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Cryptococcus neoformans serotype A strains, the major cause of cryptococcosis, are distributed worldwide, while serotype D strains are more concentrated in Central Europe. We have previously shown that deletion of the global regulator TUP1 in serotype D isolates results in a novel peptide-mediated, density-dependent growth phenotype that mimics quorum sensing and is not known to exist in other fungi. Unlike for tup1Δ strains of serotype D, the density-dependent growth phenotype was found to be absent in tup1Δ strains of serotype A which had been derived from several different genetic clusters. The serotype A H99 tup1Δ strain showed less retardation in the growth rate than tup1Δ strains of serotype D, but the mating efficiency was found to be similar in both serotypes. Deletion of TUP1 in the H99 strain resulted in significantly enhanced capsule production and defective melanin formation and also revealed a unique regulatory role of the TUP1 gene in maintaining iron/copper homeostasis. Differential expression of various genes involved in capsule formation and iron/copper homeostasis was observed between the wild-type and tup1Δ H99 strains. Furthermore, the H99 tup1Δ strain displayed pleiotropic effects which included sensitivity to sodium dodecyl sulfate, susceptibility to fluconazole, and attenuated virulence. These results demonstrate that the global regulator TUP1 has pathobiological significance and plays both conserved and distinct roles in serotype A and D strains of C. neoformans.
The fungal Tup1 proteins function as global repressors which regulate a large number of genes associated with growth, morphological differentiation, and sexual and asexual reproduction. As a consequence, tup1 mutants are known to display numerous phenotypes (9, 19, 42). The deletion of TUP1 in Candida albicans results in constitutive filamentous growth with no budding yeast cells and is accompanied by loss of virulence (2, 32). In Penicillium marneffei, the only dimorphic species known in the genus Penicillium, deletion of the TUP1 homolog, tupA, confers reduced filamentation and abnormality in yeast morphogenesis (38). In the filamentous fungi Aspergillus nidulans and Neurospora crassa, deletion of the TUP1 homologs, rcoA and rco-1, respectively, severely affects growth and sexual and asexual reproduction (12, 46).
Cryptococcus neoformans is a bipolar heterothallic basidiomycetous yeast with two serotypes, A and D, and the function of Tup1 has been studied only for serotype D strains (26, 27). While disruption of TUP1 in strains of serotype D did not affect yeast or hyphal cell morphology, it resulted in mating-type-dependent differences, including temperature-dependent growth, sensitivity to 0.8 M KCl, and expression of genes in several other biological pathways (26). Most importantly, tup1Δ strains displayed a peptide-mediated quorum-sensing-like phenomenon in both mating types of serotype D strains which has not been reported for any other fungal species (27).
According to genome sequence data, the serotype A reference strain H99 shares 95% sequence identity with the serotype D reference strain JEC21 (29). However, serotype-specific differences between the two strains have been demonstrated in two major signaling pathways, the pheromone-responsive Cpk1 mitogen-activated protein kinase and cyclic AMP (cAMP) (5, 13, 41, 47). In addition, the high-osmolarity glycerol (HOG) pathway also showed regulatory disparity between the two serotypes (1, 8). Since the regulation of peptide-mediated quorum sensing by TUP1 is reported only for serotype D strains, we sought to determine whether the deletion of TUP1 in serotype A strains would have similar consequences. Surprisingly, we found striking differences in the phenotypes manifested by tup1Δ strains of the two serotypes. We report here the serotype-specific differences in TUP1 regulation between A and D strains and the novel regulatory role of TUP1 in maintaining iron/copper homeostasis in C. neoformans.
Serotype A strains used in this study included H99 (MATα) (35), CHC186 (MATα) (6), VNBt63 (MATα) (6), WM148 (MATα) (30), KN99a (MATa) (33), and WSA1156 (MATa) (20). The first four strains were chosen from different genetic clusters among the strains of VNI, the global molecular type within serotype A strains. The other two strains, KN99a and WSA1156, are MATa strains that are isogenic to H99 and were received from J. Heitman and B. Wickes, respectively. Strains HL112 and HL132 are tup1Δ and tup1Δ+TUP1 strains derived from strain H99. HL14 (MATα) and HL40 (MATa) are serotype D tup1Δ strains derived from strains LP1 (MATα) and LP2 (MATa), respectively, as described before (26).
