1

1

Regulatory diversity of TUP1 in Cryptococcus neoformans. 2

3

Hyeseung Lee, Yun C. Chang, Ashok Varma, and Kyung J. Kwon-Chung* 4

5

Molecular Microbiology Section, Laboratory of Clinical Infectious Diseases, 6

National Institute of Allergy and Infectious Diseases, NIH, Bethesda, MD 20892, 7

USA 8

9

Running Head: Tup1 regulation in Cryptococcus neoformans 10

11

12

*Corresponding author 13

K. J. Kwon-Chung, Ph.D. 14

Phone : (+1) 301-496-1602 15

Fax : (+1) 301-480-3240 16

E-mail: june_kwon-chung@nih.gov 17

Copyright © 2009, American Society for Microbiology and/or the Listed Authors/Institutions. All Rights Reserved.Eukaryotic Cell doi:10.1128/EC.00256-09 EC Accepts, published online ahead of print on 9 October 2009

2

ABSTRACT 1

2

Cryptococcus neoformans serotype A strains, the major cause of cryptococcosis, are 3

distributed world-wide while serotype D strains are more concentrated in Central Europe. 4

We have previously shown that deletion of the global regulator TUP1 in serotype D 5

isolates results in a novel peptide-mediated density-dependent growth phenotype that 6

mimics quorum sensing not known to exist in other fungi. Unlike tup1∆ strains of 7

serotype D, the density-dependent growth phenotype was found to be absent in tup1∆ 8

strains of serotype A which had been derived from several different genetic clusters. 9

Serotype A H99 tup1∆ strain showed slight retardation in the growth rate when compared 10

to tup1∆ strains of serotype D but the mating efficiency was found to be similar in both 11

serotypes. Deletion of TUP1 in the H99 strain resulted in significantly enhanced capsule 12

production, defective melanin formation, and also revealed a unique regulatory role of the 13

gene in maintaining iron/copper homeostasis. Differential expression of various genes 14

involved in capsule formation and iron/copper homeostasis was observed between wild 15

type and the tup1∆ strains of H99. Furthermore, the tup1∆ strain of H99 displayed 16

pleiotropic effects which included sensitivity to SDS, susceptibility to fluconazole, and 17

attenuated virulence. These results demonstrate that the global regulator, TUP1, has 18

pathobiological significance and plays both conserved and distinct roles between serotype 19

A and D strains of C. neoformans. 20

21

22

23

3

INTRODUCTION 1

2

The fungal Tup1 proteins function as a global repressor which regulates a large 3

number of genes associated with growth, morphological differentiation and sexual and 4

asexual reproduction. As a consequence, tup1 mutants are known to display numerous 5

phenotypes (10, 19, 42). The deletion of TUP1 in Candida albicans results in constitutive 6

filamentous growth with no budding yeast cells and is accompanied by loss of virulence 7

(2, 32). In Penicillium marneffei, the only dimorphic species known in the genus 8

Penicillium, deletion of the TUP1 homolog, tupA, confers reduced filamentation and 9

abnormality in yeast morphogenesis (38). In filamentous fungi Aspergillus nidulans and 10

Neurospora crassa, deletion of TUP1 homologs, rcoA and rco-1, respectively, severely 11

affects growth and sexual and asexual reproduction (12, 46). 12

Cryptococcus neoformans is a bipolar heterothallic basidiomycetous yeast with 13

two serotypes, A and D, and the function of Tup1 has only been studied in serotype D 14

strains (26, 27). While disruption of TUP1 in strains of serotype D did not affect yeast or 15

hyphal cell morphology, it resulted in mating type-dependent differences including 16

temperature-dependent growth, sensitivity to 0.8M KCl and expression of genes in 17

several other biological pathways (26). Most importantly, tup1∆ strains displayed a 18

peptide-mediated quorum sensing-like phenomenon in both mating types of serotype D 19

strains which has not been reported in any other fungal species (27). 20

According to genome sequence data, the serotype A reference strain H99 shares 21

95% sequence identity with the serotype D reference strain JEC21 (29). However, 22

serotype-specific differences between the two strains have been demonstrated in two 23

4

major signaling pathways, MAPK (the pheromone-responsive Cpk1 mitogen-activated 1

protein kinase) and cAMP (cyclic AMP) (5, 13, 41, 47). In addition, HOG (high 2

osmolarity glycerol) pathway also showed regulatory disparity between the two serotypes 3

(1, 8). Since the regulation of peptide-mediated quorum sensing by TUP1 is reported only 4

in serotype D strains, we sought to determine whether the deletion of TUP1 in serotype A 5

strains would manifest similar consequences. Surprisingly, we found striking differences 6

in the phenotypes manifested by tup1∆ strains of the two serotypes. We report here the 7

serotype-specific differences of TUP1 regulation between A and D strains and the novel 8

regulatory role of TUP1 in maintaining iron/copper homeostasis in C. neoformans. 9

10

MATERIALS AND METHODS 11

12

Strains and Media 13

Serotype A strains used in this study included H99 (MATα)(35), CHC186 (MATα)(6), 14

