1 Department of Bioactive Molecules, National Institute of Infectious Diseases, 1-23-1 Toyama, Shinjuku-ku, Tokyo 162-8640, Japan
2 United Graduate School of Agricultural Science, Tokyo University of Agriculture and Technology, 3-5-8 Saiwai-cho, Fuchu-city, Tokyo 183-8509, Japan
3 Life Science Research Center, College of Bioresource Sciences, Nihon University, 1866 Kameino, Fujisawa, Kanagawa 252-8510, Japan
Correspondence
Masakazu Niimi
niimi{at}nih.go.jp
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ABSTRACT |
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Present address: Research Center for Pathogenic Fungi and Microbial Toxicoses, Chiba University, 1-8-1 Inohana, Chuo-ku, Chiba 260-8673, Japan.
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INTRODUCTION |
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Protein phosphorylationdephosphorylation is an essential element in the regulation of a wide variety of cellular mechanisms, including cell signalling, gene expression and mitosis. Protein phosphatases are a family of enzymes that catalyse phosphate hydrolysis of phosphoprotein. Protein phosphatases are classified into three classes by substrate specificity: protein serine/threonine phosphatases, which hydrolyse phosphoserine and phosphothreonine residues of proteins; protein tyrosine phosphatases, which hydrolyse a phosphotyrosine residue of proteins; and dual-specific protein phosphatases (DSPs), which hydrolyse both phosphoserine/threonine and phosphotyrosine residues of proteins. Protein serine/threonine phosphatases are further classified by the inhibitory mode of the phosphatase inhibitor or ion requirement into several subclasses, such as PP1, PP2A, PP2B, PP2C, PP4 and PP5 (Zolnierowicz & Bollen, 2000). In the model yeast Saccharomyces cerevisiae, YVH1, one of the DSPs, was first identified as a vaccinia VH1 homologue (Guan et al., 1992
). Deletion of YVH1 in S. cerevisiae causes defects in vegetative growth (particularly at lower temperatures), sporulation and glycogen accumulation, and transcription of YVH1 is induced by low temperature and nitrogen starvation (Beeser & Cooper, 2000
; Guan et al., 1992
; Park et al., 1996
; Sakumoto et al., 1999
, 2001
). However, even in S. cerevisiae, the exact function of YVH1 remains to be elucidated.
The published C. albicans genome database (http://sequence-www.stanford.edu/group/Candida/) has revealed that there are 29 protein phosphatases in the genome, which were identified on the basis of BLAST search and the annotation of the Incyte MycopathPD database (https://www.proteome.com/proteome/). C. albicans protein phosphatases so far reported include Cyr1p/Orf19.5148 (Jain et al., 2003; Mallet et al., 2000
), Cpp1p/Orf19.4866 (Csank et al., 1997
), Cmp2p/Orf19.6033 (Blankenship et al., 2003
; Sanglard et al., 2003
) and Sit4p/Orf19.5200 (Lee et al., 2004
), which were characterized from the phenotypes of disruptants of these genes. CYR1 encodes an adenyl cyclase, which functions in the Efg1-mediated pathway of morphological transition, although it is similar to the protein phosphatase 2C subclass (Jain et al., 2003
). CPP1 encodes a DSP, which is involved in the repression of the hyphal signalling pathway, and may directly act on MAP kinase Cek1p (Csank et al., 1997
). CMP2 encodes calcineurin, which is essential for cell tolerance to various growth inhibitors (Sanglard et al., 2003
), and cell viability in a serum-based growth medium (Blankenship et al., 2003
). SIT4 encodes PP2A, which plays important roles during hyphal growth in C. albicans through the regulation of cell wall biogenesis, osmosensing and protein translation (Lee et al., 2004
). In an attempt to disrupt a series of other genes encoding C. albicans protein phosphatases, the YVH1 gene disruptant was obtained, which showed slow growth. In this study, we analysed the
yvh1 disruptant in order to investigate its relationship with cell morphology and virulence. In addition, we demonstrated that the expression of YVH1 was delayed during hyphal development compared with yeast growth.
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METHODS |
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Plasmid construction.