Yeast extract-peptone-dextrose (YEPD) and RPMI agar were described previously (4). Minimal medium (SD) contains 6.7 g of yeast nitrogen base (Difco) without amino acids and 20 g of glucose per liter. YES medium contains 0.5% (wt/vol) yeast extract plus 3% glucose and, as supplements, 225 μg/ml each of uracil, adenine, leucine, histidine, and lysine (31). V8 juice agar was used for mating assays (24).
A serotype A TUP1 homolog of C. neoformans was identified by BLAST search of the serotype A (H99) genome (http://www.broad.mit.edu/annotation/fungi/cryptococcus_neoformans/index.html). The TUP1 gene was deleted by biolistic transformation in four serotype A strains of the VNI molecular type with the construct generated by PCR fusion using a strategy similar to that described for Clostridium difficile (22). The left end of the locus was amplified with primers TND-C1 and TND-C2G418; the right end of the locus was amplified with primers TND-D1G418 and TND-D2. G418-A1 and G418-B2 were used to amplify the NEO (neomycin phosphotransferase II) selectable marker from the plasmid pJAF1 (a gift from J. Heitman) (see Table S1 in the supplemental material). The upstream and downstream flanking regions of the TUP1 gene were amplified from the genomic DNA of each strain using the same primers. The amplified products were gel purified and used as templates to produce a 4.2-kb tup1::NEO deletion construct containing the flanking regions of the TUP1 gene connected by the NEO gene. The linear disruption cassette was then used to homologously integrate into the strains by biolistic transformation (39). Transformants were screened to identify the tup1Δ strains by colony PCR. Deletion of TUP1 was confirmed by Southern blot hybridization (see Fig. S1 in the supplemental material).
To obtain the H99 TUP1 gene, a 4.8-kb DNA fragment containing the 1.3-kb flanking region on both sides was PCR amplified from H99 genomic DNA, sequenced, and cloned into the pAI3 vector (a gift from J. Heitman) containing the NAT selectable marker to obtain pHL110. pHL110 was linearized with SmaI and transformed into the H99 tup1Δ strain by the biolistic method. PCR was used to identify integrative transformants containing the intact TUP1 gene, and Southern blot analysis was used to confirm the integration event (see Fig. S1 in the supplemental material).
Isolation and analysis of genomic DNA were carried out as described previously (4). For gene expression analysis, overnight cultures of wild-type (H99) and tup1Δ strains were refreshed and grown in RPMI for 6 h. RNA was extracted from yeast cells using Trizol (Invitrogen, Carlsbad, CA), treated with RNase-free DNase (Ambion, Austin, TX) for the removal of genomic DNA, and purified with the RNeasy MinElute cleanup kit (Qiagen, Valencia, CA). cDNA was synthesized using a high-capacity cDNA archive kit (Applied Biosystems, Foster City, CA) and used in real-time reverse transcription-PCR (RT-PCR) with TaqMan universal PCR master mix and the ABI Prism 7700 sequence detection system (Applied Biosystems, Foster City, CA). The primers used in RT-PCR are listed in Table S2 in the supplemental material. Data were normalized with actin levels and expressed as the relative amount in the tup1Δ strain compared to that in H99. In addition, the transcription level of CNAG_03012.2 was normalized with γ-tubulin as an internal control.
For mating assays, strains were grown on YEPD agar slants for 2 days. The cells of MATa and MATα strains were mixed on V8 juice agar medium, incubated, and monitored for evidence of mating. Melanin production was estimated after spotting serially diluted yeast cells onto norepinephrine-containing medium (2× dilutions starting at 1.8 × 105 cells/spot). The plates were then incubated for 2 days at 30°C in the dark (23). Capsule formation by the yeast cells was determined by microscopic examination of slides prepared with India ink.