VNBt63 (MATα)(6), WM148 (MATα)(30), KN99a (MATa)(33) and WSA1156 15

(MATa)(20). The first four strains were chosen from different genetic clusters among the 16

strains of VNI, the global molecular type within serotype A strains. The other two strains, 17

KN99a and WSA1156, are MATa strains isogenic to H99 received from J. Heitman and 18

B. Wickes, respectively. The strains HL112 and HL132 are tup1∆ and tup1∆ +TUP1 19

strains derived from the strain H99. HL14 (MATα) and HL40 (MATa) are serotype D 20

tup1∆ strains derived from LP1 (MATα) and LP2 (MATa) strains, respectively, as 21

described before (26). 22

5

Yeast extract-peptone-dextrose (YEPD) and RPMI agar were described previously (4). 1

Minimal media (SD) contains 6.7g of yeast nitrogen base (Difco) without amino acids 2

and 20 g of glucose per liter. YES medium contains 0.5% [w/v] yeast extract plus 3% 3

glucose and supplements, 225µg/ml each of uracil, adenine, leucine, histidine, and lysine 4

(31). V8-juice agar was used for mating assay (24). 5

6

Construction of TUP1-deleted strains 7

A serotype A TUP1 homolog of C. neoformans was identified by Blast search of the 8

serotype A (H99) genomes 9

(http://www.broad.mit.edu/annotation/fungi/cryptococcus_neoformans/index.html). 10

The TUP1 gene was deleted by biolistic transformation in four serotype A strains of the 11

VNI molecular type with the construct generated by PCR fusion using a strategy similar 12

to that described for Clostridium difficile (22). The left end of the locus was amplified 13

with TND-C1 and TND-C2G418; the right end of the locus was amplified with primers 14

TND-D1G418 and TND-D2. G418-A1 and G418-B2 were used to amplify the NEO 15

(Neomycin phosphotransferase II) selectable marker from the plasmid pJAF1 (a gift from 16

J. Heitman, Table S1 in the supplemental material). The upstream and downstream 17

flanking regions of the TUP1 gene were amplified from the genomic DNA of each strain 18

using the same primers. The amplified products were gel purified and used as templates 19

to produce a 4.2kb tup1::NEO deletion construct containing the flanking regions of the 20

TUP1 gene connected by the NEO gene. The linear disruption cassette was then used to 21

homologously integrate into the strains by biolistic transformation (39). Transformants 22

6

were screened to identify the tup1∆ strains by colony PCR. Deletion of TUP1 was 1

confirmed by Southern blot hybridization (see Fig. S1 in the supplemental material). 2

To obtain the H99 TUP1 gene, a 4.8kb DNA fragment containing the 1.3kb flanking 3

region on both sides was PCR-amplified from H99 genomic DNA, sequenced, and cloned 4

into pAI3 vector (a gift from J. Heitman) containing NAT selectable marker to obtain 5

pHL110. pHL110 was linearized with SmaI and transformed into H99 tup1∆ strain by 6

biolistic method. PCR was used to identify integrative transformants containing the intact 7

TUP1 gene and Southern blot analysis was used to confirm the integration event (see Fig. 8

S1 in the supplemental material). 9

10

Preparation and analysis of nucleic acid 11

Isolation and analysis of genomic DNA was carried out as described previously (4). For 12

gene expression analysis, overnight cultures of wild type (H99) and tup1∆ strains were 13

refreshed and grown in RPMI for 6hrs. RNA was extracted from yeast cells using Trizol 14

(Invitrogen, Carlsbad, CA), treated with RNAse-free Dnase (Ambion, Austin, TX) for the 15

removal of genomic DNA, and purified with RNeasy MinElute cleanup kit (Qiagen, 16

Valencia, CA). cDNA was synthesized using high-capacity cDNA archive kit (Applied 17

Biosystems, Foster City, CA) and used in real time reverse transcription PCR(RT-PCR) 18

with TaqMan universal PCR master mix and the ABI PRISM 7700 sequence detection 19

system (Applied Biosystems, Foster City, CA). The primers used in RT-PCR are listed in 20

Table S2. Data was normalized with ACTIN levels and expressed as the relative amount 21

in tup1∆ strain compared to that in H99. In addition, the transcription level of 22

CNAG_03012.2 was normalized with γ-tubulin as an internal control. 23

7

Assays for mating, melanization and capsule formation 1

For mating assays, strains were grown on YEPD agar slants for 2 days. The cells of 2

MATa and MATα strains were mixed on V8-juice agar medium, incubated, and 3

monitored for the evidence of mating. Melanin production was estimated after spotting 4

serially diluted yeast cells onto norepinephrine-containing medium (2X dilution starting 5

1.8x105

cells/spot). The plates were then incubated for 2 days at 30ºC in the dark (23). 6

