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Identification and characterization of a functional Candida albicans homolog of the Saccharomyces cerevisiae TCO89 gene

Changlong Zheng, Zhihui Yan, Wei Liu, Linghuo Jiang
DOI: http://dx.doi.org/10.1111/j.1567-1364.2007.00210.x 558-568 First published online: 1 June 2007

Abstract

As one of the components of target of rapamycin complex 1 (TORC1), ScTco89p is involved in rapamycin sensitivity and cellular integrity in Saccharomyces cerevisiae. Here we provide evidence showing that deletion of ScTCO89 causes yeast cells to be hypersensitive to salt stress in a high osmolarity glycerol pathway-independent fashion. In addition, we have identified and characterized a functional Candida albicans homolog (CaTCO89) of ScTCO89, which encodes a protein of 708 amino acids that shows overall 15% identity with ScTco89p at the amino acid level. However, CaTCO89 could complement the functions of ScTCO89 in rapamycin sensitivity, salt tolerance, and cellular integrity. Candida albicans cells disrupted for CaTCO89 are also sensitive to rapamycin and lithium salt, but not susceptible to challenges to cellular integrity.

Key words
  • Candida albicans
  • TOR
  • TCO89
  • rapamycin
  • salt tolerance
  • budding yeast

Introduction

Rapamycin was originally obtained from Streptomyces hygroscopicus in a screen for antimicrobial activity against Candida albicans, and was later found to have potent immunosuppressive activity (Vezina, 1975). Rapamycin acts through binding to its intracellular receptor, FK506-binding protein (FKBP), in yeast and mammalian cells (Harding, 1989; Siekierka, 1989; Heitman, 1991). The FKBP–rapamycin complex binds and inhibits the activity of a phosphatidylinositol-3-kinase-related kinase, the target of rapamycin (TOR), which is the central controller of cell growth in eukaryotes (Schmelzle & Hall, 2000). There are two highly homologous TOR proteins in the budding yeast Saccharomyces cerevisiae, Tor1p and Tor2p (Cafferkey, 1993; Kunz, 1993). In addition to its rapamycin-sensitive function, Tor2p also regulates actin cytoskeletal dynamics during polarized cell growth in a rapamycin-insensitive way (Helliwell, 1998).

Recent biochemical studies have elucidated two functionally distinct TOR complexes in S. cerevisiae (Loewith, 2002; Reinke, 2004; Wullschleger, 2005). TOR complex 1 (TORC1) contains TOR1 or TOR2, KOG1, TCO89 and LST8, whereas TOR complex 2 (TORC2) includes TOR2, AVO1, AVO2, AVO3, BIT61 and LST8. TORC1 mediates the TOR-shared, rapamycin-sensitive pathway, and TORC2 mediates the TOR2-unique, rapamycin-insensitive pathway. As one of the components of TORC1 in S. cerevisiae, ScTco89p is involved in multiple cellular functions. Deletion of TCO89 caused yeast cells to be hypersensitive to rapamycin and susceptible to challenges to cellular integrity (Reinke, 2004).

Candida albicans is the most important fungal pathogen in humans, and can cause superficial infection as well as life-threatening systemic infection in immunodeficient individuals (Odds, 1987; Whiteway, 2000; Calderone & Fonzi, 2001). Recent studies on C. albicans have been focused on understanding its pathogenesis and on identifying drug targets (Berman & Sudbery, 2002). A single TOR homolog has been identified in C. albicans (Cruz, 1999). In addition, the C. albicans RBP1 gene, encoding the C. albicans FKBP12 protein, was previously identified (Ferrara, 1992). The antifungal activity of rapamycin towards C. albicans was shown to be mediated via conserved complexes with FKBP12 and TOR kinase homologs (Cruz, 1999). These indicate that TOR signaling is conserved in C. albicans. In this study, we have identified a Candida sequence homolog of ScTCO89, and shown that CaTCO89 could complement the functions of ScTCO89 in cellular integrity and rapamycin salt sensitivity. Disruption for CaTCO89 renders C. albicans cells sensitive to rapamycin and lithium salt, but not susceptible to challenges to cellular integrity. In addition, we provide evidence that deletion of ScTCO89 causes yeast cells to be hypersensitive to salt stress in a high osmolarity glycerol (HOG)-independent fashion, and that this could also be reversed by CaTCO89.

