-type strain was grown for 16 h in YG at 37°C and
transferred for fresh YG with either 0, 10, 50 or 100 mM FOH and
grown for further 2 h. The relative quantification of (A) bipA
(AN2062.4), pdiA (AN7436.4) and pdiB (AN0248.4), and (B) hsf1
(AN8035.4), hsp90 (AN8269.4) and hsp104 (AN0858.4) and tubulin
gene expression was determined by a standard curve (i.e. C
T
values plotted against logarithm of the DNA copy number). The
results are the means standard deviation of four sets of
experiments.
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while DcasB was more sensitive than the wild-type strain
(Fig. 5A). In contrast, DcasA was as resistant to 2-DG as
the wild-type while DcasB and DcasA DcasB were more
sensitive than the wild-type strain (Fig. 5B). Exposing the
germ tubes to 20 or 50 mm FOH for 2 h revealed that
DcasA was significantly more tolerant to FOH than the
wild-type, while DcasB and DcasA DcasB mutant strains
were less tolerant to FOH than the wild-type and DcasA
mutant strain (Table 1 and Fig. 5C). We constructed by
sexual crosses a double mutant alcA::pkcA DcasA and
evaluated its sensitivity to FOH. We overexpressed the
pkcA gene in this strain and compared its tolerance to
FOH with the alcA::pkcA and DcasA mutant strains
(Table 1 and Fig. 5D). The absence of casA confers
increased tolerance to FOH when pkcA is overexpressed
(Table 1 and Fig. 5D). The complemented metacaspase
mutants showed the same phenotypes of the wild-type
strain (Table 1; Figs S2 and S4).
The results demonstrated FOH-induced upregulation of
both casA-B genes and that FOH-induced cell death may
be dependent on metacaspases. Furthermore, the over-
expression of pkcA increases both mRNA accumulation
and metacaspases activity, and there is a genetic interac-
tion between PkcA and CasA.
FOH susceptibility is mediated via PKC/MAPK
Mitochondria and ROS participate in FOH-induced apo-
ptosis in A. nidulans (Semighini et al., 2006; Savoldi
et al., 2008). Two-component systems (Histidine-to-
Aspartate phosphorelay) are signal transduction mecha-
nisms involved in several stimuli, including oxidative
stress responses (Wuichet et al., 2010; Bahn, 2008). A.
nidulans has a histidine kinase (NikA) and two response
regulators (SskA and SrrA) implicated in oxidative stress
responses (Hagiwara et al., 2007; Vargas-Pérez et al.,
2007). In A. nidulans, the HogA/MAPK kinase cascade
plays important roles downstream of the NikA-SskA
phosphorelay (Moye-Rowley, 2003; Furukawa et al.,
2005; Ikner and Shiozaki, 2005). In this species, it is
Fig. 4. Poly-Caspases FLICA showed the
presence of increased green fluorescence
when PkcA mRNA expression is increased.
A. Wild-type strain was grown for 16 h in YG
at 37°C and transferred for fresh YG with
either 0, 10, 50 or 100 mM FOH and grown for
further 2 h. The relative quantification of
casA-B and tubulin gene expression was
determined by a standard curve (i.e. C
T
values plotted against logarithm of the DNA
copy number). The results are the
means standard deviation of four sets of
experiments.
B. Wild-type and alcA::pkcA strains were
grown for 16 h in MM + glycerol 2% at 37°C
and transferred for MM + glycerol
2% + threonine 100 mM and grown for further
6 h. The relative quantification of casA-B and
tubulin gene expression was determined by a
standard curve (i.e. C
T
values plotted
against logarithm of the DNA copy number).
The results are the means standard
deviation of four sets of experiments.
Aspergillus nidulans
, farnesol and protein kinase C
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Fig. 5. for cont.
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known that NapA and AtfA are transcription factors acti-
vated by the MAPK cascade that are involved in stress
responsive transcriptional regulation to oxidative stress
(Asano et al., 2007; Hagiwara et al., 2008; Balázs et al.,
2010). Thus, we investigated the involvement of SskA-
SrrA, AtfA
YAP1
(the YAP1 homologue), and NapA signal
transduction pathway on FOH tolerance by verifying
their viability after exposure to FOH (Table 1 and Fig. 6).
Fig. 5. A. nidulans metacaspases CasA and CasB are involved in FOH-induced cell death.
A and B. (A) Wild-type, DcasA, DcasB, DcasA DcasB mutant strains were grown for 96 h at 37°C in YG medium in the presence of DTT 0.5 or
1.0 mM, or (B) MM + 2-DG 0.3 or 0.5 mM.
C and D. Aliquots (5 ml) of 10-fold dilutions derived from a starting suspension of 1.0 ¥ 10
8
conidia ml
-1
of the indicated strain were spotted on
YG-agar plates supplemented with the 0, 0.5 or 1.0 mM FOH. The plates were incubated at 37°C for 48 h.
