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Eukaryotic Cell, January 2007, p. 60-72, Vol. 6, No. 1
1535-9778/07/$08.00+0     doi:10.1128/EC.00214-06
Copyright © 2007, American Society for Microbiology. All Rights Reserved.

Peroxisome Function Regulates Growth on Glucose in the Basidiomycete Fungus Cryptococcus neoformans{triangledown}

Alexander Idnurm,1 Steven S. Giles,2 John R. Perfect,1,3 and Joseph Heitman1,3*

Departments of Molecular Genetics and Microbiology,1 Cell Biology,2 Medicine, Duke University Medical Center, Durham, North Carolina 277103

Received 6 July 2006/ Accepted 3 October 2006


   ABSTRACT
Top
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The function of the peroxisomes was examined in the pathogenic basidiomycete Cryptococcus neoformans. Recent studies reveal the glyoxylate pathway is required for virulence of diverse microbial pathogens of plants and animals. One exception is C. neoformans, in which isocitrate lyase (encoded by ICL1) was previously shown not to be required for virulence, and here this was extended to exclude also a role for malate synthase (encoded by MLS1). The role of peroxisomes, in which the glyoxylate pathway enzymes are localized in many organisms, was examined by mutation of two genes (PEX1 and PEX6) encoding AAA (ATPases associated with various cellular activities)-type proteins required for peroxisome formation. The pex1 and pex6 deletion mutants were unable to localize the fluorescent DsRED-SKL protein to peroxisomal punctate structures, in contrast to wild-type cells. pex1 and pex6 single mutants and a pex1 pex6 double mutant exhibit identical phenotypes, including abolished growth on fatty acids but no growth difference on acetate. Because both icl1 and mls1 mutants are unable to grow on acetate as the sole carbon source, these findings demonstrate that the glyoxylate pathway can function efficiently outside the peroxisome in C. neoformans. The pex1 mutant exhibits wild-type virulence in a murine inhalation model and in an insect host, demonstrating that peroxisomes are not required for virulence under these conditions. An unusual phenotype of the pex1 and pex6 mutants was that they grew poorly with glucose as the carbon source, but nearly wild type with galactose, which suggested impaired hexokinase function and that C. neoformans peroxisomes might function analogously to the glycosomes of the trypanosomid parasites. Deletion of the hexokinase HXK2 gene reduced growth in the presence of glucose and suppressed the growth defect of the pex1 mutant on glucose. The hexokinase 2 protein of C. neoformans contains a predicted peroxisome targeting signal (type 2) motif; however, Hxk2 fused to fluorescent proteins was not localized to peroxisomes. Thus, we hypothesize that glucose or glycolytic metabolites are utilized in the peroxisome by an as yet unidentified enzyme or regulate a pathway required by the fungus in the absence of peroxisomes.


   INTRODUCTION
Top
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The paucity of applicable antimicrobial agents and the growing resistance to existing agents raise concern about the long-term control of infectious diseases, particularly those caused by fungi. The search for novel genes that lack human counterparts and are essential for pathogen survival or virulence and the design of molecules that target these genes or encoded proteins represent an approach to new drug discovery. An exciting discovery towards this goal was the identification of the glyoxylate pathway as playing a role in microbial virulence (reviewed in reference 43). This pathway catalyzes the conversion of isocitrate to malate and succinate and is essential for the utilization of two-carbon molecules (e.g., ethanol and acetate) as carbon sources. The first enzyme in the pathway, isocitrate lyase, is required for virulence of Mycobacterium tuberculosis and Candida albicans in mice (44, 47, 50), as well as for the fungi Colletotrichum lagenarium, Leptosphaeria maculans, and Magnaporthe grisea in plants (2, 27, 78). The second enzyme in the pathway, malate synthase, is involved in virulence in the bacterium Rhodococcus fascians and the fungus Stagonospora nodorum towards plants (67, 75). The pathway is absent in vertebrates; hence, drugs targeting it, perhaps aided by the crystal structure (5, 65, 66) or gene-specific inactivation (40), may therefore be broad-spectrum and safe human therapeutics. Although plants contain glyoxylate pathway enzymes, there is also potential for application in agricultural settings, given the temporal expression of the pathway enzymes in plants (e.g., during seed germination and plant senescence).

Peroxisomes are single-membrane-bound organelles associated with a suite of cellular functions, including peroxide detoxification, ß-oxidation of fatty acids, and utilization of D-amino acids (reviewed in reference 46). The glyoxylate pathway in plants and fungi is most often a component of peroxisome function (53, 81). Peroxisomes are also essential for human health. Twelve complementation groups have been identified in humans with deficiencies in peroxisome function, most of which are lethal or lead to early death or debilitating disease (79). In otherwise healthy individuals, peroxisomes are required for metabolism of fatty acids and produce reactive oxygen as part of this process. Many aspects of the functions of the human proteins required for peroxisome formation have been elucidated by using fungi, particularly Saccharomyces cerevisiae, as model systems for peroxisome function, including the identification of the human genes mutated in 9 of the 12 complementation groups (reviewed in reference 22).