Materials and methods

Strains and media

The C. albicans SC5314 and RM1000 (MATa/αura3Δ::λimm434/ura3Δ:: λimm434 his1::hisG/his1::hisG) strains (Jiang, 2001; Lee, 2004), as well as the Sa. cerevisiae BY4741 (MATa his3Δ1 leu2Δ0 met15Δ0 ura3Δ0) (Jiang, 2004) strain, were maintained at 30°C on YPD medium. Deletion strain tco89Δ was derived from BY4741. The synthetic drop-out medium was supplemented with appropriate nutrients for plasmid selection and maintenance (Sherman, 1986). Rapamycin (A.G. Scientific) stock solution (1 mg mL−1) was prepared in ethanol containing 10% Tween-20. Saccharomyces cerevisiae transformation was performed by the lithium acetate procedure (Jiang, 2002).

DNA manipulation and plasmid construction

Standard DNA techniques were used (Sambrook, 1989). Primers CaTCO89-F (GGG GTA CCT TGA ATA AAG CCG AGG TGG G, KpnI site underlined) and CaTCO89-R (CGG GAT CCA GCC AAT AAG AGG CTT C, BamHI site underlined) were designed for amplification of a DNA fragment containing the 1000-base upstream region, the 2100-base ORF and the 300-base downstream region of CaTCO89. The PCR products were cut with KpnI and BamHI, and cloned into the centromeric plasmid pRS316, yielding pRS316-CaTCO89. A 3.5-kb DNA fragment containing the 761-base upstream region, the 2360-base ORF and the 329-base downstream region of ScTCO89 was PCR amplified with primers ScTCO89-F (5′-CGGGATCCAT CCTACCCCTA ACCTC-3′, BamHI site underlined) and ScTCO89-R (5′-CGCGAGCTCG TCCGTGAGGA ATACTTACC-3′, SacI site underlined). This DNA fragment was cloned into the BamHI and SacI sites of pRS316, yielding pRS426-ScTCO89. Insert sequences in plasmid constructions were verified by DNA sequencing. The full-length CaTCO89 was excised with KpnI and BamHI from pRS316-CaTCO89, and cloned into the KpnI and BamHI sites of pCR4 (Rocha, 2001), yielding pCR4-CaTCO89.

Complementation tests

The Candida and Saccharomyces cells containing plasmids were maintained on SD-URA medium, and grown at 30°C to mid-log phase for their assays. For temperature-sensitive growth, cells were grown at both 30 and 40°C. For rapamycin and salt sensitivity assays, cells were serially diluted 10-fold, and each dilution of 3 μL was spotted sequentially onto the appropriate media.

Disruption of the two alleles of CaTCO89

The disruption cassette was constructed in plasmid p5921 containing a hisG–URA3–hisG cassette with URA3 as the selectable maker (Fonzi & Irwin, 1993). First, a DNA fragment containing the 1030-bp promoter region of CaTCO89 was amplified with primers 1 and 2 (Fig. 1), and this fragment was digested with KpnI and cloned into the KpnI site at one side of the hisG–URA3–hisG cassette in p5921, yielding pTJU1. The correct direction of this fragment in pTJU1 was determined by PCR primer 2 and primer FUSE-F (from the flanking region of the KpnI site in the backbone vector of p5921). Similarly, a 920-bp DNA fragment, containing part of the C-terminal region of CaTco89p and the 3′-region of CaTCO89, was amplified with primers 3 and 4 (Fig. 1). This DNA fragment was digested with BamHI and cloned into the BamHI site at another side of the hisG–URA3–hisG cassette in pTJU1, yielding pTJU2. The correct direction of this fragment in pTJU2 was determined by PCR with primers 3 and PUC-R (from the flanking region of the BamHI site in the backbone vector of p5921).

1

The nucleotide sequence and deduced amino acid sequence of the Candida albicans TCO89 gene. Nucleotides are numbered on the left, and amino acid residues are numbered on the right. Primers CaTCO89-F and CaTCO89-R are indicated below their sequences and underlined.

