Spt3 Plays Opposite Roles in Filamentous Growth in Saccharomyces cerevisiae and Candida albicans and Is Required for C. albicans Virulence

Corresponding author: Fred Winston, Harvard Medical School, 200 Longwood Ave., Boston, MA 02115., winston{at}rascal.med.harvard.edu (E-mail)

Communicating editor: A. P. MITCHELL

	ABSTRACT

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

Spt3 of Saccharomyces cerevisiae is required for the normal transcription of many genes in vivo. Past studies have shown that Spt3 is required for both mating and sporulation, two events that initiate when cells are at G₁/START. We now show that Spt3 is needed for two other events that begin at G₁/START, diploid filamentous growth and haploid invasive growth. In addition, Spt3 is required for normal expression of FLO11, a gene required for filamentous growth, although this defect is not the sole cause of the spt3/spt3 filamentous growth defect. To extend our studies of Spt3's role in filamentous growth to the pathogenic yeast Candida albicans, we have identified the C. albicans SPT3 gene and have studied its role in C. albicans filamentous growth and virulence. Surprisingly, C. albicans spt3/spt3 mutants are hyperfilamentous, the opposite phenotype observed for S. cerevisiae spt3/spt3 mutants. Furthermore, C. albicans spt3/spt3 mutants are avirulent in mice. These experiments demonstrate that Spt3 plays important but opposite roles in filamentous growth in S. cerevisiae and C. albicans.

THE Saccharomyces cerevisiae transcription factor Spt3 was identified originally by mutations that suppress the transcriptional defects caused by Ty and Ty LTR insertion mutations (WINSTON et al. 1984A ; reviewed in WINSTON and SUDARSANAM 1998 ). Spt3 is required for the normal transcription of 3% of S. cerevisiae genes (LEE et al. 2000 ). Both genetic and biochemical evidence suggest that Spt3 activates transcription by the recruitment of TATA-binding protein (TBP) to particular promoters (EISENMANN et al. 1992 ; DUDLEY et al. 1999 ; BHAUMIK and GREEN 2001 ; LARSCHAN and WINSTON 2001 ). Other evidence suggests that Spt3 can also repress transcription (BELOTSERKOVSKAYA et al. 2000 ; LEE et al. 2000 ). The SPT3 gene is functionally conserved among yeasts and other eukaryotes, including humans (MADISON and WINSTON 1998 ; MARTINEZ et al. 1998 ; OGRYZKO et al. 1998 ; YU et al. 1998 ).

Spt3 is a component of the SAGA (Spt-Ada-Gcn-Acetyltransferase) coactivator complex (GRANT et al. 1998B ; WINSTON and SUDARSANAM 1998 ). SAGA is a 1.8-MD protein complex that contains >20 proteins. Both genetic and biochemical evidence have demonstrated that SAGA possesses distinct activities with respect to transcriptional control (HORIUCHI et al. 1997 ; ROBERTS and WINSTON 1997 ; STERNER et al. 1999 ). For example, while Spt3 is required to recruit TBP, another SAGA member, Gcn5, has histone acetlytransferase activity (BROWNELL et al. 1996 ; GRANT et al. 1997 ). In addition to Gcn5 and Spt3, SAGA contains several other classes of proteins, including those required for integrity of the complex (Spt7, Spt20, and Ada1) and for recruitment of the complex by transcriptional activators (Tra1; GRANT et al. 1998A ; BROWN et al. 2001 ; ROTH et al. 2001 ). In this article we focus primarily on the role of Spt3 in filamentous growth in S. cerevisiae and Candida albicans.

S. cerevisiae and C. albicans can each grow in both yeast and filamentous forms. The form of S. cerevisiae filamentous growth most intensively studied is diploid pseudohyphal growth (GIMENO et al. 1992 ; PAN et al. 2000 ; GANCEDO 2001 ). Closely related to this form of growth is haploid invasive growth (ROBERTS and FINK 1994 ). In addition, a functionally unrelated form of haploid pseudohyphal growth has recently been demonstrated (HOLLENHORST et al. 2000 ; ZHU et al. 2000 ). C. albicans, the most widespread yeast pathogen of humans, is responsible for clinical problems ranging from thrush and vaginal yeast infections to life-threatening systemic infections in immunocompromised patients (ODDS 1988 ; FIDEL and SOBEL 1996 ). C. albicans has three forms of growth: one yeast-like form, called blastospores, and two filamentous forms, pseudohyphae and hyphae. The transition from blastospores to the filamentous forms appears to be required for pathogenicity (KOBAYASHI and CUTLER 1998 ; MITCHELL 1998 ). Several factors have been identified that contribute, either positively or negatively, for the transition from the blastospore to filamentous forms (BRAUN et al. 2001 ; KADOSH and JOHNSON 2001 ; KHALAF and ZITOMER 2001 ; MURAD et al. 2001 ; NAVARRO-GARCIA et al. 2001 ). Many of these factors were identified on the basis of their homology to factors required for diploid pseudohyphal growth of S. cerevisiae (KOBAYASHI and CUTLER 1998 ; BROWN and GOW 1999 ; WHITEWAY 2000 ; KHALAF and ZITOMER 2001 ). An understanding of the regulatory pathways and transcription factors that control the C. albicans transition from blastospore growth to filamentous growth should aid in the development of treatments for C. albicans infections.

Two aspects of Spt3 function led us to examine whether Spt3 and other SAGA components are involved in S. cerevisiae diploid pseudohyphal growth. First, spt3 mutants are defective in mating (HIRSCHHORN and WINSTON 1988 ) and sporulation (WINSTON et al. 1984B ), which, like filamentous growth, are events that initiate at G₁/START of the cell cycle. Second, it seemed likely that Spt3 is required for transcription of the FLO11 gene, which is required for diploid pseudohyphal growth (LO and DRANGINIS 1996 ). This possibility became apparent because Spt3 is strongly required for transcription of Ty1 elements (WINSTON et al. 1984B ), and Ty1 and FLO11 transcription are regulated by many of the same factors (MADHANI et al. 1997 ; CONTE and CURCIO 2000 ).