Capsule formation by the yeast cells was determined by microscopic examination of 7

slides prepared with India Ink. 8

9

Virulence study 10

Female BALB/c mice (6-8 weeks old) were injected via the lateral tail vein with 0.2ml of 11

a suspension of each yeast strain (5X106/ml) as described previously (4) and the mortality 12

was monitored. Kaplan-Meier analysis of survival was performed with JMP software for 13

Macintosh (SAS Institute, Cary, NC). To measure the growth rate of each strain in the 14

brain, mice were injected with yeast cells (105 cells) as described above, and then 3 mice 15

per yeast strain were sacrificed at several intervals after injection (2 days as the starting 16

point and 6, 9, 13 days postinjection). The brains were homogenized with a mortar and 17

pestle, diluted, and then plated onto YEPD agar. Colonies were counted after 2 days of 18

incubation at 30°C. 19

20

Spot Assay 21

Exponentially growing cultures (OD600 = 0.5-1.0) were washed, resuspended in 0.9% 22

NaCl and adjusted to OD600= 0.1 for wild type or 0.2 for tup1∆ strain to compensate for 23

8

its slow growth rate. Adjusted cell suspensions were serially diluted, spotted onto 1

indicated media, and incubated for 3-4 days at 30°C. Limited iron medium (LIM) was 2

identical to CDM (chemically defined medium) (34) except that the salts of polyvalent 3

metals were dissolved in Chelex-100-treated water (Bio-Rad), and other components 4

were purified by treating with Chelex-100. When more stringent control of iron or 5

copper concentration was needed, 0.056 mM ethylenediamine-diacetic acid (EDDA; 6

Sigma-Aldrich) or 1mM bathocuproine sulphonate (BCS ; Sigma-Aldrich ) was added to 7

the medium, respectively. LIM+Fe was prepared by adding 0.1mM ferric EDTA 8

(FeEDTA ; Sigma-Aldrich EDFS) to LIM. 9

10

Microarray analysis 11

The previous microarray study with TUP1 in serotype D strain was done with mini-12

microarray since whole genome cryptococcus array was not available at that time (26). 13

Recently whole genome array containing 7738 70mer-oligomers was constructed by 14

academic consortium at the University of Washington-St. Louis. It has been shown that 15

H99 and JEC21 share 95% identity in their genome sequence (29). Although arrays were 16

designed based on serotype D strain, JEC21, the arrays could be useful to assess the 17

deletion effect of a specific gene in serotype A as long as the corresponding serotype A 18

wild type control strain is employed as a reference. RNA was extracted from H99 and 19

HL112 grown in RPMI liquid media for 3hr and 6hr and microarray was performed and 20

analyzed as described before (25). Two arrays were performed for each time point and all 21

the genes whose average expression was affected by greater than 2 fold in tup1∆ strain 22

9

compared with wild type strain when grown for 3hr (group A) or 6hr (group B) in RPMI 1

media were presented (Table S3). 2

3

RESULTS 4

5

Differences in growth and quorum sensing-like phenotype between tup1∆∆∆∆ strains of 6

serotype A and D. 7

TUP1 deletion in serotype D strains of C. neoformans resulted in growth retardation as 8

reported in tup1∆ strains of other fungal species but without any defects in yeast cell 9

morphology or flocculation (26). In order to investigate the effect of TUP1 deletion in 10

serotype A strains, the TUP1 gene was deleted and then complemented in the strain H99. 11

Although the Tup1 proteins from serotype A (H99) and D (JEC21) strains share 94% 12

amino acid identity, deletion of the TUP1 gene resulted in distinct phenotypic differences 13

between the strains. While the H99 tup1∆ strain showed slight growth retardation 14

compared to the wild type strain, it was less severe than what had been observed for the 15

serotype D tup1∆ strain (Fig. S2, (26)). The doubling times of the wild-type H99, tup1∆ 16

(HL112) and complemented (HL132) strains were 2.23hr, 3.9hr, and 2.28hr, respectively, 17

at 30°C and 4.08hr, 5.82hr, and 3.78hr, respectively, at 37°C in YES liquid medium. The 18

slight reduction in the growth rate of the tup1∆ strain was also observed on solid agar 19

media such as YES and SD (Fig. S2 and data not shown). Therefore, TUP1 does not 20

appear to influence cell proliferation in the H99 strain as much as in serotype D strains. 21

One of the striking phenotypes observed previously with serotype D tup1∆ strains was 22

the inoculum size threshold as a pre-requisite for normal growth which mimics the 23

10

quorum sensing phenomenon (27). As shown in figure 1, a cell density of about 5x106 1

was required for the strain HL14, the serotype D tup1∆ strain, to grow on SD media (Fig. 2

1, left panel). Tests to determine whether cell density would similarly influence growth of 3

tup1∆ strain in serotype A, however, did not show a density-dependent growth phenotype 4

in H99 tup1∆ strain (HL112) (Fig. 1, right panel). 5

To investigate whether the lack of density-dependent growth in H99 is strain-dependent 6

or common among serotype A strains, we deleted TUP1 in several other serotype A 7

strains. Three serotype A strains, CHC186, VNBt63, and WM148, that are genetically 8

diverse based on their molecular genotype such as mini and macro satellite DNA, IGS 9

and rDNA sequences, as well as MLST sequences of marker genes (6) were chosen to 10

construct tup1∆ strains. TUP1 was also deleted in WSC1156, the MATa strain isogenic to 11