To disrupt the first CaTCO89 allele, the plasmid pTJU2 DNA was digested with SalI, and the linearized plasmid DNA was transformed into C. albicans strain RM1000, as described previously (Lee, 2004). Ura+ transformants (TJU1) were selected on SD plates without uridine, and subsequently streaked onto YPD plates containing 5-fluoroorotic acid to permit loss of the URA3 marker by cis recombination between the flanking hisG repeats, resulting in TJU2. To disrupt the second CaTCO89 allele, the TJU2 strain with a genotype of TCO89/tco89ΔhisG was transformed with the SalI-linearized plasmid pTJU2 DNA. Similarly, Ura+ transformants (TJU3) were selected and streaked onto 5-fluoroorotic acid plates to obtain single Ura colonies, generating the TJU4 strains. The genotypes of TJU2 and TJU4 strains were examined by PCR (Fig. 2). To generate the revertant strain, we transformed pCR4-CaTCO89 (and the pCR4 vector as the control) into the TJU4 strain. Similarly, we also transformed pCR4-CaTCO89 into the RM1000 and the TJU2 strains for epistasis analysis.

2

Disruption of the Candida albicans CaTCO89 gene. (a) The strategy of disrupting the two alleles of CaTCO89. The CaTCO89 gene is depicted as a 3.4-kb genomic fragment including its 2.1-kb ORF. Two CaTCO89 alleles were subsequently replaced with the tco89ΔhisG–URA3–hisG disruption cassette, and this was followed by removal of – URA3-hisG with 5-fluoroorotic acid. The primers indicated on RM1000, TJU2 and TJU4 were used to verify the genotypes of TJU2 and TJU4 strains. (b) PCR confirmation of the genotypes of TJU2 and TJU4 strains with primers CaTCO89F and CaTCO89R. A 3.4-kb DNA fragment from wild-type CaTCO89 was amplified from genomic DNA of both the RM1000 and the heterozygous mutant TJU2 strains (lanes 3 and 2), but not from genomic DNA of the homozygous mutant TJU4 strain (lane 1). In contrast, a 3.1-kb fragment from the tco89ΔhisG mutant allele was amplified from genomic DNA of both the homozygous mutant TJU4 and the heterozygous mutant TJU2 strains (lanes 1 and 2 in Fig. 4b), but not from genomic DNA of the RM1000 strain (lane 3). (c) PCR confirmation of the genotypes of TJU2 and TJU4 strains with another set of primers, HisDF, TCO89DF and TCO89DR. A 2.1-kb DNA fragment from wild-type CaTCO89 was amplified from genomic DNA of both the RM1000 and the heterozygous mutant TJU2 strains (lanes 1 and 2), but not from genomic DNA of the homozygous mutant TJU4 strain (lane 3). However, a 1.3-kb fragment from the tco89ΔhisG mutant allele was amplified from genomic DNA of both the homozygous mutant TJU4 and the heterozygous mutant TJU2 strains (lanes 3 and 2), but not from genomic DNA of the RM1000 strain (lane 1).

Results

ScTco89p has a sequence homolog in C. albicans but they are divergent in amino acid sequence

From the C. albicans genomic database (http://www.candidagenome.org), we obtained a C. albicans sequence homolog (CaO19.761) of ScTCO89 with the ScTco89p amino acid sequence as query. The ORF of CaO19.761 is 2127 bp in length, encoding a protein of 708 amino acids (Fig. 1). Sequence alignment indicates that CaO19.761 protein shows only an overall 15% sequence identity and 26% similarity with ScTco89p (Fig. 3). Interestingly, those identical residues are scattered over the entire amino acid sequence, and no region with a stretch of more than five continuous identical residues exists between the two proteins (Fig. 3). Furthermore, through blast against the GenBank database, we found that only three homologous sequences from C. glabrata CBS138 (GenBank accession number CAG58144), Ashbya gossypii ATCC 10895 (accession number AAS53318) and Kluyveromyces lactis (accession number XP_454803) showed excellent E-values, 7e-42, 5e-38 and 1e-28, respectively, with ScTco89p, and CaO19.761 only showed an E-value of 3.7. It is not surprising to find the homologous sequence for ScTco89p in the genome of Ash. gossypii, because more than 90% of Ash. gossypii genes (4718 in total) show both homology and a particular pattern of synteny with S. cerevisiae (Dietrich, 2004). These results suggest that the amino acid sequence of the C. albicans homolog CaO19.761 is divergent from ScTco89p.