In this article we show that Spt3 is required for both diploid pseudohyphal growth and haploid invasive growth of S. cerevisiae. Consistent with these spt3 phenotypes, spt3 mutants have decreased FLO11 mRNA levels. However, our results demonstrate that the defect in FLO11 transcription is not the sole cause of the spt3 filamentous and invasive growth defects. In addition, we have identified the C. albicans SPT3 gene and have constructed a C. albicans spt3/spt3 mutant. In contrast to the S. cerevisiae spt3/spt3 nonfilamentous phenotype, the C. albicans spt3/spt3 mutant is hyperfilamentous. Furthermore, C. albicans spt3/spt3 mutants are avirulent in mice. Taken together, our data demonstrate that C. albicans Spt3 acts as a repressor of filamentous growth and that it is also required for virulence.

	MATERIALS AND METHODS

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

Yeast strains:
All S. cerevisiae strains used in these studies (Table 1) are in the 1278b genetic background (SIDDIQUI and BRANDRISS 1988 ). The spt3, spt7, spt20, and gcn5 deletion mutations were each recombined into strain 10560-6B (LO et al. 1997 ) by standard gene replacement methods (GUTHRIE 1991 ), using the appropriate restriction fragments. The restriction fragments used were as follows: for spt3203::TRP1, a 2.5-kb SspI fragment from pAH100 (HAPPEL 1989 ); for spt7402::LEU2, a 2.7-kb MluI-SphI fragment from pLG59 (GANSHEROFF et al. 1995 ); for spt20100::URA3, a 2.3-kb ClaI-XbaI fragment from pSR56 (ROBERTS and WINSTON 1996 ); and for gcn5::LEU2, a 3.1-kb SphI-SacI fragment from YCplac111-gcn5::LEU2 (kindly provided by Kevin Struhl). The deletions were confirmed by PCR. These strains were then tested for several mutant phenotypes previously identified in the S288C background, including inositol auxotrophy, hydroxyurea sensitivity, the ability to use galactose as a carbon source, and slow growth on minimal media. In all cases, the mutant phenotypes correlated well between the two genetic backgrounds. One difference noted between the two genetic backgrounds is that an unusual cell morphology, previously observed in spt7 and spt20 mutants, is more severe in the 1278B background. The cells appear to be elongated and swollen and the buds do not always separate from the mother cell. However, these cells do not form distinct chains as do pseudohyphal cells (data not shown).

The C. albicans strains (Table 2) constructed for this study are derived from strains BWP17 (WILSON et al. 1999 ) and constructed by transformation (HULL and JOHNSON 1999 ). The C. albicans spt3/spt3 homozygous diploid was constructed in two steps. First, the XcmI-StuI fragment of plasmid pLP13, containing the spt3::ARG4 allele, was used to transform BWP17 to Arg⁺. Second, the XcmI-StuI fragment of plasmid pLP12, containing the spt3::URA3 allele, was used to transform the SPT3/spt3::ARG4 strain FWC6 to Ura⁺ using Sc-Arg-Ura plates. A wild-type copy of SPT3 was integrated into strains FWC7, FWC8, and FWC9 (spt3::URA3/spt3::ARG4) with NruI-digested pLP14, resulting in integration of SPT3 at HIS1. These transformants were selected on SC-Ura-Arg-His medium. The heterozygote strain FWC16 was created by transforming NruI-digested pGEMT-HIS1 and ClaI-digested pRSARG4 into strain FWC5. For all C. albicans transformants, the correct integration event was confirmed by Southern blot analysis (AUSUBEL et al. 1988 ).

Media and growth conditions:
Rich (YPD), minimal (SD), and synthetic complete (SC) media were prepared as previously described (ROSE et al. 1990 ). SLAD media, for testing pseudohyphal growth of S. cerevisiae strains, was prepared as previously described (GIMENO et al. 1992 ). For growth of C. albicans, YPD was supplemented with 80 mg of uridine per liter. Two types of media were used for testing filamentous growth of C. albicans: first, medium 199 (GIBCO BRL, Gaithersburg, MD), made as previously described (SAPORITO-IRWIN et al. 1995 ) and supplemented with 1.35% agar and 80 mg uridine per liter; second, liquid YPD was supplemented with fetal bovine calf serum (Sigma, St. Louis) to a final concentration of 20%. Haploid invasion and pseudohyphal growth assays were done as previously described (ROBERTS and FINK 1994 ). For copper induction of CUP1-FLO11 the SLAD plates were supplemented with 0.05 mM and 0.1 mM CuSO₄. C. albicans strains were grown on YPD plates for 3 days at 30° and then the colonies were visualized and photographed using a Leica MZFLIII Microscope at x4 magnification. Colonies grown on medium 199 were visualized and photographed after 1 day at 37° using a Nikon Eclipse E1000 Microscope with a x20 DIC objective. All other cells were visualized and photographed with a Nikon Eclipse E1000 Microscope with a x40 DIC objective. For growth in YPD with 20% fetal calf serum, 10 ml of YPD (20% serum) was inoculated and cultures were grown for 4 hr at 37°.

Cloning and DNA sequence analysis of the C. albicans SPT3 gene:
C. albicans SPT3 was cloned by complementation of the S. cerevisiae spt3 mutant phenotypes. The host strain, FY293, contains two Ty1-derived insertion mutations, his4-917 and lys2-173R2. In an SPT3 wild-type strain these insertion mutations confer His^- Lys⁺ phenotypes; in an spt3 mutant such as FY293, the phenotypes are reversed to His⁺ Lys^-. To clone C. albicans SPT3, strain FY293 was transformed with 0.5 µg of a YEp352 C. albicans genomic library (NAVARRO-GARCIA et al. 1998 ). Approximately 80,000 Ura⁺ transformants were then replica plated to screen for transformants that had an Spt⁺ phenotype (His^- Lys⁺). Of 221 candidates, plasmid DNA was isolated from 12 and used to retransform FY293. Eight of the 12 transformants had an Spt⁺ (His^- Lys⁺) phenotype. The DNA sequences of two candidates were determined by standard sequencing methods and universal M13 and synthetic primers. Sequencing was performed in the Biopolymers Facility in the Department of Genetics, Harvard Medical School. By Southern hybridization analysis, the cloned C. albicans SPT3 gene hybridizes to C. albicans genomic DNA but not to S. cerevisiae genomic DNA.

