Department of Medicine and Department of Molecular Genetics and Microbiology, Duke University Medical Center, Durham, NC, USA
Correspondence
J. Andrew Alspaugh
andrew.alspaugh{at}duke.edu
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ABSTRACT |
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The GenBank accession number for the C. neoformans RAM1 gene sequence reported in this paper is AY162319.
Present address: Departamento de Biologia Celular, IB, UnB, Brasilia, DF.
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INTRODUCTION |
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In order for small G proteins such as Ras and Rho to localize to the cell membrane and to function properly, they must undergo prenylation, a post-translational modification in which hydrophobic groups are added to the C-terminus of the protein (Casey et al., 1996). Three prenyltransferases have been described in eukaryotic cells that are responsible for these protein modifications: protein farnesyltransferase (FTase) and protein geranylgeranyltransferase (GGTase) types I and II. FTase catalyses the addition of 15-carbon (farnesyl) groups to proteins destined for cell membranes (Reiss et al., 1990
), and GGTases catalyse a similar reaction with 20-carbon (geranylgeranyl) groups (Seabra et al., 1991
). Since activating Ras mutations have been demonstrated in many human malignancies (Schafer et al., 1989
), and since prenylation is required for Ras function (Casey et al., 1989
; Hancock et al., 1989
), these prenyltransferases have been extensively studied as potential targets for cancer chemotherapy (Hill et al., 2000
; Lerner et al., 1995
; Prendergast, 2000
).
FTase and GGTase I are related heterodimeric proteins that consist of a common subunit and distinct
subunits. In Saccharomyces cerevisiae, the RAM2 gene encodes the common
subunit of FTase and GGTase I, and the RAM1 and CDC43 genes encode the
subunits of FTase and GGTase I, respectively (He et al., 1991
; Mayer et al., 1992
). RAM2 and CDC43 are essential genes, in contrast to the FTase
-subunit gene RAM1, which is not essential for growth (He et al., 1991
; Mayer et al., 1992
). However, ram1 mutant strains are not able to grow at 37 °C or survive other stressful conditions (He et al., 1991
). Therefore, for optimal cell growth, both FTase and GGTase I activities are required for S. cerevisiae survival.
We hypothesized that cellular functions that depend on small G proteins in the pathogenic fungus C. neoformans, such as cell polarization, high-temperature growth, and differentiation, would be defective in strains with inhibited prenylation. Here we describe the identification of a gene encoding a homologue of a protein farnesyltransferase subunit in C. neoformans. Using genetic and pharmacologic inhibition of FTase activity, we have investigated the role of this prenyltransferase in the growth and differentiation of this human fungal pathogen.
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METHODS |
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Nucleic acid manipulation.
Prior to DNA or RNA extractions, yeast cells were pelleted and frozen on dry ice, followed by lyophilization. Genomic DNA was extracted as described by Pitkin et al. (1996). Total RNA was extracted using Trizol (Invitrogen), following the manufacturer's instructions.
DNA sequencing.
Sequencing was performed at the Duke DNA Analysis Facility at Duke University.
Northern and Southern blot analysis.
For Northern blots, 15 µg total RNA was separated in a formaldehyde 1·2 % agarose gel and transferred to a nylon membrane. For Southern blots, 5 µg genomic DNA was digested with EcoRV, followed by size fractionation in a 1 % agarose gel and transfer to a nylon membrane (Nytran Super Charge, Schleicher & Schuel). The probe comprised the region of the RAM1 gene amplified by primers AA19 and AA20 (see below), using JEC21 genomic DNA as template. The probe was labelled with [32P]dCTP (New England Nuclear) using the Rediprime II Kit (Amersham). Gel size fractioning, transfer, hybridization and washes for both Northern and Southern blots were performed as described by Sambrook & Russel (2001)
.
Gene deletion.
A portion of the RAM1 gene (nucleotides 2321531, corresponding to amino acid residues 77440) was replaced by the URA5 gene by the PCR overlap-extension technique (Davidson et al., 2002). The primers for the PCR-based construct are shown in Table 1
.
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C. neoformans colony PCR.