H99. Consistent with the observation in H99, deletion of TUP1 in the background of all 12

these serotype A strains showed slight growth retardation but without the density-13

dependent growth phenotype (data not shown). 14

In serotype D tup1∆ strains, the inability to grow at low cell densities could be rescued by 15

supplementing the growth media with the culture filtrate from a high cell density tup1∆ 16

culture. The active molecule in the culture supernatant responsible for density-dependent 17

growth phenotype was identified to be an oligopeptide, QSP1 (quorum sensing-like 18

peptide1) (27). QSP1 is an 11-amino acid-peptide processed from CQS1 gene product 19

(Fig. 1B). A CQS1 homolog, CNAG_03012.2, is present in the H99 genome which 20

encodes a hypothetical protein of 45 amino acids. The sequence of H99 Cqs1 was found 21

to be homologous to the JEC21 Cqs1 with only two amino acids (amino acid position 28, 22

34) substitutions (Fig. 1B). Deletion of TUP1 results in the transcriptional induction of 23

11

CQS1 in serotype D strains (27). The expression levels of CNAG_03012.2 were 1

measured by real-time RT-PCR and found to be 2.2± 0.2-fold higher in HL112 than in 2

H99 suggesting that the CQS1 homolog in H99 is also repressed by Tup1. 3

The biological activity in the culture filtrate from H99 tup1∆ (HL112) was determined 4

with regards to the possible accumulation of QSPs. Since HL112 did not show the 5

density-dependent growth phenotype, the serotype D tup1∆ strain (HL40) was used for 6

the activity assays. A 25% mix of the culture filtrate from H99 tup1∆ combined with 7

fresh medium (v/v) was used in these assays since a similar proportion of culture filtrate 8

from serotype D HL40 had shown strong biological activity (27). The HL112 culture 9

filtrate failed to rescue the growth of HL40 at low cell density (data not shown). The H99 10

tup1∆ strain apparently did not produce enough QS-like molecule or the molecule may 11

not have been biologically active. We postulate that strains which can grow regardless of 12

cell density do not require QS-like molecule(s) for cell proliferation at low densities. 13

Therefore, deletion of the TUP1 gene in C. neoformans affects growth differently 14

between strains of serotype A and D and density-dependent growth phenotype appears to 15

be serotype D specific. 16

17

TUP1 deletion affects mating and capsule formation. 18

One of the conserved roles of TUP1 in many fungi, including C. neoformans serotype D 19

strains, is the regulation of sexual reproduction (26, 42, 46). We tested the effect of TUP1 20

deletion on mating in the serotype A strain H99. Mating of the wild type H99 strain with 21

the tester strain KN99a on V-8 juice agar produced extensive hyphae after 3 days of 22

incubation (Fig 2A, left panel). In a cross between HL112 (tup1∆) and KN99a, however, 23

12

the production of hyphae was reduced significantly (Fig 2A, right panel). Similar 1

observations of reduced mating have been reported between tup1∆ strains of serotype D 2

(26). Therefore, the role of TUP1 in sexual reproduction appears to be conserved in both 3

serotype A and D strains. 4

Although we did not see any effect of TUP1 deletion on capsule formation in serotype D 5

strains, tup1∆ strains in serotype A background produced significantly enlarged capsules 6

compared to wild type strains. Figure 2B shows the markedly increased capsule size in 7

HL112, the tup1∆ strain of H99, compared to HL132, the tup1∆ + TUP1 strain, and the 8

wild type strain H99. Deletion of TUP1 in other genetically unrelated serotype A strains 9

also showed significant increases in capsule size (data not shown). These observations 10

clearly associate the hypercapsular phenotype with the deletion of TUP1 in serotype A 11

strains. The enlarged capsule formation in serotype A tup1∆ strains was consistent 12

regardless of growth media both at 30ºC and 37ºC. Among all the media tested, RPMI 13

induced the most pronounced difference in capsule size between wild type and tup1∆ 14

strains (Fig. 2B). 15

16

TUP1 regulates expression of genes involved in capsule formation and iron/copper 17

homeostasis in serotype A, H99. 18

Given the prominent hypercapsular phenotype of serotype A tup1∆ strains, it is possible 19

that TUP1 might alter the expression levels of genes involved in capsule formation. Gene 20

profile analysis was undertaken as a preliminary screen to identify the putative genes 21

whose expressions were affected by TUP1 deletion. Microarray analysis was performed 22

on the H99 and tup1∆ strains grown for 3hr and 6hr in RPMI liquid media at 30ºC. The 23