Peroxisomes can be required for microbial virulence. Deletion of pex6 in the fungus Colletotrichum lagenarium reduces disease on bean leaves by impairing appressorium function during the initial stages of infection (36). Recently, a PEX6 homolog and a peroxisome-targeted acetyltransferase have been shown to be required for appressorium formation and pathogenicity of the major rice pathogen Magnaporthe grisea (4, 60). Similarly, hex1 mutations impair appressorium formation in M. grisea (68). Hex1 is a protein required for formation of Woronin bodies, which are specialized peroxisomes found only in filamentous ascomycete fungi, which function to block septal pores in damaged hyphae. The Kinetoplastida parasites, such as those in the Trypanosoma and Leishmania genera, contain an unusual type of peroxisome named the glycosome because up to seven of the nine enzymes for glycolysis are targeted within them, and no Kreb's cycle occurs within these organisms. Reduced expression of genes required for peroxisome assembly is lethal to trypanosomes as a consequence of enzyme mislocalization to the cytoplasm, and as such, the glycosomes are proposed drug targets for these parasites (15, 23, 49).

One concern with developing a drug that targets the glyoxylate pathway enzymes was the discovery that Cryptococcus neoformans does not require isocitrate lyase for virulence, despite the observation that the transcript is highly up-regulated in the central nervous system during infection of mammals (61). In addition, in a Saccharomyces cerevisiae virulence study, an icl1 mutant also showed no reduction in virulence (21). C. neoformans is a basidiomycete fungus that causes disease, most commonly in immunocompromised people. The genome sequences of five different isolates are available, and together with the development of a suite of genetic approaches, this fungus serves as an excellent model system to study eukaryote biology and microbial pathogenesis (7, 26). The initial aim of this research was to investigate further the role of the glyoxylate pathway and peroxisome function in this fungus. We discovered that peroxisomes are not required for glyoxylate pathway activity, but rather interact in a novel way with growth in the presence of glucose.


   MATERIALS AND METHODS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Fungal strains. Cryptococcus neoformans serotype A strains H99 (MAT{alpha}), KN99a, KN99{alpha}, KN3B7#12 (MATa, the eighth backcross to generate serotype A congenic parents KN99a and KN99{alpha}) (52), and icl1 (MAT{alpha}) mutant (61) were used. Serotype D strains JEC20 (MATa) and JEC21 (MAT{alpha}) were used to assay the mating type of serotype A strains (39). Saccharomyces cerevisiae BY4743 wild-type reference and icl1{Delta} and pex1{Delta} mutant diploid strains were obtained from the S. cerevisiae gene deletion set (18). The C. neoformans strains generated in this study are listed in Table 1.


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  		TABLE 1. C. neoformans strains used in this study

 
Database analyses of the C. neoformans genome. The protein sequences encoded by the S. cerevisiae MLS1, PEX1, PEX5, PEX6, and PEX7 genes were used to search the C. neoformans genome database (www.tigr.com) (42) with BLASTp and tBLASTn using default parameters. The sequences of the nine enzymes required for glycolysis in S. cerevisiae were likewise used in searches against the C. neoformans genome databases. The subcellular localizations of the putative C. neoformans homologs were predicted using PSORT II software and manual searches of peroxisomal targeting signals.

Creation of C. neoformans mutant strains. Mutations of the HXK2, MLS1, PEX1, PEX5, PEX6, and PEX7 genes were isolated by replacing 95 to 100% of the coding regions of these genes with a cassette conferring resistance to nourseothricin (NAT) (28). A disruption allele was generated in which approximately 1.5 kb of DNA flanking the NAT cassette was obtained by overlap PCR (14) using the primers in Table 2 and transformed using a biolistic apparatus into H99 or KN99{alpha} cells (Bio-Rad model PDS-1000/He biolistic particle delivery system) (72). Mutation was confirmed by PCR and Southern blot analysis with DNA extracted using cetyltrimethylammonium bromide buffer (58). A pex1 mutant was crossed to the congenic isolate KN3B7#12, and basidiospores from the cross were isolated by micromanipulation and analyzed to obtain a pex1 MATa strain. Crosses were conducted on V8 medium (5% Campbell's V8 juice, 0.5 g/liter KH2PO4, 40 g/liter Bacto-agar, pH 5) for up to 3 weeks in the dark. Mating was unaffected in pex1 mutants, including pex1 x pex1 crosses. The pex1 MATa strain was crossed to a pex6 strain, and the progeny of this cross were analyzed by Southern blotting to identify a double pex1 pex6 mutant strain. An mls1::NAT disruption allele was created by overlap PCR and subcloning. The cassette was cloned into plasmid pPZP-201BK and introduced into cells of Agrobacterium tumefaciens strain LBA4404 or EHA105 by electroporation. Agrobacterium-mediated transformation of C. neoformans was performed as described previously (28). Attempts to mutate MLS1 using Agrobacterium-mediated transformation were unsuccessful. The mls1::NAT construct was therefore amplified by PCR using the plasmid as template and used successfully to delete the MLS1 gene via biolistic transformation. The pex1 mutation was complemented by the ectopic introduction of a wild-type copy of the PEX1 gene fused to a cassette conferring resistance to neomycin via biolistic transformation (14). The hxk2 mutation was complemented by the ectopic introduction of a wild-type copy of the HXK2 gene fused to the cassette conferring resistance to neomycin (pPZP-NEO1) (76) via Agrobacterium-mediated transformation.