Table 2 lists the primers used in this work. A DNA fragment containing YVH1 allele A or B (see Results) was PCR amplified using two primers, YVH1-N and YVH1-C, with TUA4 chromosomal DNA as a template, digested with BamHI and SphI, and then cloned into the BamHI and SphI sites of p3HA-ACT1 (Umeyama et al., 2005
) to generate pCaYVH1A or pCaYVH1B. The nucleotide sequences of cloned fragments were confirmed.
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Strain construction.
Table 1 lists the strains constructed in this work. Strain TUA6, used as the wild-type strain, was constructed by integration of p3HA-ACT1 as URA3 complementation, and an ARG4 DNA fragment amplified with primers ARG4-5' and ARG4-3', into the UraArg strain TUA4.
To disrupt YVH1, two different markers were used for two different alleles (see Fig. 3A). First, a 500 bp DNA fragment corresponding to the 5' or 3' end of YVH1 was amplified using primers disYVH1-1 and disYVH1-2, and primers disYVH1-3 and disYVH1-4, to yield DNA fragments disYVH1-A and disYVH1-B, respectively (Fig. 3A, a
). For the first allele, two DNA fragments, named disYVH1-R and disYVH1-L, were amplified using pUC19-Hph200-URA3 (Umeyama et al., 2005
) as a template. For amplification of disYVH1-R, DNA fragment disYVH1-A, and primers disYVH1-1 and URA3-3', were used. For amplification of disYVH1-L, DNA fragment disYVH1-B, and primers disYVH1-4 and URA3-5', were used (Fig. 3A, b
). DNA fragments disYVH1-R and disYVH1-L were simultaneously used to transform the C. albicans UraArg strain TUA4 (Fig. 3A, c
). After selection on SD-URA medium [6·7 g l1 YNB without amino acids (Difco), 2% glucose, CSM-URA (QBiogene)], the resulting Ura+ transformants (YVH101) were used for a second transformation. A disruption cassette containing ARG4 auxotroph marker was amplified in a manner similar to that described above, with primers disYVH1-1 and disYVH1-4, DNA fragments disYVH1-A and disYVH1-B, and plasmid pUC19-ARG4 as a template (Fig. 3A, b
). This was then used to transform an Arg strain YVH101 to generate YVH102 (Fig. 3A, d
). The resulting Ura+Arg+ transformants of YVH102 were plated on a medium containing 5-fluoroorotic acid to isolate the Ura segregants (YVH103). To confirm the gene disruption, genomic DNA was isolated from each strain, digested with SpeI and NcoI, run in 0·7 % agarose gel, and then transferred onto a Hybond-N+ nylon membrane (Amersham Biosciences). Southern hybridization was done using an AlkPhos direct labelling kit and CDP-Star reagent (Amersham Biosciences). The YVH1 gene was reintroduced into the CaRP10 locus of the null mutant using the StuI-digested pCaYVH1A or pCaYVH1B to transform strain YVH103, to generate YVH105A and YVH105B, respectively. The StuI-digested empty vector p3HA-ACT1 was integrated into strain YVH103 as a control (YVH104).
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Northern analysis.
The cells were collected by centrifugation, washed twice with ice-cold water, frozen with liquid nitrogen, and stored at 80 °C until used. For RNA extraction, the cells were suspended in TES buffer (10 mM Tris/HCl, pH 7·5, 10 mM EDTA, 0·5 % SDS), and incubated at 65 °C for 45 min with acid phenol (Sigma). The aqueous phase solution was purified with acid phenol and chloroform, and then precipitated with ethanol. Each 10 µg total RNA was loaded on formamide agarose gel [0·9 % agarose and 5 % formaldehyde in 1xMOPS (20 mM MOPS, 5 mM sodium acetate, 1 mM EDTA)] and transferred to a Hybond-N+ nylon membrane. Northern hybridization was performed using an AlkPhos direct labelling kit. A DNA probe for detection of HWP1 was amplified with primers HWP1-5' and HWP1-3', using TUA4 genomic DNA as a template; for HYR1, HYR1-5' and HYR1-3'; for HGC1, HGC1-5' and HGC1-3'; and for ACT1, ACT1-5' and ACT1-3'. All primers used for probe construction are listed in Table 1. Alternatively, we performed quantitative realtime RT-PCR analysis for these hypha-specific genes. To synthesize cDNA, we used SuperScript III reverse transcriptase (Invitrogen). mRNA quantification by realtime PCR was performed using ABI PRISM 7000 (Applied Biosystems) and SYBR Premix ExTaq (Takara, Japan). All primers used for realtime PCR are listed in Table 1
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Virulence study.