Plasmids:
All plasmids were constructed by standard procedures with exceptions noted below (AUSUBEL et al. 1988 ). Plasmid pLP15, used to overexpress FLO11, contains the FLO11 open reading frame amplified from genomic DNA by PCR using Pfu polymerase (Stratagene, La Jolla, CA). The EcoRI-digested FLO11 DNA was then ligated to EcoRI-digested and phosphatased pYSK7 (BUTT et al. 1984 ). The ligations were then used to transform yeast, and strains containing the correct orientation of FLO11 were confirmed by PCR, using primers internal to the CUP1 and FLO11 sequences. The resulting yeast strain was used in crosses to segregate the CUP1-FLO11 plasmid into the strains of interest. Plasmids that contain the spt3::URA3 and spt3::ARG4 deletion constructs were each constructed in three steps. The spt3::URA3 construct was made by using primer pairs to amplify 346 bp upstream of SPT3 and 371 bp downstream of SPT3 and another set to amplify URA3 from pGEMURA3 (WILSON et al. 1999 ). The primers were designed such that the resulting PCR products would overlap in subsequent PCR reactions to create a DNA fragment consisting of URA3 flanked by sequences adjacent to the SPT3 open reading frame (AMBERG et al. 1995 ). The C. albicans ARG4 gene was PCR amplified from pRSARG4SpeI (WILSON et al. 1999 ), using primers that contain only 60 bp of homology to C. albicans SPT3. Each resulting PCR product was then used to construct a plasmid containing extensive homology to SPT3 flanking sequences. This was done by mixing each PCR product with ClaI-linearized pLP11 (Yep352-SPT3 from the C. albicans genomic library) and using this DNA to transform strain FY293. Ura⁺ transformants were screened for an Spt^- phenotype, indicating that the spt3 construct had recombined to replace SPT3 on the plasmid. Plasmids were then rescued from yeast and confirmed by standard procedures (AUSUBEL et al. 1988 ). These plasmids are pLP12 (YEp352-spt3::URA3) and pLP13 (YEp352-spt3::ARG4). Plasmid pLP14, used to rescue the spt3 homozygous mutant phenotype, was constructed from SacI-NsiI-digested pGEMT-HIS1 (WILSON et al. 1999 ) and a SacI-NsiI fragment from pLP11 (SPT3).

Overexpression of FLO11:
The strains containing either the CUP1-FLO11 plasmid or the CUP1 vector only were grown overnight in SC-Trp liquid media with 0, 0.05, or 0.1 mM copper. The cultures were then plated for 100 colonies per plate on SLAD containing 0, 0.05, or 0.1 mM copper. The plates were then incubated at 30° for 10 days and the colonies were scored each day for their pseudohyphal growth using a light microscope.

RNA isolation and Northern analysis:
RNA isolation and Northern analysis were performed as previously described (SWANSON et al. 1991 ). The haploid strains were grown in YPD to 1–2 x 10⁷ cells/ml. To check expression of the CUP1-FLO11 plasmid in diploid strains, the cultures were pregrown overnight in SC-Trp containing 0, 0.05, or 0.1 mM copper. Then the cultures were diluted back in fresh SC-Trp media with 0, 0.05, or 0.1 mM copper and grown to a cell density of 1–2 x 10⁷ cells/ml. The FLO11 levels were quantified using an SPT15 normalization probe (SUDARSANAM et al. 1999 ). The FLO11 probe was PCR amplified (RUPP et al. 1999 ). All probes were labeled with [³²P]dATP by random priming (AUSUBEL et al. 1988 ).

Mouse studies:
C. albicans strains used in infection experiments were grown in YPD + Uri to OD₆₀₀ between 0.9 and 1, corresponding to 0.5–1.0 x 10⁸ cells/ml, depending upon the particular strain. The C. albicans strains used were SC5314, HLC54, FWC15, FWC13, FWC11, FWC10, FWC14, and FWC12. C. albicans cells were counted and washed once with 0.5 ml of phosphate-buffered saline (PBS) pH 7. BALB/cJ mice 5–6 weeks of age were obtained from Jackson Laboratories and maintained in a barrier facility at Harvard Medical School. All mice were infected by injection of 300 µl of PBS suspension containing 5 x 10⁶ cells into lateral tail veins. Four mice were tested for each of the three spt3/spt3 mutant classes, their respective rescue strains, wild type, and a cph1 efg1 double mutant used as a negative control (LO et al. 1997 ). Progression of the disease was monitored several times a day in accordance to the Animal Experimentation Protocol approved by the HMS Standing Committee on Animals.

	RESULTS

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

Spt3 is required for both pseudohyphal and invasive growth in S. cerevisiae:
To study pseudohyphal filamentous growth in SAGA mutants, we first constructed strains that contain deletion mutations of four SAGA genes, SPT3, SPT7, SPT20, and GCN5, in a genetic background permissive for S. cerevisiae filamentous growth, 1278b (GIMENO et al. 1992 ; MATERIALS AND METHODS). Then, diploids homozygous for each SAGA mutation to be tested were analyzed after incubation on solid medium that normally induces filamentous growth (SLAD medium). After growth on SLAD plates for 4 days, the wild-type colonies displayed normal filamentous growth, with long chains of pseudohyphae growing outward from the colony (Fig 1). In contrast, the SAGA mutants displayed a range of filamentous growth defects (Fig 1). The clearest defect was observed for the spt3/spt3 mutant, which was completely defective for filamentous growth. The colonies had completely smooth edges and all cells examined were in the budding yeast form. The gcn5/gcn5 mutant showed only a modest defect—after 4 days of incubation it displayed reduced filamentous growth, but after 8 days it was indistinguishable from the wild-type strain. Effects of the spt7 and spt20 mutations on filamentous growth were difficult to interpret as both mutants grew poorly and formed irregular cells and colonies under all growth conditions in the 1278b genetic background (Fig 1 and data not shown). Our results demonstrate that at least one SAGA component, Spt3, is essential for filamentous growth, while another, Gcn5, may play a minor role.