Homologous construct integration was verified using one primer located within the RAM1 locus but outside of the ram1 : : URA5 disruption construct (primer AA21, 5'-TTCTTGGCATAGGACCTACGC-3', RAM1 position 1073 to 1053 bp) and one primer located within the URA5 sequence (AA16, 5'-CGCGATATCGTTCTTGATTGGAGGCG-3', URA5 position 147 bp to 122 bp). This allowed an amplicon of 1·2 kb to be generated only from a strain in which the endogenous RAM1 allele was precisely replaced by the ram1 : : URA5 mutant allele. One yeast colony was used as template for a PCR reaction as follows: 1 cycle of 95 °C for 12 min; 35 cycles of 95 °C for 20 s, 56 °C for 20 s, and 72 °C for 4 m; 1 cycle of 72 °C for 10 min.
cDNA amplification and RACE.
The 5' and 3' ends of the RAM1 cDNA were determined by the 5', 3' RACE System for Rapid Amplification of cDNA Ends, Version 2.0 (Gibco). Primer AA138 (5'-CGCTTCGATGGACTGTCCTTCAACAGGG-3') is the gene-specific primer used to determine the 5' end, and primer AA139 (5'-GCGACGAACCCCGGTTGAAGGTAGGTGG-3') is the gene-specific primer used to determine the 3' primer end. The RACE assays were performed according to the manufacturer's instructions.
MIC testing.
MIC testing was performed according to the NCCLS standard assay, with slight modification (Galgiani et al., 1997). Briefly, five C. neoformans colonies grown on a YPD plate for 48 h at 37 °C were inoculated in 5 ml sterile saline (0·85 % NaCl) to an OD530 of 0·23 to 0·27 (about 1x1065x106 cells ml1). A final cell suspension was made in RPMI 1640 plus 2 % glucose that contained 1x103 cells ml1. Cells were inoculated in a final concentration of 100 cells per well. Serial twofold dilutions of the drugs tested for MIC were made from stock solutions in 96-well tissue-culture plates. Cell growth was analysed at 72 h. The stock solution of FPT Inhibitor III (Calbiochem) was diluted in RPMI 1640 to a final concentration of 5 mM. The FK506 (Fujisawa) stock solution was made by diluting the drug in RPMI 1640 to a final concentration of 25 µg ml1. Fluconazole (Pfizer) was diluted with saline (0·85 % NaCl) to concentrations ranging from 800 µg ml1 to 25 µg ml1. The 96-well plates were incubated in an air incubator at 35 °C, or as otherwise stated.
Genetic analysis.
Sporulation of the transformed diploid strains was performed on V8 mating medium in the dark at room temperature, as described by Sia et al. (2000). The basidiospores were separated by micromanipulation using a Nikon Eclipse 400 microdissecting microscope. To test for specific auxotrophies, the basidiospores were incubated on YDP medium at 30 °C and transferred to synthetic media lacking uracil, adenine or lysine. Mating type was determined by crossing the basidiospores with either JEC21 (MAT
) or JEC20 (MATa) on V8 mating medium at room temperature in the dark, and assessing for mating filaments at 35 days.
RAM1 overexpression construct.
The RAM1 gene was PCR amplified with primer AA237 (5'-ACGTCCGCGGGCGGCCGCGGGCTAGCAGTGCGAGTGGG-3', position 1389 bp to 1372 bp, NotI site underlined), and AA238 (5'-ACGTTCTAGAACTAGTGGAGACCATGAACAATGTCGG-3', position 2734 bp to 2754 bp, SpeI site underlined), which added NotI and SpeI restriction sites to the 5' and 3' ends, respectively. This gene was cloned into pHYG7-KB1, which contains the hph gene conferring resistance to hygromycin B as a selectable marker (Cox et al., 1996). The resulting plasmid was completely sequenced to confirm the absence of PCR-induced sequence errors. This plasmid was transformed by biolistic transformation into serotype A wild-type strain H99, and transformants were selected on YPD containing 300 µg hygromycin B ml1. In order to select for stable genomic integration of the construct, the transformants were passaged multiple times on a non-selective medium. Those that retained resistance to hygromycin B were analysed further. Two transformants demonstrated marked overexpression of RAM1 by Northern analysis, and were designated MVC72 and MVC73. The level of expression remained stable, despite repeated incubations on non-selective media.
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RESULTS |
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The predicted C. neoformans Ram1 protein shows homology to S. cerevisiae Ram1p (48 % similarity), as well as to mammalian protein farnesyltransferase subunits (46 % similarity to rat fntb). The RAM1 gene sequence was submitted to GenBank under accession number AY162319.