13

expression levels of a few genes involved in capsule biosynthesis and iron/copper 1

homeostasis were affected by deletion of TUP1 in H99 (see Table S3 in the supplemental 2

material). To confirm the expression patterns, mRNA levels of several genes were 3

examined by real time RT-PCR. Quantitative PCR results showed the transcriptional 4

levels of three genes involved in capsule synthesis, CAP10, CAP64, and CAS35, were 5

about 3-fold higher in the tup1∆ strain than in H99 (Fig. 3). Conversely, the expression 6

levels of three genes with an annotated function involved in iron/copper homeostasis, 7

CTR4, FRT1, and SIT2, were 2 to 9 fold lower in the tup1∆ strain (Fig. 3)(28). In 8

addition, the expression of CIG1 which encodes a product believed to be an extracellular 9

mannoprotein involved in the retention of iron at the cell surface and/or in the uptake of 10

siderophore-bound iron (28) was down-regulated by 2-fold in the tup1∆ strain. However, 11

the expression of CFT2, an ortholog of S. cerevisiae high-affinity iron permease FTR1 in 12

C. neoformans was not affected by the deletion of TUP1 (17). 13

Since TUP1 affected the expression of genes involved in iron and copper 14

uptake/homeostasis, growth of the tup1∆ strain was tested on both iron-chelated media 15

(LIM+EDDA) and iron-replete media (LIM+Fe). Interestingly, growth of the tup1∆ 16

strain was reduced on LIM+EDDA media compared to the wild type strain and iron 17

repletion (LIM+Fe) restored growth of the tup1∆ strain (Fig. 4A). Furthermore, on LIM 18

media treated with a chelator to remove copper (LIM+BCS+EDDA), the growth 19

difference between the wild type and tup1∆ strains was even more drastic (Fig. 4B, right 20

panel). These results indicated that TUP1 regulates the utilization of iron and copper, 21

which corroborates with the expression data of several iron/copper homeostasis genes 22

affected by the deletion of TUP1 in H99. Melanin production is one of the known 23

14

virulence factors in C. neoformans and iron/copper homeostasis can affect melanin 1

production (14). Melanin production in the tup1∆ strains, examined on norepinephrine-2

containing media, was observed to be notably reduced compared to the wild type and 3

complemented strains (Fig. 4C, left panel). Laccase is a key enzyme for melanin 4

biosynthesis in C. neoformans. Since it requires four bound copper ions (43, 44) and 5

copper is known to suppress the defect in melanin formation caused by mutation in genes 6

involved in metal ion homeostasis (48, 49), the effect of Cu+2

in melanin phenotype of 7

the tup1∆ strain was studied. Melanin production was apparently restored in the tup1∆ 8

strain by supplementing the growth media with 10µM CuSO4. These results suggest that 9

tup1∆ strain is defective in copper homeostasis affecting melanin production (Fig. 4C, 10

right panel). 11

Insufficient iron concentration in the growth environment is known to induce large 12

capsules in C. neoformans (15). Although the tup1∆ strain already showed an enlarged 13

capsule in RPMI media, it would be interesting to determine if iron levels still influence 14

the capsule size. The capsule size was measured after growing cells on RPMI, LIM, and 15

LIM+Fe agar plates (Fig. 4D). In concordance with previous studies, the strain H99 16

produced larger capsules in iron-limited medium (LIM; 2.51±0.71 µm; n=33) compared 17

to RPMI (RPMI; 1.87±0.39 µm; n=25) and iron replete medium (LIM+Fe; 0.64±0.22 18

µm; n=24)(Fig 4D, upper panel). In the tup1∆ strain, cells grown on RPMI were already 19

hypercapsulated compared to H99 cells but the capsules became even larger when 20

cultured on LIM media. The capsule size was significantly reduced upon addition of Fe 21

to the LIM media (RPMI; 5.34µm±1.21, n=14; LIM; 6.14µm ±2.98, n=26 and LIM+Fe; 22

2.47µm ±0.62, n=49, respectively, Fig. 4D middle panel). The TUP1-complemented 23

15

strain (HL132) behaved similar to the wild type strain in all growth conditions (Fig. 4D, 1

bottom panel). Thus, deletion of TUP1 results in the formation of an enlarged capsule in 2

non-iron-limiting conditions, which can be altered further in tup1∆ cells by iron levels in 3

the environment. 4

5

TUP1 deletion affects cell wall integrity and susceptibility to fluconazole 6

Cir1 is another cryptococcal transcriptional regulator involved in iron homeostasis. CIR1, 7

CNAG_04864, is important for capsule formation and negatively regulates laccase 8

expression in H99 (18). In addition, it has been shown that cir1 mutants are sensitive to 9

SDS and the azole drug fluconazole, suggesting that Cir1 is involved in cell wall integrity 10

and membrane functions (18). Since capsule and melanin production were affected in the 11

tup1∆ strain, the effect of TUP1 deletion was examined relative to changes in CIR1 12

expression. Quantitative RT-PCR results showed CIR1 expression to be mildly affected 13

in the tup1∆ strain compared H99 in which the relative expression level was only 14

1.36±0.05 fold higher in tup1∆ strain. Furthermore, expression of iron permease genes, 15

such as CFT1 and CFT2, which are regulated by CIR1, was not affected by the deletion 16

of TUP1 according to our microarray data (Table S3 in the supplemental material). 17

