1 Molecular Genetics Program, Wadsworth Center, New York State Department of Health, State University of New York, Albany, NY 12208, USA
2 Mycology Laboratory, Wadsworth Center, New York State Department of Health, State University of New York, Albany, NY 12208, USA
3 Department of Biomedical Sciences, School of Public Health, State University of New York, Albany, NY 12208, USA
Correspondence
Steven D. Hanes
hanes{at}wadsworth.org
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ABSTRACT |
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The GenBank/EMBL/DDBJ accession number for the sequence reported in this paper is AF533511.
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INTRODUCTION |
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Peptidyl-prolyl cis/trans isomerases (PPIases) catalyse the interconversion between cis and trans forms of the peptide bond preceding proline residues in proteins (Fischer, 1994; Fischer et al., 1998
; Schiene & Fischer, 2000
). Conformational isomerization by PPIases is thought to control the activity of target proteins and their ability to interact with other proteins to form complexes (Schmid et al., 1993
; Hunter, 1998
). Three families of PPIases that differ in structure and substrate specificity are known: the cyclophilins, the FK506-binding proteins (FKBPs) and the parvulins (Dolinski & Heitman, 1997
; Arévalo-Rodríguez et al., 2004
). The PPIase activity of cyclophilin A is potently inhibited by the immunosuppressive drug cyclosporin A (CsA) (Handschumacher et al., 1984
; Takahashi et al., 1989
). In C. neoformans, there are two cyclophilin A homologues, encoded by linked genes, CPA1 and CPA2 (Wang et al., 2001
). Mutations in CPA1 and CPA2 confer a spectrum of cell growth, mating and virulence phenotypes that indicate the homologues have distinct but overlapping roles in C. neoformans (Wang et al., 2001
). The second class of PPIases, the FKBPs, are inhibited by the immunosuppressants FK506 and rapamycin, drugs which also show antifungal activity. In C. neoformans, disruption of the FKBP12 gene confers rapamycin and FK506 resistance but has no effect on growth, differentiation or virulence of C. neoformans (Cruz et al., 1999
, 2001
). Prior to this report, no parvulin-class PPIases had to our knowledge been identified in C. neoformans.
Parvulin-class PPIases are named after an Escherichia coli protein called parvulin (Rahfeld et al., 1994). The first eukaryotic parvulin to be discovered was the Ess1 protein from Saccharomyces cerevisiae (Hanes et al., 1989
; Hani et al., 1995
). Ess1 is the only PPIase that is essential for growth in S. cerevisiae (Hanes et al., 1989
; Dolinski et al., 1997
). Cells depleted of Ess1 arrest in mitosis and undergo nuclear fragmentation (Lu et al., 1996
; Wu et al., 2000
). Some evidence suggests that Ess1 and its homologue in humans, Pin1, interact with cell cycle proteins to control mitotic progression (Crenshaw et al., 1998
; Shen et al., 1998
; Stukenberg & Kirschner, 2001
), while other evidence shows that Ess1 binds to the carboxy-terminal domain (CTD) of the large subunit of RNA polymerase II and is important for mRNA transcription (Morris et al., 1999
; Wu et al., 2000
, 2001
). It is thought that Ess1-mediated isomerization of the CTD controls multiple steps in transcription, including initiation and elongation, and pre-mRNA processing (Shaw, 2002
; Wu et al., 2003
; Xu et al., 2003
; Wilcox et al., 2004
).
Ess1 homologues have been studied in metazoans such as Drosophila melanogaster (Maleszka et al., 1996; Hsu et al., 2001
), Xenopus (Winkler et al., 2000
), mice and humans (Fujimori et al., 1999
; Lu et al., 1996
). In most of these organisms, Ess1/Pin1 is not essential for growth. However, in two other fungi, Candida albicans and Aspergillus nidulans, Ess1 was shown to be essential (Devasahayam et al., 2002
; Joseph et al., 2004
). In Ca. albicans, mutants with a reduced gene dosage showed defects in filamentation (Devasahayam et al., 2002
) and reduced virulence in a mouse model of candidiasis (Li et al., 2005
). Here, we sought to identify an Ess1/Pin1 homologue in C. neoformans, a distantly related human fungal pathogen, and to characterize its requirement for growth, differentiation and virulence. Unlike the case for S. cerevisiae and Ca. albicans, disruption of ESS1 in C. neoformans was not lethal. No defects were observed in growth rate, capsule formation, response to mating pheromones, or haploid fruiting. However, ess1
mutants were avirulent when tested in a murine model of cryptococcosis, and showed reduced levels of melanin production and urease activity, factors known to be important for virulence (Salas et al., 1996
; Cox et al., 2000
). These results, together with studies in Ca. albicans, suggest that the Ess1 PPIase might be a useful target for the development of broad-spectrum antifungal drugs.