Fig. 6. FOH-susceptibility of mutants from the MAP kinase pathway. Aliquots (5 ml) of 10-fold dilutions derived from a starting suspension of
1.0 ¥ 10
8
conidia ml
-1
of the corresponding wild-type, DsskA, DsrrA, DsskA DsrrA (A), DnapA and DatfA (B), and DmpkA, DmpkC and DhogA
(C) strains were spotted on YG-agar plates supplemented with the 0, 0.5 or 1.0 mM FOH. The plates were incubated at 37°C for 48 h.
Aspergillus nidulans
, farnesol and protein kinase C
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When exposed to lower concentrations of FOH (20 and
50 mM), the ssrA, sskA, ssrA sskA, napA and atf deletion
mutants are as sensitive to FOH as the wild-type strain
(Table 1). However, when exposed to higher concentra-
tions of FOH (0.5 and 1.0 mM), the ssrA, sskA, ssrA
sskA and atf deletion mutants are more sensitive to FOH
than the wild-type strain (Fig. 6A and B). There are four
MAPK kinases in A. nidulans: MpkA-C and SakA/HogA
(for a review see May, 2008). We evaluate the FOH sen-
sitivity of deletion mutants for three of these kinases,
MpkA, MpkC and HogA (Table 1 and Fig. 6C). Interest-
ingly, the DmpkA mutant shows increased tolerance
while DmpkC and DhogA showed increased sensitivity
to FOH (Table 1 and Fig. 6C). These results suggest
SsrA, SskA, AtfA and MAP kinases are involved in the
signal transduction in response to the effects caused by
FOH.
FOH activates the CWI pathway
In S. cerevisiae PKC1 is involved in the regulation of cell
construction via the reactivation of MPK1 and activation
of the transcription factor Rlm1p, which regulates the
expression of genes whose products are important in
cell wall biosynthesis (Jung and Levin, 1999). Fujioka
et al. (2007) demonstrated that A. nidulans rlmA and
mpkA are functional orthologues of S. cerevisiae RLM1
and MPK1. Considering that DmpkA mutant is more
resistant to FOH, we speculated if FOH is activating the
CWI pathway. Therefore, we tested the mRNA accumu-
lation of agsA (AN5885.3; encoding one of the A. nidu-
lans a-1,3-glucan synthases), rlmA (AN2984.3) and
mpkA (AN5666.3) genes when A. nidulans is exposed to
different concentrations of FOH (Fig. 7A). All three
genes showed increased mRNA accumulation upon A.
nidulans exposure to different FOH concentrations. We
also tested if the mRNA levels of these genes could be
controlled directly or indirectly by PkcA by overexpress-
ing PkcA even in the absence of FOH. Again all three
genes showed increased mRNA accumulation when A.
nidulans PkcA was overexpressed (Fig. 7B). Previously,
it was shown that cell wall stress results in the phos-
phorylation of A. nidulans MpkA (Fujioka et al., 2007).
We checked if FOH can activate the MpkA phosphory-
lation by exposing A. nidulans cultures to different FOH
concentrations (Fig. 7C). Addition of Congo red induced
phosphorylation of A. nidulans MpkA (Fig. 7C, first row).
Interestingly, increasing FOH concentrations also
induced A. nidulans MpkA phosphorylation (Fig. 7C,
second row). MpkA was also phosphorylated when A.
nidulans was exposed to DTT (Fig. 7C, third row). These
results strongly indicate that FOH is able to activate the
CWI pathway and that there is a possible cross-talk
between CWI and UPR pathways.
Discussion
In mammalian cells, the PKC family consists of 10 related
serine/threonine kinases, which are important controllers
of cell proliferation, survival and cell death (Reyland,
2009). The PKC1 gene was originally identified in S. cer-
evisiae (Levin, 2005) and is a close homologue of the
human PKC gene. In A. nidulans, the PKC/MAPK pathway
is involved in the CWI, polarized growth and morphogen-
esis (Ronen et al., 2007; Teepe et al., 2007), utilization of
polyalcohol sugars (Reyes et al., 2006), osmotic and oxi-
dative stresses (Davenport et al., 1995; Han and Prade,
2002; Kawasaki et al., 2002; Furukawa et al., 2005),
sexual development and spore viability (Kawasaki et al.,
2002; Ichinomiya et al., 2007), and penicillin production
(Herrmann et al., 2006). Here, we showed a completely
novel feature of the A. nidulans PKC/MAPK pathway
related to FOH and the UPR. Interestingly, Binder et al.
(2009) have recently reported that the antifungal protein
PAF also interferes with PKC/MAPK pathway.