These data suggested that TUP1 does not regulate iron homeostasis through the CIR1 18

regulatory circuit. However, growth of the tup1∆ strain was significantly hampered in the 19

presence of 0.01% SDS and the tup1∆ strain displayed increased sensitivity to 20

fluconazole (Fig. 4E). These data suggested that TUP1 is also involved in cell wall 21

integrity and membrane functions. 22

23

16

TUP1 deletion reduced the virulence 1

Since deletion of TUP1 in H99 resulted in both positive and negative effects with respect 2

to the three major C. neoformans virulence factors which include growth at 37°C, 3

formation of melanin and capsule, its effect on virulence was investigated. Groups of 10 4

mice were challenged with different yeast stains via tail vein injection. Figure 5A shows 5

that all mice challenged with wild type or the complemented strains succumbed to 6

infection 9 days post injection while it took 20 days for the tup1∆-infected mice (p < 7

0.001, compared to wild type infected mice), indicating that deletion of TUP1 causes 8

attenuation of virulence in C. neoformans. 9

To examine the pathobiological differences in mice infected with wild type or tup1∆ 10

strains, the brain fungal burden and capsule size was determined at different stages of 11

infection. Significant differences in the number of colony-forming unit (CFU) were 12

observed between the H99 and the tup1∆ strain (5.93×103 versus 5.3×10

2 per brain) in as 13

early as 2 days after injection (Fig. 5B). The number of CFU, however, increased 14

exponentially and differed even more at 6 days after injection. These data suggest that 15

TUP1 is important for growth in vivo although the tup1∆ strain only showed a marginal 16

reduction in growth at 37°C in vitro. Another noteworthy observation was that fungal 17

burden analyzed on the day of death in a mouse injected with tup1∆ strain (1.25×107 at 18

day 13) was 80-fold lower than that of a mouse injected with H99 (8.35×108 at day 9) 19

(Fig. 5B). It was possible that the larger capsule size in the tup1∆ strain in vitro (Fig. 2A) 20

might have contributed to such a difference. Surprisingly, the capsule size of the yeast 21

cells in the brain smear from mice infected with tup1∆ strain was similar to that of mice 22

17

infected with H99 (Fig. 5C). These findings clearly indicate that Tup1 plays an important 1

role in pathobiology of C. neoformans. 2

3

DISCUSSION 4

This study investigated the function of TUP1 in C. neoformans serotype A strains 5

including H99 and presents another example of serotype-specific difference in gene 6

regulation. TUP1 plays a conserved role with respect to growth and mating but distinctly 7

different roles in strains of serotype A vs D. Serotype A-specific phenotypes of tup1∆ 8

strains include a lack of density-dependent growth, an enlarged capsule size, reduced 9

melanin production, and a defect in iron/copper homeostasis. 10

The prominent capsule size in tup1∆ strain under non-inducing conditions indicated the 11

important role of TUP1 in capsule formation. Many environmental factors have been 12

shown to influence the size of capsule in C. neoformans. Low concentrations of glucose 13

and iron, high concentrations of carbon dioxide, and the presence of serum components 14

have been shown to enhance capsule formation (15). However, limited information is 15

available concerning the regulation of capsule formation. Gpa1-cAMP-PKA signaling 16

pathway has been thoroughly studied in its effect on capsule production. Both pka1∆ and 17

gpa1∆ strains exhibited a marked defect in capsule production while pkr1∆ strain 18

overproduced the capsule (9). Several observations indicate that TUP1 affects capsule 19

production independent of the cAMP-PKA signaling pathway. First, our preliminary 20

microarray data did not show any significant change in the gene expression of cAMP-21

PKA pathway components such as CAC1 (adenyl cyclase), PKA1 and PKR1 (data not 22

shown). Second, the addition of cAMP to growth media did not alter the hypercapsular 23

18

phenotype of the tup1∆ strain (data not shown). Third, the hypercapsular phenotype of 1

pkr1∆ was observed not only in vitro but also in vivo resulting in hypervirulence while 2

the tup1∆ strain exhibited reduced virulence and yet its capsule size in vivo was 3

comparable to that of the wild type strain. Another signaling pathway involved in capsule 4

formation is the HOG pathway. HOG1 negatively regulates synthesis of capsule and 5

melanin in the serotype A strain H99, but not in the serotype D strain JEC21(1). Deletion 6

of TUP1 or HOG1 has a similar effect on capsule production and their serotype-specific 7

regulatory function offers the possibility that Tup1 and Hog1 might share downstream 8

regulatory targets either in a parallel signaling pathway or by direct interaction. The 9

HOG1 pathway in S. cerevisiae is activated by osmotic stress and modulates diverse 10

osmo-adaptive gene expression through the recruitment of the general transcription 11

repressor complex Tup1-Ssn6 and the sequence-specific DNA-binding protein Sko1 (36). 12