Deletion of RAM1 gene in a diploid strain
Using the PCR overlap-extension technique (Davidson et al., 2002), we replaced the majority of the RAM1 coding region (nucleotides 2321532, corresponding to amino acid residues 77440) with the URA5 gene. The linear ram1 : : URA5 construct was biolistically transformed into two strains: JEC43 (haploid, MAT
ura5) and RAS10 (diploid, MATa/MAT
, ura5/ura5, ade2/ADE2, lys1/LYS1, lys2/LYS2). PCR and Southern blot analysis of 29 haploid JEC43 transformants revealed that no homologous recombination had occurred. In contrast, among 32 transformants for the diploid RAS10 background, by both colony PCR and Southern blot analysis, three demonstrated a precise replacement of one RAM1 allele with the ram1 : : URA5 mutant construct (Fig. 1
a).
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Genetic analysis was performed to confirm that the basidiospores were products of meiosis. The genetic markers segregated among these strains in a manner consistent with meiotic recombination. Mating type segregated 1 : 1, with 4 MAT and 4 MATa strains among the eight germinated spores. Exactly one half of the germinated spores were adenine auxotrophs, and one half were prototrophic for adenine. Only one of these strains was prototrophic for lysine, consistent with the presence of the lys1 and lys2 mutations in the original diploid strain.
In contrast, all eight strains failed to grow on synthetic medium lacking uracil, indicating that they likely lacked the ram1 : : URA5 mutant allele and were all RAM1 wild-type strains. Southern blot analysis confirmed that all eight basidiospores indeed contained only the wild-type RAM1 allele (Fig. 1a). Together, these data confirm that RAM1 is an essential gene in C. neoformans.
MIC testing and RAM1 overexpression
Since the gene encoding a homologue of the farnesyltransferase subunit is essential in C. neoformans, we hypothesized that pharmacologic inhibition of farnesylation would result in growth arrest. A commercially available protein farnesyltransferase inhibitor III (FPT Inhibitor III, Calbiochem) had been previously investigated for its ability to inhibit the growth of malignant cells (Prendergast, 2000
). Using standardized protocols for MIC testing (Galgiani et al., 1997
), we tested FPT Inhibitor III for anti-cryptococcal activity. At 35 °C in RPMI medium, FPT Inhibitor III completely inhibited growth of the clinically derived serotype A strain H99 at concentrations of 400500 µM (Fig. 2
). We tested other C. neoformans strains, including the serotype D congenic strains JEC20 and JEC21, and they were inhibited by similar concentrations of this compound at 72 h incubation.
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Compared to the wild-type strain, the RAM1-overexpression strain demonstrated a greater than fourfold increase in the MIC for FPT Inhibitor III in six independent experiments. Limited drug solubility in the growth medium at higher concentrations prevented the determination of an exact MIC for the overexpression strain. This result supports our hypothesis that the Ram1 protein is a limiting component of the cellular target for FPT Inhibitor III in C. neoformans. The identification of other FTase inhibitors with increased solubility and improved fungal-cell entry may allow a more precise delineation of the differences in MIC between the wild-type and RAM1-overexpressing strains.
FPT Inhibitor III inhibits C. neoformans mating
C. neoformans has a bipolar mating system in which a and strains fuse and undergo meiosis under appropriate environmental conditions. After the initial fusion of the opposite mating partners, the heterokaryon forms mating hyphae. These filamentous structures typically undergo terminal differentiation to form a basidium, in which meiosis and sporulation occur (Kwon-Chung, 1975
). The mating process and the associated steps of differentiation are dependent upon the pheromone response and Ras1 signal transduction pathways (Alspaugh et al., 2000
; Davidson et al., 2003
; Wang et al., 2000
). Since components of each of these pathways, including the
mating factor and the small G protein Ras1, are presumed to require prenylation in order to be functional (He et al., 1991
), we hypothesized that pharmacological inhibition of farnesylation would also inhibit mating.
The congenic serotype D strains JEC20 and JEC21 were co-incubated in a mating reaction on V8 mating medium containing various concentrations of FPT Inhibitor III. In the absence of drug, the co-incubated strains underwent a rapid filamentous mating response, with initial hyphae evident by 48 h, and mature mating structures visible by 47 days. However, the addition of FPT Inhibitor III inhibited this mating process (Fig. 3). Mating filamentation was almost completely inhibited by 100200 µM FPT Inhibitor III, concentrations that are permissive for growth of these strains in a liquid medium. The mating mixture cells grew as well on V8 medium containing these concentrations of the drug as they did on V8 mating medium alone, suggesting that a general growth inhibition was not the cause of decreased mating. At lower concentrations of FPT Inhibitor III, a doseresponse relationship was observed between the concentration of FPT Inhibitor III in the medium and the degree of mating inhibition observed (Fig. 3
). Mating reactions similar to wild-type, with completely normal mating structures, occurred in the presence of 10µM FPT Inhibitor III (Fig. 3
).