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METHODS |
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S. cerevisiae strain construction.
To construct haploid strain YPR-57 (ess1 : : HIS3 pGD-CaESS1), a high-copy plasmid carrying Ca. albicans ESS1, pGD-CaESS1 (URA3) (Devasahayam et al., 2002
), was transformed into the heterozygous ESS1/ess1
: : HIS3 (YSH-55) strain. Cells were induced to sporulate, tetrads dissected, and His+ Ura+ segregants were selected. To generate YPR-34 (ess1
: : URA3/ESS1), a PCR product of the URA3 gene flanked by 46 nucleotides of homology to 5' and 3' ESS1 flanking sequences was transformed into diploid S. cerevisiae strain W303-1AxB and uracil prototrophs were selected. The ess1 : : URA3 disruption was confirmed by PCR.
Complementation experiments.
The temperature-sensitive S. cerevisiae strain ess1H164R (YGD-ts22W; Wu et al., 2000) was transformed with the plasmid pCnESS1-
I, a positive-control vector (pRS424-ESS1, which contains a BamHIXhoI fragment of S. cerevisiae ESS1 from plasmid pRS413-ESS1), or vector alone (pJG4-1
E), and the ability of cells to grow at the restrictive temperature (37 °C) was tested by streaking on appropriate solid media. For plasmid-curing experiments, a haploid S. cerevisiae ess1 deletion strain, YPR-57, carrying a plasmid-borne copy of ESS1 was transformed with pCnESS1-
I. Transformants were serially passaged in liquid CSM lacking tryptophan for 3 days, i.e. selecting for the pCnESS1-
I (TRP1) but not for the pGD-CaESS1 (URA3) plasmid. Cells were plated and the phenotypes of individual colonies were scored by replica-plating to appropriate selective media. For segregation analysis, the heterozygous disruption strain YPR-34 (ess1
: : URA3/ESS1) was transformed with pCnESS1-
I. Cells were induced to undergo sporulation on 1 % potassium acetate plates, tetrads were dissected and haploid segregants were grown on rich medium. Growth was scored after 3 days, and segregation of the Ura+ and Trp+ phenotypes monitored by replica-plating to detect presence of the ess1 : : URA3 disruption and the pCnESS1-
I plasmid (TRP1) respectively.
Disruption of ESS1 in C. neoformans and reconstitution of mutant strains.
The ESS1 gene was disrupted by homologous recombination using an ess1 : : URA5 disruption allele. To generate the disruption allele the following strategy was used. A 2·0 kb fragment containing the C. neoformans URA5 gene was PCR amplified from C. neoformans B-3501 genomic DNA. The primers used incorporated SpeI and MfeI sites at the 5' and 3' ends of URA5, respectively. The fragment was digested with these enzymes and cloned into the same sites of plasmid pUC-CnESS1-SpeI. pUC-CnESS1-SpeI contains a modified version of ESS1 in which the initiator codon was destroyed and replaced with stop codons in all three frames and included a SpeI cloning site (P. Ren & S. D. Hanes, unpublished). The final disruption construct, pUC-Cness1-URA5, replaces the first 124 nucleotides of the ESS1 ORF with the URA5 gene. To use this disruption construct, a 4·1 kb fragment was released by EcoRI and XbaI digestion and used for biolistic transformation of JEC43. Uracil-prototrophic transformants were selected and colony purified. Approximately 60 transformants per µg DNA were obtained. As determined by PCR and Southern analysis, about 1 % of the transformants carried homologous gene replacements at the ESS1 locus, resulting in strain CnPR68.