View larger version (62K): [in this window] [in a new window] [Download PPT slide]   Figure 1. Pseudohyphal growth of SAGA mutants. Comparison of wild-type (L5366), spt3 (L962), gcn5 (L976), and spt7 (L970) strains grown on SLAD plates at 30° for 4 days and 8 days.

A process closely related to diploid filamentous growth is haploid invasion, characterized by filament formation and agar invasion (ROBERTS and FINK 1994 ). Previous studies have demonstrated that both types of filamentous growth are controlled by common factors (ROBERTS and FINK 1994 ). Therefore, we also tested haploid SAGA mutants for this growth property. Our results (Fig 2) show that, similar to the diploid pseudohyphal growth defects, spt3 mutants are defective for haploid invasive growth compared to wild type (Fig 2). The gcn5, spt7 (Fig 2), and spt20 (data not shown) mutants are also defective for haploid invasion, although the defect appears to be less severe than for spt3.

View larger version (69K): [in this window] [in a new window] [Download PPT slide]   Figure 2. SAGA mutants are defective for haploid invasion. (Left) Wild-type (L5684), spt3 (L959), spt7 (L965), and gcn5 (L974) strains after growing for 3 days at 30° and then an additional 2 days at 25°. (Right) The same strains after washing the cells off the plate under running water.

Analysis of FLO11 expression and its relationship to the spt3/spt3 filamentous growth defect:
Expression of the FLO11 gene is necessary for S. cerevisiae filamentous and invasive growth (LO and DRANGINIS 1998 ). Transcription of both FLO11 and Ty1 elements depends upon a DNA sequence element called the filamentation response element that is cooperatively bound by the factors Ste12 and Tec1 (LO and DRANGINIS 1996 ; MADHANI et al. 1997 ; RUPP et al. 1999 ; CONTE and CURCIO 2000 ). Since several of the Spt proteins in SAGA are required for Ty1 transcription, we reasoned that Spt3, Spt7, Spt20, and Gcn5 may also be required for FLO11 transcription. We initially attempted to measure FLO11 mRNA levels in diploid strains. However, FLO11 mRNA levels are very low in diploids, making accurate measurements difficult (data not shown). Therefore, we measured FLO11 mRNA levels in haploid strains. Northern analysis (Fig 3) demonstrates that in spt3 mutants, FLO11 mRNA levels are reduced approximately fivefold compared to wild-type levels. In gcn5 mutants, FLO11 mRNA levels are reduced twofold, while in spt20 and spt7 mutants there is a severe reduction in FLO11 mRNA levels (Fig 3 and data not shown). The relative effects among these three classes of SAGA mutants are similar to other SAGA mutant phenotypes that have been examined (HORIUCHI et al. 1997 ; ROBERTS and WINSTON 1997 ; STERNER et al. 1999 ).

View larger version (49K): [in this window] [in a new window] [Download PPT slide]   Figure 3. FLO11 mRNA levels are reduced in SAGA mutants. Total RNA was prepared from wild-type (L5684), spt3 (L959), spt20 (L973), and gcn5 (L975) strains. SPT15 serves as a loading control. mRNA levels are normalized to the wild-type strain.

To determine if the strong spt3 pseudohyphal and invasive growth defects are conferred solely by reduced FLO11 mRNA levels, we expressed FLO11 under the control of an Spt3-independent promoter, CUP1 (CUP1-FLO11), and determined if ectopic FLO11 expression suppresses the spt3/spt3 defects. Our results show that CUP-FLO11 expression is unable to suppress the pseudohyphal growth defect of an spt3/spt3 mutant (data not shown). As a positive control for FLO11 expression, CUP1-FLO11 was shown to complement the filamentous growth defect of the flo11 mutant. In these experiments FLO11 mRNA levels were approximately equal in the flo11 and spt3 strains and at a level greater than those in wild-type diploids without CUP1-FLO11 (data not shown). Thus, Spt3's role in filamentous growth is not limited to a role in FLO11 expression.

Cloning and characterization of the C. albicans SPT3 gene:
Several factors known to be required for S. cerevisiae filamentous growth are also required for C. albicans filamentous growth (BRAUN and JOHNSON 1997 ; LO et al. 1997 ; KADOSH and JOHNSON 2001 ). Therefore, we decided to identify the C. albicans SPT3 gene and test if it is also required for C. albicans filamentous growth. First, we cloned the C. albicans SPT3 gene by complementation of an S. cerevisiae spt3 mutation (MATERIALS AND METHODS). Sequence analysis identified a 986-bp open reading frame homologous to S. cerevisiae SPT3. This sequence is also now present in the C. albicans genome sequence database (http://genolist.pasteur.fr/CandidaDB/). The predicted C. albicans Spt3 protein is 59% identical and 79% similar to S. cerevisiae Spt3. The two histone-fold motifs that are conserved among previously analyzed Spt3 protein sequences from human, S. cerevisiae, and other yeasts (BIRCK et al. 1998 ; MADISON and WINSTON 1998 ) are also conserved in the C. albicans Spt3 protein. The C. albicans SPT3 gene strongly complements all S. cerevisiae spt3 mutant phenotypes tested, including the spt3/spt3 pseudohyphal growth defect (Fig 4A), the spt3 growth defect on galactose as a carbon source, and the spt3 Spt^- phenotypes (suppression of the his4-917 and lys2-173R2 insertion mutations; data not shown). These results demonstrate strong functional and sequence conservation between the S. cerevisiae and C. albicans SPT3 genes.

View larger version (70K): [in this window] [in a new window] [Download PPT slide]   Figure 4. (A) C. albicans SPT3 complements the S. cerevisiae spt3 filamentous growth defect. Plasmid pRS416 (left) and plasmid pLP11 (right) were used to transform strain L962. Transformants were then tested for pseudohyphal growth. Shown are representative colonies after 4 days of incubation at 30°. (B) C. albicans spt3/spt3 mutants have a slow-growth phenotype. Comparison of wild type (SC5314), spt3 class 1 mutant (FWC15), spt3/SPT3+ heterozygous strain (FWC16), and an spt3 class 1 mutant with a copy of SPT3 integrated at HIS1 (FWC10) after growth on YPD media at 30° for 2 days.