3

Amino acid sequence comparison of CaTco89p with other ScTco89p sequence homologs. The amino acid sequences of Candida albicans CaO19.761 (CaTco89p), ScTco89p and its homologous sequences from Candida glabrata (CgTco89p), Ashbya gossypii (AgTco89p) and Kluyveromyces lactis (KlTco89p) were aligned with the NTI program. The amino acid sequences are numbered on the right. Identical amino acids are shadowed in black, and conserved substitution residues are shadowed in gray. A glutamine-rich motif from Q366 to Q416 in CaTco89p is underlined under its sequence. Nuclear localization signals [pat4 (K132PRR), pat7 (P133RRSKST) and pat7 (P308YDKEKK)] and a mitochondrial presequence motif of the R-10 type (F29RQ FS) in ScTco89p are also underlined under their sequences.

CaTCO89 rescues the cellular integrity defect and rapamycin sensitivity caused by deletion of ScTCO89

Deletion of ScTCO89 causes yeast cells to have a defect in cellular integrity (Reinke, 2004). To test the functionality of CaO19.761, we transformed pRS316-CaTCO89 into the S. cerevisiae tco89Δ cells. Growth of tco89Δ cells is sensitive to a temperature of 40°C [plate (b) in the top panel of Fig. 4], which is consistent with previous observations (Reinke, 2004). This temperature sensitivity could be rescued by introduction of pRS316-CaTCO89 [plate (b) in the top panel of Fig. 4] or by addition of 1 M sorbitol to the growth medium [plate (c) in the top panel of Fig. 4]. Taken together, these results suggest that CaO19.761 could complement the function of ScTCO89 in maintaining cellular integrity.

4

CaTCO89 is a functional homolog of ScTCO89. Top panel: CaTCO89 reverses the cell-wall integrity defect of tco89Δ cells. (a) BY4741 and tco89Δ cells containing pRS316 vector, as well as tco89Δ cells containing pRS316-CaTCO89, were streaked onto YPD medium and grown at 30°C (a) or at 40°C in the absence (b) or the presence (c) of 1 M sorbitol. Middle panel: partial complementation of ScTCO89 function in rapamycin sensitivity by CaTCO89. BY4741 or tco89Δ cells containing pRS316 vector, as well as tco89Δ cells containing pRS316-CaTCO89, were serially diluted and spotted onto YPD plates in the absence (a) or the presence of 3 ng mL−1 rapamycin (b) and grown at 30°C. Bottom panel: lithium sensitivity of tco89Δ cells is rescued by CaTCO89. BY4741 or tco89Δ cells with pRS316, as well as tco89Δ cells containing pRS316-CaTCO89, were streaked onto YPD medium (a), and grown at 30°C in the absence (b) or presence of 0.2 M LiCl (c) or 0.4 M LiCl (d).

Deletion of ScTCO89 also causes yeast cells to be hypersensitive to rapamycin (Reinke, 2004; Wullschleger, 2005). We tested whether CaO19.761 could complement the function of ScTCO89 in this regard. As expected, tco89Δ cells were hypersensitive to rapamycin in comparison to the wild-type cells [spot array (b) in the middle panel of Fig. 4]. However, after CaTCO89 was introduced into tco89Δ cells, this hypersensitivity was rescued, albeit partially [spot array (b) in the middle panel of Fig. 4]. Taken together, these results suggest that the cellular integrity defect and rapamycin hypersensitivity due to the deletion of ScTC089 could be rescued by CaO19.761. Therefore, CaO19.761 is a functional homolog of ScTC089, and is designated CaTC089 hereafter.

Saccharomyces cerevisiae cells deleted for ScTCO89 are sensitive to salt stress, and this sensitivity can be rescued by CaTCO89

ScTOR1 is required for yeast cell growth under saline stress, as the TOR signaling pathway controls the transcription of ENA1, which encodes a lithium and sodium transporter in yeast (Crespo, 2001). Deletion of ScTOR1 or ScTCO89 causes yeast cells to have similar defects in cellular integrity (Reinke, 2004). In addition, the C-terminus of ScTco89p has been shown to be important for its function in glycerol uptake-mediated recovery from salt stress (Holst, 2000). Therefore, we tested whether deletion of ScTCO89 could also cause yeast cells to be sensitive to salt stress. In addition to being hypersensitive to rapamycin [plate (b) in Fig. 5], tco89Δ cells were sensitive to 0.3 M lithium and 1 M NaCl [plates (c) and (d) in Fig. 5], but not to 1 M KCl and 1 M sorbitol [plates (e) and (f) in Fig. 5]. These sensitivities could be reversed by the introduction of ScTCO89 back to tco89Δ cells (Fig. 5). Taken together, these results indicate that ScTCO89 is required for yeast cell growth under saline stress, but that this function is independent of the HOG pathway.