A CnPR68 ura5 prototrophic revertant, CnPR37, was obtained by counterselection using 5-fluoroorotic acid (5-FOA) medium (Kwon-Chung et al., 1992b). Strain CnPR37 was used to generate a reconstituted strain by biolistic transformation with plasmid pCn-tel-CnESS1 that was linearized with NotI. pCn-tel-CnESS1 was constructed by insertion of a 2·1 kb EcoRIBamHI fragment (consisting of 1·4 kb of upstream untranslated region plus 0·7 kb of the C. neoformans ESS1 gene) into plasmid pCn-tel1. pCn-tel contains a URA5 selection marker and was kindly supplied by Ping Wang and Joseph Heitman (Duke University, Durham, NC, USA; Davidson et al., 2000
; Edman & Kwon-Chung, 1990
). The 2·1 kb fragment had been obtained by PCR from C. neoformans B-3501 genomic DNA. The DNA sequence of the C. neoformans ESS1 gene in this construct was confirmed by sequence analysis. In the reconstituted strain, CnPR170, the NotI-linearized pCn-tel-CnESS1 was integrated at random into the genome (i.e. not at the ess1 : : ura5 locus). The integrated copy of ESS1 contained its own promoter region so that it could be expressed normally. The presence of intact ESS1 in the reconstituted strain was confirmed using four PCR reactions with different sets of primers.
Cell morphology, capsule formation, melanin production and urease biosynthesis assays.
To observe the cell morphology, cells were incubated in YPD broth at 25 °C to mid-exponential phase and photographed at x400 magnification using a compound microscope equipped with a digital camera. For capsule formation, the wild-type, ess1 mutant and ess1+ESS1 reconstituted strains were inoculated into 10 ml limited-iron medium with 100 µM EDTA and 100 µM bathophenanthroline disulphonic acid, and incubated at 30 °C for 5 to 7 days with agitation. Cells from these cultures were mixed with a standard India ink preparation and photographed (x400 magnification). To detect melanin or urease production, wild-type, ess1 mutant and ess1+ESS1 reconstituted strains were freshly cultured to mid-exponential phase in YPD broth, and 5 µl of each culture was spotted onto Niger seed agar medium and incubated for 4 days at 30 °C, or onto urea agar medium for 2 days at 30 °C, respectively.
Virulence in a mouse model of cryptococcal meningitis.
Cells of wild-type strain (JEC21), the ess1 mutant strain (CnPR68) and the reconstituted strain (CnPR170) were grown to mid-exponential phase in liquid YPD medium and washed and resuspended in 15 mM PBS. BALB/c male mice (Jackson Laboratory, Bar Harbour, ME; USA, six in a group for each strain) weighing 2025 g (about 68 weeks old) obtained from the Griffin Laboratory (Wadsworth Center) were infected by lateral tail vein injection using 100 µl C. neoformans at 107 cells ml1. The cell concentrations were determined using a haemocytometer before injection and confirmed by plating 100 µl of 104 dilutions of cell suspensions onto YPD plates. Mice were monitored once daily and those that were moribund or in apparent discomfort were sacrificed by CO2 inhalation. Survival was analysed using the KaplanMeier method with SAS software, version 6.12 (SAS Institute, Cary, NC, USA). Brain tissue pieces dissected from the dead mice were spread on Niger seed agar medium and incubated at 30 °C for 5 to 7 days to detect the presence of C. neoformans by microscopic examination after staining with India ink. To confirm the presence of the ess1 : : URA5 disruption allele in strains from infected brains, colony isolates were subjected to diagnostic PCR.
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RESULTS |
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To examine the evolutionary relatedness of the predicted C. neoformans Ess1 protein to Ess1/Pin1 homologues in other organisms, the protein sequences were analysed using the neighbour-joining method. We chose seven other fungal and metazoan species for which at least some functional data on Ess1/Pin1 exist. In the resulting phylogenetic tree (Fig. 1b), the Ess1/Pin1 sequences clearly show the fungal clades to be distinct from those of the fly and mammals. Within the four ascomycetes species, S. cerevisiae and Ca. albicans were grouped in one clade and Schizosaccharomyces pombe and A. nidulans were grouped in another clade. That C. neoformans (a basidiomycete) appeared as a monophyletic group, a sister to the other four fungal species, was as expected. These results support the idea that C. neoformans Ess1 is homologous to Ess1/Pin1 proteins from other organisms.
