A putative dual-specific protein phosphatase encoded by YVH1 controls growth, filamentation and virulence in Candida albicans

The opportunistic fungal pathogen Candida albicans usually resides as a commensal in humans. However, in immunocompromised patients, such as cancer or transplant patients undergoing immunosuppressive treatment, this fungus can cause severe systemic invasion, leading to life-threatening circumstances (Calderone, 2002). A notable feature associated with the virulence of C. albicans is its ability to switch between several morphological states: it can adopt either a budding yeast form or a filamentous form. The filamentous form includes pseudohyphae, chains of elongated cells which have constrictions at the septation sites, and true hyphae, which have no constrictions at the septation sites. When C. albicans develops true hyphae, the hyphal cells evaginate, not bud, from the mother cells to form germ tubes, the name for the parallel-walled structure that is formed prior to formation of the first septum. After the first cytokinesis in a growing hypha, the hyphal tip continues to elongate, with the apical cell entering the next cell cycle. The time period of one hyphal cell cycle, from one cytokinesis to the next, is very similar to that of the yeast cell cycle (Hazan et al., 2002), indicating that yeast and hyphal growth, not germ tube formation, are controlled by a similar cell-cycle engine. Germ tube formation can be triggered by a variety of inducers, specifically temperature, pH, nitrogen supply, carbon source and serum. For example, serum induces rapid true hyphal growth at 37 °C.

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 () 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 (). 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 Δyvh1 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.

Strains, growth conditions and basic techniques.
Table 1 lists the C. albicans strains used in this study. Cells were grown in YPD (adjusted to pH 5·6; Qbiogene) or SD-AU [6·7 g l¹ yeast nitrogen base (YNB) without amino acids (Difco), 2 % glucose, CSM-ARG-URA (Qbiogene)] with shaking to induce the yeast form, or in YPD (adjusted to pH 7·2) plus 10 % serum at 37 °C with shaking to induce hyphae. The speed of growth was measured as OD₆₆₀ by using a Biophotorecorder TN-1506 (Advantec). For filamentous growth on solid medium, strains were grown for 7 days at 37 °C on 10 % calf serum solidified with 2 % agar.

Table 1. C. albicans strains used or constructed in this study

Escherichia coli XL-1 Blue and cloning vector pUC19 (Yanisch-Perron et al., 1985) were used for DNA manipulation. General recombinant DNA procedures were performed as described by Sambrook & Russell (2001). C. albicans was transformed by the method described by Umeyama et al. (2005). An Applied Biosystems model 310 automated capillary sequencer was used for nucleotide sequencing. Western analysis using anti-HA or anti-PSTAIRE antibody was performed as described by Umeyama et al. (2005). Microscopic observation was performed by using a conventional fluorescence microscope (Olympus IX81) equipped with a DP70 digital camera (Olympus). Cells were fixed with ethanol, and DNA and chitin were stained with 4',6-diamidino-2-phenylindole (DAPI) and Calcofluor white, respectively. Imaging software ImageJ () was used to measure hyphal length, or to count the number of nuclei or chitin rings.

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.

Table 2. Oligonucleotide primers used in this study

A plasmid, pUC19-ARG4, was constructed for gene replacement. A DNA fragment containing an ARG4 marker was obtained by digestion of plasmid pRS-ARG4ΔSpeI (Wilson et al., 1999) with SacI and KpnI and cloned into the SacI and KpnI sites of pUC19 to generate pUC19-ARG4.

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).

(49K): Fig. 3. Gene disruption of CaYVH1. (A) Disruption strategy by PCR-mediated cassettes and split Ura-blaster. See Methods for details. (B) Restriction maps for each modified allele. N, NcoI; S, SpeI. (C) Confirmation of disruption constructs by Southern hybridization using the three probes indicated in (B).