C. albicans spt3/spt3 mutants are hyperfilamentous:
To test the role of Spt3 in C. albicans filamentous growth, we constructed an spt3/spt3 mutant. Since all C. albicans strains are diploids, we deleted both copies of SPT3 in C. albicans strain BWP17, replacing one copy of SPT3 with URA3 and the second copy of SPT3 with ARG4 (described in MATERIALS AND METHODS). Both gene replacements were confirmed by Southern analysis. The homozygous deletion mutant, spt3::URA3/spt3::ARG4 (hereafter referred to as spt3/spt3), has several obvious growth phenotypes: (1) slow growth compared to wild-type strains in both liquid and solid media (Fig 4B); (2) flocculence in liquid media; and (3) a wrinkled colony morphology on YPD, SD, and SC solid media.

During purification of different initial isolates of spt3/spt3 mutants, we noted that the growth and other mutant phenotypes varied over a moderate range. Fourteen spt3/spt3 isolates that fell throughout this range of phenotypes were studied in greater detail. These isolates define three phenotypic classes (classes 1, 2, and 3). Class 1 mutants (seven isolates) grew as uniformly small, wrinkled colonies on YPD plates; class 2 mutants (four isolates) were more variable, with mostly small colonies, but also some larger smooth colonies; and class 3 mutants (three isolates) gave rise to a larger proportion of large, smooth colonies. As described below, these classes also vary with respect to filamentous growth, and the colony morphologies depended upon the auxotrophies in the strains. The different classes appear to have arisen during the strain constructions and their phenotypes remained stable during purification. Furthermore, these three classes were repeatedly observed when reconstructing the spt3/spt3 homozygous mutant. The different classes might be caused by second-site mutations that modify the severity of the spt3/spt3-conferred growth defect. To confirm that the phenotypes observed in all three classes are caused by the spt3/spt3 deletions, we integrated a wild-type copy of SPT3 at the HIS1 locus in a representative of each class. These transformants all have a wild-type growth rate and colony morphology. They are also wild-type with respect to most of the filamentous phenotypes, as described below. The only phenotype observed was a slightly larger cell size compared to the wild-type strain, a property we also saw for the SPT3/spt3 heterozygote (Fig 5B). Thus, the phenotypes observed in these spt3/spt3 mutants are caused by loss of Spt3 function.

View larger version (85K): [in this window] [in a new window] [Download PPT slide]   Figure 5. C. albicans spt3 mutants are constitutive for filamentous growth. Column 1 shows a wild-type strain (SC5314); column 2 shows an spt3/SPT3+ heterozygous strain (FWC16); column 3 shows an spt3 class 1 mutant (FWC15); column 4 shows an spt3 class 2 mutant (FWC13); column 5 shows an spt3 class 3 mutant (FWC11); column 6 shows an spt3 class 1 mutant with a copy of SPT3 integrated at HIS1 (FWC10; rescue strain). (A) Colonies grown on YPD plates. The strains were incubated at 30° for 3 days and photographed using a x4 objective. (B) Individual cells from a colony grown on a YPD plate at 30° for 3 days were resuspended in water and photographed using a x40 DIC objective. (C) Colonies grown on medium 199 plates. The colonies were streaked from the permanent frozen stock and incubated at 37° for 1 day and then photographed using a x20 DIC objective. (D) Individual cells from a colony grown on a medium 199 plate at 37° for 1 day were resuspended in water. The cells were then visualized using a x40 DIC objective.

To test whether C. albicans spt3/spt3 mutants have defects in filamentous growth, we first tested them under conditions noninducing for filamentous growth, on YPD solid media at 30°. For this analysis, we compared strains that were prototrophic, to avoid any possible effects of auxotrophies on growth. When the three classes of spt3/spt3 mutants were in a prototrophic background, we no longer saw the colony size heterogenity that was observed for the original isolates. However, differences in the degree of filamentous growth were still observed, as described below. Under these growth conditions, wild-type C. albicans formed smooth and round colonies (Fig 5A) that contained all budding yeast (Fig 5B). In strong contrast, the spt3/spt3 mutants formed wrinkled, irregularly shaped colonies that contained filaments (Fig 5A and Fig B). The spt3/spt3 class 1 mutants formed colonies that were composed almost entirely of filamentously growing cells. The class 2 and class 3 mutants also formed wrinkled colonies. In these cases, the colonies contained some filaments, but had a greater proportion of budding yeast. The filamentous forms observed in these experiments consisted of both hyphae and pseudohyphae. All of the hyperfilamentous phenotypes observed in the spt3/spt3 mutants on YPD were complemented by the introduction of a copy of wild-type SPT3 (Fig 5A and Fig B; see column labeled "rescue"). Thus, although we observed a range in the severity of the hyperfilamentous phenotype, we conclude that the loss of Spt3 function in C. albicans results in filamentous growth under noninducing conditions.

We also examined filamentous growth of wild-type and spt3/spt3 strains on M199 media, which induces wild-type C. albicans filamentous growth (SAPORITO-IRWIN et al. 1995 ). After one day of incubation at 37°, the wild-type colonies were smooth (Fig 5C) and contained cells that were mostly in the budding yeast form, with some germ tubes beginning to form (Fig 5D). After 2 days, more germ tubes began to appear and true hyphae appeared by 3–4 days of growth (data not shown). To test the spt3/spt3 mutants, cells were plated from YPD-grown cultures that consisted of a mix of budding and hyphal forms for all three classes. In contrast to the wild-type strain, the spt3/spt3 class 1 mutant exhibited entirely hyphal growth after only 1 day of incubation (Fig 5C and Fig D). The spt3/spt3 class 2 and 3 mutants exhibited a more intermediate mixture of cell types, containing both filamentous and budding forms. When a copy of wild-type SPT3 was integrated into representatives of the three classes of spt3/spt3 mutants, we observed complementation of the hyperfilamentous growth (Fig 5D). In conclusion, on inducing media, spt3/spt3 mutants also exhibit hyperfilamentous growth.