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To assess the effects of farnesyltransferase inhibition on haploid fruiting, we supplemented the filament agar containing galactose with subinhibitory concentrations of FPT Inhibitor III (0400 µM). The STE12-overexpressing strain was incubated on this medium for 14 days. No hyphae were observed in the presence of 25 µM FPT Inhibitor III, a concentration of the drug that is 10-fold lower than the MIC (Fig. 4
). At lower concentrations of FPT Inhibitor III, haploid fruiting was inhibited in a dose-dependent manner (Fig. 4
). Therefore, this farnesyltransferase inhibitor blocks both forms of hyphal differentiation in C. neoformans, mating and haploid fruiting.
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The immunosuppressive drug FK506 pharmacologically inhibits calcineurin signalling, and C. neoformans cells treated with this drug are unable to grow at high temperatures. However, FK506 has little effect on cells incubated at 2530 °C (Odom et al., 1997). A ras1 mutant strain demonstrates increased susceptibility to FK506 at the permissive temperature of 30 °C. The wild-type (H99), ras1 mutant (LCC1), and ras1+RAS1 reconstituted (LCC2) strains were tested for growth inhibition by FK506. In contrast to the wild-type and LCC2 strains, which grew in the presence of 1000 ng FK506 ml1 at 30 °C, the growth of the LCC1 strains (ras1) was inhibited by 60 ng FK506 ml1 at this temperature (Fig. 5
).
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At various temperatures, we performed a checkerboard MIC test to compare the combined activity of FPT Inhibitor III and FK506 to inhibit the growth of C. neoformans. As demonstrated in Fig. 6, FK506 inhibits C. neoformans growth at 37 °C at concentrations between 50 and 100 ng ml1. As noted previously, no growth inhibition by FK506 is observed at 30 °C, and incubation at 35 °C resulted in an intermediate effect. However, subinhibitory concentrations of FK506 dramatically decrease the MIC of FPT Inhibitor III at 37 °C. Thus, there is a synthetic effect on C. neoformans growth with the combination of calcineurin inhibition and treatment with an FTase inhibitor. Slightly less growth inhibition by FK506 was observed at 35 °C as compared to 37 °C, both alone and in combination with FPT Inhibitor III. There was no change in the MIC to FPT Inhibitor III at these different temperatures.
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DISCUSSION |
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Protein farnesylation in S. cerevisiae is specifically required for the proper functioning of Ras proteins as well as for the a factor mating pheromone (He et al., 1991). In contrast, GGTase I demonstrates specificity for other small G proteins, such as Rho1 and Cdc42 (Ohya et al., 1993
). The S. cerevisiae RAM2 gene, encoding the
subunit common to FTase and GGTase I, and the CDC43 gene, encoding the
subunit of GGTase I, are both essential (Mayer et al., 1992
). Strains with null mutations of the RAM1 gene, which encodes the
subunit of FTase, are viable, but display a temperature-sensitive lethality and grow poorly at a permissive temperature of 30 °C (He et al., 1991
). These results suggest that S. cerevisiae proteins that are typically farnesylated may undergo alternative prenylation and retain partial function in the absence of FTase, but that alternative prenylation cannot compensate for defective protein-geranylgeranyltransferase activity in this organism.
The closely related yeast Candida albicans is a prominent human pathogen. In contrast to S. cerevisiae, GGTase-I activity in Ca. albicans is not required for viability. Ca. albicans strains with mutations in the GGTase-I -subunit gene CDC43 are viable, although lacking GGTase-I activity (Kelly et al., 2000
). These mutant strains demonstrate similar growth rates to wild-type strains in early- and mid-exponential-phase cultures, but they arrest in later-phase cultures. Ca. albicans cdc43 mutants also exhibit altered cellular morphology. Interestingly, the levels of two Ca. albicans proteins that undergo geranylgeranylation under normal circumstances, Rho1 and Cdc42, are increased in GGTase-I-null (cdc43 mutant) strains. These two proteins are also mislocalized in the cdc43 mutant, being present in cytosolic rather than membrane fractions. This observation led the investigators to hypothesize that Rho1 and Cdc43 may undergo alternative prenylation (farnesylation) in the absence of GGTase I in Ca. albicans (Kelly et al., 2000
). However, prenylation is required for viability and pathogenicity in Ca. albicans, since the RAM2 gene, encoding the
subunit of FTase and GGTase I, is essential (Song & White, 2003
)
In these studies, we demonstrated that the RAM1 gene, encoding a homologue of an FTase subunit of C. neoformans, encodes an essential function. Even though the S. cerevisiae ram1 mutant is viable, it grows poorly even under optimal conditions, and does not grow at all at 37 °C. Therefore, farnesylation is clearly required for S. cerevisiae to survive under stressful conditions such as elevated temperatures. Together, these observations suggest that intact microbial protein farnesylation may be required for the pathogenesis of human fungal infections.
