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Infection and Immunity, August 2005, p. 5022-5030, Vol. 73, No. 8
0019-9567/05/$08.00+0     doi:10.1128/IAI.73.8.5022-5030.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.

Identification and Characterization of an SKN7 Homologue in Cryptococcus neoformans

F. L. Wormley Jr.,* G. Heinrich, J. L. Miller, J. R. Perfect, and G. M. Cox

Duke University Medical Center, Durham, North Carolina 27710

Received 23 November 2004/ Returned for modification 27 December 2004/ Accepted 1 April 2005


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ABSTRACT
 
Cryptococcus neoformans is an encapsulated fungal pathogen that primarily infects the central nervous system of immunocompromised individuals, causing life-threatening meningoencephalitis. The capacity of C. neoformans to subvert host defenses and disseminate by intracellular parasitism of alveolar macrophages in the immune-compromised host has led to studies to evaluate genes associated with C. neoformans resistance to oxidative stress. In the present study, we identify and characterize a C. neoformans homologue to SKN7, a transcription factor in Saccharomyces cerevisiae that regulates the oxidative stress response, cell cycle, and cell wall biosynthesis. To examine the contribution of SKN7 in the pathogenesis of fungal infections, we created skn7 mutants via targeted disruption. The skn7 mutants were observed to be more susceptible to reactive oxygen species in vitro and were significantly less virulent than the wild-type strain and a reconstituted strain as measured by cumulative survival in the mouse inhalational model. The Skn7 protein was observed to be important for expression of thioredoxin reductase in response to oxidative challenge. Interestingly, skn7 mutants were also observed to flocculate following in vitro culture, a novel phenotype not observed in skn7 mutants derived from other fungi. These findings demonstrate that SKN7 contributes to the virulence composite but is not required for pathogenicity in C. neoformans. In addition, flocculation of C. neoformans skn7 mutants suggests a potentially unique function of SKN7 not previously observed in other cryptococcal strains or skn7 mutants.


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INTRODUCTION
 
Cryptococcus neoformans, the etiological agent of cryptococcosis, is an encapsulated fungal pathogen that infects the central nervous system of immunocompromised individuals, causing life-threatening meningoencephalitis (31). Cryptococcosis occurs in approximately 5 to 25% of AIDS patients worldwide (37), and studies have shown that 2.8% of organ transplant recipients can develop C. neoformans infections, resulting in an overall death rate of 42% (16). Although highly active antiretroviral therapy has contributed to a significant decrease in the incidence of cryptococcosis in AIDS patients in developed countries (2), increases in organ transplant recipients and patients undergoing extensive corticosteroid therapy forecast a rise of cryptococcosis in other high-risk populations. In medically advanced countries the acute mortality rate is between 10 and 25% (37), and conventional antifungal agents are often excessively toxic, lack potent fungicidal properties, or are being rendered less effective by the emergence of drug-resistant strains. Therefore, continued studies are needed to identify novel targets for the development of drugs or vaccines to combat cryptococcal infections.

Obligate aerobic microorganisms, such as C. neoformans, are under persistent exposure to endogenous oxidative stress caused by the incomplete reduction of oxygen to water, which yields reactive oxygen species (ROS) such as H2O2, the hydroxyl radical (HO–), and superoxide anions (O2–) (15, 45). Exposure to ROS at excessively high levels can induce toxic damage to DNA, lipids, and proteins, resulting in cell death. In addition, fungi pathogenic to humans are exposed to exogenous sources of ROS from host immune cells (4).

Given the deleterious effects that ROS can have on cellular constituents, it is not surprising that yeast cells possess both enzymatic (e.g., catalase, superoxide dismutase, and thioredoxin peroxidase) and nonenzymatic (e.g., glutathione and thioredoxin) mechanisms that counteract the effects of ROS (17, 29, 33). When the level of ROS within a cell exceeds the capacity of the available antioxidant defenses to inactivate the ROS, yeast cells respond with the de novo synthesis of antioxidant proteins in a process termed the oxidative stress response (OSR). Previous studies in C. neoformans have shown that inactivation of genes participating in the OSR render the strains more susceptible to macrophage-mediated fungistasis and attenuates virulence (3, 9, 18, 30, 35).

The SKN7 gene encodes a transcription factor that has been shown in Saccharomyces cerevisiae (21, 23, 32) and Candida albicans (41) to have an important role in the cellular response to oxidative stress. SKN7 was initially isolated as a multicopy suppressor of a kre9 mutation affecting cell wall biosynthesis (5) and elsewhere cloned as POS9 (positive for peroxide sensitivity) in a screen for S. cerevisiae mutants with elevated sensitivity to hydrogen peroxide (20). The S. cerevisiae AP-1 homologue YAP1 and SKN7 have been shown to cooperate in the transcriptional regulation of the OSR by the induction of thioredoxin (TRX2) and thioredoxin reductase (TRR1) in response to oxidative stress (22, 23, 32). Given the precedence of other studies showing the importance of the yeast OSR in virulence, we investigated whether SKN7 has a similar role in C. neoformans. We have identified a gene within C. neoformans strain H99 with sequence homology to the SKN7 genes of S. cerevisiae and C. albicans. In the present study we created specific C. neoformans skn7 mutants using targeted gene disruption and demonstrate that SKN7 contributes to the OSR and is involved in virulence. In addition, skn7 mutants were found to have a flocculation phenotype not previously described in skn7 mutants of other fungi.