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  		TABLE 2. Oligonucleotide primers used to make gene disruption alleles by overlap PCR

 
Fluorescent protein constructs and microscopy. To assess organelle localization, overlap fusion products were created for green or red fluorescent proteins expressed from the C. neoformans histone H3 promoter using primers listed in Table 3. Green fluorescent protein (GFP) (S65T) was provided by Tian Lian and Jim Kronstad, and red fluorescent protein (DsRED) was provided by Connie Nichols as a derivative of pDsRED-Express (BD Biosciences, Palo Alto, CA). A peroxisome-targeted fluorescent protein (DsRED-SKL) driven by the histone H3 promoter and terminator was fused to the neomycin resistance marker using overlap PCR and introduced into C. neoformans strain KN99{alpha} by biolistic transformation. The same strategy was used to create a DsRED construct without the -SKL-terminal amino acids. Overlap fusion products to express GFP or DsRED were subcloned into the SacI site of pPZP-NATcc (76), as illustrated in Fig. 1A. To visualize the cell wall, strains were grown in YPD (yeast extract, peptone, dextrose) medium with Calcofluor white (40 µg/ml) for 5 min and washed three times in phosphate-buffered saline. Cells were mounted in Vectashield (Vector Laboratories, California). DsRED or GFP protein and Calcofluor fluorescence was examined using a Zeiss Axioskop 2 Plus fluorescence microscope and photographed with an AxioCam MRM digital camera. For fluorescence, the Zeiss filters for 4',6'-diamidino-2-phenylinodle (DAPI) (Calcofluor white staining; excitation {lambda}max, 365 nm), GFP gr (excitation, 450 to 480 nm), and both tetramethyl rhodamine isothiocyanate (TRITC) (DsRED; excitation 510 to 560 nm) and Texas red (DsRed; excitation, 530 to 580 nm) were used. As an independent measure of fluorescence, strains grown overnight in liquid YPD were sorted for size and fluorescence by flow cytometry.


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  		TABLE 3. Oligonucleotide primers for overlap PCR to make DsRed or GFP constructs or plasmids

 

Figure 1
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  		FIG. 1. (A) Construct design of the T-DNA region in plasmids for expression of GFP and DsRED protein fusions in C. neoformans. (B) Flow cytometry of the fluorescence levels of 10,000 cells from each strain analyzed for green or red fluorescence (fluorescein isothiocyanate [FITC] or Texas red filters, respectively).

 
In C. neoformans, GFP and DsRED have been used previously to study protein localization or expression (10, 80). However, use of GFP has been problematic and few published vectors are available for expression in C. neoformans. The constructs were introduced in A. tumefaciens strain EHA105, and these strains were used to transform C. neoformans. Weak autofluorescence was observed in wild-type cells, particularly associated with the plasma membrane. While cytoplasmic red fluorescence was observed with the DsRED construct, green fluorescence was very weak with the GFP construct, despite the same promoter being used to drive expression (Fig. 1B). The reasons are unknown; however, fusion of the C. neoformans Hxk2 protein to the N terminus dramatically enhanced levels of fluorescence (Fig. 1B and 8C), suggesting a requirement for C. neoformans nucleotide bias or introns for stable expression. A similar result has been observed for GFP expression in other basidiomycete fungi, including Agaricus bisporus and Coprinus cinereus, in which inclusion of introns is hypothesized to stabilize protein expression (6). Recently, the GFP gene has been optimized for C. neoformans codon usage to enhance fluorescence (41).


Figure 8
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  		FIG. 8. Hexokinase 2 is required for efficient utilization of glucose but is not localized to peroxisomes. (A) Growth of 10-fold dilutions of strains on acetate, galactose (30°C), or glucose (22°C) medium for 3 days. (B) Growth of strains in liquid yeast nitrogen base supplemented with 2% glucose, galactose, or acetate at 30°C. Error bars are the standard error of the mean (n = 3). (C) Intensity and localization of DsRED or GFP expressed from the histone H3 promoter fused or unfused to the Hxk2 protein in the wild-type background. Micrographs were overexposed (10,000 ms) for the green and red channels to highlight the lack of fluorescence or autofluorescence, except for +Hxk2-GFP (1,400-ms green channel), +DsRed (3,200-ms red channel) and +Hxk2-DsRed (1,400-ms red channel).

 
In vitro phenotypic analysis. The growth of strains was compared on YPD (yeast extract, peptone, dextrose) and YNB (yeast nitrogen base) agar medium supplemented with different carbon sources: glucose (0.2%, 0.5%, 1%, or 2%), fructose (2%), mannose (2%), galactose (2%), sodium acetate (1% or 2%), and oleic acid (5 mM plus 1% tergitol). Melanin and capsule were assayed on L-DOPA medium (100 mg/liter) and in low-iron medium (with the chelator EDDHA). Growth curves were conducted on cells that were grown overnight in liquid YPD medium, washed, and added to yeast nitrogen base with different carbon sources in shaking or rotating cultures at 30°C or 37°C, using starting inocula at an optical density at 600 nm (OD600) of ~0.1.