Female BALB/c mice (6 to 8 weeks old) were injected via the lateral tail vein with 0.2 ml of a suspension of each yeast strain (5 × 106/ml) as described previously (4), and the mortality was monitored. Kaplan-Meier analysis of survival was performed with JMP software for Macintosh (SAS Institute, Cary, NC). To measure the growth rate of each strain in the brain, mice were injected with yeast cells (105 cells) as described above, and then three mice per yeast strain were sacrificed at several intervals after injection (at 2 days as the starting point and at 6, 9, and 13 days postinjection). The brains were homogenized with a mortar and pestle, diluted, and then plated onto YEPD agar. Colonies were counted after 2 days of incubation at 30°C.
Exponentially growing cultures (optical density at 600 nm [OD600] of 0.5 to 1.0) were washed, resuspended in 0.9% NaCl, and adjusted to an OD600 of 0.1 for the wild type or 0.2 for the tup1Δ strain (to compensate for its low growth rate). Adjusted cell suspensions were serially diluted, spotted onto the indicated media, and incubated for 3 to 4 days at 30°C. Limited-iron medium (LIM) was identical to chemically defined medium (34) except that the salts of polyvalent metals were dissolved in Chelex-100-treated water (Bio-Rad), and other components were purified by treating with Chelex-100. When more stringent control of iron or copper concentration was needed, 0.056 mM ethylenediamine-diacetic acid (EDDA) (Sigma-Aldrich) or 1 mM bathocuproine sulfonate (BCS) (Sigma-Aldrich) was added to the medium, respectively. LIM-Fe was prepared by adding 0.1 mM ferric EDTA (Sigma-Aldrich EDFS) to LIM.
The previous microarray study with TUP1 in a serotype D strain was done with a mini-microarray since a whole-genome cryptococcus array was not available at that time (26). Recently, a whole-genome array containing 7,738 70-mer oligomers was constructed by an academic consortium at the University of Washington, St. Louis. It has been shown that H99 and JEC21 share 95% identity in their genome sequences (29). Although arrays were designed based on serotype D strain JEC21, the arrays can be useful to assess the deletion effect of a specific gene in serotype A as long as the corresponding serotype A wild-type control strain is employed as a reference. RNA was extracted from H99 and HL112 grown in RPMI liquid medium for 3 h and 6 h, and microarray analysis was performed as described before (25). Two arrays were used for each time point, and all the genes whose average expression was affected by greater than twofold in the tup1Δ strain compared with the wild-type strain when grown for 3 h (group A) or 6 h (group B) in RPMI medium were presented (see Table S3 in the supplemental material).
TUP1 deletion in serotype D strains of C. neoformans resulted in growth retardation, as reported for tup1Δ strains of other fungal species, but without any defects in yeast cell morphology or flocculation (26). In order to investigate the effect of TUP1 deletion in serotype A strains, the TUP1 gene was deleted and then complemented in strain H99. Although the Tup1 proteins from serotype A (H99) and D (JEC21) strains share 94% amino acid identity, deletion of the TUP1 gene resulted in distinct phenotypic differences between the strains. While the H99 tup1Δ strain showed slight growth retardation compared to the wild-type strain, it was less severe than what had been observed for the serotype D tup1Δ strain (see Fig. S2 in the supplemental material) (26). The doubling times of the wild-type H99, tup1Δ (HL112), and complemented (HL132) strains were 2.23 h, 3.9 h, and 2.28 h, respectively, at 30°C and 4.08 h, 5.82 h, and 3.78 h, respectively, at 37°C in YES liquid medium. The slight reduction in the growth rate of the tup1Δ strain was also observed on solid agar media such as YES and SD (see Fig. S2 in the supplemental material, and data not shown). Therefore, TUP1 does not appear to influence cell proliferation in the H99 strain as much as in serotype D strains.