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MATERIALS AND METHODS
 
Strains and media. C. neoformans strain H99 (serotype A, Mat{alpha}) and strain H99R (a spontaneous ura5 auxotroph derived from H99 by plating on 5-fluoroorotic acid agar) were recovered from 15% glycerol stocks and stored at –80°C prior to use in the experiments described herein. The strains were maintained on yeast extract-peptone-dextrose (YPD) medium (1% yeast extract, 2% peptone, and 2% dextrose). Transformants were selected on uracil dropout medium containing 1 M sorbitol (10, 11), and reconstituted (REC) strains were selected on YPD medium supplemented with 100 µg of nourseothricin (clonNAT; Werner Bioagents, Jena, Germany) per ml as previously described (27).

Identification and disruption of SKN7. The predicted amino acid sequence of S. cerevisiae SKN7 (www.yeastgenome.org) was used to query the C. neoformans strain H99 genomic database (cneo.genetics.duke.edu) to identify the cryptococcal homologue. Primers (SKN7F, 5'-GCTGGAGGACTTGATGAGTA-3', and SKN7R, 5'-CAAGGTGCTTGTCGAGGATA-3') spanning the genomic locus were used to amplify a 2,413-bp genomic fragment that was subcloned into a plasmid. Sequencing confirmed the identity of the cloned fragment. The disruption construct was created by insertion of a 2,029-bp genomic URA5 fragment into the single HpaI site located in the coding region. The disruption construct was used to transform strain H99R using biolistic delivery as described previously (10, 11). Stable prototrophs were selected on ura dropout medium and analyzed using colony PCR and primers flanking the URA5 insertion (SKN7UF, 5'-CCGTATGCACTTGATGGAAG-3', and SKN7UR, 5'-GAGCAATGTTGCTGTGGTA-3') to identify strains containing a disrupted SKN7 gene. Confirmation of the disruption was done by Southern blotting of genomic DNA digested with XhoI and BamHI and probed with a [32P]dCTP-labeled SKN7 fragment. The reconstitution construct was generated using primers (SKN7RecF, 5'-CCGATTCATTCACTGTCGCC-3', and SKN7 RecR, 5'-CCCGGGTAAATACATCTGTAGCCATA-3') homologous to regions forward and rear of the predicted SKN7 gene and including a SmaI restriction enzyme site (underlined) The amplicon was subcloned into a plasmid, and the nourseothricin resistance cassette (27) was inserted into the SmaI site. The resulting plasmid was used to transform skn7 mutants using biolistic delivery, and stable reconstituted transformants were selected on YPD-nourseothricin agar.

Phenotypic assays. Prior to testing, C. neoformans strains H99, skn7, and REC were grown for 16 to 20 h at 30°C with shaking in YPD medium, harvested, and washed three times in sterile phosphate-buffered saline (PBS). For all studies requiring the enumeration of the skn7 mutant, the suspension was dispersed several times through a 1-ml syringe equipped with a 27-gauge needle which completely disrupted all flocculent cells. Temperature sensitivity of each strain was analyzed by growth on YPD agar at 30°C and 37°C and in Dulbecco's modified eagle medium (DMEM; GIBCO, Grand Island, NY) at 37°C with 5% CO2. Melanin production was assayed by growth on Niger seed agar at 30°C. C. neoformans capsules were observed by microscopic examination of India ink preparations of yeast cells following growth overnight in DMEM (GIBCO) at 30°C with 5% CO2 to stimulate capsule production. Flocculation was evaluated in liquid YPD medium alone or supplemented with 10% mouse serum (Equitech-Bio Inc., Kerrville, TX), human serum or fetal bovine serum (Gibco), 0.4% bovine serum albumin (Sigma), 1 M mannose, or 1 M glucose (Sigma), or in DMEM equilibrated to pH 5, 7, or 8.5. Sensitivity to tert-butyl hydroperoxide (t-BOOH; Sigma) was assayed by culture of 1 x 105 yeast cells in 1 ml of YPD medium containing t-BOOH at a final concentration of 0.25 mM for 48 h at 30°C with shaking followed by quantitative culture on YPD agar. Additionally, yeast cells (1 x 104) were cultured in the wells of a 96-well plate in 200 µl of YPD medium containing 1.0 mM, 0.5 mM, 0.025 mM, 0.0125 mM, and 0.00625 mM of t-BOOH for 48 h. Aliquots (5 µl) of the cultures were then spotted onto YPD agar and incubated at 30°C for 72 h. Growth of wild-type, skn7, and REC strains was also evaluated in YPD medium containing menadione (4 µg/ml; Sigma) and DETA NoNOate {(Z)-1-[2(2-aminoethyl)-N-(2-ammonioethyl)amino]diazen-1-ium-1,2-diolate; 1 mM; Sigma}, caffeine (12.5 mM; Sigma), sodium orthovanadate (25 mM; Sigma), and Calcoflour white (10 µg/ml; Sigma) followed by quantitative culture on YPD agar. The concentrations of reactive oxygen (t-BOOH and menadione) and nitrogen (DETA NoNOate) species as well as cell wall inhibitors (caffeine, sodium orthovanadate, and Calcoflour white) used in these experiments were predicated on previous studies evaluating similar functions in yeast (12, 18, 19, 24, 35, 41). The MIC of the C. neoformans strains to caspofungin was determined as previously described (44). Caspofungin was a generous gift from Merck & Co., Inc.