C. neoformans ESS1 is the functional homologue of S. cerevisiae ESS1
Although the primary sequence features suggest that C. neoformans ESS1 encodes a homologue of Ess1/Pin1, it was necessary to demonstrate a conserved function. To do this, we tested whether C. neoformans ESS1 would functionally complement S. cerevisiae ess1 mutants. This was done using several different methods. After the two introns were deleted from the original isolate of the C. neoformans ESS1 gene (see Methods), the intron-less version, carried on an episomal plasmid (pCnESS1-I), was introduced into a conditional-lethal strain of S. cerevisiae (ess1H164R; Wu et al., 2000
). This strain cannot grow at the restrictive temperature of 37 °C due to a mutation in ESS1 (ess1H164R) that renders it temperature-sensitive. The no-growth phenotype of the S. cerevisiae temperature-sensitive strain was fully complemented at the restrictive temperature (37 °C) by the C. neoformans ESS1 gene (data not shown).
While these results are highly suggestive of functional homology, it could be argued that the C. neoformans ESS1-encoded protein (Ess1) was simply stabilizing or restoring the activity of the mutant ess1H164R protein. To rule out this possibility, we tested whether C. neoformans ESS1 could complement S. cerevisiae strains bearing a complete deletion of the ESS1 gene. Since ESS1 is essential in S. cerevisiae, certain manipulations were necessary to create the appropriate genetic background. First, the pCnESS1-I plasmid was introduced into a heterozygous ess1 mutant strain of S. cerevisiae (ess1
: : URA3/ESS1). The diploid cells were induced to undergo sporulation and the resulting tetrads were dissected. As expected, cells transformed with the vector alone showed a 2 : 2 segregation for viable : inviable spores (Table 2
). In contrast, about half of the tetrads derived from diploids transformed with the pCnESS1-
I plasmid yielded a 4 : 0 segregation of viable : inviable spores, indicating that the C. neoformans ESS1 gene complements ess1
haploid cells to allow spore outgrowth and cell viability. Note that not all of these tetrads showed 4 : 0 segregation for viability. This was presumably due to failure of the plasmid to be uniformly maintained during sporulation, a commonly observed phenomenon. As expected, all of the cells that carried the ess1 : : URA3 disruption (i.e. were Ura+) also carried the pCnESS1-
I plasmid (i.e. were Trp+; data not shown).
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The reconstituted strain CnPR170 expressed ESS1 mRNA, but at a much lower level than that of wild-type cells (Fig. 2b, right panel). Other reconstituted strain isolates did not show detectable ESS1 mRNA (data not shown). The reason for the low expression is not known, but might be due to missing 5' or 3' regulatory sequences (or introns) in the construct we used, or to local repressive effects at the site(s) of insertion. The finding that even low levels of ESS1 expression in CnPR170 can rescue certain in vitro and in vivo phenotypes (see below) may not be surprising given results in S. cerevisiae, which show that Ess1 protein is present in vast excess over the levels required for its essential function (Gemmill et al., 2005
).
ess1 mutants show normal growth, morphology and capsule formation
We next compared the C. neoformans mutants lacking a functional ESS1 gene with control strains (wild-type and the reconstituted strain) for general growth properties and the ability to express a standard set of virulence-associated traits. First, we examined the growth rate since slow-growing cells might not be expected to retain virulence in a host organism. The growth rate of ess1 mutants at three different temperatures appeared to be the same compared to the wild-type and the reconstituted strains (Fig. 3a). This can best be seen by comparing the slopes over the 34 log units that represent the linear portion of the growth curves. However, at higher temperatures (30 °C and 37 °C) the ess1 mutant cells appeared to reach saturation at slightly lower concentrations than did the wild-type or reconstituted strains. Second, cells were examined microscopically for possible defects in overall morphology. No obvious defects could be discerned; cells still appeared normal in size and shape (Fig. 3b
, upper panels). Third, we assayed the ability of ess1 mutants to undergo capsule formation, a key differentiation step required for virulence in vivo. Cells were induced to form capsules by growth on standard inducing medium and then stained with India ink. No defects in capsule formation were observed; the size of the capsules appeared to be the same between mutant and control strains (Fig. 3b
, lower panels). Finally, we tested for the ability of ess1 mutants to undergo mating and haploid fruiting, and to generate pheromone-induced conjugation tubes. No changes compared to control strains were observed (data not shown). Thus, ess1 mutant cells behaved normally for several basic properties of growth and differentiation.