The PKC/MAPK pathway is required for transducing the
response to FOH in mammalian cells, S. cerevisiae, C.
albicans and A. nidulans (Voziyan et al., 1995; Yang and
Kazanietz, 2003; Taylor et al., 2005; Fairn et al., 2007;
Uppuluri et al., 2007; Savoldi et al., 2008; Román et al.,
2009). However, it is not very clear how PKC/MAPK could
mediate this response. We observed that a mutation in the
pkcA (calC2), which confers sensitivity to calcofluor and
SDS, conferred FOH tolerance. By investigating a condi-
tional mutant of pkcA and other MAP kinase mutants, we
identified a relationship between the CWI, UPR and FOH.
Actually, there are several pieces of evidence in the litera-
ture showing a connection among these pathways. Mam-
malian PKC q is required for autophagy in response to
stress in the ER (Sakaki and Kaufman, 2008; Sakaki
et al., 2008), while PKC d communicates ER stress to the
mitochondria (Qi and Mochly-Rosen, 2008). In S. cerevi-
siae, ER stress has been shown to trigger signalling
through the CWI/MAPK signalling cascade (Bonilla and
Cunningham, 2003; Chen et al., 2005; Nita-Lazar and
Lennarz, 2005; Pal et al., 2007; Wright et al., 2007;
Shaner et al., 2008). More recently, strong evidences con-
solidated the hypothesis that cellular responses to ER and
cell wall stress are co-ordinated to buffer the cell against
these two unrelated cellular stresses (Krysan, 2009;
Scrimale et al., 2009). Another interesting connection
between ER stress and MAP kinases was shown by Bick-
nell et al. (2010). These authors demonstrated that S.
cerevisiae
Hog1 MAP kinase becomes phosphorylated
during the late stage of ER stress. S. cerevisiae Hog1
co-ordinates a multifaceted response to persistent ER
stress activating autophagy by enhancing the stability of
Atg8, a critical autophagy protein. Furthermore, the A.
fumigatus DhacA mutant was unable to activate the UPR
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in response to ER stress and was hypersensitive to
agents that disrupt ER homeostasis or the cell wall (Richie
et al., 2009). Mutations of HAC1 homologue in Candida
albicans also lead to increased susceptibility to the clini-
cally used, cell wall-directed antifungal caspofungin
(Wimalasena et al., 2008). Our results strongly suggest
that FOH is inducing the UPR in A. nidulans and this
response is mediated via PKC/MAPK pathway.
FOH-induced apoptosis in human lung carcinoma cells
analysed by microarray analysis revealed that many genes
implicated in ER stress were highly upregulated, suggest-
ing that FOH-induced apoptosis in these cells is coupled to
Fig. 7. FOH activates the cell wall integrity (CWI) pathway.
A. Wild-type strain was grown for 16 h in YG at 37°C and transferred for fresh YG with either 0, 10, 50 or 100 mM FOH and grown for further
2 h. The relative quantification of agsA, rlmA and mpkA and tubulin gene expression was determined by a standard curve (i.e. C
T
values
plotted against logarithm of the DNA copy number). The results are the means standard deviation of four sets of experiments.
B. Wild-type and alcA::pkcA strains were grown for 16 h in MM + glycerol 2% at 37°C and transferred for either MM + glucose 4% (G), or
MM + glycerol 2% (Gly), or MM + glycerol 2% + ethanol 2% (GE) and grown for further 6 h. The relative quantification of agsA, rlmA and mpkA
and tubulin gene expression was determined by a standard curve (i.e. C
T
values plotted against logarithm of the DNA copy number). The
results are the means standard deviation of four sets of experiments.
C. FOH activates the CWI pathway. A. nidulans overnight cultures grown at 37°C were exposed to the indicated concentrations of the
indicated agents. Protein extracts were analysed by Western blotting using antibodies directed against phosphorylated MpkA or b-tubulin
(TubC) for loading control.
Aspergillus nidulans
, farnesol and protein kinase C
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Fig. 7. cont.
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the activation of an ER stress response pathway and the
UPR (Joo et al., 2007). The precise mechanism how FOH
induces ER stress has not been elucidated yet (Joo and
Jetten, 2010). Actually, the UPR is not the single target for
FOH either in mammalian cells or fungi (Miquel et al., 1996;
Semighini et al., 2006; Savoldi et al., 2008; Langford et al.,
2009; Joo and Jetten, 2010). In mammalian cells, these
include regulation of 3-hydroxy-3-methylglutaryl-CoA
reductase and CTP:phosphocholine cytidylyltransferase
alpha, rate-limiting enzymes in the mevalonate pathway
and phosphatidylcholine biosynthesis, respectively, the
generation of ROS, and the activation of the NF-kappaB
signalling pathway and a number of NF-kappaB target
genes (Joo and Jetten, 2010). Previously, we investigate
which pathways are influenced through FOH by examining
the transcriptional profile of A. nidulans exposed to this
isoprenoid (Savoldi et al., 2008). We observed decreased
mRNA abundance of several genes involved in RNA pro-
cessing and modification, transcription, translation, riboso-
mal structure and biogenesis, amino acid transport
and metabolism, and ergosterol biosynthesis. We also
observed increased mRNA expression of genes encoding
a number of mitochondrial proteins and characterized in
detail one of them, the aifA, encoding the apoptosis-
inducing factor (AIF)-like mitochondrial oxidoreductase.