Macrophage assays. The J774.16 macrophage-like cell line (American Type Culture Collection, Manassas, VA) was maintained at 37°C in 5% CO2 in culture medium consisting of DMEM supplemented with 10% heat-treated fetal bovine serum, 1x nonessential amino acids, 100 µg/ml penicillin-streptomycin, and 10% NCTC-109 medium. Macrophages were harvested by mechanical dislocation, washed three times in Hanks' balanced salt solution (GIBCO), and cell viability and number were quantified using trypan blue dye exclusion. The macrophage cell concentration was adjusted to 1 x 106 cells/ml in culture medium. Approximately 18 to 24 h prior to addition of yeast, the macrophages were primed with 100 U/ml of murine gamma interferon (IFN-{gamma}; Roche Diagnostics GmbH, Mannheim, Germany), stimulated with 0.3 µg/ml of lipopolysaccharide (LPS; Sigma), plated (100 µl) into the wells of a 96-well plate, and incubated at 37°C in 5% CO2. Cryptococci grown for 16 to 20 h at 30°C with shaking in YPD medium were harvested and washed three times in sterile PBS, and cell viability and number were quantified by trypan blue dye exclusion (Sigma). Yeast cells (1 x 106/ml) were incubated with 10 µg of 18B7 (immunoglobulin G1 anti-GXM monoclonal antibody; a generous gift from Arturo Casadevall) per ml at 30°C for 1 h and subsequently added to the wells containing macrophages. The macrophage-yeast cell mixtures were incubated at 37°C in 5% CO2 for 1 h, washed with two exchanges of sterile PBS to remove any extracellular yeast cells, and incubated in 200 µl of culture medium for an additional 24 h. Medium was aspirated from each well, and macrophages were lysed with 100 µl of 0.5% sodium dodecyl sulfate. The wells were washed with sterile H2O, and aspirate, lysate, and washes were combined for quantitative cultures on YPD agar. Wells containing yeast cells alone served as controls in all experiments. All experiments were performed using triplicate wells.

Murine model. Female A/Jcr mice, 4 to 6 weeks of age, were obtained from the National Cancer Institute (NCI/Charles River Laboratories) and housed at the Duke University Medical Center vivarium. Mice were infected with 5 x 104 H99, skn7, or REC strain cells in 50 µl of sterile PBS by nasal inhalation as previously described (10, 11). The inocula were verified by quantitative culture on YPD agar. The mice were fed ad libitum and were monitored by inspection twice daily. Mice that appeared moribund or were not maintaining normal grooming were sacrificed by CO2 inhalation. All mice were handled according to guidelines approved by the Duke University Institutional Animal Care and Use Committee. Survival data from the mouse studies were analyzed using the Kruskal-Wallis test.

Real-time PCR. C. neoformans strain H99 was grown overnight at 30°C with shaking in YPD medium. Exponentially growing yeast cells were then diluted 1:10 in YPD medium and treated with 1 mM t-BOOH, 1 mM DETA NoNOate, or 1 mg/ml menadione (all obtained from Sigma) and allowed to grow for an additional 3 h. Untreated yeast cells were similarly allowed to grow at 30°C for an additional 3 h. Cultures were transferred to 50-ml centrifuge tubes (Corning Corp., Corning, NY) and subsequently centrifuged (800 x g) at 4°C to pellet yeast cells lyophilized until completely dry. Each sample was then vigorously vortexed with glass beads (3 mm) until a fine powder was created. Total RNA was then isolated using TRIzol reagent (Invitrogen) and DNase (Invitrogen) treated to remove possible traces of contaminating DNA according to manufacturer's instructions. First-strand cDNA was synthesized using the oligo(dT) primer and reagents supplied in the SuperScript III RT kit (Invitrogen) according to the manufacturer's instructions. The cDNA was used as template for analysis by real-time PCR using iQ SYBR Green Supermix (Bio-Rad, Hercules, CA) according to the manufacturer's instructions. All real-time PCRs were performed using the iCycler iQ Multicolor real-time detection system (Bio-Rad). For each real-time PCR, a master mix was prepared on ice with 4 mM of primer and 2 µl of a 1:5 dilution of cDNA. Primer sequences for SKN7 (Skn7RTF 5'-GCACTCGCGAAATCTAGGTC-3' and Skn7BTR 5'-GTAAGGCATCGTCGTTGGTT-3'), TRX2 (TRX2RTF 5'-TCCACGCTTCTCCTATTTCG-3' and TRX2RTR 5'-CACCTTGTACTTGGCAGCAA-3'), TRR1 (TRR1RTF 5'-CAAGGTTACCGTCCTTTGGA-3' and TRR1RTR 5'-TGTATCCATCGCTGTCAAGC-3'), SOD1 (SOD1RTF 5'-ACGTCCACGAGTTTGGAGAC-3' and SOD1RTR 5'-TTCGACCAATGATGGAGTGA-3'), and GLR1 (GLR1RTF 5'-GCTTCCTACGGCATCACTTC-3' and GLR1RTR 5'-CATGCAAGGAACCAAGACCT-3') were derived from sequence information obtained from The Institute for Genomic Research C. neoformans database (http://www.tigr.org/tdb/e2k1/cna1/) and were compared to that from the Duke Center for Genome Technology database (http://cneo.genetics.duke.edu/BLAST.html) to ensure accuracy of the primer sequences to amplify C. neoformans strain H99 sequences with the exception of the SKN7 primers. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (GAPDHF, 5'-AGTACTCCACACATGGTCG-3', and GAPDHR, 5'-AGACCAACATCGGAGCATCAGC-3') was used as an internal control. The software program Primer 3 (40) was used to design real-time primer oligonucleotides. The thermal cycling parameters contained an initial denaturing cycle of 95°C for 3 min followed by 40 cycles of 95°C for 20 s and 55°C for 45 s followed by an 80-cycle melt curve to confirm the amplification of a single product. A series of fivefold dilutions of cDNA template was amplified by real-time PCR to verify efficiency of each reaction. Results of the real-time PCR data were derived using the comparative Ct method as previously described (1, 7, 49) to detect the relative gene expression. The parameter Ct is defined as the point at which the amplification plot passes a fixed threshold above baseline. Each reaction was run in duplicate in separate tubes and normalized to a control, endogenous gene, GAPDH. The following formula was used to quantify the fold differential expression of a specific cryptococcal gene following treatment of the yeast compared to untreated cryptococci: 2–{Delta}{Delta}Ct, where {Delta}{Delta}Ct = [{Delta}Ct untreated sample -{Delta}Ct GAPDH of untreated sample] – [{Delta}Ct treated sample -{Delta}Ct GAPDH of treated sample]. {Delta}Ct represents the mean Ct value of each sample duplicate. The {Delta}{Delta}Ct range was determined by calculating the expression 2 –{Delta}Ct ± s, where s is the standard deviation of the difference calculated from the Ct standard deviation of both the gene of interest and GAPDH. Therefore, the result represents the fold increase or decrease in the expression of the gene in question following treatment of each C. neoformans strain compared to untreated cryptococci.