One of the striking phenotypes observed previously with serotype D tup1Δ strains was the inoculum size threshold as a prerequisite for normal growth, which mimics the quorum-sensing phenomenon (27). A cell density of about 5 × 106 was required for the strain HL14, the serotype D tup1Δ strain, to grow on SD medium (Fig. (Fig.1A,1A, left panel). Tests to determine whether cell density would similarly influence growth of the tup1Δ serotype A strain, however, did not show a density-dependent growth phenotype in the H99 tup1Δ strain (HL112) (Fig. (Fig.1A,1A, right panel).
To investigate whether the lack of density-dependent growth in H99 is strain dependent or common among serotype A strains, we deleted TUP1 in several other serotype A strains. Three serotype A strains, CHC186, VNBt63, and WM148, that are genetically diverse based on their molecular genotype, such as mini-and macrosatellite DNA, intergenic sequence, and ribosomal DNA sequences as well as multilocus sequencing type sequences of marker genes (6), were chosen to construct tup1Δ strains. TUP1 was also deleted in WSC1156, the MATa strain isogenic to H99. Consistent with the observations for H99, deletion of TUP1 in the backgrounds of all these serotype A strains caused slight growth retardation but without the density-dependent growth phenotype (data not shown).
In serotype D tup1Δ strains, the inability to grow at low cell densities could be rescued by supplementing the growth medium with the culture filtrate from a high-cell-density tup1Δ culture. The active molecule in the culture supernatant responsible for the density-dependent growth phenotype was identified as an oligopeptide, quorum-sensing-like peptide 1 (QSP1) (27). QSP1 is an 11-amino-acid peptide that is processed from the CQS1 gene product (Fig. (Fig.1B).1B). A CQS1 homolog, CNAG_03012.2, which encodes a hypothetical protein of 45 amino acids, is present in the H99 genome. The sequence of H99 Cqs1 was found to be homologous to that of JEC21 Cqs1, with only two amino acid substitutions (at amino acid positions 28 and 34) (Fig. (Fig.1B).1B). Deletion of TUP1 results in the transcriptional induction of CQS1 in serotype D strains (27). The expression levels of CNAG_03012.2 were measured by real-time RT-PCR and found to be 2.2- ± 0.2-fold higher in HL112 than in H99, suggesting that the CQS1 homolog in H99 is also repressed by Tup1.
The biological activity in the culture filtrate from H99 tup1Δ (HL112) was determined with regard to the possible accumulation of QSPs. Since HL112 did not show the density-dependent growth phenotype, the serotype D tup1Δ strain (HL40) was used for the activity assays. A 25% (vol/vol) mix of the culture filtrate from H99 tup1Δ combined with fresh medium was used in these assays, since a similar proportion of culture filtrate from serotype D strain HL40 had shown strong biological activity (27). The HL112 culture filtrate failed to rescue the growth of HL40 at low cell density (data not shown). The H99 tup1Δ strain apparently did not produce enough of the quorum-sensing-like molecule, or the molecule may not have been biologically active. We postulate that strains which can grow regardless of cell density do not require a quorum-sensing-like molecule(s) for cell proliferation at low densities. Therefore, deletion of the TUP1 gene in C. neoformans affects growth differently in strains of serotypes A and D, and the density-dependent growth phenotype appears to be serotype D specific.
One of the conserved roles of TUP1 in many fungi, including C. neoformans serotype D strains, is the regulation of sexual reproduction (26, 42, 46). We tested the effect of TUP1 deletion on mating in the serotype A strain H99. Mating of the wild-type H99 strain with the tester strain KN99a on V-8 juice agar produced extensive hyphae after 3 days of incubation (Fig. (Fig.2A,2A, left panel). In a cross between HL112 (tup1Δ) and KN99a, however, the production of hyphae was reduced significantly (Fig. (Fig.2A,2A, right panel). Similar observations of reduced mating have been reported for tup1Δ strains of serotype D (26). Therefore, the role of TUP1 in sexual reproduction appears to be conserved in both serotype A and D strains.