To tag Yvh1p with a 3xHA (haemagglutinin) tag in the genomic locus, a DNA fragment containing the 3' region of YVH1, a three-tandem repeat of the HA tag, the ACT1 terminator, the URA3 marker, and the downstream region of YVH1 was amplified by PCR with two primers, iYVH1short-5' and disYVH1-4, and the DNA fragment disYVH1-B, using pCaYVH1A as a template, and transformed into Ura strain TUA4, to generate YVH1HA. To verify the strain construction, direct-colony PCR with primers iCheckYVH1-5' and ACT1T-3' was performed, after which the nucleotide sequence of the PCR fragment was confirmed.

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.

Virulence study.
An animal experiment using mice was performed as described previously (Umeyama et al., 2005). For each strain tested, six mice were used.

Identification of C. albicans YVH1 homologue and its allelic differences
The S. cerevisiae YVH1 gene encodes a DSP, which has specificity for phosphoserine/threonine and phosphotyrosine. A database search of the C. albicans genomic sequences () revealed one gene with homology to S. cerevisiae YVH1, designated CaYVH1 [orf19.4401 (encoded in contig19-10203) and orf19.11879 (contig19-20203)]. The respective gene products share 45 % identity between Yvh1p phosphatases of C. albicans and S. cerevisiae. The amino acid alignment between them is shown in Fig. 1.

(55K): Fig. 1. Amino acid alignment of Yvh1p phosphatases between S. cerevisiae (ScYVH1) and C. albicans (CaYVH1A and B) performed by the program CLUSTALW. Identical residues among more than two proteins are shown in white on black.

The YVH1 coding sequence was amplified by PCR, and cloned into expression vector p3HA-ACT1. Nucleotide sequencing revealed that the clones obtained could be classified into two alleles of YVH1 (YVH1A and YVH1B). Furthermore, comparison of the nucleotide sequences of the PCR fragment amplified from the parental strain TUA4, and the heterozygous disruptant YVH102, demonstrated that the YVH1 coding sequence has nine single-nucleotide polymorphisms (SNPs), including six amino acid differences (Table 3). All the SNPs were conserved among SC5314, CAF2 and CAI4 in our laboratory (data not shown).

Table 3. SNPs in the YVH1 coding sequence

Regulation of YVH1 expression
To investigate the Yvh1p protein expression profile, YVH1 was tagged with three repeats of the HA tags at its C-terminus in the genomic locus in strain TUA5 by PCR-mediated integration. When the strain harbouring Yvh1p-3HA, which is expressed from its own promoter, was grown on YPD agar, and then in a liquid YNB (without glucose) medium overnight at 30 °C, more than 90 % of the cells were unbudded. The single cells without buds were inoculated into fresh YPD (pH 5·6) for maintaining the yeast growth, or into YPD (pH 7·2) containing 10 % serum for inducing the hyphal growth. An aliquot was taken every 30 min, and a crude extract was prepared from each sample. Western analysis using anti-HA antibody revealed no expression of Yvh1p-3HA in the unbudded cell fraction (Fig. 2). Moreover, expression of Yvh1p-3HA in the hyphal form started around 90 min after that in the yeast form, indicating that the expression of Yvh1p-3HA might be repressed during the initial stage of germ tube formation. Northern analysis or realtime RT-PCR analysis also demonstrated an expression pattern similar to that of Western analysis (data not shown).

(16K): Fig. 2. Expression of Yvh1p-3HA in yeast and hyphal cells. Protein was extracted from the 3xHA-tagged Yvh1p-expressing strain (YVH1HA) after the indicated times of growth in liquid YPD medium at 30 °C (Yeast) or YPD+10 % serum at 37 °C (Hyphae). Western blotting using anti-PSTAIRE antibody was performed as a loading control. NT, protein from an untagged strain (SC5314).