5

Sensitivity of Saccharomyces cerevisiae tco89Δ cells to salt stress. BY4741 cells containing pRS316 vector and tco89Δ cells containing pRS316 or pRS316-ScTCO89 were serially diluted 10-fold, spotted onto YPD plates, YPD plates containing 3 ng mL−1 rapamycin, YPD plates containing 0.3 M LiCl, YPD plates containing 1 M NaCl, YPD plates containing 1 M KCl and YPD plates containing 1 M sorbitol, and grown at 30°C for 3–5 days.

Next, we examined whether CaTCO89 could complement the function of ScTCO89 in salt sensitivity. To do so, we introduced CaTCO89 into tco89Δ cells. We found that lithium sensitivity of tco89Δ cells was reversed by CaTCO89 [plates (c) and (d) in the bottom panel of Fig. 4]. This suggests that CaTCO89 could complement the function of ScTCO89 in salt stress.

Deletion of CaTCO89 renders C. albicans cells sensitive to rapamycin and lithium

To examine the functions of CaTCO89, we subsequently disrupted the two alleles of CaTCO89 in C. albicans. Disruption for CaTCO89 did not affect cell growth in YPD (left plate in Fig. 6), which indicates that CaTCO89 is not required for cell survival in C. albicans. Candida cells disrupted for both alleles of CaTCO89 were not susceptible to challenges to cellular integrity (data not shown). However, the heterozygous cells disrupted for one allele of CaTCO89 were sensitive to 10 ng mL−1 rapamycin in comparison to the wild-type cells, and the homozygous deletion mutant cells were even more sensitive to rapamycin than the heterozygous deletion mutant cells (middle plate in Fig. 6). The rapamycin sensitivity of both the heterozygous and the homozygous mutant cells could be reversed by introduction of CaTCO89 back to the mutants (Fig. 6).

6

Deletion of CaTCO89 renders Candida albicans cells sensitive to rapamycin and lithium. Plasmid pCR4-CaTCO89 (or the pCR4 vector as control) was transformed into the RM1000 (CaTCO89/CaTCO89), the TJU2 (CaTCO89/catco89) and the TJU4 (catco89/catco89) strains, respectively, for experimental analysis. RM1000 cells containing the pCR4 vector, and TJU2 and TJU4 cells containing pCR4 or pCR4-CaTCO89, were serially diluted and spotted on YPD (left plate), YPD containing 10 ng mL−1 rapamycin (middle plate), or YPD containing 0.4 M LiCl (right plate), and grown at 30°C.

In comparison to the wild-type cells, cells that were heterozygous for CaTCO89 were not sensitive to 0.4 M LiCl, but the homozygous mutant cells were (right plate in Fig. 6). Introduction of CaTCO89 back to the homozygous mutant not only reversed the lithium sensitivity of the homozygous mutant cells, but also increased the cell growth of the homozygous mutant, as manifested by the greater colony size for the homozygous mutant in comparison to the wild-type strain (bottom row in the right plate of Fig. 6). However, this effect was not observed in the heterozygous mutant cells containing the pCR4-CaTCO89 plasmid (third row from the top in the right plate of Fig. 6) and in the wild-type cells containing the pCR4-CaTCO89 plasmid (data not shown).

Discussion

Recent studies have shown that there are two functionally distinct TOR complexes, TORC1 and TORC2, in S. cerevisiae (Loewith, 2002; Reinke, 2004; Wullschleger 2005). As one of the TORC1 components in S. cerevisiae, ScTco89p is involved in multiple cellular functions. Deletion of ScTCO89 causes yeast cells to be hypersensitive to rapamycin and susceptible to challenges to cellular integrity (Reinke, 2004). In agreement with a previous observation on ScTOR1 (Crespo, 2001), we here provide evidence that deletion of ScTCO89 also causes yeast cells to be hypersensitive to salt stress, which is consistent with its role of encoding ScTco89p as one of the TORC1 components.