5'-RACE. The 5' end of the SKN7 gene was confirmed by the RNA ligase-mediated rapid amplification of cDNA ends (RLM-RACE) method, using a GeneRacer kit (Invitrogen) designed to obtain full-length 5' cDNA ends as per the manufacturer's instructions. First-strand cDNA synthesis was performed using 1 µg of total RNA from C. neoformans serotype A strain H99 grown in liquid YPD medium and treated with 1 mM of t-BOOH. The initial PCR was performed using a reverse gene-specific internal primer for SKN7 (SKN7UR, 5'-GAGCAATGTTGCTGTGGTA-3') and a GeneRacer 5' primer (homologous to the GeneRacer RNA Oligo supplied in the kit). A nested PCR was subsequently performed using a nested reverse gene-specific primer for SKN7 (SKN7Nested, 5'-AGTTTTGTGCGACAGCTGAA3') and a nested 5' GeneRacer primer (homologous to an internal region of the GeneRacer RNA Oligo provided in the kit). The PCR program was 3 min at 95°C, 35 cycles of 20 s at 95°C, 20 s at 55°C, 2 min at 72°C, and a final 10-min extension step at 72°C. The resultant PCR RACE product was ligated into the TA vector (Invitrogen) and sequenced. Sequence information derived from the 5'-RLM-RACE was used to develop primer sets to obtain further SKN7 cDNA fragments that were also cloned and sequenced. The predicted full-length amino acid sequence was obtained using the sequence information gathered from the RLM-RACE cDNA fragments and cDNA sequence information obtained from the Cryptococcus neoformans cDNA Sequencing Project database (http://www.genome.ou.edu/cneo.html).

Nucleotide sequence accession number. Sequence data were submitted to GenBank with accession number AY766114.


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RESULTS
 
Identification and disruption of C. neoformans SKN7. The predicted amino acid sequence of the S. cerevisiae SKN7 gene was used to search the C. neoformans strain H99 genomic sequence database. A single homologous cryptococcal sequence was identified and cloned. The corresponding cDNA was amplified and sequenced using RLM-RACE, and this information was used to identify introns and the putative amino acid sequence. We found that the C. neoformans strain H99 SKN7 gene was 3,081 bp long and contained three introns. The predicted amino acid sequence was 1,027 amino acids long and displayed similarities to Saccharomyces cerevisiae, Candida albicans, Schizosaccharomyces pombe, and Emericella nidulans of 69, 69, 65, and 63%, respectively. Search for conserved domains using the National Center for Biotechnology Information database (http://www.ncbi.nlm.nih.gov/BLAST/) identified areas within the SKN7 amino acid sequence with a high degree of homology to the DNA binding domain of heat shock factor 1 (residues 206 to 344), a coiled-coil region (residues 467 to 550) that is possibly responsible for protein-protein interactions, and a signal receiver domain of bacterial two-component response regulators (residues 691 to 804). Similarities of C. neoformans SKN7 to the C. albicans and S. cerevisiae SKN7 genes are predominantly restricted to the heat shock factor 1 DNA binding and the signal receiver domains.

We used the genomic SKN7 fragment to create mutants by using site-directed gene disruption. Disruption of the SKN7 gene in C. neoformans strain H99 was confirmed by PCR using primers flanking the URA5 insertion in the disruption construct (Fig. 1B), and Southern blot hybridization confirmed that a single insertion into the genome occurred and that the native band was displaced to the expected position in the mutant strain (Fig. 1C). Reconstitution of skn7 to the wild-type phenotype was accomplished by integration of a plasmid containing the wild-type SKN7 gene and a NATr selectable marker. Interestingly, PCR and Southern blot hybridization analysis of REC strains showed that integration of the reconstitution construct occurred at the native locus via a single crossover event (Fig. 1B and C). As expected with reintegration to the native locus, the REC strains were ura5 auxotrophs and resistant to nourseothricin. REC strains were restored to prototrophy by ectopic integration with a plasmid containing the wild-type URA5 gene.