However, surprisingly we have not observed any gene
in the CWI and UPR with increased mRNA accumulation,
except for chaperone-encoding genes (Savoldi et al.,
2008). This could be due to the low resolution of
the microarray slides used for these experiments (A. nidu-
lans oligonucleotide slides version 1 for microarray
hybridizations; for details see http://pfgrc.jcvi.org/index.
php/microarray/array_description/aspergillus_nidulans/
version1.html).
Interestingly, A. nidulans PkcA overexpression acti-
vates the UPR in the absence of FOH. This suggests that
PkcA activity already induces some major stresses on the
cells independently of FOH. FOH exposure has an addi-
tive lethal effect what strongly indicates there is a connec-
tion among PkcA, CWI and UPR in A. nidulans.Itiswell
known that FOH increases ROS production in A. nidulans
(Semighini et al., 2006; Savoldi et al., 2008; Dinamarco
et al., 2010). We have not observed increased ROS sus-
ceptibility when PkcA is overexpressed (data not shown).
However, pkcA mRNA accumulation is increased in the
presence of oxidative stress (Fig. S3). Gerik et al. (2008)
observed that besides its role in the activation of CWI,
Cryptococcus neoformans PKC1 is essential for defence
against both oxidative and nitrosative stresses. Vilella
et al. (2005) also showed the involvement of Pkc1 in
oxidative stress but could not detect a role for this protein
in the nitrosative stress response. Thus it is possible that
the connection among PkcA and UPR could be mediated
through ROS formation upon FOH exposure, by not only
activating PkcA activity but also the UPR. The environ-
ment of the ER is oxidizing, supporting the formation of
intra- and interchain disulphide bonds that serve to stabi-
lize the folding and assembly of nascent proteins (Shimizu
and Hendershot, 2009). Each disulphide bond that forms
during oxidative folding should produce a single ROS
(Shimizu and Hendershot, 2009). Increased ROS produc-
tion through FOH will create an intense oxidized environ-
ment in the ER with increased production of mis-folded
proteins, activating the UPR.
Recently, Dichtl et al. (2010) provided evidence that
FOH is able to interfere with A. fumigatus CWI signalling.
These authors showed that FOH inhibits the activation of
the CWI pathway and weakens the cell wall, leading to the
exit of AfRho1 and AfRho3 from the hyphal tip. They also
observed a displacement of the actin-binding protein tro-
pomyosin from the apical tip. Furthermore, they showed
that Dmkk2 and DmpkA mutants were highly susceptible
to FOH. We have also seen that A. nidulans DmpkC and
DhogA mutants are FOH-sensitive while DmpkA is resis-
tant to FOH. These results suggest a different mechanism
of regulation for FOH-cell death induced in A. fumigatus
and A. nidulans. However, in both organisms the signal
transduction for FOH-cell death is mediated via MAP
kinases. The A. nidulans YAP1 homologue, DatfA is also
more susceptible to FOH, suggesting that the FOH
response could be mediated through PKC/MAPK/YAP1
pathway. There is also MpkA phosphorylation and
increased mRNA accumulation of A. nidulans genes
involved in the CWI pathway, such as agsA, rlmA and
mpkA, both upon FOH exposure and PkcA
overexpression. However, we have not addressed here
more specifically how FOH is activating the A. nidulans
CWI pathway.
Caspases are members of a family known as cysteine
proteases that are actively involved in cell death in
eukaryotes (for a review see Li and Yuan, 2008). Although
S. cerevisiae does not have caspases in its genome, a
caspase-like protein named YCA1, fitting into the type I
category of metacaspases, was identified and character-
ized (Uren et al., 2000; Madeo et al., 2002; Mazzoni and
Falcone, 2008). Previously, Richie et al. (2007) have iden-
tified A. fumigatus metacaspases CasA and CasB. These
authors have not observed any difference in sensitivity to
a range of pro-apoptotic stimuli, but the double casA casB
deletion mutant showed a growth disadvantage in the
presence of agents that disrupt ER, such as 2-DG and
DTT. ER stress caused by an increase of unfolded or
misfolded proteins activates apoptotic pathways (Momoi,
2004; Rasheva and Domingos, 2009). A subset of
caspases, some of them located in the ER, are activated
during ER stress (Momoi, 2004; Rasheva and Domingos,
2009). These results suggested a protective role for A.