The question remains why GGTase I and FTase appear to be able to partially compensate for each other's absence in the ascomycetous yeasts S. cerevisiae and Ca. albicans, while FTase activity is absolutely required for viability in the distantly related basidiomycetous fungus C. neoformans. Perhaps the C. neoformans FTase and GGTase I proteins have differentiated sufficiently that they are no longer able to serve redundant functions. Interestingly, analysis of the completed C. neoformans genome databases using a BLAST search with the S. cerevisiae Cdc43p reveals no close match for genes encoding GGTase-I subunits. Therefore, it is also possible that no GGTase-I activity is present in C. neoformans, explaining why the FTase
-chain gene is essential. Biochemical analysis of C. neoformans protein-prenyltransferase activities will help to address whether GGTase-I activity is indeed absent in C. neoformans. Additionally, as genome projects from other basidiomycetes are completed, it will be interesting to determine whether both FTase and GGTase-I
-subunit genes are present in these organisms.
The possibility that C. neoformans possesses FTase activity without GGTase-I activity might also explain the C-terminal amino-acid sequences of the Ras1, Ras2 and Rho1 proteins. All of these proteins have a CVVL amino-acid sequence at the C-terminus, a typical consensus sequence for geranylgeranylation (Zhang et al., 1996). This is in contrast to Ras proteins in the ascomycete group of fungi, such as S. cerevisiae, Ca. albicans, Aspergillus fumigatus and Neurospora crassa, whose Ras proteins possess C-terminus CAAX motifs with terminal amino-acid residues such as cysteine, serine or methionine, sequences typically associated with farnesylation (Zhang et al., 1996
). Interestingly, similar to C. neoformans, two other basidiomycetes, Ustilago maydis and Lentinula edodes, display leucine as the terminal amino acid of the CAAX motif of their Ras proteins (U. maydis Ras1, CNIL; L. edodes LE ras, CVVL). Therefore, the prenylation process of small G proteins may differ substantially between the ascomycete and basidiomycete groups of fungi.
Pharmacological inhibition of prenylation
As discussed above, the S. cerevisiae and Ca. albicans RAM2 genes, encoding the subunit of both GGTase I and FTase, are essential. This indicates that simultaneously abolishing both FTase and GGTase-I activity results in lethality. Also, FTase activity is required for viability in C. neoformans and for growth of S. cerevisiae at human physiological temperature. Therefore, protein prenylation remains an interesting target for further antimicrobial drug development.
Compounds that inhibit protein prenylation have been developed and studied as potential agents in treating human malignancies. This field was motivated by the observation that activated Ras mutations were associated with a significant number of human cancers. Initial trials indicated that inhibiting prenylation could result in a reduction in the growth rate of some tumour lines (Kohl et al., 1995).
In limited initial experiments, these agents have also been considered for antifungal effects. Ca. albicans was treated with the protein farnesyltransferase inhibitor FPT Inhibitor III and a related compound, FTase inhibitor II, but no antifungal activity was observed at FPT Inhibitor III concentrations as high as to 400 µM (McGeady et al., 2002; Song et al., 2003
). However, slightly lower concentrations of FTase inhibitor II were able to inhibit prenyltransferase enzymic activity in cell extracts: Ca. albicans FTase activity was inhibited in vitro by 100 µM FTase Inhibitor II (Song et al., 2003
). Similarly, in our studies, high concentrations of FPT Inhibitor III were required for growth inhibition of C. neoformans, precluding its use in clinical studies. Therefore, this drug is clearly not a sufficiently potent inhibitor of fungal FTase, and other compounds may demonstrate significantly lower inhibitory concentrations. Related farnesyl diphosphate-based compounds inhibit the mammalian FTase enzyme at concentrations ranging from 75 nM to 200 µM (Patel et al., 1995
), suggesting that chemical modification of these compounds may yield enhanced antifungal activity. Additionally, the compound may be degraded by cellular factors in fungi, or perhaps this drug does not efficiently enter the fungal cell. The identification of prenylation inhibitors with enhanced specificity for fungal protein prenyltransferases and optimized for entry into fungal cells might offer novel antimicrobial therapeutic options.