An animal experiment using mice was performed as described previously (Umeyama et al., 2005). For each strain tested, six mice were used.
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RESULTS |
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Morphological phenotype of YVH1 mutant strains
By calculation from the OD660, deletion of both alleles of C. albicans YVH1 resulted in slow growth rates when grown in YPD or SD at 30 or 37 °C (Table 4). The generation times of YVH105A and YVH105B were similar, indicating that alleles A and B are equally able to restore the growth defect of the null mutant. Expression of YVH1 under the control of the ACT1 promoter in strain YVH105A or YVH106A led to a significantly faster growth rate than that of the wild-type (Table 4
). By estimation from Western analysis using anti-HA antibody (data not shown), expression of Yvh1p-3HA in YVH105A was actually greater than that in strain YVH1HA, which was expressed from its own promoter, indicating that the expression level of Yvh1p may affect the growth rate of C. albicans. The morphology or size of the yeast cell was not affected by gene deletion. Furthermore, there was no significant difference in susceptibility to fluconazole, amphotericin B or micafungin among the wild-type, the disruptant and the revertant.
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DISCUSSION |
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We constructed the yvh1 disruptant by combining a PCR-amplified deletion cassette and a split Ura-blaster technique. Finally, both alleles of YVH1 were replaced with hph200 and ARG4. Under conditions inducing the yeast form, such as YPD (pH 5·6), the disruptant YVH104 grew slower than the wild-type (Table 4
). When grown on hyphal-inducing solid media, the null mutant extended very few hyphae radiating from the colonies (Fig. 5
). Similarly, when grown in liquid media inducing a hyphal form, the disruptant elongated at a slower rate (Fig. 4
), although the germ tubes evaginated normally. Since the deletion of YVH1 in S. cerevisiae causes defects in vegetative growth (Guan et al., 1992
), the role of Yvh1p phosphatase was not different between S. cerevisiae and C. albicans. Hazan et al. (2002)
demonstrated that germ tube formation is regulated by an independent cell cycle programme; therefore YVH1 may affect yeast and hyphal growth, but not germ tube formation. This idea was also supported by the fact that expression of Yvh1p was undetectable during germ tube formation (Fig. 2
). Apart from germ tube formation, hyphal development, yeast growth and cell cycle progression, as functions of generation time, nuclear division, chitin ring and septum formation were also delayed significantly in the YVH1 disruptant as compared with the wild-type. These data indicate that the role of YVH1 in C. albicans may be connected to the cell cycle progression, but not to regulation of germ tube formation.
It is very interesting, but incomprehensible, that the revertant strain YVH105A grew faster as a yeast form (Table 4), elongated faster as a hyphal cell (Fig. 4
), and killed the host faster (Fig. 7
) than did the wild-type strain TUA6. When a YVH1-expressing plasmid was introduced into TUA5, the rate of growth of this strain was almost identical to that of YVH105A (Table 4
), indicating that the elevated copy number of YVH1 mRNA might contribute to faster growth of the revertant.
The virulence of C. albicans was also found to be dependent on YVH1. It is possible to consider that the rate of growth is a virulence factor, while the null mutant retained the ability to kill the host. SIT4, encoding protein phosphatase class 2A (Lee et al., 2004), is also involved in virulence: SIT4 disruptants show reduced virulence and slow growth. Although only two examples are known so far, it is easy to believe that slow-growing mutants are generally avirulent. C. albicans has 29 possible protein phosphatases in its genome, deduced from the C. albicans genome database (http://sequence-www.stanford.edu/group/candida/). Hitherto, four genes encoding protein phosphatase have been reported with phenotypic data of null mutants, indicating the importance of protein phosphatases in morphogenesis and virulence. Deletion of other non-reported protein phosphatases should provide further interesting information on signal transduction for C. albicans morphology and virulence.
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ACKNOWLEDGEMENTS |
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REFERENCES |
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Received 1 March 2005;
revised 24 March 2005;
accepted 11 April 2005.
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