PCR-based gene disruption of YVH1
Disruption of the YVH1 gene was achieved by a PCR-amplified disruption cassette, and the split-Ura-blaster technique (Fig. 3A). A longer region for homologous recombination is obviously more efficient for C. albicans transformation, and a split Ura-blaster can be used for C. albicans gene replacement by three-point homologous recombination (de Hoogt et al., 2000). By combining these advantages, we developed a gene-disruption method using a split Ura-blaster, which has a 500 bp 5' or 3' region for recombination of the gene, and an ARG4 marker. The resultant URA3 or ARG4 marker was sequentially transformed into the UraArg strain TUA4. For the first allele, YVH1 was replaced with an hph200URA3hph200 cassette (YVH101), and the remaining allele was replaced by ARG4. With Southern hybridization, no YVH1 was confirmed to remain in strain YVH102 (Fig. 3B, C), demonstrating that YVH1 is not an essential gene in C. albicans. The URA3 gene was excised from YVH102, generating the Ura-auxotrophic derivative, YVH103. To confirm that loss of the YVH1 function was responsible for the observed phenotypes, the YVH1 expression plasmids pCaYVH1A and pCaYVH1B, and the p3HA-ACT1 vector alone, were reintroduced into the CaRP10 locus (Murad et al., 2000) of the Δyvh1 mutant strain YVH103, yielding strains YVH105A, YVH105B and YVH104, respectively.

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.

Table 4. Generation times of strains constructed in this study The genetic background of each strain is indicated in Table 1. All values were calculated from three independent experiments and are shown as means±SD.

How C. albicans develops into hyphal form is one of the greatest interests in C. albicans research. In order to observe hyphal formation of the disruptant, overnight-cultured cells were inoculated into liquid YPD (pH 7·2) plus 10 % serum. Time course observation of growth at 37 °C with shaking demonstrated that hyphal growth of Δyvh1 was obviously slower than that of the wild-type, although there was no significant difference in germ tube formation (Fig. 4): more than 90 % of cells induced germ tube formation within 60 min. On agar medium containing 10 % serum, the filamentation of the YVH1 disruptant was somewhat defective (Fig. 5). Similarly, as with the yeast growth, the revertant, in which YVH1 was expressed under the control of the ACT1 promoter in Δyvh1, extended its hyphal cells faster than did the wild-type. In fact, the hyphal length measured from captured images of cells cultured for 3 h was 50·9±11·7 µm (TUA6; n=142), 37·0±9·3 µm (YVH104; n=132; vs TUA6, P<0·001) and 65·22±12·4 µm (YVH105A; n=193; vs TUA6, P<0·001). To examine cell cycle progression of the YVH1 disruptant, we visualized the position of the nuclei and chitin rings, and counted their number in one hyphal cell. In the absence of YVH1, nuclear division and septum formation of hyphal cells were delayed (Table 5). Overall, these results suggest that deletion of the YVH1 gene hinders yeast or hyphal vegetative growth, but does not impair germ tube formation. To understand the slower hyphal growth of the YVH1 disruptant more clearly, we compared the expression of hypha-specific genes HWP1 (Staab & Sundstrom, 1998) and HYR1 (Bailey et al., 1996), and hypha-specific cyclin HGC1 (Zheng & Wang, 2004), by Northern analysis. There was no significant difference, except that the amount of HGC1 transcript increased, and the HYR1 transcription was retarded in the gene disruptant compared with the wild-type (Fig. 6). The ACT1 transcription at time 0 was significantly reduced in YVH105A compared with YVH104 and TUA6, possibly because YVH105A reached stationary phase earlier than YVH104 and TUA6, at which growth phase the expression level of ACT1 was very low (data not shown). Real-time RT-PCR analysis gave results similar to those obtained by Northern analysis (data not shown).

(52K): Fig. 4. Δyvh1 shows slow filamentation in liquid media. Photographs of hyphal elongation of TUA6 (wild-type), YVH104 (Δyvh1 disruptant) and YVH105A (revertant) were taken at the indicated times. Bars, 15 µm.

(35K): Fig. 5. Δyvh1 (YVH104) was defective in filamentation in a solid medium. Cells (104) of the indicated strain were spotted on 10 % serum agar and incubated at 37 °C for 7 days.