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  		FIG. 1. (A) Map of genomic fragment containing SKN7. The URA5 gene was inserted into the HpaI site in SKN7 in order to create the disruption construct. Sites of the PCR primers used to verify disruption are indicated by arrows, and the sizes of the relevant pieces of DNA are also indicated. (B) PCR analysis of genomic DNA from the wild-type strain H99, the skn7 strain, and the REC strain performed with primers indicated in panel A. Disruption of the native SKN7 was indicated by the single amplicon at approximately 4,400 kb for the skn7 strain, and amplification of the native and restored SKN7 gene in the wild-type and REC strains, respectively, was indicated by amplification of a single amplicon at approximately 2,400 kb. (C) Southern blot of genomic DNAs from the same three strains (in the same order as in panel B) that were digested with XhoI and BamHI and probed with a labeled SKN7 genomic fragment.

Characterization of skn7 phenotypes. The skn7 mutant had no significant gross differences in capsule production as assessed by India ink examination, growth on solid YPD medium at 30 and 37°C, or melanin formation as assessed by growth on Niger seed agar (data not shown). However, the skn7 mutant appeared to be flocculent upon gross (Fig. 2A) and microscopic (Fig. 2B) examination following overnight incubation in liquid YPD medium at 30°C. The phenotype was consistently observed in several independent skn7 mutant strains. Similar to wild-type C. neoformans, the REC strain was observed to grow as free-floating cells following overnight culture at 30°C in liquid YPD medium (Fig. 2A and B). To determine the mechanism for the flocculation observed in the skn7 mutant, we examined the effects of potential flocculation inhibitors. Specifically, we examined possible inhibitors of flocculation mediated by lectin-like interactions, such as 1 M glucose and 1 M mannose. In addition, we used several possible inhibitors of flocculation mediated by protein-receptor interactions, such as growth in liquid YPD medium containing 10% mouse, human, or fetal bovine serum or 0.4% bovine serum albumin. We also assessed whether flocculation was affected by changes in pH by culture of the skn7 mutant in DMEM equilibrated to various pH levels (pH 5 to 8.5). These assays were, in part, predicated on conditions and principles suggested in previous reports (6, 43). Flocculation of the skn7 mutant was not observed upon gross (Fig. 2A) or microscopic (Fig. 2B) examination following culture of the yeast in liquid YPD medium containing 10% mouse serum. Flocculation of the skn7 mutant was also not observed following growth at 30°C in liquid YPD medium containing 10% human or fetal bovine serum or 0.4% bovine serum albumin. However, culture in liquid YPD medium equilibrated to various pH levels or containing 1 M mannose or 1 M glucose did not suppress the flocculation phenotype. To test whether the flocculation phenotype of the skn7 mutant is associated with a deleterious defect in cell wall integrity, we examined the susceptibility of the skn7 mutants to several inhibitors of cell wall structure/synthesis, including caspofungin, caffeine, sodium orthovanadate, and Calcoflour white, compared to wild-type and REC strains. Results demonstrated no significant differences in susceptibility of the skn7 mutant following exposure to the cell wall inhibitors tested compared to wild-type and REC strains (data not shown).



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  		FIG. 2. Flocculation of skn7 mutant. C. neoformans H99 and REC strains were cultured overnight in YPD liquid medium along with the skn7 mutant strain, which was cultured overnight in liquid YPD medium alone or supplemented with 10% mouse serum (MS) and observed by gross (A) and microscopic (B) examination. Magnification, x200.

Contribution of SKN7 in the response of C. neoformans to oxidative stress. To determine the resistance of the skn7 mutant to oxidative stress, quantitative cultures were prepared from wild-type, skn7 mutant, and REC strains cultured for 48 h in liquid YPD medium containing t-BOOH. As shown in Fig. 3A and B, growth of the skn7 strain in YPD medium containing 0.025 mM of t-BOOH for 48 h at 30°C was significantly retarded compared to wild-type (P < 0.001) and REC (P < 0.001) strains. The growth of wild-type and REC strains under these conditions was similar. There was no effect on the growth of the wild-type, skn7, or REC strains when cultured in YPD medium containing lower concentrations of 0.0125 or 0.00625 mM t-BOOH (Fig. 3A). Growth of the skn7 mutant in liquid medium containing menadione and DETA NoNOate was similar to that of wild-type and REC strains (data not shown).



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  		FIG. 3. Susceptibility of skn7 mutant to oxidative stress. (A) C. neoformans H99, skn7 mutant, and REC strains were cultured in liquid YPD medium containing t-BOOH at the indicated concentrations, spotted (5 µl) onto YPD agar, and incubated at 30°C for 72 h. (B) C. neoformans H99, skn7 mutant, and REC strains were cultured in liquid YPD medium containing t-BOOH (0.025 mM), and yeast viability was evaluated by quantitative culture on YPD agar. Asterisks indicate where significant differences were observed compared to wild-type H99 (*) and REC (**) strains incubated in liquid YPD medium containing 0.025 mM t-BOOH. The line indicates the initial yeast inoculum.