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Production of the enzyme urease, which is known to be required for virulence in animal models (Cox et al., 2000), was also reduced in ess1 mutant cells. A spot-test assay for urease activity showed that the ess1 mutant strains produce a much smaller zone of substrate utilization than do the wild-type or the reconstituted strain (Fig. 4b
). Consistent with this decrease in activity, expression of the URE1 gene, which encodes urease, is reduced slightly (about twofold) in ess1 mutants vs wild-type cells (Fig. 4c
, lower panel). As expected, URE1 levels were partially restored in the reconstituted strain.
In addition to direct transcriptional effects on LAC1 and URE1 genes, mutation of ESS1 might affect laccase and urease enzyme activity indirectly, for example, by affecting expression or function of genes such as VPH1, which encodes an intracellular vesicular proton pump. Both laccase and urease are metalloenzymes whose activities are greatly reduced in vph1 mutants (Erickson et al., 2001). The vph1 defect can be overcome by the addition of excess copper, which, for example, restores laccase activity allowing melanin production (Zhu et al., 2003
). Addition of copper sulphate did not seem to restore melanin production to ess1 mutants (Fig. 4a
, lower panel), indicating that the defect is not due to post-transcriptional defects in laccase metallation. The results suggest that VPH1 may not be affected in ess1 mutants, consistent with our finding that ess1 mutants generate normal-looking capsules, whereas no capsules are formed in vph1 mutants. Thus, some other defect(s) must occur in the pathway leading to melanin production.
These results and those shown in Fig. 3 show that some, but not all, of the standard virulence factors in C. neoformans are affected by disruption of the ESS1 gene. Note that the levels of melanin and urease production are not completely restored in the reconstituted strain. This was not surprising given that the ESS1 mRNA levels in the reconstituted strain were much lower than in the wild-type (Fig. 2b
).
ESS1 is required for virulence
We tested if ESS1 is required for virulence of C. neoformans in a murine model of cryptococcosis. Each BALB/c animal was infected by lateral tail vein injection with 106 C. neoformans cells. The mean survival of these mice with the wild-type serotype D strain JEC21 was 56 days and all injected mice were moribund or dead after 72 days. In contrast, all mice injected with the ess1 mutant strain were still viable with no signs of sickness even after 100 days, at which time the experiment was ended. These results indicate that the virulence of the ess1 mutant strain is severely attenuated compared with the wild-type (KaplanMeier analysis log-rank, P<0·001). Consistent with the idea that the loss of virulence is due to the loss of ESS1 function, virulence was largely restored in the reconstituted strain (ess1 : : ura5+ESS1) compared with the mutant (P<0·001). Results showed that the mean survival of infection of the mice injected with the reconstituted strain was 74 days, and that all mice were moribund or dead after 80 days (Fig. 5). In summary, the results indicate that the C. neoformans ESS1 gene is required for virulence in a murine model for cryptococcosis.
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Ess1 and cyclophilins may have some overlapping functions
Previous work in S. cerevisiae had shown that overexpression of cyclophilin A suppresses ess1 mutations, and that under some conditions ess1 mutants are hypersensitive to the effects of the CsA, an inhibitor of cyclophilin A (Arévalo-Rodríguez et al., 2000; Wu et al., 2000
). C. neoformans is known to have two cyclophilin A homologues (Wang et al., 2001
). We therefore tested whether CsA affected the growth of C. neoformans ess1 mutant cells. As shown in Fig. 6(a)
, CsA strongly inhibited the growth of ess1 mutant cells as compared to wild-type control cells. At least some of the inhibitory effects of CsA were reversed by adding back a wild-type copy of the ESS1 gene, as in the reconstituted strain. We obtained similar results at 28 °C (data not shown). In this plate assay, ess1 mutant cells grew slightly slower than the wild-type or the reconstituted strain, even without the addition of drug. However, the magnitude of this difference does not fully account for the CsA inhibitory effect. Note that this experiment was conducted at 25 °C because calcineurin (a target of cyclophilins) becomes essential at 3037 °C, and therefore CsA inhibits cell growth even in wild-type cells at elevated temperature (Odom et al., 1997
).
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DISCUSSION |
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The mechanism by which the Ess1 protein contributes to virulence in C. neoformans is not yet known. Our findings showed that mice infected with the ess1 mutant continued to carry at least some viable C. neoformans cells, despite having no clinical signs of illness. These results are intriguing and indicate that ess1 mutant cells can still colonize the brain, at least to some degree, but are unable to cause disease over the time-frame of our experiments (100 days). Thus, we suggest that ess1 mutant cells have defects in differentiated functions related to disease progression, but this idea will require further investigation to confirm.