Virulence assays. For murine assays, C. neoformans cells were used to infect 4- to 6-week-old female A/Jcr mice (NCI/Charles River Laboratories) by nasal inhalation. Ten animals each were inoculated with a 50-µl inoculum containing 1 x 105 yeast cells of KN99a, H99, mls1, pex1, or pex1 PEX1 complemented strains. Animals were examined daily and sacrificed when signs of morbidity were observed. The experiment was double blinded, such that the strain genotypes remained anonymous during inoculum preparation and administration into animals, as well as until all animals were sacrificed. The murine experimental protocol was approved by the Duke University Animal Use Committee.

The wax moth virulence assay followed the previous protocol (51), with Galleria mellonella larvae purchased from Vanderhorst, Inc. (St. Marys, Ohio). The inoculum was 1 x 105 yeast cells, and larvae were incubated at 37°C postinoculation. Larvae were examined daily, and those not responding to touch were scored as inviable.


   RESULTS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
A malate synthase mutant is virulent, demonstrating the glyoxylate pathway is not required for virulence. The glyoxylate pathway enzyme isocitrate lyase is essential for virulence in diverse pathogenic organisms. One exception is C. neoformans: despite high transcription of ICL1 in the host, no reduction in virulence was observed when the gene was mutated (61). Here a mutation was isolated in the gene encoding the second enzyme unique to this pathway, malate synthase (MLS1), to test if this gene played a role in virulence. We hypothesized that the high transcription of ICL1 in vivo in an mls1 mutant background may cause metabolic perturbation to the fungus and that icl1 and mls1 mutations might confer different phenotypes. Like the icl1 mutant, the mls1 mutant was unable to grow on acetate as the sole carbon source (Fig. 2A). A wild-type strain and an mls1 mutant strain were inoculated into mice or an alternative insect host (wax moth larvae), and survival was monitored daily. Mice or wax moths infected with either strain succumbed to lethal infection with the same efficiency as the wild type, indicating that MLS1 plays no role in virulence under these conditions (Fig. 2B and C). Thus, the two components of the glyoxylate pathway, isocitrate lyase and malate synthase, have similar functions in C. neoformans to promote growth on the two-carbon substrate acetate, but appear to play no role in virulence in a murine inhalation or insect assay.


Figure 2
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  		FIG. 2. Malate synthase of C. neoformans is required for growth on acetate as the sole carbon source, but is not required for virulence. (A) Dilutions (10-fold) of yeast cells were inoculated onto minimal medium plates containing glucose or acetate as the sole carbon source, and grown for 2 days at 30°C. Yeast cells (1 x 105) of wild-type (WT) and the mls1 mutant strains were inoculated into mice (B) or the wax moth Galleria mellonella (C), and survival was monitored daily.

 
Protein localization to peroxisomes is impaired by mutating the PEX1 or PEX6 genes. The glyoxylate pathway is generally considered to be localized within the peroxisomes of plants and fungi (53, 81). We therefore aimed to disrupt peroxisomes to test their function with respect to the glyoxylate pathway and virulence. The completed genome sequence of C. neoformans (35) was searched for homologs of highly conserved peroxin (PEX) genes that have been identified from organisms as required for peroxisome formation or function. To test the function of peroxisomes in the biology of C. neoformans, two key genes required for peroxisome function in other organisms were disrupted. Pex1 and Pex6 homologs are functionally-related AAA (ATPases associated with various cellular activities)-type proteins with a role in the assembly of small peroxisomal vesicles into peroxisomes, aiding vesicle fusion in an ATP-dependent manner, and/or protein import into the peroxisomes (32, 59, 64, 71). The PEX1 and PEX6 genes of C. neoformans were mutated by targeted disruption, and a pex1 pex6 double mutant was obtained as a meiotic segregant from a pex1 x pex6 cross. The pex1 mutation was complemented by reintroduction of a wild-type copy of the PEX1 gene.

Peroxisome function was assayed based on localization of a fluorescent protein into these organelles. Addition of a peroxisome targeting signal 1 (PTS1), such as variants of the tripeptide -SKL, to the C-terminal end of proteins can enable their translocation into the peroxisome (16). Even conjugation of the -SKL tripeptide to gold beads targets their import into peroxisomes (77). C. neoformans Icl1 terminates with -HKL. When a DsRed-SKL protein was expressed in a wild-type background, a punctate fluorescence pattern was observed, indicating that the DsRED protein was localized to peroxisomes (Fig. 3). Punctate fluorescence was observed in cells growing on normal carbon sources (i.e., glucose) and did not require induction by fatty acid carbon sources, which is consistent with peroxisomes being present in the related Cryptococcus humicolus species under glucose conditions (30) but in contrast to several other fungi in which peroxisomes are either not formed or are degraded in the presence of glucose (1, 24, 73).


Figure 3
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  		FIG. 3. Disruption of peroxisome function in C. neoformans causes mislocalization of DsRed-SKL. C. neoformans cells were transformed with constructs to express the red fluorescent DsRED protein with or without an additional -SKL tripeptide at the C-terminal end. The protein localizes to peroxisomes as punctate structures in wild-type strains, while the pex1 and pex6 mutant strains show a cytoplasmic distribution.

