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Acta Biochim Biophys |
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doi:10.1111/j.1745-7270.2006.00144.x |
CaMac1, a Candida albicans
Copper Ion-sensing Transcription Factor, Promotes Filamentous and Invasive
Growth in Saccharomyces cerevisiae
Guang-Hua HUANG, Xin-Yi
NIE, and Jiang-Ye CHEN*
State Key Laboratory of Molecular
Biology,
Received:
November 15, 2005
Accepted:
December 23, 2005
This work
was supported by the grants from the National Natural Science Foundation of
author: Tel, 86-21-54921251; Fax, 86-21-54921011; E-mail,
[email protected]
Abstract Molecular mechanisms of
morphogenesis share many common components between Candida albicans and Saccharomyces
cerevisiae. The Kss1-associated MAPK cascade and the cAMP/PKA pathway are
two important signal transduction pathways that control morphogenesis in S.
cerevisiae. A C. albicans copper ion-sensing transcription factor
gene, CaMAC1, was cloned from C. albicans SC5314. Ectopic
expression of CaMAC
Key words CaMac1; MAPK pathway; cAMP/PKA
pathway; morphogenesis; Candida albicans; Saccharomyces cerevisiae
Candida albicans is a
frequently isolated opportunistic pathogen in humans, causing both mucosal and
systemic infections, especially in immunocompromised individuals [1]. The study
of this fungus has been hindered by its obligate diploid genome and lack of a
sexual phase [2]. Saccharomyces cerevisiae is a well-characterized model
system. The molecular mechanisms of morphogenesis in the two yeasts share many
common components. Therefore, we are able to use S. cerevisiae mutants
to study the function of C. albicans genes.
The Kss1-associated MAPK
cascade and the cAMP/PKA pathway work together to control the morphogenesis of S.
cerevisiae. Mutations in the genes, such as STE7, STE12, TEC1 and
FLO8 of the two cascades, impair the ability of diploid filamentous and
haploid invasive growth in S. cerevisiae [3–5]. The two cascades converge on the promoter of FLO11,
a gene encoding for a cell wall protein, which is critical for pseudohyphae
formation in S. cerevisiae [6]. Many important components of the MAPK
and cAMP/PKA pathways have been identified in C. albicans [7–10]. Several hyphal development regulators
have been isolated in C. albicans by functional complementation [11,12].
Copper is an essential
cofactor for a variety of enzymes, including cytochrome c oxidases,
copper,zinc-SOD1 and multicopper oxidases [13]. Metal binding activator ScMac1
is a copper ion-sensing transcription factor, whose activity is inhibited by
copper ions [14,15]. Loss-of-function mutants of ScMac1 are slow growing,
respiratory deficient and hypersensitive to heat or exposure to cadmium.
Biochemical studies have been reported to map residues and motifs, which are
important to the activity of the ScMac1 protein [16,17]. However, its function
in the morphogenesis of S. cerevisiae has not been elucidated.
In this report, we
cloned CaMAC1, a homologue of ScMAC1, from the C. albicans SC5314
genome and studied the functional role of CaMac
Materials and Methods
Strains and culture
conditions
The yeast strains SC5314
(wild type) [18], L5528 (MATa ura3-52 his3::hisG), HLY367 (MATa
ste7::LEU2 ura3-52 leu2::hisG), HLY362 (MATa ste12::LEU2 ura3-52
leu2::hisG) [3], HLY2000 (MATa tec1::HIS3 ura3-52) [19], HLY850 (MATa
flo8::hisG ura3-52) [4], CG31 (MATa/a ura3-52/ura3-52) [20], HLY351 (MATa/a
ste7::LEU2/ste7::LEU2 ura3-52/ura3-52 leu2::hisG/leu2::hisG),
HLY352 (MATa/a ste12::LEU2/ste12::LEU2 ura3-52/ura3-52
leu2::hisG/leu2::hisG), HLY2002 (MATa/a
tec1::HIS3/tec1::HIS3 ura3-52/ura3-52) and HLY852
(MATa/a flo8::hisG/flo8::hisG ura3-52/ura3-52),
YPD medium (1% yeast extract, 2% peptone, 2% glucose) and synthetic medium (SD)
were used for yeast growth. Transformation of S. cerevisiae was
performed, as described by Ito et al. [21]. Uracil-deficient synthetic complete
medium (SC-Ura) was used for selection of URA+ yeast transformants. Synthetic low ammonia medium (SLAD) was used
for pseudohyphal growth of S. cerevisiae. Invasive growth of S.
cerevisiae was carried out, as described previously [11].
Cloning of CaMAC1
and construction of plasmids
Genomic DNA of C.
albicans SC5314 was extracted and used as the template for amplifying CaMAC1.