We also explored the possibility of simultaneously inhibiting two distinct signalling pathways that control the growth of C. neoformans at human physiological temperature. We observed that co-inhibition of the calcineurin and Ras1 pathways results in a synthetic effect on the growth of this fungus. Additionally, the simultaneous inhibition of calcineurin signalling and farnesylation also resulted in a synthetic growth arrest. Such strategies may offer new possibilities for novel targets for antifungal therapy.
Although the farnesylation inhibitor FPT Inhibitor III does not impair growth of Ca. albicans at low concentrations, this compound is able to inhibit hyphal development of this pathogenic yeast (McGeady et al., 2002). This observation is clinically relevant, since the yeasthyphal transition in this organism has been closely associated with pathogenicity. Defined signalling and structural molecules are required for hyphal formation in Ca. albicans, including Ras proteins and components of the MAP-kinase and cAMP-signalling cascades (Feng et al., 1999
; Kohler et al., 1996
; Rocha et al., 2001
). Several of these proteins, including small G proteins, would be expected to require prenylation in order to function properly, providing a rational, hypothetical mechanism for the ability of FPT Inhibitor III to inhibit hyphal development.
Our studies similarly demonstrated that FPT Inhibitor III inhibited hyphal differentiation in the pathogenic fungus C. neoformans. We demonstrated that two hyphal processes, mating and haploid fruiting, were inhibited by FPT Inhibitor III. Several proteins in C. neoformans are known to be required for mating and haploid fruiting. The pheromone response pathway is activated by peptide pheromones to initiate the mating process (Moore et al., 1993), and the Mf
1 pheromone contains consensus farnesylation sequences. Moreover, Mf
1 synthesized heterologously in bacterial cells has been demonstrated to require both farnesylation and carboxy-methylation in order to be functional and induce hyphal formation in MATa cells (Davidson et al., 2000b
). The pheromone response pathway is also activated by the Ras1 protein, which also likely requires prenylation to be membrane-associated and functional (Waugh et al., 2003
). It is likely that one or several of these proteins is affected by FPT Inhibitor III, resulting in the failure of C. neoformans to undergo hyphal differentiation when treated with this drug. Thus, this prenylation inhibitor blocks steps required for hyphal development in both ascomycetes and basidiomycetes.
In conclusion, these studies have explored the role of protein farnesylation in the pathogenic fungus C. neoformans. Although individual prenyltransferases may demonstrate varying substrate specificity in different fungal species, these proteins regulate central processes controlling growth and differentiation in these micro-organisms. Novel therapeutic targets can be identified by understanding the differences between prenylation in microbial pathogens and in the human host.
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ACKNOWLEDGEMENTS |
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REFERENCES |
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Alspaugh, J. A., Cavallo, L. M., Perfect, J. R. & Heitman, J. (2000). RAS1 regulates filamentation, mating and growth at high temperature of Cryptococcus neoformans. Mol Microbiol 36, 352365.[CrossRef][Medline]
Casey, P. J. & Seabra, M. C. (1996). Protein prenyltransferases. J Biol Chem 271, 52895292.
Casey, P. J., Solski, P. A., Der, C. J. & Buss, J. E. (1989). p21ras is modified by a farnesyl isoprenoid. Proc Natl Acad Sci U S A 86, 83238327.[Abstract]
Chang, Y. C. & Penoyer, L. A. (2000). Properties of various Rho1 mutant alleles of Cryptococcus neoformans. J Bacteriol 182, 49874991.
Cox, G. M., Toffaletti, D. L. & Perfect, J. R. (1996). Dominant selection system for use in Cryptococcus neoformans. J Med Vet Mycol 34, 385391.[Medline]
Davidson, R. C., Cruz, M. C., Sia, R. A., Allen, B., Alspaugh, J. A. & Heitman, J. (2000a). Gene disruption by biolistic transformation in serotype D strains of Cryptocococus neoformans. Fungal Genet Biol 29, 3848.[CrossRef][Medline]
Davidson, R. C., Moore, T. D., Odom, A. R. & Heitman, J. (2000b). Characterization of the MFalpha pheromone of the human fungal pathogen Cryptocococus neoformans. Mol Microbiol 38, 10171026.[CrossRef][Medline]
Davidson, R. C., Blankenship, J. R., Kraus, P. R., de Jesus, B. M., Hull, C. M., D'Souza, C., Wang, P. &, Heitman. J. (2002). A PCR-based strategy to generate integrative targeting alleles with large regions of homology. Microbiology 148, 26072615.