fumigatus metacaspases during ER stress rather than a
Aspergillus nidulans
, farnesol and protein kinase C
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© 2010 Blackwell Publishing Ltd, Molecular Microbiology, 78, 1259–1279
death-inducing function (Richie et al., 2007). Accordingly,
we have also not observed any growth differences among
the A. nidulans wild-type, single and double casA-B dele-
tion mutants for several apoptotic stimuli, except for FOH
and ER-disrupting agents. Curiously, the observed effects
in the presence of these agents for DcasA and DcasB
were absolutely contrasting, i.e. DcasA mutation confers
increased survival to them while DcasB mutation
decreased its viability. These results suggest A. nidulans
CasA and CasB have different functions. The decreased
susceptibility of A. nidulans DcasA mutant to ER-
disrupting agents and FOH is not due to a protective role
but rather to an active cell death role for CasA. Thus, it is
tempting to speculate that its paralogue, CasB, would
keep a function as a protecting agent against ER stress
while CasA could have evolved a different function as a
cell death effector. Our findings reveal that FOH induces a
caspase-mediated process of apoptosis in A. nidulans.
The same caspase dependency of FOH-induced cell
death was recently observed for C. albicans (Shirtliff
et al., 2009). An interesting and novel aspect of our work
was to show a genetic interaction between PkcA and
metacaspases.
Experimental procedures
Strains, media and culture methods
Aspergillus nidulans strains used are described in Table S1.
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 supplemented with
1.2gl
-1
(each) of uracil and uridine], and liquid YG or
YG + UU medium with the same composition (but without
agar). The minimal media were a modified minimal medium
(MM; 1% w/v glucose, original high-nitrate salts, trace ele-
ments, 2% w/v agar, pH 6.5). Expression of pkcA gene under
the control of alcA promoter was regulated by carbon source:
repression on glucose 4% w/v, derepression on glycerol, and
induction on ethanol or threonine. Therefore, MM + Glycerol
and MM + Ethanol were identical to MM, except that glycerol
(2% v/v) and/or ethanol (2% v/v) or threonine (100 mM) were
used, respectively, in place of glucose as the sole carbon
source. Trace elements, vitamins, and nitrate salts were
included as described by Kafer (1977). Strains were grown at
37°C unless indicated otherwise.
For the FOH viability assay (Noventa-Jordão et al., 1999),
1 ¥ 10
6
conidia ml
-1
were incubated in YG + UU for 5 h at
30°C in a reciprocal shaker (250 r.p.m.). After this period,
FOH 50 mM was added to the germlings and they were
allowed to grow for additional 120 min, respectively, at the
same conditions. Conidia were conveniently diluted and
plated in YAG + UU plates. Plates were incubated at 37°C for
48 h. Viability was determined as the percentage of colonies
on treated plates compared with untreated controls. For the
viability assays using 10-fold dilutions of a starting suspen-
sion of conidia, conidia were collected from 5-day-old cul-
tures grown on YAG or YUU agar plates. The amount of
conidia was calculated using a counting chamber. We spotted
10
8
,10
7
,10
6
and 10
5
conidia in a volume of 5 ml on YUU agar
plates that were incubated at 37°C for 48 h.
In vivo detection of caspase activation
Activated caspases in A. nidulans germlings were detected
microscopically using a Poly-Caspases FLICA apoptosis
detection kit (Immunochemistry Technologies, LLC) accord-
ing to the manufacturer’s recommendation. Briefly, 16-hour-
old A. nidulans germlings were exposed to FOH 50 mM for 0
and 30 min at 30°C. Germlings with intracellular active
caspases fluoresce green, whereas non-apoptotic cells
appear unstained. Thirty microlitres of FLICA reagent was
dropped on coverslips with adherent germlings and incu-
bated at 37°C for 1 h in the dark. Then, they were briefly
rinsed with PBS buffer (140 mM NaCl, 2 mM KCl, 10 mM
NaHPO
4
, 1.8 mM KH
2
PO
4
, pH 7.4), and the slides were
treated with a 10 mg ml
-1
solution of RNase A for 1 h at 37°C
and stained with a solution containing 1.25 mgofPIper
millilitre. For the positive control with PI, coverslips containing
germlings were fixed for 3 min at room temperature in a
solution containing 3.7% formaldehyde, 0.2% Triton X-100,
and 50 mM phosphate buffer, pH 7 (Bhabhra et al., 2004).
The slides were visualized with an epifluorescence micro-
scope, using a Carls Zeiss (Germany) microscope using
100¥ magnification oil immersion objective lens (EC Plan-
Neofluar, NA 1.3) equipped with a 100W HBO mercury lamp
epifluorescence module. Phase contrast for the brightfield
images and fluorescent images were captured with a
AxioCan camera (Carl Zeiss), processed using the AxioVision
software version 3.1 and saved as tiff files. Further process-
ing was performed using Adobe Photoshop 7.0 (Adobe
Systems Incorporated, CA, USA).
Molecular techniques
Standard genetic techniques for A. nidulans were used for all
strain constructions and transformations (Kafer et al., 1977).
DNA manipulations were according to Sambrook and Russell
(2001). All PCR reactions were performed using Platinum Taq
DNA Polimerase High Fidelity (Invitrogen).