Induction of antioxidant transcripts in response to tert-butyl hydroperoxide. To characterize the expression of antioxidant transcripts of C. neoformans in response to oxidative stress, wild-type, skn7 mutant, and REC strains were cultured overnight at 30°C in liquid YPD medium and subsequently at 30°C in YPD medium with or without 1.0 mM of t-BOOH for 3 h. Total RNA was extracted, reverse transcribed, and used as template for analysis by real-time PCR and compared to GAPDH as an internal control. The expression levels of genes in strains treated with t-BOOH were calculated and expressed relative to gene expression in their untreated counterparts. Table 1 indicates little induction of SKN7 transcripts in C. neoformans strain H99 following treatment with 1 mM t-BOOH. As expected, no SKN7 transcripts were detected in cDNA derived from untreated or treated skn7 mutant cultures, and the induction of SKN7 transcripts in the REC strain in response to 1 mM t-BOOH was similar to the levels observed in the wild-type C. neoformans strain H99.


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  		TABLE 1. Induction of SKN7, TRX2, TRR1, SOD1, and GLR1 transcripts in response to t-BOOH treatment compared to untreated counterparts

To determine any putative role that SKN7 may have in the control of other genes involved in antioxidant defenses, we evaluated the induction of thioredoxin 2 (TRX2), thioredoxin reductase 1 (TRR1), superoxide dismutase 1 (SOD1), and glutathione reductase 1 (GLR1) transcripts following 3-h t-BOOH treatment relative to untreated yeast by real-time PCR in skn7 mutant yeasts compared to wild-type and REC yeast. Table 1 shows little to no induction of TRX2 transcript expression beyond basal levels in wild-type and REC strains in response to t-BOOH treatment. There appeared to be a slight decrease in TRX2 transcript expression in skn7 mutant cells in response to t-BOOH treatment compared to untreated skn7 mutant cells. Evaluation of TRR1 transcript induction of wild-type, skn7 mutant, and REC yeasts in response to t-BOOH treatment relative to its expression at 30°C demonstrated that TRR1 was induced approximately three- and twofold in wild-type and REC strains, respectively (Table 1), and the levels of TRR1 transcripts in skn7 mutant cells were decreased in response to t-BOOH treatment. SOD1 transcripts were induced in wild-type, skn7 mutant, and REC strains following t-BOOH treatment relative to growth at 30°C. However, SOD1 levels in the skn7 mutant were not induced at the levels observed for the wild-type and REC strains. Interestingly, GLR1 transcript levels were observed to be decreased to the same extent in wild-type, skn7 mutant, and REC strains following treatment with t-BOOH relative to GLR1 transcript expression at basal levels. No changes in the induction pattern of SKN7, TRX2, TRR1, SOD1, and GLR1 transcripts were observed following t-BOOH treatment for 1 h relative to untreated yeast by real-time PCR in skn7 mutant yeast compared to wild-type and REC yeast cells at 1 h.

Virulence of skn7 mutant in murine model of pulmonary cryptococcosis. To determine whether SKN7 was required for the virulence of C. neoformans, we compared the survival of mice inoculated with wild-type, skn7, and REC strains by nasal inhalation. As shown in Fig. 4, mice inoculated with the skn7 strain survived significantly longer than mice inoculated with the wild-type and REC strains (P < 0.001). The median survival of mice inoculated with the skn7 strain was 47 days (43 to 53 days), compared to 19 days (18 to 23 days) and 21 days (20 to 23 days) for mice inoculated with the wild-type and REC strains, respectively. There were no significant differences in survival between the wild-type and REC strains. Histological analysis of lung tissues from mice experimentally infected with 105 CFU of the skn7 mutant or wild-type C. neoformans strain H99 were examined on day 14 postinoculation to investigate whether the skn7 mutant was flocculent in vivo. Figure 5 shows several budding yeast cells in the lungs of mice inoculated with C. neoformans strain H99 (Fig. 5A) or the skn7 mutant (Fig. 5B). However, the skn7 mutant does not appear to occur in aggregates in the lungs of mice, as observed in vitro. To determine a possible mechanism (i.e., reduced intracellular growth) for the attenuated virulence of the skn7 strain, yeast cells were tested for growth within the J774.16 macrophage-like cell line following stimulation with recombinant murine IFN-{gamma} and LPS. Results showed that the intracellular survival of the skn7 mutant within a macrophage cell line in vitro was similar to wild-type and REC strains. Growth of the skn7 mutant in DMEM complete culture medium containing recombinant murine IFN-{gamma} and LPS was similar to wild-type and reconstituted strains.



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  		FIG. 4. Virulence of skn7 mutant in murine inhalation model. A/J mice (10/inoculation) were infected with 5 x 104 cells of C. neoformans strain H99, skn7 mutant, or the REC strain via nasal inhalation. The mice were monitored twice daily after infection, and mice that appeared to be morbidly ill were sacrificed.



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  		FIG. 5. Silver staining of lung tissue during experimental pulmonary crytococcosis. A/J mice were given an intranasal inoculation with 105 CFU of C. neoformans strain H99 (A) or the skn7 mutant (B). Whole-lung tissues were excised on day 14 postinoculation, fixed in zinc-formalin fixative, paraffin embedded, sectioned, and silver stained to visualize the yeast. The figure shows representative staining of several mice from four experiments. Arrows point to budding yeast. Magnification, x200.