C. albicans spt3/spt3 mutants are avirulent in mice:
Previous studies of C. albicans mutants that are either nonfilamentous or hyperfilamentous have shown that they are avirulent (BRAUN et al. 2001 ; KADOSH and JOHNSON 2001 ; MURAD et al. 2001 ; NAVARRO-GARCIA et al. 2001 ). To test the virulence of C. albicans spt3/spt3 mutants, we used them to infect mice and monitored mouse survival over a period of 30 days. In these experiments, we tested the three classes of spt3/spt3 mutants. We also tested each of these mutants containing a wild-type copy of SPT3 integrated at HIS1. For each infection, 5 x 10⁶ cells were injected in the tail veins of BALB/cJ mice. Four mice were tested for each yeast strain. Our results (Fig 6) demonstrate that all three classes of spt3/spt3 mutants are avirulent as all of the mice infected with the three classes of spt3/spt3 mutants remained viable and healthy over the 30 days. In contrast, infection with the wild-type C. albicans strain resulted in the death of all four mice within 2 days. The three classes of spt3/spt3 mutants that also contained a single copy of wild-type SPT3 integrated at HIS1 displayed partial complementation with an intermediate level of virulence, suggesting that virulence is sensitive to the level of Spt3. Taken together, these results demonstrate that virulence of C. albicans is dependent upon Spt3.

View larger version (24K): [in this window] [in a new window] [Download PPT slide]   Figure 6. C. albicans spt3 mutants are avirulent. Shown are survival plots over 30 days for 6-week-old BALB/cJ mice infected with 5 x 106 C. albicans cells. The strains used are as follows: (A) wild type, SC5314; cph1 efg1, HLC54; spt3 class 1 mutant, FWC15; and spt3 class 1 mutant rescue (with SPT3+ at HIS1), FWC10; (B) wild type, SC5314; cph1 efg1, HLC54; spt3 class 2 mutant, FWC13; and spt3 class 2 mutant rescue (with SPT3+ at HIS1), FWC14; (C) wild type, SC5314; cph1 efg1, HLC54; spt3 class 3 mutant, FWC11; and spt3 class 3 mutant rescue (with SPT3+ at HIS1), FWC12.

	DISCUSSION

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

Our results show that Spt3 plays critical, but opposite roles in filamentous growth in both S. cerevisiae and C. albicans. In S. cerevisiae, an spt3 mutation causes severe defects in both pseudohyphal and invasive growth, suggesting that Spt3 plays a positive role in filamentous growth. In contrast, in C. albicans, an spt3/spt3 mutation causes hyperfilamentous growth, strongly suggesting that Spt3 plays a negative role in filamentous growth. Furthermore, a C. albicans spt3/spt3 mutant is avirulent in mice. Since the C. albicans SPT3 gene can fully complement all tested S. cerevisiae spt3 mutant phenotypes, including the filamentous growth defect, we conclude that the opposite roles of Spt3 between the two yeasts are not caused by differences between the genes themselves, but rather by differences between the S. cerevisiae and C. albicans regulatory systems that control filamentous growth. The apparent negative role of Spt3 in C. albicans filamentous growth is consistent with evidence that Spt3 plays both positive and negative roles in transcription in S. cerevisiae (BELOTSERKOVSKAYA et al. 2000 ; LEE et al. 2000 ).

Our results suggest that an S. cerevisiae spt3 mutant has multiple transcriptional defects that impair filamentous growth. In this work, we have shown that Spt3 is required for normal levels of FLO11 mRNA. Possibly, a direct activator of FLO11 recruits Spt3, as part of the SAGA complex, to the FLO11 promoter to allow Spt3-dependent activation. Such an activity for Spt3 has been demonstrated at the GAL1 promoter (DUDLEY et al. 1999 ; BHAUMIK and GREEN 2001 ; LARSCHAN and WINSTON 2001 ). Consistent with the idea that Spt3 acts directly at the FLO11 promoter is genome-wide expression analysis that has shown that mRNA levels for three known regulators of FLO11 transcription, TEC1, STE12, and FLO8 (RUPP et al. 1999 ), are not significantly altered in an spt3 mutant (LEE et al. 2000 ). However, the spt3 effect on FLO11 mRNA levels cannot be the only defect impairing filamentous growth, as ectopic expression of FLO11 does not suppress the filamentous defect in spt3/spt3 mutants. Genome-wide expression analysis of spt3 mutants grown in media that induces pseudohyphal growth will help to identify the extent of the requirement for Spt3 during filamentous growth.

Current evidence suggests that C. albicans Spt3 likely functions as the part of a protein complex in C. albicans that is similar to the SAGA complex of S. cerevisiae. Previous studies have shown that Spt3 and SAGA are conserved throughout eukaryotes (MADISON and WINSTON 1998 ; OGRYZKO et al. 1998 ; YU et al. 1998 ; MARTINEZ et al. 2001 ). Furthermore, the C. albicans SPT3 gene fully complements an S. cerevisiae spt3 mutation, demonstrating strong functional conservation. In addition, sequences homologous to the SAGA genes SPT7, SPT8, SPT20, ADA2, and GCN5 have also been found in the partially completed C. albicans genome sequence (http://genolist.pasteur.fr/CandidaDB/). Therefore, it seems likely that a C. albicans SAGA complex plays critical roles in transcription and filamentous growth. Since at least one SAGA member, Spt7, is only weakly conserved between yeast and humans (MARTINEZ et al. 2001 ), a C. albicans SAGA complex may be a valuable drug target for impairing C. albicans growth and virulence.

One complication in our studies of C. albicans spt3/spt3 mutants was that we observed three phenotypic classes that exhibited different degrees of hyperfilamentous growth. These different phenotypes may be caused by a second mutation in another gene that partially suppresses the spt3/spt3 mutant phenotype. On the basis of studies in S. cerevisiae, the strongest candidates for such a gene are SPT15 and SPT8, as mutations in these genes have been shown to have allele-specific interactions with mutations in SPT3 (EISENMANN et al. 1992 , EISENMANN et al. 1994 ). Unfortunately, the inability to perform standard genetic analysis in C. albicans makes this hypothesis difficult to test.