 
The DsRED-SKL strain with punctate peroxisomal fluorescence was crossed to the pex1 mutant, and meiotic progeny (a total of 18) were isolated. Half of those that were fluorescent (4/9) showed a pattern of discrete localization, whereas the other half (5/9) showed fluorescence throughout the cell. Those five progeny with diffuse cytoplasmic fluorescence all bore a disruption of the pex1 gene. In a similar genetic approach (38 progeny analyzed: 12/22 localized, wild type for PEX6 wild type; 10/22 nonlocalized, pex6 deletion), no distinct localization was observed when the DsRED-SKL protein was expressed in the pex6 background (Fig. 3). Thus, pex1 or pex6 deletions impair the transport of a peroxisomal marker protein and likely impact the function of peroxisomes in C. neoformans.

PEX1 and PEX6 are required for growth on minimal medium containing glucose, but not acetate. The phenotypes of the pex1 and pex6 mutant strains were compared to those of the wild-type strain (H99), the pex1 PEX1 complemented strain, and the icl1 and mls1 mutants (Fig. 4; Table 4). As noted above, the mls1 and icl1 strains were unable to grow on acetate as their sole carbon source. The pex1, pex6, and pex1 pex6 mutant strains of C. neoformans showed growth equivalent to that of the wild type on YNB with acetate. Wild-type C. neoformans exhibits slow growth on many fatty acids (19); the icl1, mls1, and pex mutants all show a marked reduction in growth on YNB with the fatty acid oleic acid as the carbon source (Table 4). Curiously, pex mutant strains all grew poorly on YNB media with glucose as the sole carbon source. Growth of pex1 or pex6 mutant strains was also inhibited on medium containing a mixture of glucose and acetate, which can be interpreted as the presence of glucose either is toxic to the cells or causes carbon catabolite repression of genes required for acetate utilization (Table 4). To ensure that the phenotype of the pex1 and pex6 mutants in the presence of glucose was not due to an artifact of medium preparation, growth of the C. neoformans strains was compared to that of S. cerevisiae icl1 and pex1 mutant strains. The S. cerevisiae pex1 mutant showed equal growth to the reference strain on YNB with either glucose or acetate, while the icl1 mutant was unable to grow on acetate (Fig. 4).


Figure 4
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  		FIG. 4. The PEX1 or PEX6 gene is required for metabolism on minimal media with glucose as an additional carbon source, but is not required for growth on acetate. Shown are phenotypes of C. neoformans (C. n.) and S. cerevisiae (S. c.) strains growing on different carbon sources.

 

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  		TABLE 4. Qualitative growth rates of C. neoformans strains grown on yeast nitrogen base agar supplemented with 2% glucose, acetate, or galactose, or 1% of each in mixed carbon media

 
Pex5 functions in -SKL targeting to peroxisomes, while Pex7 functions in the growth defect seen in pex1 or pex6 mutants. To elucidate which proteins are responsible for the glucose-defective growth phenotype of pex1 and pex6 mutants, two genes that control targeting of proteins to peroxisomes were mutated: PEX5 and PEX7, which were identified based on their similarity to the S. cerevisiae homologs. Pex5 is a conserved protein that binds to the peroxisome-targeting signal 1 (PTS1) located at the C-terminal end of proteins and assists in their import into the peroxisome. There are two putative homologs of PEX5 in C. neoformans, similar to the situation in humans, while there is only one copy in ascomycete fungi (34). There is a single copy of PEX7, which encodes the protein that binds a second class of peroxisome targeting signal (PTS2). The gene encoding the DsRed-SKL protein was crossed into the pex5 and pex7 mutant backgrounds. Localization was severely impaired in pex5 cells. However, occasional punctate localization was observed, which may be consistent with a partially functional second homolog of PEX5 in the C. neoformans genome (Fig. 5B). Punctate localization equivalent to that seen in DsRED-SKL in wild-type cells was observed in the pex7 mutant background, indicating that Pex7 is dispensable for targeting this type of protein to the peroxisomes.


Figure 5
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  		FIG. 5. Function of the Pex5 and Pex7 proteins for targeting proteins to peroxisomes or growth on minimal media, respectively. (A) Serial dilutions (10-fold) of strains were grown for 3 days on YNB with 2% glucose (at 22°C) or 2% acetate (at 30°C). (B) Punctate localization of DsRED-SKL is impaired in the pex5 mutant background, but not in the pex7 mutant background. (C) Growth of the strains shown in panel A on glucose or galactose (at 30°C).

 
Growth rates of the wild-type and pex1, pex5, and pex7 mutant strains were compared on minimal media supplemented with glucose or acetate (Fig. 5A) and other carbon sources (Table 4). The pex5 mutant grew like the wild type, while the pex7 mutant exhibited reduced growth on YNB with glucose, although not as dramatically as that seen with the pex1 mutant (Fig. 5A). To further illustrate this reduction in growth rate, strains were assessed in YNB media with glucose or galactose as the carbon sources. The pex7 mutant exhibits a modest reduced growth rate over the first 24 h of culture (Fig. 5C). The pex5 mutant showed an equivalent reduction in growth on oleic acid, as seen in strains bearing pex1 or pex6 deletions. In contrast, deletion of pex7 had no effect on growth on oleic acid. These data suggest that Pex5 mediates the targeting of the PTS1 proteins to the peroxisomes, including those for fatty acid utilization. Second, the data suggest that the PTS2 (Pex7-dependent) pathway of protein import into the peroxisomes could in part mediate the reduced growth rates seen in pex1 or pex6 mutant backgrounds on glucose medium.