For the DNA-mediated transformation, the deletion cas-
settes were constructed by in vivo recombination in S. cer-
evisiae as previously described by Colot et al. (2006). Briefly,
about 1.5 kb regions on either side of the ORFs were
selected for primer design. For every construction, the
primers were named as 5F and 5R in that de 5F primer
contains a short homologue sequence to the MCS of the
plasmid pRS426. Both primers were used to amplify the
5-UTR flanking region of the targeted ORF. Likewise, the
primers 3F and 3R were used to amplify the 3-UTR ORF
flanking region and the 3R primer also contains a short homo-
logue sequence to the MCS of the plasmid pRS426 (small
letters indicated in Table S2). Both fragments 5- and 3-UTR
were PCR-amplified from genomic DNA using as templates
the A4 strain for A. nidulans cassettes. The pyrG used in the
A. nidulans cassettes for generating the strain DcasB was
used as marker for auxotrophy and was amplified from
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pCDA21 plasmid (Chaveroche et al., 2000), and pyroA for
DcasA was amplified from A. fumigatus Af293 (for details see
Fig. S4). For the the construction of alcA::pkcA, 1000 bp of
the pkcA encoding region was cloned downstream to the alcA
promoter into the pMCB17apx vector. This construction was
further transformed in A. nidulans to replace the endogenous
pkcA promoter yielding the strain alcA::pkcA.
Deletion cassettes generation was achieved by transform-
ing each fragment for each construction along with the
plasmid pRS426 BamHI/EcoRI cut in the in S. cerevisiae
strain SC94721 by the lithium acetate method (Schiestl and
Gietz, 1989). The DNA of the yeast transformants was
extracted by the method described by Goldman et al. (2003),
dialysed and transformed by electroporation in Escherichia
coli strain DH10B to rescue the pRS426 plasmid harbouring
the cassettes. The cassettes were PCR-amplified from these
plasmids and used for transformation of A. nidulans accord-
ing to the procedure of Osmani et al. (1987). Transformants
were scored for their ability to grow on minimal medium. PCR
or Southern blot analyses were used throughout of the manu-
script to demonstrate that the transformation cassettes had
integrated homologously at the targeted A. nidulans loci
(Fig. S4).
RNA isolation, real-time PCR reactions and UPR
PCR assay
For total RNA isolation, the germlings were disrupted by
grinding in liquid nitrogen with pestle and mortar and total
RNA was extracted with Trizol reagent (Invitrogen, USA). Ten
micrograms of RNA from each treatment was then fraction-
ated in 2.2 M formaldehyde, 1.2% w/v agarose gel, stained
with ethidium bromide, and then visualized with UV light. The
presence of intact 25S and 18S ribosomal RNA bands was
used as a criterion to assess the integrity of the RNA. RNase-
free DNase I treatment for the real-time PCR experiments
was carried out as previously described (Semighini et al.,
2002).
All the PCR reactions were performed using an ABI 7500
Fast Real-Time PCR System (Applied Biosystems, USA) and
Taq-Man Universal PCR Master Mix kit (Applied Biosystems,
USA). The reactions and calculations were performed
according to Semighini et al. (2002). The primers and Lux
fluorescent probes (Invitrogen, USA) used in this work are
described in Table S2.
The hacA mRNA splicing was assayed using a modification
of a reported protocol (Bicknell et al., 2007). Overnight cul-
tures were grown and treated with FOH 0, 10, 50 or 100 mM
for 2 h or DTT 1 mM for 60 min. The cells were frozen and
harvested, and total RNA was isolated using Trizol. Twenty
micrograms of total RNA was treated with DNase, purified
using a RNA easy kit (Qiagen), and cDNA was generated
using the SuperScript III First Strand Synthesis system (Invit-
rogen) and oligo(dT) primers according to the manufacturer’s
protocol. The hacA uninduced (hac
u
100 bp long) and hacA
induced (hac
i
80 bp long) cDNAs were amplified by PCR
using the primers hacAF and hacAR (described in Table S2),
with the following PCR conditions: 94°C, 1 min; 25 cycles of
94°C, 30 s; 60°C, 30 s; 72°C, 30 s; 72°C, 10 min. Amplicons
were analysed by electrophoresis on 5% polyacrilamid plus
0.5% agarose gels. The cDNA loading was normalized by
tubC (encoding b-tubulin) PCR amplification under the same
conditions by using primers tubC 918F and tubC 1075R
(Table S2). The images generated were subjected to densi-
tometric analysis of pixel intensity using the ImageJ software
(http://rsbweb.nih.gov/ij/index.html).