Several different types of genes have been shown to be required for C. albicans virulence, including transcription factors that play positive and negative roles in controlling filamentous growth (NAVARRO-GARCIA et al. 2001 ). In addition to Spt3, three other negative regulators of filamentous growth, Tup1, Rgt1, and Nrg1, have been previously identified and shown to be required for virulence (BRAUN and JOHNSON 2000 ; BRAUN et al. 2001 ; KADOSH and JOHNSON 2001 ; KHALAF and ZITOMER 2001 ; MURAD et al. 2001 ). At least two of these factors, Tup1 and Rfg1, play a positive role in S. cerevisiae filamentous growth and a negative role in C. albicans filamentous growth (BRAUN and JOHNSON 1997 ; KADOSH and JOHNSON 2001 ), similar to Spt3. However, in S. cerevisiae the regulatory roles of Spt3 and the SAGA complex are distinct from Tup1, Rox1, and Nrg1 (DERISI et al. 1997 ; LEE et al. 2000 ), suggesting that Spt3 defines an independent regulatory pathway in C. albicans filamentous growth. Furthermore, the poor growth of C. albicans spt3/spt3 mutants suggests that Spt3 plays an important role in controlling gene expression in C. albicans. Genome-wide expression analysis of C. albicans spt3/spt3 mutants should help to elucidate further the role of Spt3 in C. albicans transcription and filamentous growth.

	FOOTNOTES

Sequence data from this article have been deposited with the EMBL/GenBank Data Libraries under accession no. AF142757.

	ACKNOWLEDGMENTS

We are grateful to Gerald Fink, William Fonzi, Steve Hanes, Julie Kohler, Aaron Mitchell, Shannon Roberts, Kevin Struhl, and Cora Styles for strains and advice. We thank Aimée Dudley and Andrea Duina for very helpful comments on the manuscript and Sheldon Rowan for help with the microscopy. This work was supported by National Institutes of Health grant GM-45720 to F.W. W.F.D. is an Assistant Investigator of the Howard Hughes Medical Institute. V.L.B. is a fellow of the Irvington Institute for Immunological Research.

Manuscript received December 6, 2001; Accepted for publication February 26, 2002.

	LITERATURE CITED

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

AMBERG, D. C., D. BOTSTEIN, and E. M. BEASLEY, 1995  Precise gene disruption in Saccharomyces cerevisiae by double fusion polymerase chain reaction. Yeast 11:1275-1280[Medline].

AUSUBEL, F. M., R. BRENT, R. E. KINGSTON, D. D. MOORE, J. G. SEIDMAN et al., 1988 Current Protocols in Molecular Biology. Greene Publishing Associates/Wiley-Interscience, New York.

BELOTSERKOVSKAYA, R., D. E. STERNER, M. DENG, M. H. SAYRE, and P. M. LIEBERMAN et al., 2000  Inhibition of TATA-binding protein function by SAGA subunits Spt3 and Spt8 at Gcn4-activated promoters. Mol. Cell. Biol. 20:634-647[Abstract/Free Full Text].

BHAUMIK, S. R. and M. R. GREEN, 2001  SAGA is an essential in vivo target of the yeast acidic activator Gal4p. Genes Dev. 15:1935-1945[Abstract/Free Full Text].

BIRCK, C., O. POCH, C. ROMIER, M. RUFF, and G. MENGUS et al., 1998  Human TAF(II)28 and TAF(II)18 interact through a histone fold encoded by atypical evolutionary conserved motifs also found in the SPT3 family. Cell 94:239-249[Medline].

BRAUN, B. R. and A. D. JOHNSON, 1997  Control of filament formation in Candida albicans by the transcriptional repressor TUP1. Science 277:105-109[Abstract/Free Full Text].

BRAUN, B. R. and A. D. JOHNSON, 2000  TUP1, CPH1 and EFG1 make independent contributions to filamentation in Candida albicans.. Genetics 155:57-67[Abstract/Free Full Text].

BRAUN, B. R., D. KADOSH, and A. D. JOHNSON, 2001  NRG1, a repressor of filamentous growth in C. albicans, is down-regulated during filament induction. EMBO J. 20:4753-4761[Medline].

BROWN, A. J. and N. A. GOW, 1999  Regulatory networks controlling Candida albicans morphogenesis. Trends Microbiol. 7:333-338[Medline].

BROWN, C. E., L. HOWE, K. SOUSA, S. C. ALLEY, and M. J. CARROZZA et al., 2001  Recruitment of HAT complexes by direct activator interactions with the ATM-related Tra1 subunit. Science 292:2333-2337[Abstract/Free Full Text].

BROWNELL, J. E., J. ZHOU, T. RANALLI, R. KOBAYASHI, and D. G. EDMONDSON et al., 1996  Tetrahymena histone acetyltransferase A: a homolog to yeast Gcn5p linking histone acetylation to gene activation. Cell 84:843-851[Medline].

BUTT, T. R., E. J. STERNBERG, J. A. GORMAN, P. CLARK, and D. HAMER et al., 1984  Copper metallothionein of yeast, structure of the gene, and regulation of expression. Proc. Natl. Acad. Sci. USA 81:3332-3336[Abstract/Free Full Text].

CONTE, D., JR. and M. J. CURCIO, 2000  Fus3 controls Ty1 transpositional dormancy through the invasive growth MAPK pathway. Mol. Microbiol. 35:415-427[Medline].

DERISI, J. L., V. R. IYER, and P. O. BROWN, 1997  Exploring the metabolic and genetic control of gene expression on a genomic scale. Science 278:680-686[Abstract/Free Full Text].

DUDLEY, A. M., C. ROUGEULLE, and F. WINSTON, 1999  The Spt components of SAGA facilitate TBP binding to a promoter at a post-activator-binding step in vivo. Genes Dev. 13:2940-2945[Abstract/Free Full Text].

EISENMANN, D. M., K. M. ARNDT, S. L. RICUPERO, J. W. ROONEY, and F. WINSTON, 1992  SPT3 interacts with TFIID to allow normal transcription in Saccharomyces cerevisiae. Genes Dev. 6:1319-1331[Abstract/Free Full Text].

EISENMANN, D. M., C. CHAPON, S. M. ROBERTS, C. DOLLARD, and F. WINSTON, 1994  The S. cerevisiae SPT8 gene encodes a very acidic protein that is functionally related to SPT3 and TATA-binding protein. Genetics 137:647-657[Abstract].