Peroxisome function is not required for C. neoformans virulence in two animal host models. The pex1 or pex6 mutants show a growth defect on YNB medium, which is the standard minimal medium used for this fungus. We hypothesized that this defect might compromise the virulence of these strains. The C. neoformans mutants were examined for in vitro changes in traits most commonly associated with virulence. Growth at mammalian temperature (37°C) was equal to that at 30°C. Melanin and capsule were produced normally in the C. neoformans mutants. The pex1 mutant strain was tested for virulence in the murine nasal inhalation model. Surprisingly, wild-type, pex1 mutant, and pex1 PEX1 complemented strains showed equivalent virulence in this assay (Fig. 6A). As a second test of virulence, the wax moth larvae model was used. As with the murine inhalation model, no decrease in virulence was observed in the pex1 mutant background (Fig. 6B). Thus, in two diverse models there is no evidence for a role of peroxisomes in C. neoformans virulence.


Figure 6
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  		FIG. 6. The pex1 mutant is virulent, despite an in vitro growth defect. Yeast cells (1 x 105) of the wild-type (WT), pex1 mutant, and pex1 PEX1 complemented strains were inoculated into mice (A) or wax moth Galleria mellonella (B), and survival was monitored daily.

 
C. neoformans peroxisomes are required for efficient growth on glucose and other monosaccharides. The most unexpected and curious phenotype of the C. neoformans pex1 or pex6 mutants was represented by their growth defects on minimal medium (YNB). Peroxisomes have numerous and diverse functions in different organisms (46). Disruption of peroxisome function does not usually impair fungal growth under rich growth conditions (25, 32, 36): these data on C. neoformans provide a rare exception. A second exception is the ascomycete fungus Penicillium chrysogenum. In this fungus, a strain with a mutation in the PEX5 gene, required for transport of peroxisomally targeted proteins, shows poor growth, while isolation of mutants with mutation in PEX1 or PEX6 was not possible, suggesting that these genes may be essential (31, 33). A recent report from another ascomycete, M. grisea, suggests that growth rate of the pex6 mutant strain is also slightly reduced (60).

There are two possible explanations for the adverse effects of glucose on growth of the pex1 and pex6 mutants. The first is a defect in response to oxidative stress, e.g., that the mitochondria are producing reactive oxygen intermediates during respiration that cannot be detoxified in the absence of the peroxisomes. The second hypothesis is that some component of primary metabolism, such as nitrogen/amino acid or carbon metabolism, is localized to the peroxisomes of C. neoformans.

First, the possible role of reactive oxygen detoxification by peroxisomes was examined. To test this hypothesis, we examined the four catalases in the C. neoformans genome. One catalase (encoded by CAT2) contains a PTS2 motif, suggesting it could be targeted to the peroxisomes in a Pex7-dependent manner. Cat2 also clusters within the peroxisomal catalase clade by phylogenetic analysis (20). The catalase CAT2 gene was mutated (20), and the cat2 mutant strain exhibited wild-type growth in the presence of glucose, in contrast to a pex1 or pex6 mutant, showing that this gene is not required for the phenotype. However, the C. neoformans quadruple catalase mutant also grows like the wild type on glucose and has no in vitro phenotype (20), and the recent finding of the absence of catalases in N. crassa peroxisomes suggests that this enzymatic marker may not be representative of peroxisome function (63). The pex1 and pex6 mutants are no more hypersensitive to H2O2 than the wild type, and addition of the antioxidant ascorbic acid (vitamin C) did not improve growth of pex mutants in YNB medium (data not shown). Further evidence against an oxidative stress phenotype is that no change in growth was observed at 37°C, a high temperature that can trigger increased reactive oxygen species (Fig. 7B) (data not shown). Taken together, these observations suggest that the glucose-specific phenotype is not attributable to a change in oxidative stress tolerance.


Figure 7
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  		FIG. 7. Peroxisome function is carbon source specific. (A) Ten-fold serial dilutions of strains were grown on YPD or YNB supplemented with glucose, mannose, fructose, galactose (all 2%), or glucose plus galactose (1% each) and photographed. Growth was for 2 days at 30°C. (B) Growth curves of C. neoformans wild-type, pex1 mutant, and pex1 PEX1 complemented strains in glucose and galactose at 30°C and 37°C.