Although we did not see any effect of tup1 deletion on capsule formation in serotype D strains, tup1Δ strains in a serotype A background produced capsules that were significantly enlarged compared to those of wild-type strains. Figure Figure2B2B shows the markedly increased capsule size in HL112, the tup1Δ strain of H99, compared to HL132, the tup1Δ+TUP1 strain, and the wild-type strain H99. Deletion of TUP1 in other genetically unrelated serotype A strains also showed significant increases in capsule size (data not shown). These observations clearly associate the hypercapsular phenotype with the deletion of TUP1 in serotype A strains. The enlarged capsule formation in serotype A tup1Δ strains was consistent regardless of growth media at both 30°C and 37°C. Among all the media tested, RPMI induced the most pronounced difference in capsule size between wild-type and tup1Δ strains (Fig. (Fig.2B2B).
Given the prominent hypercapsular phenotype of serotype A tup1Δ strains, it is possible that TUP1 might alter the expression levels of genes involved in capsule formation. Gene profile analysis was undertaken as a preliminary screen to identify the putative genes whose expression was affected by TUP1 deletion. Microarray analysis was performed on the H99 and tup1Δ strains grown for 3 h and 6 h in RPMI liquid medium at 30°C. The expression levels of a few genes involved in capsule biosynthesis and iron/copper homeostasis were affected by deletion of TUP1 in H99 (see Table S3 in the supplemental material). To confirm the expression patterns, mRNA levels of several genes were examined by real-time RT-PCR. Quantitative PCR results showed that the transcriptional levels of three genes involved in capsule synthesis, CAP10, CAP64, and CAS35, were about threefold higher in the tup1Δ strain than in H99 (Fig. (Fig.3).3). Conversely, the expression levels of three genes with an annotated function involved in iron/copper homeostasis, CTR4, FRT1, and SIT2, were two- to ninefold lower in the tup1Δ strain (Fig. (Fig.3)3) (28). In addition, the expression of CIG1, which encodes a product believed to be an extracellular mannoprotein involved in the retention of iron at the cell surface and/or in the uptake of siderophore-bound iron (28), was downregulated by twofold in the tup1Δ strain. However, the expression of CFT2, an ortholog of the Saccharomyces cerevisiae high-affinity iron permease gene FTR1 in C. neoformans, was not affected by the deletion of TUP1 (17).
Since TUP1 affected the expression of genes involved in iron and copper uptake/homeostasis, growth of the tup1Δ strain was tested on both iron-chelated medium (LIM+EDDA) and iron-replete medium (LIM+Fe). Interestingly, growth of the tup1Δ strain was reduced on LIM+EDDA medium compared to that of the wild-type strain, and iron repletion (LIM+Fe) restored growth of the tup1Δ strain (Fig. (Fig.4A).4A). Furthermore, on LIM medium treated with a chelator to remove copper (LIM+BCS+EDDA), the growth difference between the wild-type and tup1Δ strains was even more drastic (Fig. (Fig.4B,4B, right panel). These results indicated that TUP1 regulates the utilization of iron and copper, which corroborates the expression data for several iron/copper homeostasis genes affected by the deletion of TUP1 in H99. Melanin production is one of the known virulence factors in C. neoformans, and iron/copper homeostasis can affect melanin production (14). Melanin production in the tup1Δ strains, examined on norepinephrine-containing medium, was observed to be notably reduced compared to that in the wild-type and complemented strains (Fig. (Fig.4C,4C, left panel). Laccase is a key enzyme for melanin biosynthesis in C. neoformans. Since it requires four bound copper ions (43, 44) and copper is known to suppress the defect in melanin formation caused by mutation in genes involved in metal ion homeostasis (48, 49), the effect of Cu2+ on the melanin phenotype of the tup1Δ strain was studied. Melanin production was apparently restored in the tup1Δ strain by supplementing the growth medium with 10 μM CuSO4. These results suggest that the tup1Δ strain is defective in copper homeostasis affecting melanin production (Fig. (Fig.4C,4C, right panel).