Table 5. Number of nuclei or chitin rings in C. albicans cells grown in hypha-inducing conditions The genetic background of each strain is indicated in Table 1. Cells were incubated in YPD+10 % serum at 37 °C for 180 min, and fixed with ethanol. Nuclei were stained with DAPI, and chitin rings were stained with Calcofluor white.

(48K): Fig. 6. Expression of hypha-specific genes in TUA6, YVH104 and YVH105A. Cells grown in YPD for overnight (∼14 h) were diluted in YPD+10 % serum, and grown at 37 °C for the indicated times. RNA samples were prepared from each strain, and Northern analysis was performed with probes to the indicated genes.

Δyvh1 strains are less virulent
To determine the role of Yvh1p in virulence, mice were intravenously injected with the wild-type (TUA6), Δyvh1-null mutant (YVH104) or the revertant (YVH105A), and monitored for survival. Infection with 10⁶ c.f.u. of the wild-type strain resulted in 100 % mortality after 16 days (Fig. 7). In contrast, 100 % mortality of the mice injected with an equal inoculum of Δyvh1 null mutant cells was not observed until day 40. Mice that received the revertant strain were all killed within day 12 post-infection, which was faster than that of the wild-type; this suggested that the expression of YVH1 from the ACT1 promoter in the Δyvh1 mutant might accelerate virulence, as well as yeast or hyphal growth. A position effect of the URA3 marker could be excluded as the reason for the higher virulence of the revertant, because TUA6, which was used as the control strain, contains the URA3 marker of p3HA-ACT1 at the CaRP10 locus, like YVH104 and YVH105A. There were no significant differences in the tissue burden of C. albicans recovered from kidney among TUA6, YVH104 and YVH105A, with mean burdens of 4·5±0·2, 4·3±0·07 and 4·8±0·2 log₁₀(c.f.u. g¹), respectively.

(13K): Fig. 7. Virulence study of the Δyvh1 disruptant in a systemic candidiasis mouse model. Survival of mice (six for each group) was monitored after injection of 106 C. albicans cells into the tail vein. Wild-type (TUA6, •), yvh1 mutant (YVH104, ) and revertant (YVH105A, ) were injected. Statistical analysis showed significant survival differences (TUA6 vs YVH104, *, P<0·01; TUA6 vs YVH105A, #, P<0·05) by the log-rank test.

In this study, we characterized a S. cerevisiae YVH1 homologue as a regulator of growth in the opportunistic fungal pathogen C. albicans. Yvh1p encodes a putative DSP of 322 aa. The YVH1 coding sequence has nine SNPs, six of which affect the encoded amino acids on the strains examined, although no SNPs of the YVH1 locus were reported in the Stanford genome database (). The two alleles, designated YVH1A and YVH1B, may be functionally identical, because both alleles A and B were able to complement growth defects of the YVH1 disruptant. It remains to be determined whether the gene products of both alleles have the same enzymic activity as protein phosphatases.

We constructed the Δyvh1 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 (). 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.

We thank Aki Kaneko, Koichi Tanabe, Yuki Utena and Yukie Takano (National Institute of Infectious Diseases) for their technical assistance and valuable comments. This work was supported by a grant from the Systemic Fungal Infection Forum (SFIF), and a Grant-in-Aid for Scientific Research (no. 15780064) from the Ministry of Education, Culture, Sports, Science and Technology (MEXT). It was also supported by Health Science Research Including Drug Innovation (KH53315), the Japan Health Sciences Foundation (JHSF), and the Health Science Research Grants for Research on Emerging and Reemerging Infectious Diseases, Ministry of Health, Labour and Welfare of Japan.

Footnotes

†Present address: Research Center for Pathogenic Fungi and Microbial Toxicoses, Chiba University, 1-8-1 Inohana, Chuo-ku, Chiba 260-8673, Japan.