 
Second, the role of nutrient availability was examined. Consistent with normal growth on acetate, but not glucose, growth rates on a wide selection of synthetic omission media were the same for the strains (data not shown), suggesting that poor growth was not due to a role for the peroxisomes in biosynthesis of a specific amino acid or nucleotide. Next, different carbon sources were investigated. Of four monosaccharides tested, glucose, fructose, and mannose showed similar reduced growth rates of pex1 or pex6 mutant strains compared to wild-type cells. In contrast, growth of the pex1 and pex6 mutant strains on galactose was nearly identical to that of the wild type (Fig. 7A and B). This was apparent in both solid agar and liquid cultures. All four monosaccharides enter glycolysis to produce fructose 6-phosphate as a common intermediate. The key difference between galactose with fructose, glucose, and mannose is that the later three require hexokinase activity to enter the glycolytic pathway. Yeast extract-peptone base is a rich medium. While we observed a slight decrease in growth rate of the pex1 and pex6 strains on YPD medium that contains glucose, we note that C. neoformans exhibits robust growth on yeast extract-peptone in the absence of glucose, whereas no difference in growth was observed between the pex mutants and the wild type. In addition, hexokinases have various specificities towards different sugars, which may account for the slight variations in growth observed on different carbon sources (Fig. 7A).

The C. neoformans genome was searched for hexokinase homologs using the S. cerevisiae HKX1 and HXK2 genes and the glucose-specific glucokinase (GLK1) gene. Two matches were obtained: one (HXK1) most similar to HXK1 and HXK2 of S. cerevisiae and the other (HXK2) with highest similarity to GLK1. The predicted protein sequences of the two C. neoformans genes were examined for potential peroxisome targeting sequences. There is no evidence of a C-terminal PTS1-type sequence (like -SKL) for either. For C. neoformans Hxk2, there was an N-terminal stretch of nine amino acids (KVVDIVKHF) similar to the most recently described consensus for PTS2 (R/K)(L/V/I/Q)XX(L/V/I/H/Q)(L/S/G/A/K)X(H/Q)(L/A/F) (56). We hypothesized that impaired function of Hxk2 in the peroxisome mutants could be due to mislocalization of this protein to the cytoplasm, leading to inactivity or a new deleterious function.

Hexokinase 2 (Hxk2) deletion partially suppresses the pex1 phenotype, but Hxk2 is not localized to the peroxisomes. Based on the potential PTS sequence in hexokinase and aided by the new fluorescence protein vectors, the HXK2 gene was studied. The HXK2 gene was deleted, and a double hxk2 pex1 mutant was isolated by crosses and confirmed by Southern blot analyses (data not shown). The hxk2 mutant showed reduced growth on glucose, but equal growth on galactose, relative to the wild-type strain (Fig. 8A and B; Table 4). However, growth in the hxk2 mutant was not as severely reduced as that in the pex1 mutant, providing an opportunity to assess the effects of deletion of both genes. The hxk2 pex1 double mutant had a growth rate like that of the hxk2 single mutant, not the pex1 mutant, in the presence of glucose, as well as in media containing glucose and acetate or galactose. Thus, a partial suppression of the growth defects of the pex1 mutation by deletion of HXK2 suggests that the defects observed in pex1 mutants could be due to incorrect localization of Hxk2.

The HXK2 gene was fused to the DsRED or GFP genes and expressed in C. neoformans cells from a constitutively active promoter (from histone H3; fluorescence was too low from the native HXK2 promoter [data not shown]). In most cells, the protein was localized to the cytoplasm and a single structure in the cell (Fig. 8C). However, when the Hxk2-DsRED protein was expressed in a pex5 or pex1 background, no change was seen in this punctate localization (data not shown), suggesting it may be an artifact of overexpression or fusion to the DsRED protein. In further confirmation, the HXK2 gene was fused to the GFP gene and introduced into C. neoformans cells. In this case, localization was solely cytoplasmic, with no evidence of any punctate localization (Fig. 8C). Thus, we conclude that Hxk2 is not localized to the peroxisomes in C. neoformans and that the fungus therefore does not contain the equivalent of a glycosome.


   DISCUSSION
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
C. neoformans is a fungus, pathogenic to humans, that lives in both the environment and the mammalian host. Therefore, the fungus must be able to adapt to diverse nutrient conditions to be successful. Here we investigated the role of peroxisomes in the biology of C. neoformans. To assess peroxisome function, two genes were selected initially for targeted mutation. Pex1 and Pex6 homologs are well studied as functionally-related AAA-type proteins with a role in the assembly of small peroxisomal vesicles into mature peroxisomes and import of proteins into peroxisomes (59). Pex1 and Pex6 interact physically in fungal and mammalian cells and function in early stages of peroxisome formation (13, 17, 32, 69, 71). Disruption of the interaction between Pex1 and Pex6 is a common pathophysiological mechanism in patients with Zellweger syndrome, which is a class of peroxisomal defects causing early death due to accumulation of toxic intermediates usually metabolized in the peroxisome (17). A homolog of PEX6 is essential for virulence of the fungi Colletotrichum lagenarium on bean and Magnaporthe grisea on rice or barley (36, 60). In both fungi, the PEX6 homologs are required for fatty acid utilization, such as growth on Tween 80 or olive oil. Mutants are unable to use fatty acids during formation of the specialized infection structure, the appressorium, but can infect plants when inoculated into a wound site.