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DISCUSSION
 
The present study evaluated the role of the SKN7 gene in the adaptation of C. neoformans strain H99 to oxidative stress and its contribution to pathogenesis by using an experimental mouse model of pulmonary cryptococcosis. Previous studies in S. cerevisiae and C. albicans have demonstrated an important role for SKN7 in the cellular response to oxidative stress (21, 23 32, 41). Since the release of reactive oxidant species (ROS; i.e., H2O2, HO–, and O2–) is a critical component for the microbicidal activity of effector phagocytic cells, we hypothesized that disruption of the SKN7 gene by targeted gene disruption would result in attenuation of virulence in C. neoformans.

Phenotypic analysis of the C. neoformans skn7 strain demonstrated that this gene does not contribute to the known virulence phenotypes of capsule, growth at 37°C, and melanin production. We did observe, however, that skn7 strains cultured at 30°C in liquid YPD medium were flocculent, a phenotype not previously observed in other Cryptococcus mutant strains or skn7 mutants derived from other fungi. Flocculation was observed in multiple independent mutants, suggesting that the phenotype was not an artifact. In addition, reconstitution of the native locus resulted in a reversal of the phenotype, and culture of the skn7 strain in liquid YPD medium containing serum (mouse or human) or 0.4% bovine serum albumin suppressed the flocculation phenotype, suggesting some protein receptor interaction may contribute to flocculation. As expected, the skn7 mutant was not observed to be flocculent in vivo or under other conditions simulating physiologic protein concentrations. Interestingly, SKN7 was first identified as a suppressor of the kre9 mutation in S. cerevisiae, which resulted in defective cell wall biosynthesis (5). In addition, the predicted amino acid sequence of Skn7p shows similar areas of homology to the DNA binding domains of yeast heat shock factor protein (Hsf1p) (42, 51), which is also induced in response to oxidants (26, 38) and, more notably, to a yeast suppressor gene for flocculation, SFL1 (14) in S. cerevisiae. Mutational analyses of the S. cerevisiae SFL1 gene suggested a role for it in the regulation of cell surface assembly, and mutations in SFL1 do lead to flocculation. A defect in cell wall assembly of the C. neoformans skn7 mutant was not found, as assessed by exposure to several inhibitors of cell wall structure/synthesis (caspofungin, caffeine, sodium orthovanadate, and Calcoflour white). Furthermore, we were unable to identify cryptococcal homologues to genes associated with flocculation, including SFL1 (14), FLO1 (36), and FLO11 (50), in the available cryptococcal gene databases. Thus, there appears to be little conservation of the pathways involved in flocculation between C. neoformans, S. cerevisiae, and C. albicans. Nonetheless, this phenotype is probably not directly associated with the virulence composite.

Oxidative metabolism within yeasts necessitates dedicated defense mechanisms to protect their cellular components from endogenous reactive oxidants and further exogenous oxidants in the host. The increased susceptibility of the skn7 strain to the oxidant t-BOOH in vitro compared to wild-type and REC strains indicated a clear role for SKN7 in resistance of C. neoformans to oxygen radicals. Culture of the skn7 mutant in liquid YPD medium containing 0.025 mM t-BOOH appeared to have a microbicidal effect on the yeast, as colony counts of the mutant strain were significantly lower than with the initial inoculum. However, the similar resistance of the skn7 mutant to treatment with menadione, a superoxide anion (O2–) generator, compared to wild-type and REC strains suggests either some redundancy in the OSR or that disruption of the SKN7 gene affects specific ROS. C. albicans skn7 mutants also demonstrated a susceptibility to t-BOOH, but not menadione, compared to wild-type yeast (41). The lack of any effects of DETA NoNOate, an NO donor, on the skn7 mutant supports the hypothesis that SKN7 function is limited to reactive oxidative and not nitrosative species. Interestingly, although t-BOOH was clearly shown to inhibit the growth of skn7 strains in vitro, no significant induction of SKN7 transcripts was measured following treatment of wild-type and REC strains with the oxidant. An explanation for our observation may be that SKN7 transcripts are readily and rapidly converted to protein upon sensing an oxidative threat and, therefore, changes in the induction of SKN7 transcripts expression are difficult to observe, or that posttranslational modifications occur. Alternatively, since the mechanisms responsible for adaptation of cryptococci to oxidative stress are currently incomplete, other known and/or yet-to-be-described cryptococcal antioxidant genes may be preferentially expressed in response to oxidative challenge compared to SKN7.

The evaluation of the effect that SKN7 disruption may have on other known factors involved in the OSR, namely, TRX2, TRR1, GLR1, and SOD1, yielded variable results. Studies in S. cerevisiae showed a clear role for SKN7 in the induction of TRX2 and TRR1 in response to oxidative stress (39). However, our results in C. neoformans did not demonstrate a significant role for SKN7 in the induction of TRX2 transcripts following treatment with t-BOOH over basal levels. Previous experimental studies have shown that residual TRX2 induction is produced in S. cerevisiae skn7 mutants in response to oxidative stress, suggesting the presence of another induction mechanism (32, 39). Indeed, an AP-1-like transcription factor identified as either YAP1, CAP1, or PAP1 in S. cerevisiae, C. albicans, and Schizosaccharomyces pombe, respectively, is also involved in the oxidative stress-induced expression of thioredoxin genes (34, 47, 48). AP-1-like transcription factors, such as mammalian c-Jun, contain a leucine zipper domain and an adjacent basic region that are important for dimerization and DNA binding, respectively. However, C. albicans YAP1 null mutants and YAP1/SKN7 double null mutants demonstrate residual oxidative stress-induced expression of thioredoxin, further suggesting the presence of another possible transcriptional regulator of thioredoxin (32, 39). Search of the C. neoformans H99 gene sequence did not reveal an obvious YAP1 homologue and suggested the presence of another factor that is involved in the induction of thioredoxin in C. neoformans.