Ma5 (5‘-atCTCGAGatgatactaatagatgatatcaaa-3‘)
and Ma3 (5‘-taCTGCAGttatttggtcttttttgagcaacatg-3‘)
were used as polymerase chain reaction (PCR) primers. PCR products were
digested with XhoI and PstI, and inserted into the XhoI/PstI
site of pVT102-U to generate the S. cerevisiae expression plasmid,
pVT-CaMAC1. Sequence analysis was performed using the DNAStar and GeneDoc
programs (DNAStar,
Results and Discussion
Cloning of CaMAC1
and sequence analysis
A sequence (orf19.7068)
which shared high similarity with ScMac1 was found by searching the C.
albicans genome database (http://www-sequence.stanford.edu/group/Candida/search.html).
The orf19.7068 encoded for a protein of 471 amino acids, with isoelectric point
7.39. According to the sequence similarity, the orf19.7068 was
designated CaMac1. Compared with ScMac1, CaMac1 contained a conserved
“copper-fist” motif in the N-terminus, which was postulated to be a
DNA-binding domain. In addition, CaMac1 contained two conserved cysteine-rich
sequences corresponding to the REPI and REPII motifs of ScMac1 [14]. Sequence
analysis indicated that CaMac1 was a homolog of ScMac
Ma3, were used to amplify CaMAC1 from genomic DNA of C. albicans SC5314.
The PCR fragment was subcloned into the XhoI/PstI site of a S.
cerevisiae expression vector, pVT102-U, to generate pVT-CaMAC1. The
construction was confirmed by restriction digestion and sequencing (Fig. 2).
Ectopic expression of CaMAC1
promotes filamentous and invasive growth in S. cerevisiae
Many regulatory proteins
responding to morphological transition are conserved between C. albicans and
S. cerevisiae. We investigated the role of CaMac
Activation of CaMac
Because both diploid
filamentous growth and haploid invasive growth were controlled by the
Kss1-associated MAPK cascade and the cAMP/PKA pathway, we were interested in
examining whether or not CaMac1 plays its role in S. cerevisiae cells
via the two pathways. The plasmids were introduced into the ste7, ste12, tec1 and
flo8 mutants. Ectopic expression of CaMAC1 suppressed the
filamentous growth defect of the ste7, ste12, tec1 and flo8 mutants (Fig. 4).
Data indicated that the activating function of CaMac
Activation of CaMac
To investigate whether
or not CaMac1 also functions in invasive growth, the plasmid pVT-CaMAC1
was transformed into S. cerevisiae wild-type, ste7, ste12, tec1 and flo8 haploid
mutants. Ectopic expression of CaMAC1 suppressed the invasive growth
defect of the MAPK cascade mutants including ste7, ste12 and tec1, but not the flo8 mutant (Fig. 5). Results
indicated that activation of CaMac
Copper ions inhibit
filamentous and invasive growth of S. cerevisiae
The morphogenesis of
yeast was affected by a variety of environmental signals. In response to
nitrogen starvation, the diploid cells develop into pseudophae and form
filamentous colonies on the SLAD plate. Under glucose starvation, the haploid
cells invade into agar. Metal ions are also important for fungal morphogenesis.
To examine the role of copper ions in the morphogenesis of S. cerevisiae,
we added copper salt to the growth media. It was observed that copper ions had
an inhibitory role on filamentous and invasive growth of yeast (Table 1). The effect is
dependent on the copper ion concentration; a high concentration of CuSO4 blocks filamentous growth in diploid cells
and invasive growth in haploid cells. Copper homeostasis in yeast is maintained
by Cu-regulation of the biosynthesis of several proteins. The transcription
factor ScMac1 is sensitive to copper ions, and its activity was repressed by
copper ions. We investigated if the activity of CaMac1 was also inhibited by
copper ions. Addition of 20 mM CuSO4 attenuated the activating effect of CaMac
reported the role of CaMac
important cause of candidiasis infections. The dimorphic switch in C.
albicans has been proven to be a crucial virulence factor for successful
infection. C. albicans can develop into three forms: budding yeast, true
hyphae and pseudohyphae. True hyphae and pseudohyphae are critical for tissue
penetration and the colonization of organs [22], because they are invasive.
Although S. cerevisiae is a simple system, the mechanism involved in
morphogenesis could be referred by the other organisms. The phenomenon that
CaMac1 promotes pseudohyphal and invasive growth of S. cerevisiae suggests
that CaMac1 might play a similar role in C. albicans. The
activation of CaMac1 was repressed by copper ions, reflecting a potential
copper response pathway that may be involved in the regulation of morphological
transition in C. albicans.
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