The exact function of C. neoformans Ess1 is not known. However, studies in the model organism S. cerevisiae would suggest that the molecular nature of the ess1 defect is likely to involve gene-specific changes in transcription regulation. In S. cerevisiae, Ess1 binds to and regulates the function of RNA polymerase II (reviewed by Shaw, 2002; Arévalo-Rodríguez et al., 2004
). It seems plausible, therefore, that expression of certain virulence-associated genes in C. neoformans might be affected by disruption of ESS1. Indeed, expression of URE1, which encodes an enzyme required for synthesis of urease, was reduced (albeit only twofold) in ess1 mutant cells (Fig. 4c
). In contrast, expression of LAC1, which is required for melanin production, showed an unexpected increase in ess1 mutants. We note that it is possible that expression of the actin gene, which was used as a control, might also vary in ess1 mutants. A more complete study of gene expression of virulence factors affected by ess1 deletion could be accomplished by microarray analysis using the C. neoformans strains generated in this study.
ESS1 was first identified as a gene essential for growth in S. cerevisiae (Hanes et al., 1989). However, in addition to its non-essentiality in C. neoformans, ESS1 homologues are not essential in several other organisms (reviewed by Arévalo-Rodríguez et al., 2004
), including fungi such as Sch. pombe (Huang et al., 2001
). While functional studies of ESS1/PIN1 have been undertaken only in a limited number of organisms, phylogenetic analysis using this small dataset (Fig. 1b
) reveals that within the ascomycetes group, fungi are divided into two subgroups, one in which Ess1 is essential and one in which it is not essential. Since C. neoformans is a basidiomycete and is outside this group, perhaps it should not have been surprising that ESS1 is not essential in this organism.
One explanation for the fact that ESS1 is essential in some organisms but not others is that under some circumstances, cyclophilin A can substitute for Ess1 (Arévalo-Rodríguez et al., 2000; Wu et al., 2000
). These and other studies indicate that Ess1 and cyclophilin A exhibit crosstalk, i.e. that they possess partially overlapping functions (Fujimori et al., 2001
). C. neoformans is very unusual in that it has two distinct cyclophilin A homologues, Cpa1 and Cpa2 (Wang et al., 2001
). It is possible, therefore, that in ess1 mutant strains, the two cyclophilin A homologues might compensate for many, although not all, of the functions of Ess1 lost by gene disruption. Such an overlap in function might explain why ESS1 is not essential for growth in C. neoformans, despite playing an important role in virulence. Consistent with this idea, ess1 mutant cells were more sensitive than wild-type cells to the cyclophilin inhibitor CsA.
Interestingly, both ess1 and cpa1 cpa2 double mutants are defective in melanin synthesis, and in virulence in a mouse model (this study; Wang et al., 2001). These similarities, and the fact that both Ess1 and cyclophilin A homologues have been implicated in transcription (Arévalo-Rodríguez et al., 2000
, 2004
; Wu et al., 2000
), support the idea of functional overlap. However, there are important differences. For example, unlike ess1 mutants, cpa1 cpa2 double mutants are also defective in capsule formation and mating, and are resistant to the effects of CsA (Wang et al., 2001
). Thus, while some pathway overlap probably occurs, Ess1 and cyclophilins clearly have distinct functions in vivo. Further genetic analysis would be useful to investigate these differences.
Based on studies using another human fungal pathogen, Candida albicans, we proposed that parvulin-class PPIases such as Ess1 might be valuable targets for antifungal drug development (Devasahayam et al., 2002). Indeed, ESS1 appears to be important for virulence of Ca. albicans (G. Devasahayam, V. Chaturvedi & S. D. Hanes, unpublished). The present study demonstrates that ESS1 is also required for virulence of C. neoformans in at least one animal model system. It seems reasonable to predict that inhibitors of ESS1 might attenuate the virulence of C. neoformans in humans. Since Pin1, the vertebrate Ess1 homologue, does not appear to be essential in mammals (Fujimori et al., 1999
), targeting this family of PPIases might be feasible for human therapeutic applications.
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ACKNOWLEDGEMENTS |
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Received 24 November 2004;
revised 20 January 2005;
accepted 24 January 2005.
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