Since the tup1∆ strains also show sensitivity to 2mM H2O2 as observed with the hog1∆ 13

strains (1)(data not shown), this possibility was considered. However, the hog1∆ strain 14

derived from H99 exhibited enhanced melanization and temperature sensitivity at 40°C, 15

neither of which are shared in the tup1∆ strains. Furthermore, the H2O2-sensitive 16

phenotype of the tup1∆ strain was rescued by addition of copper or iron to the media, 17

suggesting that the low intracellular iron/copper content and not a defect in the HOG 18

pathway resulted in the H2O2 sensitivity (data not shown). Therefore, the involvement of 19

TUP1 in regulating capsule formation does not share all the common characteristics with 20

the aforementioned known pathways. 21

Given that TUP1 affects capsule production independent of previously known regulatory 22

pathways, we tried to identify the targets of TUP1. Preliminary microarray and RT-PCR 23

19

experiments revealed that several genes involved in iron/copper homeostasis were down 1

regulated in tup1∆ strains. The requirement of iron/copper in the growth of tup1∆ strain 2

and restoration of melanin production by copper in tup1∆ strain further supports the 3

importance of TUP1 in iron/copper homeostasis in serotype A strain, H99. A role of 4

TUP1 in iron metabolism was first suggested by the identification of ferric reductase 5

gene, RBT2, as one of the genes repressed by TUP1 in C. albicans (2). Subsequent study 6

showed that ferric reductase activity in ∆tup1/∆tup1 cells was constitutively elevated and 7

iron-dependent transcriptional alteration of CaFTR1 and CaFTR2 mRNA was abrogated 8

in ∆tup1/∆tup1 mutant (21). Furthermore, examples of the physical interaction between a 9

co-repressor and an iron-sensing factor controlling the expression of iron uptake genes 10

have been shown in Schizosaccharomyces pombe (50). S. pombe fep1+ encodes GATA 11

transcription factor that represses the expression of iron transport genes in response to 12

elevated iron conditions. Using yeast two hybrid analysis, it has been shown that Tup11, 13

a Tup1 homolog of S. pombe, and Fep1 physically interact with each other (50). Whether 14

C. neoformans Tup1 directly interacts with the proteins involved in iron homeostasis is 15

yet to be determined. 16

The importance of copper homeostasis in C. neoformans is suggested by the copper 17

dependency of two well-known virulence factors, the Cu/Zn superoxide dismutase (7), 18

and laccase, a key enzyme in melanin synthesis (37, 43). Both enzymes require copper as 19

a cofactor for their function. Deletion of CNLAC1 encoding the laccase or the mutation in 20

the copper binding site of the gene resulted in significant reduction in virulence (37, 43). 21

Copper also induces laccase transcription in wild-type cells and can restore the laccase 22

activity in vph1∆ mutants (48). In addition, the close relationship between copper and 23

20

iron homeostasis has been reviewed (16). For example, copper homeostasis also affects 1

iron since Fet3, the high-affinity iron transporter, requires the incorporation of four 2

copper ions for the function (16). Thus, ineffective copper loading of Fet3 due to a defect 3

in copper homeostasis can also lead to lower intercellular levels of iron, which affects 4

capsule and melanin production. In fact, mutation in CCC2 (copper transporter) or ATX1 5

(copper chaperone) resulted in large capsules in iron-replete conditions and showed 6

impaired growth under iron-limiting condition (40). Although our microarray data did not 7

show significant changes in CCC2 and ATX1 gene expression (Table S3), reduced 8

expression of CTR4 (copper transporter 4) in tup1∆ strain and additive growth defect of 9

tup1∆ strain in iron/copper-chelated media lend additional support to the regulatory role 10

of TUP1 in both iron and copper homeostasis. 11

Another intriguing result of our study is that Tup1 appears to function as both a repressor 12

and an activator in C. neoformans. In contrast to the prevailing view of Tup1 as a global 13

repressor, our results showed that many genes were also down-regulated in the absence of 14

TUP1 suggesting that Tup1 functions as an activator for the expression of those genes. 15

An analogy is seen with Hap1 in S. cerevisiae which was originally identified as a heme-16

dependent transcriptional activator but was reported to function also as transcriptional 17

repressor depending on oxygen levels (11). Also mammalian nuclear hormone receptors 18

are examples of factors that can act both positively and negatively through the 19

recruitment of coactivators and corepressor complexes, respectively (45). Conversely, it 20

is also possible that the down-regulated genes in tup1∆ strain are due to the indirect effect 21

of Tup1. For instance, Tup1 could interact with a negative regulator and inactivation of 22

Tup1 could lead to the activation of a negative regulator, which in turn would cause the 23

21

observed down-regulation of genes in the tup1∆ strain. Additional experiments are 1

required to identity the direct target(s) of Tup1 and possible interacting partners, if any, to 2

understand the mechanism of Tup1 regulation in C. neoformans. 3

In C. albicans, disruption of TUP1 causes its inability to switch between yeast and 4

filament forms and results in constitutive filamentous growth, which presumably is the 5

reason why the tup1∆ strain is avirulent (2, 3). Previously, virulence studies could not be 6

carried out properly with tup1∆ strains in a serotype D background because of their 7

inability to grow at low cell densities which hindered the precise determination of 8

inoculum size based on colony forming units (26, 27). Here, we showed that deletion of 9