An insufficient iron concentration in the growth environment is known to induce large capsules in C. neoformans (15). Although the tup1Δ strain already showed an enlarged capsule in RPMI medium, it was interesting to determine if iron levels still influence the capsule size. The capsule size was measured after growing cells on RPMI, LIM, and LIM+Fe agar plates (Fig. (Fig.4D).4D). In concordance with previous studies, strain H99 produced larger capsules in LIM (2.51 ± 0.71 μm; n = 33) than in RPMI (1.87 ± 0.39 μm; n = 25) and iron-replete medium (LIM+Fe) (0.64 ± 0.22 μm; n = 24) (Fig. (Fig.4D,4D, upper panels). In the tup1Δ strain, cells grown on RPMI were already hypercapsulated compared to H99 cells, but the capsules became even larger when cultured on LIM. The capsule size was significantly reduced upon addition of Fe to LIM (RPMI, 5.34 μm ± 1.21 [n = 14]; LIM, 6.14 μm ± 2.98, [n = 26], and LIM+Fe, 2.47 μm ± 0.62 [n = 49]) (Fig. (Fig.4D,4D, middle panels). The TUP1-complemented strain (HL132) behaved similarly to the wild-type strain in all growth conditions (Fig. (Fig.4D,4D, bottom panels). Thus, deletion of TUP1 results in the formation of an enlarged capsule under non-iron-limiting conditions, which can be altered further in tup1Δ cells by iron levels in the environment.
Cir1 is another cryptococcal transcriptional regulator involved in iron homeostasis. CIR1 (CNAG_04864) is important for capsule formation and negatively regulates laccase expression in H99 (18). In addition, it has been shown that cir1 mutants are sensitive to sodium dodecyl sulfate (SDS) and the azole drug fluconazole, suggesting that Cir1 is involved in cell wall integrity and membrane functions (18). Since capsule and melanin production were affected in the tup1Δ strain, the effect of TUP1 deletion relative to changes in CIR1 expression was examined. Quantitative RT-PCR results showed CIR1 expression to be mildly affected in the tup1Δ strain compared to H99, as the relative expression level was only 1.36- ± 0.05-fold higher in the tup1Δ strain. Furthermore, expression of iron permease genes, such as CFT1 and CFT2, which are regulated by CIR1, was not affected by the deletion of TUP1 according to our microarray data (see Table S3 in the supplemental material). These data suggested that TUP1 does not regulate iron homeostasis through the CIR1 regulatory circuit. However, growth of the tup1Δ strain was significantly hampered in the presence of 0.01% SDS, and the tup1Δ strain displayed increased sensitivity to fluconazole (Fig. (Fig.4E).4E). These data suggested that TUP1 is also involved in cell wall integrity and membrane functions.
Since deletion of TUP1 in H99 resulted in both positive and negative effects with respect to the three major C. neoformans virulence factors, which include growth at 37°C and formation of melanin and capsule, its effect on virulence was investigated. Groups of 10 mice were challenged with different yeast stains via tail vein injection. Figure Figure5A5A shows that all mice challenged with the wild-type or the complemented strain succumbed to infection by 9 days postinjection, while it took 20 days for all mice infected with the tup1Δ strain to succumb (P < 0.001 compared to wild-type-infected mice), indicating that deletion of TUP1 causes attenuation of virulence in C. neoformans.
To examine the pathobiological differences in mice infected with the wild-type or tup1Δ strain, the brain fungal burden and capsule size were determined at different stages of infection. Significant differences in the number of CFU were observed for the H99 and tup1Δ strains (5.93 × 103 versus 5.3 × 102 per brain) as early as 2 days after injection (Fig. (Fig.5B).5B). The number of CFU, however, increased exponentially and differed even more at 6 days after injection. These data suggest that TUP1 is important for growth in vivo, although the tup1Δ strain only showed a marginal reduction in growth at 37°C in vitro. Another noteworthy observation was that fungal burden analyzed on the day of death in a mouse injected with the tup1Δ strain (1.25 × 107 at day 13) was 80-fold lower than that in a mouse injected with H99 (8.35 × 108 at day 9) (Fig. (Fig.5B).5B). It is possible that the larger capsule size in the tup1Δ strain in vitro (Fig. (Fig.2A)2A) might have contributed to such a difference. Surprisingly, the capsule size of the yeast cells in the brain smear from mice infected with tup1Δ strain was similar to that for mice infected with H99 (Fig. (Fig.5C).5C). These findings clearly indicate that Tup1 plays an important role in the pathobiology of C. neoformans.