PKC assay and Western blotting analysis
Protein assays were performed by initially growing conidia
from the wild-type and mutant strains in a reciprocal shaker at
37°C for 16 h in liquid MM 2% glycerol medium supple-
mented with threonine 100 mM. Each sample was harvested
by filtration through a Whatman filter number 1, washed thor-
oughly with sterile water, quickly frozen in liquid nitrogen,
and disrupted by grinding. Total protein was extracted at 4°C
with extraction buffer [25 mM Tris-HCl (pH 7.4), 0.5 mM
EDTA, 0.5 mM EGTA, 0.05% Triton X-100, 10 mM b-
mercaptoethanol, 1 mgml
-1
leupeptin, 1 mgml
-1
aprotinin,
0.5 mM of PMSF]. The lysate was centrifuged for 30 min at
4°C, 14 000 g in a microcentrifuge. The supernatant was
chromatographed on a 1 ml DEAE-cellulose anion-exchange
chromatography column that has been pre-equilibrated in
extraction buffer. The column was washed with 5 ml of extrac-
tion buffer, and the PKC-containing fraction was eluted using
1 ml of extraction buffer containing 200 mM NaCl. Protein
concentration was determined using a modified Bradford
assay (Bio-Rad, Hercules, CA, USA). The enzymatic assay
was performed according to SignaTECT PKC Assay System
protocol (Promega).
Conidia (5 ¥ 10
7
ml
-1
) were grown in appropriated liquid
medium using a reciprocal shaker at 37°C for 16 h, treated
with FOH 0, 10, 50 or 100 mM for 2 h, or DTT 1 mM for 0, 15,
30, or 60 min, or Congo red 1 mM for 0, 20 or 60 min, har-
vested, frozen and disrupted by grinding. Protein extracts
were obtained using HB buffer [25 mM Tris-HCl (pH 7.4),
0.5 mM EDTA, 0.5 mM EGTA, 0.05% Triton X-100, 10 mM
b-mercaptoethanol and mini-complete protease inhibitors
(Roche Diagnostics, Indianapolis, IN, USA)] at 4°C, and cen-
trifuged for 30 min at 4°C, 14 000 g in a microcentrifuge
Extracts were normalized by protein concentration (Bradford
assay; Bio-Rad), fractionated by SDS-PAGE electrophoresis
(12% gel), and transferred to nitrocellulose (Hybond C
+
)
membranes.
The MpkA phosphorylation was assessed by using anti-
phosphop44/42 MAPK (Erk1/2) (Thr202/Tyr204) antibodies
(1:1500 dilution, Cell Signalling Technology, USA). Primary
antibodies were detected by using a rabbit peroxidase
(HRP)-conjugated second antibody (Kirkegaard and Perry
laboratories), 1:5000 diluted in TBS-T for 1 h at room tem-
perature shaking, and ECL reagents (GE) for chemiolumi-
nescent detection. Protein concentrations were normalized
by using Mouse monoclonal (TU-06) antibody to b-tubulin
(Abcam), diluted 1:5000 em TBS-T plus skimmed milk
powder 5%, incubated with agitation for 16 h at 4°C. Beta-
tubulin was detected by using a mouse peroxidase (HRP)-
conjugated second antibody (Pierce), 1:3000 diluted in
TBS-T, incubated with agitation for 1 h at room tem-
perature, and Super signal West Pico chemiolumine-
scent substrate (Pierce) was used for chemioluminescent
detection.
Aspergillus nidulans
, farnesol and protein kinase C
1275
© 2010 Blackwell Publishing Ltd, Molecular Microbiology, 78, 1259–1279
Acknowledgements
This research was supported by the Fundação de Amparo à
Pesquisa do Estado de São Paulo (FAPESP), Conselho
Nacional de Desenvolvimento Científico e Tecnológico
(CNPq), Brazil, John Simon Guggenheim Memorial Founda-
tion, USA. We also thank Drs Jesus Aguirre, K. Abe, and T.
Mizuno for supplying us with A. nidulans mutant strains, Dr
Robert Cramer Jr. for critical reading of the manuscript, and
the four anonymous reviewers for their suggestions.
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    • "The role MsbA performs in the induction of the cell wall integrity and filamentous growth MAPK cascades was assessed via culturing the parental, ΔmsbA_1 and activated ΔMHD_1 strains in liquid minimal media and then exposing them to Congo Red or transferring the submerged mycelia from liquid cultures to solid minimal media agar plates, conditions thought to induce the CWI and FG pathways respectively (Colabardini et al., 2010; Perez-Nadales and Di Pietro, 2011). Phospho-specific and non-specific antibodies for the respective MAPKs (p44/42) were used to determine the extent of MAPK activation (Fig. 6). "
    [Show abstract] [Hide abstract] ABSTRACT: In the heterogeneous semi-solid environment naturally occupied by lignocellulolytic fungi the majority of nutrients are locked away as insoluble plant biomass. Hence, lignocellulolytic fungi must actively search for, and attach to, a desirable source of nutrients. During growth on lignocellulose a period of carbon deprivation provokes carbon catabolite derepression and scavenging hydrolase secretion. Subsequently, starvation and/ or contact sensing was hypothesised to play a role in lignocellulose attachment and degradation. In Aspergillus nidulans the extracellular signalling mucin, MsbA, influences growth under nutrient-poor conditions including lignocellulose. Cellulase secretion and activity was affected by MsbA via a mechanism that was independent of cellulase transcription. MsbA modulated both the cell wall integrity and filamentous growth MAPK pathways influencing adhesion, biofilm formation and secretion. The constitutive activation of MsbA subsequently enhanced cellulase activity by increasing the secretion of the cellobiohydrolase, CbhA, while improved substrate attachment and may contribute to an enhanced starvation response. Starvation/ contact sensing therefore represents a new dimension to the already multifaceted regulation of cellulase activity.