The effect of SKN7 disruption was more evident in the induction of TRR1 in response to oxidative stress. Induction of TRR1 transcripts was nearly abolished in skn7 mutants compared to wild-type and REC strains in response to t-BOOH, suggesting that SKN7 is indeed associated with the induction of TRR1 in response to oxidative stress. Not surprisingly, SOD1 transcripts were increased in wild-type and REC strains in response to oxidative stress and induction of SOD1 transcripts in the skn7 mutants was lower. Similarly, the synthesis rate of Sod1p in S. cerevisiae skn7 mutants was observed to be decreased compared to wild-type yeast in response to oxidative stress (39). There was no GLR1 transcript induction beyond that expressed at basal levels in all strains tested in response to t-BOOH treatment. Studies in S. cerevisiae suggest that regulation of SOD1 and GLR1 is codependent and independent, respectively, of SKN7 (39). Our results appear to support these findings with respect to the role of SKN7 in the partial regulation of SOD1 and not GLR1 in C. neoformans.

As the lungs are the principal routes of entry for C. neoformans, clearance from the alveoli is largely dependent upon the ability of resident alveolar macrophages, and soon thereafter recruited neutrophils, to engulf and degrade the yeast cells. Several studies have addressed the role of macrophages and polymorphonuclear cells in the killing of C. neoformans (8, 13, 28, 46). The fungistatic and fungicidal activities of macrophages and neutrophils are partly mediated through the generation of reactive oxygen species produced during an oxidative burst (4, 25). Furthermore, correlations have been made between various yeast antioxidant enzymes and OSR transcriptional regulators and their impact on virulence in the host (9, 18, 30, 35, 41). In our studies, we were able to demonstrate that the virulence of the skn7 mutant was significantly attenuated compared to wild-type and REC strains within the murine inhalational model. It was attractive to hypothesize that a reduced antioxidant response would impact the intracellular growth of the skn7 mutant within macrophages, in vitro, as a possible mechanism for the attenuation of its virulence in vivo. However, growth of the skn7 mutant within a stimulated (LPS and IFN-{gamma}) macrophage cell line, in vitro, compared to wild-type and REC strains was similar, which supports the hypothesis that other redundant systems aid in maintaining the cellular redox state within C. neoformans in response to external oxidative challenge; thus, skn7 has little impact on intracellular growth. Furthermore, we were able to detect TRX2 transcript expression as well as the induction of SOD1 expression in skn7 mutants exposed to oxidants in vitro. In addition, other redundant mechanisms that aid in the protection of cryptococci from macrophage killing could contribute to protection against oxidative damage within macrophages. Also, SKN7 appears to only partially regulate responses to reactive oxygen species and has not been shown to affect the yeast's response to reactive nitrogen species released by macrophages. Therefore, numerous protective mechanisms are still available to enable skn7 mutants to survive intracellular macrophage killing mechanisms in vitro. However, the increased susceptibility of skn7 mutants to t-BOOH in vitro, as well as the attenuation in virulence of skn7 mutants in the murine inhalational model, suggests that SKN7 has a role in oxidative protection and the total virulence composite of C. neoformans.

In conclusion, the results of the present study show that SKN7 contributes to the virulence composite of C. neoformans in vivo but is not required for the yeast to produce disease. Targeted disruption of SKN7 resulted in an increased susceptibility to some oxidants, in vitro, suggesting that the attenuation in vivo is related to decreases in the OSR of skn7 mutants. However, skn7 mutants were able to survive in an in vitro macrophage killing assay, suggesting that other mechanisms with which to resist both intracellular and extracellular oxidative damage may have a greater role in the pathogenesis of C. neoformans. Moreover, C. neoformans skn7 mutants were observed to be flocculent, suggesting a role for SKN7 in the regulation of some surface component in C. neoformans. Further studies are needed to understand this novel phenotype as well as the linkage of C. neoformans SKN7 to other genes that contribute to the oxidative stress response and/or cell wall integrity pathway of C. neoformans.


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ACKNOWLEDGMENTS
 
We acknowledge Quincy Gerrald for his assistance in performing the real-time PCR experiments described in this study.

This work was supported by grants 5T32 AI007392-15, AI-39156, and AI-28388 from the National Institute of Allergy and Infectious Diseases of the National Institutes of Health.


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FOOTNOTES
 
* Corresponding author. Mailing address: Department of Medicine/Division of Infectious Diseases, Duke University Medical Center, Duke South, Stead Bldg., Box 3353, Durham, NC 27710. Phone: (919) 684-2660. Fax: (919) 684-8902. E-mail: floyd.wormley{at}duke.edu. Back

Editor: T. R. Kozel


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Infection and Immunity, August 2005, p. 5022-5030, Vol. 73, No. 8
0019-9567/05/$08.00+0     doi:10.1128/IAI.73.8.5022-5030.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.



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