Davidson, R. C., Nichols, C. B., Cox, G. M., Perfect, J. R. & Heitman, J. (2003). A MAP kinase cascade composed of cell type specific and non-specific elements controls mating and differentiation of the fungal pathogen Cryptococcus neoformans. Mol Microbiol 49, 469485.[Medline]
Feng, Q., Summers, E., Guo, B. & Fink, G. (1999). Ras signaling is required for serum-induced hyphal differentiation in Candida albicans. J Bacteriol 181, 63396346.
Galgiani, J. N., Bartlett, M. S., Ghannoum, M. A., Espinel-Ingroff, A., Lancaster, M. V., Odds, F. C., Pfaller, M. A., Rinaldi, M. G. & Walsh, T. J. (1997). Reference method for broth dilution antifungal susceptibility testing of yeasts; approved standard. National Committee for Clinical Laboratory Standards 17 No. 9, 129.
Hancock, J. F., Magee, A. I., Childs, J. E. & Marshall, C. J. (1989). All ras proteins are polyisoprenylated but only some are palmitoylated. Cell 57, 11671177.[Medline]
He, B., Chen, P., Chen, S. Y., Vancura, K. L., Michaelis, S. & Powers, S. (1991). RAM2, an essential gene of yeast, and RAM1 encode the two polypeptide components of the farnesyltransferase that prenylates a-factor and Ras proteins. Proc Natl Acad Sci U S A 88, 1137311377.[Abstract]
Hill, B. T., Perrin, D. & Kruczynski, A. (2000). Inhibition of RAS-targeted prenylation: protein farnesyl transferase inhibitors revisited. Crit Rev Oncol/Hematol 33, 723.[CrossRef][Medline]
Jiang, Y., Rossi, G. & Ferro-Novick, S. (1993). Bet2p and Mad2p are components of a prenyltransferase that adds geranylgeranyl onto Ypt1p and Sec4p. Nature 366, 8486.[CrossRef][Medline]
Kelly, R., Card, D., Register, E. & 8 other authors (2000). Geranylgeranyltransferase I of Candida albicans: null mutants or enzyme inhibitors produce unexpected phenotypes. J Bacteriol 182, 704713.
Kinsella, B. T., Erdman, R. A. & Maltese, W. A. (1991). Posttranslational modification of Ha-ras p21 by farnesyl versus geranylgeranyl isoprenoids is determined by the COOH-terminal amino acid. Proc Natl Acad Sci U S A 88, 89348938.[Abstract]
Kohl, N. E., Omer, C. A., Conner, M. W. & 7 other authors (1995). Inhibition of farnesyltransferase induces regression of mammary and salivary carcinomas in ras transgenic mice. Nat Med 1, 792797.[Medline]
Kohler, J. R. & Fink, G. R. (1996). Candida albicans strains heterozygous and homozygous for mutations in mitogen-activated protein kinase signaling components have defects in hyphal development. Proc Natl Acad Sci U S A 93, 1322313228.
Kwon-Chung, K. J. (1975). Filobasidiella the perfect state of Cryptococcus neoformans. Mycologia 67, 11971200.[Medline]
Kwon-Chung, K. J., Edman, J. C. & Wickes, B. L. (1992). Genetic association of mating types and virulence in Cryptococcus neoformans. Infect Immun 60, 602605.[Abstract]
Lerner, E. C., Qian, Y., Hamilton, A. D. & Sebti, S. M. (1995). Disruption of oncogenic K-Ras4B processing and signaling by a potent geranylgeranyltransferase I inhibitor. J Biol Chem 270, 2677026773.
Mayer, M. L., Caplin, B. E. & Marshall, M. S. (1992). CDC43 and RAM2 encode the polypeptide subunits of a yeast type I protein geranylgeranyltransferase. J Biol Chem 267, 2058920593.
McGeady, P., Logan, D. A. & Wansley, D. L. (2002). A protein-farnesyl transferase inhibitor interferes with the serum-induced conversion of Candida albicans from a cellular yeast form to a filamentous form. FEMS Microbiol Lett 213, 4144.[CrossRef][Medline]
Moore, T. D. & Edman, J. C. (1993). The alpha-mating type locus of Cryptococcus neoformans contains a peptide pheromone gene. Mol Cell Biol 13, 19621970.[Abstract]
Odom, A., Muir, S., Lim, E., Toffaletti, D. L., Perfect, J. & Heitman, J. (1997). Calcineurin is required for virulence of Cryptococcus neoformans. EMBO J 16, 25762589.