    Article · Oct 2014
    • "The experiment was normalized by counting the number of cells in a Neubauer chamber.Table 2. A. nidulans strains used in this work. Strain Genotype Reference TNO2A3 (wild-type) pyroA4, pyrG89, argB2, DnKuA::argB, cho1 Nayak et al., 2006 (reference [58]) alcA::pkcA pyrG89; pyroA4; alcA::pkcA::pyr4; choA1 Colabardini et al., 2010 (reference [10]) DcnaA pyroA4 pyrG89, DcnaA::pyro, wA3 Soriani et al., 2008 (reference [34]) alcA::pkcA DcnaA alcA::pkcA DcnaA, cho1 This work doi:10.1371/journal.pone.0104792.t002 "
    [Show abstract] [Hide abstract] ABSTRACT: In filamentous fungi, intracellular signaling pathways which are mediated by changing calcium levels and/or by activated protein kinase C (Pkc), control fungal adaptation to external stimuli. A rise in intracellular Ca2+ levels activates calcineurin subunit A (CnaA), which regulates cellular calcium homeostasis among other processes. Pkc is primarily involved in maintaining cell wall integrity (CWI) in response to different environmental stresses. Cross-talk between the Ca2+ and Pkc-mediated pathways has mainly been described in Saccharomyces cerevisiae and in a few other filamentous fungi. The presented study describes a genetic interaction between CnaA and PkcA in the filamentous fungus Aspergillus nidulans. Overexpression of pkcA partially rescues the phenotypes caused by a cnaA deletion. Furthermore, CnaA appears to affect the regulation of a mitogen-activated kinase, MpkA, involved in the CWI pathway. Reversely, PkcA is involved in controlling intracellular calcium homeostasis, as was confirmed by microarray analysis. Furthermore, overexpression of pkcA in a cnaA deletion background restores mitochondrial number and function. In conclusion, PkcA and CnaA-mediated signaling appear to share common targets, one of which appears to be MpkA of the CWI pathway. Both pathways also regulate components involved in mitochondrial biogenesis and function. This study describes targets for PkcA and CnaA-signaling pathways in an A. nidulans and identifies a novel interaction of both pathways in the regulation of cellular respiration.
    Full-text · Article · Aug 2014
    • "containing 2% glucose, it is possible that the mpkA deletion mutant is resistant to farnesol at high glucose concentrations. Colabardini et al. also reported that an alcA(p)-pkcA strain was hypersensitive to farnesol under the alcA(p)-inducing condition [20]. Consistent with their result, our alcA(p)-pkcA strain was also hypersensitive to farnesol under the alcA(p)-inducing condition (Fig. 5B). "
    [Show abstract] [Hide abstract] ABSTRACT: The pkcA gene, which encodes a protein kinase C (PKC) in the filamentous fungus Aspergillus nidulans, is essential for its viability. However, little is known about its functions. To address this issue, we constructed and characterized temperature-sensitive mutants of pkcA. The conidia of these mutants swelled slightly and exhibited apoptotic phenotypes at 42°C. The apoptotic phenotypes were suppressed by an osmotic stabilizer. Under these conditions, the conidia swelled extensively and did not form germ tubes. Moreover, polarized distribution of F-actin was not observed. We then utilized deletion mutants of bckA, an ortholog of Saccharomyces cerevisiae bck1 that encodes a mitogen-activated protein (MAP) kinase kinase kinase and functions downstream of PKC in the cell wall integrity pathway. These mutants exhibited apoptotic phenotypes at 42°C, but they did not show defects in polarity establishment under osmotically stabilized conditions. These results suggest that PkcA plays multiple roles during germination under conditions of heat stress. The first of these roles is the suppression of apoptosis induction, while the other involves polarity establishment. The former depends on the MAP kinase cascade, whereas the latter does not. In addition, repolarization, which was observed after depolarization in the wild-type strain and the bckA deletion mutant under conditions of heat stress, was not observed in the pkcA-ts mutant. This suggests that PkcA also plays role in polarity establishment during hyphal growth independent of the MAP kinase cascade under these conditions.
    Full-text · Article · Nov 2012
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