Rapamycin acts on C. albicans cells through conserved components of TOR signaling, Candida homologs of TOR, and FKBP12 proteins (Ferrara, 1992; Cruz, 1999). In the present study, we have shown that C. albicans cells disrupted for CaTCO89 are hypersensitive to rapamycin, which suggests its possible role in TOR signaling in C. albicans. Similar to ScTCO89 function in salt sensitivity in S. cerevisiae, C. albicans cells disrupted for CaTCO89 are sensitive to lithium stress, but not susceptible to challenges to cellular integrity (data not shown). In addition, CaTCO89 could fully complement the salt sensitivity function and partially complement the rapamycin sensitivity function of ScTCO89, which indicates that CaTCO89 and ScTCO89 are functional homologs. It is very interesting to note that ectopic expression of CaTCO89 in the pCR4 plasmid vector promotes Candida cell growth in the homozygous mutant cells, but not in the heterozygous mutant and wild-type cells, under lithium stress condition. However, this effect of CaTCO89 has not been observed in those mutants under treatment with rapamycin. We have also introduced ScTCO89 in pRS316 or pRS426 plasmid vectors into mutant cells homozygous and heterozygous for ScTCO89, but we did not observe a similar effect for the ectopic expression of ScTCO89 in S. cerevisiae (data not shown). This suggests that, unlike ScTCO89, CaTCO89 might have another function in addition to being one of the components in the TOR complex in C. albicans.

CaTco89p shows only limited identity with ScTco89p at the amino acid level, which might be the reason why CaTCO89 could only partially complement the function of ScTCO89 in rapamycin hypersensitivity. However, it could fully reverse the cellular integrity defect and salt sensitivity of S. cerevisiae tco89Δcells. CaTco89p might form a conformational structure similar to that of ScTco89p, as certain sequence motifs are conserved in these homologous proteins (Fig. 3). The secondary conformation of Tco89p might be critical for its function in fungal cells, which is the case for other regulatory proteins, such as the members of the type 2C protein phosphatase (PP2C) family (Bork, 1996; Jiang, 2001). Nevertheless, CaTco89p showed 25% identity and 48% similarity within the C-terminal region (S484–S608) of ScTco89p, which is also conserved in other homologs of ScTco89p (Fig. 3). The C-terminal conserved P777XTRA motif could be important for ScTco89p function in glycerol uptake, as the mutant BHY21 with a transposon inserted immediately after L775 in ScTco89p (YPL180w) lacked glycerol uptake-mediated recovery from salt stress (Holst, 2000). It is also interesting to note that there are 57 glutamine residues, with almost half of them grouping at a glutamine-rich motif from Q366 to Q416 in CaTco89p (Fig. 3). In contrast, there are only 38 glutamine residues scattered over the entire amino acid sequence in ScTco89p (Fig. 3). Glutamine-rich domains exist in many transcription factors and are involved in recruiting interacting proteins (Chen & Courey, 2000; Johannessen, 2004). Therefore, it is conceivable that the glutamine-rich region in CaTco89p might play a role in its function in C. albicans cells. Nuclear localization signals of the classical type (Hicks & Raikhel, 1995) are predicted in ScTco89p–pat4 (K132PRR), pat7 (P133RRSKST) and pat7 (P308YDKEKK) – by the PSORT WWW Server (http://psort.hgc.jp), and so is a mitochondrial presequence motif of the R-10 type (F29RQ FS), with a cleavage site at F40 in ScTco89p (Fig. 3). Nevertheless, no possible vacuolar targeting motif was predicted for both CaTco89p and ScTco89p, although ScTco89p was observed in the vacuolar membrane via green fluorescent protein tagging and immunologic electronic microscopy (Huh, 2003; Reinke, 2004).

As one of the TORC1 components, Tco89p might be a potential target for therapeutic drugs to control candidiasis caused by the human yeast pathogens C. albicans and C. glabrata, as Tco89p sequence homologs exist only in a few fungal species, and not in the mammals whose genomic sequences are available in public. Therefore, we will carry out further experiments to examine the phenotypes of the heterozygous and homozygous mutants of CaTCO89 in hyphal growth and virulence. In addition, antifungal activity of rapamycin is observed in other fungi, including C. neoformans, C. stelloidea, Aspergillus fumigatus, Asp. flavus, Asp. niger, Fusarium oxysporum, and Penicillium sp. (Odom, 1997; Wong, 1998; Fang, 2000; Cruz, 2001). Understanding the mechanisms of antifungal activity of rapamycin could help us to develop novel drugs for use against diseases caused by pathogenic fungi.

Acknowledgements

We wish to thank Haihua Ruan and Jihong Wang for their valuable advice and help during the experiment. This work was supported partially by both the National Science Foundation of China (grant no. 30470850 to L. Jiang) and the National Basic Science Program (973) (grant no. 2005CB523105 to Yuan-Ying Jiang).

References

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