This study investigated the function of TUP1 in C. neoformans serotype A strains, including H99, and presents another example of serotype-specific difference in gene regulation. TUP1 plays a conserved role with respect to growth and mating but distinctly different roles in strains of serotypes A and D. Serotype A-specific phenotypes of tup1Δ strains include a lack of density-dependent growth, an enlarged capsule size, reduced melanin production, and a defect in iron/copper homeostasis.
The prominent capsule size in the tup1Δ strain under noninducing conditions indicated the important role of TUP1 in capsule formation. Many environmental factors have been shown to influence the size of the capsule in C. neoformans. Low concentrations of glucose and iron, high concentrations of carbon dioxide, and the presence of serum components have been shown to enhance capsule formation (15). However, limited information is available concerning the regulation of capsule formation. The Gpa1-cAMP-protein kinase A (PKA) signaling pathway has been thoroughly studied in regard to its effect on capsule production. Both pka1Δ and gpa1Δ strains exhibited a marked defect in capsule production, while a pkr1Δ strain overproduced the capsule (10). Several observations indicate that TUP1 affects capsule production independent of the cAMP-PKA signaling pathway. First, our preliminary microarray data did not show any significant change in the gene expression of cAMP-PKA pathway components such as CAC1 (adenyl cyclase), PKA1, and PKR1 (data not shown). Second, the addition of cAMP to growth media did not alter the hypercapsular phenotype of the tup1Δ strain (data not shown). Third, the hypercapsular phenotype of pkr1Δ was observed not only in vitro but also in vivo, resulting in hypervirulence, while the tup1Δ strain exhibited reduced virulence and yet its capsule size in vivo was comparable to that of the wild-type strain. Another signaling pathway involved in capsule formation is the HOG pathway. HOG1 negatively regulates synthesis of capsule and melanin in the serotype A strain H99 but not in the serotype D strain JEC21 (1). Deletion of TUP1 or HOG1 has a similar effect on capsule production, and their serotype-specific regulatory function offers the possibility that Tup1 and Hog1 might share downstream regulatory targets either in a parallel signaling pathway or by direct interaction. The HOG1 pathway in S. cerevisiae is activated by osmotic stress and modulates diverse osmo-adaptive gene expression through the recruitment of the general transcription repressor complex Tup1-Ssn6 and the sequence-specific DNA-binding protein Sko1 (36). Since the tup1Δ strains also show sensitivity to 2 mM H2O2 as observed with the hog1Δ strains (1) (data not shown), this possibility was considered. However, the hog1Δ strain derived from H99 exhibited enhanced melanization and temperature sensitivity at 40°C, neither of which is shared in the tup1Δ strains. Furthermore, the H2O2-sensitive phenotype of the tup1Δ strain was rescued by addition of copper or iron to the medium, suggesting that the low intracellular iron/copper content and not a defect in the HOG pathway resulted in the H2O2 sensitivity (data not shown). Therefore, the involvement of TUP1 in regulating capsule formation does not share all the common characteristics with the aforementioned known pathways.
Given that TUP1 affects capsule production independently of previously known regulatory pathways, we tried to identify the targets of TUP1. Preliminary microarray and RT-PCR experiments revealed that several genes involved in iron/copper homeostasis were downregulated in tup1Δ strains. The requirement of iron/copper in the growth of the tup1Δ strain and restoration of melanin production by copper in the tup1Δ strain further support the importance of TUP1 in iron/copper homeostasis in serotype A strain H99. A role of TUP1 in iron metabolism was first suggested by the identification of the ferric reductase gene, RBT2, as one of the genes repressed by TUP1 in C. albicans (2). Subsequent study showed that ferric reductase activity in Δtup1/Δtup1 cells was constitutively elevated and iron-dependent transcriptional alteration of C. albicans FTR1 and FTR2 mRNAs was abrogated in Δtup1/Δtup1 mutant (21). Furthermore, examples of the physical interaction between a corepressor and an iron-sensing factor controlling the expression of iron uptake genes have been shown in Schizosaccharomyces pombe (50). S. pombe fep1+ encodes a GATA transcription factor that represses the expression of iron transport genes in response to elevated iron levels. Using yeast two-hybrid analysis, it has been shown that Tup11, a Tup1 homolog of S. pombe, and Fep1 physically interact with each other (50). Whether C. neoformans Tup1 directly interacts with the proteins involved in iron homeostasis is yet to be determined.