The phenotype of the C. neoformans pex1 or pex6 mutants includes reduced growth on fatty acids; however, the mutants can use acetate as a sole carbon source. These data suggest that, like many other organisms, peroxisomes are required for utilization of some fatty acid sources. In contrast to both plants and other fungi, the ability to utilize acetate in the pex1 or pex6 mutants suggests that glyoxylate pathway components are unlikely to be localized solely in the peroxisome or are present in remnant peroxisome bodies or mislocalized (but functional) in the cytoplasm or another organelle. There is evidence that the glyoxylate pathway is localized to peroxisomes in fungi, including the ascomycetes Ashbya gossypii, Aspergillus nidulans, Candida tropicalis, Botryosphaeria dothidea, Hansula polymorpha, and Neurospora crassa (3, 11, 29, 35, 45, 74) and in the basidiomycetes Fomitopsis palustris and Coprinus species (8, 54, 62). Thus, the peroxisomes of C. neoformans function somewhat similarly to those of S. cerevisiae, which are also not required for acetate metabolism (12) (Fig. 4). Current evidence suggests Icl1 is not localized in the peroxisomes, while Mls1 is localized there only in the presence of oleic acid (9, 38, 70). In recent studies, it has been shown that mutants lacking the PEX5 or PEX6 homologs of C. albicans or A. nidulans, respectively, can also grow efficiently on acetate (25, 57), whereas at least in another ascomycete species, C. lagenarium, mutation of the PEX6 homolog reduces growth on acetate (2). While these studies do not exclude the glyoxylate pathway enzymes from being present in the peroxisomes during ß-oxidation of fatty acids, the glyoxylate pathway can function under other growth conditions in pex mutants. It is possible that the use of fatty acids as a peroxisome induction source may have biased previous reports on the localization of the enzymes in fungi.

In contrast to other fungi, C. neoformans has an unusual requirement for peroxisomes for efficient growth in the presence of monosaccharides like glucose, fructose, and mannose that are metabolized by hexokinase. Hexokinase is the first enzyme of glycolysis. The glycosomes of kinetoplasts like trypanosomes (e.g., Trypanosoma and Leishmania species) represent an unusual and unique form of peroxisome because of their role in glycolysis. Seven to nine of the enzymes for glycolysis, including hexokinase, are localized in the peroxisomes (reviewed in reference 55). Mutation of either PEX2 or PEX14 in T. brucei by double-stranded RNA interference causes the organisms to die in the presence of glucose (15, 23). The proteins of C. neoformans encoding the other nine enzymes in the glycolytic pathway were examined for possible peroxisomal targeting sequences. No evidence for a PTS1 was found, and only enolase had a potential PTS2 sequence (KIDQLLIQL), although not at the N terminus where PTS2 is usually, though not invariably, located. We were excited by the prospect that C. neoformans may also have the equivalent of a glycosome. Deletion of the HXK2 gene results in a reduced growth rate on glucose, and the hxk2 pex1 double mutants partially rescue the reduced growth rates seen in the pex1 single mutants grown in the presence of glucose. However, Hxk2 fusions to fluorescent markers were localized either in the cytoplasm or to a nonperoxisomal structure, depending on the fusion protein. Thus, we hypothesize that Hxk2 regulates a peroxisomal protein required for C. neoformans growth, either directly or via a glycolytic intermediate, such as glucose-6-phosphate, which is a signaling molecule in other fungi (48).

Our initial aim was to investigate aspects of the genetic controls of virulence of C. neoformans. Enzymes of the glyoxylate pathway (here Mls1 and previously Icl1) are dispensable for C. neoformans virulence. We were reluctant to test the virulence of the pex1 mutant towards mice because the strain showed a clear growth defect on minimal medium. Nevertheless, when tested for virulence, pex1 mutant strains were as virulent as the wild type, thus showing that (i) growth patterns on minimal medium in vitro are not reliable predictors for virulence outcome and (ii) during mammalian infection, fatty acid utilization and glucose metabolism are unlikely to be the major nutrients available for growth, in contrast to other fungi (37). It has recently been shown that the pex5 mutant of C. albicans shows virulence equivalent to that of wild-type strains in a murine infection model (57). C. neoformans associates with environmental predators, like amoeba and possibly insects. We also tested the mls1 and pex1 mutants in the wax moth system and found no reduction in virulence in this insect host. While our research aimed to identify potential new drug targets for controlling cryptococcosis, these data show that the glyoxylate pathway and peroxisomes are less than ideal targets. Future research on peroxisome function in C. neoformans, which is an outstanding system for molecular genetic analysis, will focus on the identification of the mechanisms of interaction of these organelles with glucose metabolism via proteomic or genetic analyses.


   ACKNOWLEDGMENTS
 
We thank Weihua Fan and Kirsten Nielsen for assistance with experiments, and Connie Nichols, Tian Lian, and Jim Kronstad for fluorescent protein vectors.

This research was funded in part by NIAID grants AI028388 (J.R.P.) and AI063443 (J.H.).


   FOOTNOTES
 
* Corresponding author. Mailing address: Room 322 CARL Building, Box 3546, Department of Molecular Genetics and Microbiology, Duke University Medical Center, Durham, NC 27710. Phone: (919) 684-2824. Fax: (919) 684-5458. E-mail: heitm001{at}duke.edu. Back

{triangledown} Published ahead of print on 13 October 2006. Back


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 ABSTRACT
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 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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