Ohya, Y., Qadota, H., Anraku, Y., Pringle, J. R. & Botstein, D. (1993). Suppression of yeast geranylgeranyl transferase I defect by alternative prenylation of two target GTPases, Rho1p and Cdc42p. Mol Biol Cell 4, 10171025.[Abstract]
Patel, D. V., Schmidt, R. J., Biller, S. A., Gordon, E. M., Robinson, S. S. & Manne, V. (1995). Farnesyl diphosphate-based inhibitors of Ras farnesyl protein transferase. J Med Chem 38, 29062921.[Medline]
Perfect, J. R., Lang, S. D. R. & Durack, D. T. (1980). Chronic cryptococcal meningitis: a new experimental model in rabbits. Am J Pathol 101, 177194.[Abstract]
Pitkin, J. W., Panaccione, D. G. & Walton, J. D. (1996). A putative cyclic peptide efflux pump encoded by the TOXA gene of the plant-pathogenic fungus Cochliobolus carbonum. Microbiology 142, 15571565.[Abstract]
Prendergast, G. C. (2000). Farnesyltransferase inhibitors: antineoplastic mechanism and clinical prospects. Curr Opin Cell Biol 12, 166173.[CrossRef][Medline]
Reiss, Y., Goldstein, J. L., Seabra, M. C., Casey, P. J. & Brown, M. S. (1990). Inhibition of purified p21ras farnesyl:protein transferase by Cys-AAX tetrapeptides. Cell 62, 8188.[Medline]
Rocha, C. R., Schroppel, K., Harcus, D., Marcil, A., Dignard, D., Taylor, B. N., Thomas, D. Y., Whiteway, M. & Leberer, E. (2001). Signaling through adenylyl cyclase is essential for hyphal growth and virulence in the pathogenic fungus Candida albicans. Mol Biol Cell 12, 36313643.
Sambrook, J. & Russel, D. W. (2001). Molecular Cloning: a Laboratory Manual. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory.
Schafer, W. R., Kim, R., Sterne, R., Thorner, J., Kim, S. H. & Rine, J. (1989). Genetic and pharmacological suppression of oncogenic mutations in ras genes of yeast and humans. Science 245, 379385.[Medline]
Seabra, M. C., Reiss, Y., Casey, P. J., Brown, M. S. & Goldstein, J. L. (1991). Protein farnesyltransferase and geranylgeranyltransferase share a common alpha subunit. Cell 65, 429434.[Medline]
Sherman, F. (1991). Getting started with yeast. Methods Enzymol 194, 321.[Medline]
Sia, R. A., Lengeler, K. B. & Heitman, J. (2000). Diploid strains of the pathogenic basidiomycete Cryptococcus neoformans are thermally dimorphic. Fungal Genet Biol 29, 153163.[CrossRef][Medline]
Song, J. L. & White, T. C. (2003). RAM2: an essential gene in the prenylation pathway of Candida albicans. Microbiology 149, 249259.
Wang, P., Perfect, J. R. & Heitman, J. (2000). The G-protein beta subunit GPB1 is required for mating and haploid fruiting in Cryptococcus neoformans. Mol Cell Biol 20, 352362.
Waugh, M. S., Vallim, M. A., Heitman, J. & Alspaugh, J. A. (2003). Ras1 controls pheromone expression and response during mating in Cryptococcus neoformans. Fungal Genet Biol 38, 110121.[CrossRef][Medline]
Wickes, B. L., Mayorga, M. E., Edman, U. & Edman, J. C. (1996). Dimorphism and haploid fruiting in Cryptococcus neoformans: association with the alpha-mating type. Proc Natl Acad Sci U S A 93, 73277331.
Wickes, B. L., Edman, U. & Edman, J. C. (1997). The Cryptococcus neoformans STE12 gene: a putative Saccharomyces cerevisiae STE12 homologue that is mating type specific. Mol Microbiol 26, 951960.[Medline]
Zhang, F. L. & Casey, P. J. (1996). Protein prenylation: molecular mechanisms and functional consequences. Annu Rev Biochem 65, 241269.[CrossRef][Medline]
Received 13 January 2004;
revised 9 March 2004;
accepted 12 March 2004.
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