The importance of copper homeostasis in C. neoformans is suggested by the copper dependency of two well-known virulence factors, the Cu/Zn superoxide dismutase (7) and laccase, a key enzyme in melanin synthesis (37, 43). Both enzymes require copper as a cofactor for their function. Deletion of C. neoformans LAC1, encoding the laccase, or mutation in the copper-binding site of the gene resulted in a significant reduction in virulence (37, 43). Copper also induces laccase transcription in wild-type cells and can restore laccase activity in vph1Δ mutants (48). In addition, the close relationship between copper and iron homeostasis has been reviewed (16). For example, copper homeostasis also affects iron, since Fet3, the high-affinity iron transporter, requires the incorporation of four copper ions for the function (16). Thus, ineffective copper loading of Fet3 due to a defect in copper homeostasis can also lead to lower intercellular levels of iron, which affects capsule and melanin production. In fact, mutation in CCC2 (encoding a copper transporter) or ATX1 (encoding a copper chaperone) resulted in large capsules under iron-replete conditions and impaired growth under iron-limiting condition (40). Although our microarray data did not show significant changes in CCC2 and ATX1 gene expression (see Table S3 in the supplemental material), reduced expression of CTR4 (encoding copper transporter 4) in the tup1Δ strain and additive growth defects of the tup1Δ strain in iron/copper-chelated media lend additional support for the regulatory role of TUP1 in both iron and copper homeostasis.
Another intriguing result of our study is that Tup1 appears to function as both a repressor and an activator in C. neoformans. In contrast to the prevailing view of Tup1 as a global repressor, our results showed that many genes were also downregulated in the absence of TUP1, suggesting that Tup1 functions as an activator for the expression of those genes. An analogy is seen with Hap1 in S. cerevisiae, which was originally identified as a heme-dependent transcriptional activator but was reported to function also as transcriptional repressor, depending on oxygen levels (11). Mammalian nuclear hormone receptors also are examples of factors that can act both positively and negatively through the recruitment of coactivators and corepressor complexes, respectively (45). Conversely, it is also possible that the downregulated genes in the tup1Δ strain are due to the indirect effect of Tup1. For instance, Tup1 could interact with a negative regulator, and inactivation of Tup1 could lead to the activation of a negative regulator, which in turn would cause the observed downregulation of genes in the tup1Δ strain. Additional experiments are required to identify the direct target(s) of Tup1 and possible interacting partners, if any, to understand the mechanism of Tup1 regulation in C. neoformans.
In C. albicans, disruption of TUP1 causes an inability to switch between yeast and filament forms and results in constitutive filamentous growth, which presumably is the reason why the tup1Δ strain is avirulent (2, 3). Previously, virulence studies could not be carried out properly with tup1Δ strains in a serotype D background because of their inability to grow at low cell densities, which hindered the precise determination of inoculum size based on CFU (26, 27). Here, we showed that deletion of TUP1 in H99 affected virulence. Since the TUP1 deletion displayed pleiotropic effects, it is likely that the reduced virulence of the tup1Δ strain resulted from the combination of these effects and is possibly related to iron/copper homeostasis. Given the global regulatory role of TUP1 in fungi and the manifestation of different phenotypes in serotype A and D tup1Δ strains, an in-depth analysis of TUP1 function would offer a valuable tool toward understanding the divergence of gene regulation in C. neoformans.
This study was supported by funds from the intramural program of the National Institute of Allergy and Infectious Diseases, NIH.
Published ahead of print on 9 October 2009.
†Supplemental material for this article may be found at http://ec.asm.org/.