TUP1 in H99 affected virulence. Since the TUP1 deletion displayed pleiotropic effects, it 10

is likely that the reduced virulence of tup1∆ strain resulted from the combination of these 11

effects and is possibly related to iron/copper homeostasis. Given the global regulatory 12

role of TUP1 in fungi and the manifestation of different phenotypes between A and D 13

tup1∆ strains, an in-depth analysis of TUP1 function would offer a valuable tool towards 14

understanding the divergence of gene regulation in C. neoformans. 15

16

ACKNOWLEDGMENTS 17

This study was supported by funds from the intramural program of the National Institute 18

of Allergy and Infectious Diseases, NIH. 19

20

22

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29

FIGURE LEGENDS 1

2

Figure 1. Deletion of TUP1 in H99 shows no density-dependent growth. 3

(A) Exponentially growing cultures (OD600 = 0.5-1.0) were washed, resuspended in 0.9% 4

NaCl and plated on SD media. A total of 5X106

and 106 cells for LP1 (WT) and tup1∆ 5

(serotype D strains) and 5X105

and 5X102 cells for H99 (WT) and tup1∆ (serotype A 6

strains) were plated on SD media and incubated for 2 days at 30ºC. (B) Comparison of 7

Cqs1 sequences between JEC21 (serotype D) and H99. Sequence of QSP1, the active 8

peptide purified from serotype D tup1∆ culture filtrate, is in bold. Two amino acids in 9

H99 Cqs1, which are different from JEC21 Cqs1, are underlined. 10

11

Figure 2. TUP1 disruption affects mating and capsule formation. (A) H99 (WT; left 12

panel) and HL112 (tup1∆; right panel) strain were each mixed with KN99a on the V-8 13

juice agar, incubated for 72hr, and observed for hyphal formation. (B) H99 (WT), HL112 14

(tup1∆), and HL132 (TUP1-complemented strain ; tup1∆ + TUP1) were grown on RPMI 15

agar media at 37ºC for 2 days and examined for the capsule formation by microscopic 16

examination of India ink slide preparation. 17

18

Figure 3. TUP1 positively regulates iron-related genes and negatively regulates capsule-19

related gene. Total RNA was isolated from wild type (H99) and tup1∆ strains. 20

Transcriptional changes in several genes were determined by real time RT-PCR. Data 21

were normalized with ACTIN level and expressed as the relative amount in tup1∆ 22

compared to that in H99. Genes and the annotated functions are listed. 23

30

1

Figure 4. Pleiotropic effects of TUP1 deletion. (A-E) H99 (WT), HL112 (tup1∆) and 2

HL132 (tup1∆ + TUP1). (A) Iron conditions affect growth of tup1∆ strain. Serially 3

diluted yeast cells from the indicated strains were spotted on SD medium (left panel), 4

iron-chelated medium (LIM+EDDA), iron-replete medium (LIM+Fe) and incubated at 5

30°C for 3 days. (B) tup1∆ strain shows additive growth defect on both iron- and copper-6

limited medium. Serially diluted yeast cells were spotted on YES, LIM+Fe and 7

LIM+BCS+EDDA (1mM BCS; copper chelator, 0.056mM EDDA; iron chelator) and 8

incubated at 30°C for 3 days. (C) Melanin production is affected in tup1∆ strain. Serially 9

diluted yeast cells were spotted onto norepinephrine-containing medium. The plates were 10

then incubated for 2 days at 30ºC in the dark. 10µM CuSO4 was added to the 11

norepinephrine-media (right panel). (D) Iron concentrations affect capsule size in tup1∆ 12

strain. Cells were grown on RPMI (left panel), LIM (middle panel), and LIM+Fe (right 13

panel) for 2 days at 30°C and capsule formation was examined. (E) The tup1∆ strain is 14

sensitive to SDS and fluconazole. Serially diluted yeast cells were spotted on YPD (left), 15

YPD+0.01% SDS (middle), and YPD+8µg/ml fluconazole (right) and incubated at 30°C 16

for 2 days. 17

18

Figure 5. Virulence study. (A) Survival of mice injected with H99 (WT), HL112 (tup1∆) 19

and HL132 (tup1∆ + TUP1). Groups of 10 female BALB/c mice were injected via the 20

lateral tail vein with 106 viable yeast cells and mortality was monitored. (B) Fungal load 21

in the mouse brain. Three mice per yeast strain were sacrificed at several intervals after 22

injection as indicated. The brains were homogenized and plated onto YEPD agar. 23

31

Colonies were counted after 2 days of incubation at 30°C. (C) Brain smear showing cells 1

of H99 and tup1∆ strains at 3, 9, and 14 days postinfection as indicated. Brain tissue of 2

mice injected with H99 or tup1∆ strains was smeared on a microscopic slide and 3

examined under a microscope with a Normalski interference condenser. 4