FIDEL, P. L., JR. and J. D. SOBEL, 1996  Immunopathogenesis of recurrent vulvovaginal candidiasis. Clin. Microbiol. Rev. 9:335-348[Abstract].

GANCEDO, J. M., 2001  Control of pseudohyphae formation in Saccharomyces cerevisiae.. FEMS Microbiol. Rev. 25:107-123[Medline].

GANSHEROFF, L. J., C. DOLLARD, P. TAN, and F. WINSTON, 1995  The Saccharomyces cerevisiae SPT7 gene encodes a very acidic protein important for transcription in vivo. Genetics 139:523-536[Abstract].

GILLUM, A. M., E. Y. TSAY, and D. R. KIRSCH, 1984  Isolation of the Candida albicans gene for orotidine-5'-phosphate decarboxylase by complementation of S. cerevisiae ura3 and E. coli pyrF mutations. Mol. Gen. Genet. 198:179-182[Medline].

GIMENO, C. J., P. O. LJUNGDAHL, C. A. STYLES, and G. R. FINK, 1992  Unipolar cell divisions in the yeast S. cerevisiae lead to filamentous growth: regulation by starvation and RAS. Cell 68:1077-1090[Medline].

GRANT, P. A., L. DUGGAN, J. COTE, S. M. ROBERTS, and J. E. BROWNELL et al., 1997  Yeast Gcn5 functions in two multisubunit complexes to acetylate nucleosomal histones: characterization of an Ada complex and the SAGA (Spt/Ada) complex. Genes Dev. 11:1640-1650[Abstract/Free Full Text].

GRANT, P. A., D. SCHIELTZ, M. G. PRAY-GRANT, J. R. R. YATES, and J. L. WORKMAN, 1998a  The ATM-related cofactor Tra1 is a component of the purified SAGA complex. Mol. Cell 2:863-867[Medline].

GRANT, P. A., D. E. STERNER, L. J. DUGGAN, J. L. WORKMAN, and S. L. BERGER, 1998b  The SAGA unfolds: convergence of transcription regulators in chromatin-modifying complexes. Trends Cell Biol. 8:193-197[Medline].

GUTHRIE, C., 1991 Guide to Yeast Genetics and Molecular Biology. Academic Press, San Diego.

HAPPEL, A. M., 1989 Analysis of mutations that affect Ty transcription in yeast. Ph.D. Thesis, Harvard University, Cambridge, MA.

HIRSCHHORN, J. N. and F. WINSTON, 1988  SPT3 is required for normal levels of a-factor and alpha-factor expression in Saccharomyces cerevisiae.. Mol. Cell. Biol. 8:822-827[Abstract/Free Full Text].

HOLLENHORST, P. C., M. E. BOSE, M. R. MIELKE, U. MULLER, and C. A. FOX, 2000  Forkhead genes in transcriptional silencing, cell morphology and the cell cycle: overlapping and distinct functions for FKH1 and FKH2 in Saccharomyces cerevisiae.. Genetics 154:1533-1548[Abstract/Free Full Text].

HORIUCHI, J., N. SILVERMAN, B. PINA, G. A. MARCUS, and L. GUARENTE, 1997  ADA1, a novel component of the ADA/GCN5 complex, has broader effects than GCN5, ADA2, or ADA3. Mol. Cell. Biol. 17:3220-3228[Abstract].

HULL, C. M. and A. D. JOHNSON, 1999  Identification of a mating type-like locus in the asexual pathogenic yeast Candida albicans.. Science 285:1271-1275[Abstract/Free Full Text].

KADOSH, D. and A. D. JOHNSON, 2001  Rfg1, a protein related to the Saccharomyces cerevisiae hypoxic regulator Rox1, controls filamentous growth and virulence in Candida albicans.. Mol. Cell. Biol. 21:2496-2505[Abstract/Free Full Text].

KHALAF, R. A. and R. S. ZITOMER, 2001  The DNA binding protein Rfg1 is a repressor of filamentation in Candida albicans.. Genetics 157:1503-1512[Abstract/Free Full Text].

KOBAYASHI, S. D. and J. E. CUTLER, 1998  Candida albicans hyphal formation and virulence: is there a clearly defined role? Trends Microbiol. 6:92-94[Medline].

LARSCHAN, E. and F. WINSTON, 2001  The S. cerevisiae SAGA complex functions in vivo as a coactivator for transcriptional activation by Gal4. Genes Dev. 15:1946-1956[Abstract/Free Full Text].

LEE, T. I., H. C. CAUSTON, F. C. HOLSTEGE, W. C. SHEN, and N. HANNETT et al., 2000  Redundant roles for the TFIID and SAGA complexes in global transcription. Nature 405:701-704[Medline].

LIU, H., C. A. STYLES, and G. R. FINK, 1993  Elements of the yeast pheromone response pathway required for filamentous growth of diploids. Science 262:1741-1744[Abstract/Free Full Text].

LO, H. J., J. R. KOHLER, B. DIDOMENICO, D. LOEBENBERG, and A. CACCIAPUOTI et al., 1997  Nonfilamentous C. albicans mutants are avirulent. Cell 90:939-949[Medline].

LO, W. S. and A. M. DRANGINIS, 1996  FLO11, a yeast gene related to the STA genes, encodes a novel cell surface flocculin. J. Bacteriol. 178:7144-7151[Abstract/Free Full Text].

LO, W. S. and A. M. DRANGINIS, 1998  The cell surface flocculin Flo11 is required for pseudohyphae formation and invasion by Saccharomyces cerevisiae.. Mol. Biol. Cell 9:161-171[Abstract/Free Full Text].

MADHANI, H. D., C. A. STYLES, and G. R. FINK, 1997  MAP kinases with distinct inhibitory functions impart signaling specificity during yeast differentiation. Cell 91:673-684[Medline].

MADISON, J. M. and F. WINSTON, 1998  Identification and analysis of homologues of Saccharomyces cerevisiae Spt3 suggest conserved functional domains. Yeast 14:409-417[Medline].

MARTINEZ, E., T. K. KUNDU, J. FU, and R. G. ROEDER, 1998  A human SPT3-TAFII31-GCN5-L acetylase complex distinct from transcription factor IID. J. Biol. Chem. 273:23781-23785[Abstract/Free Full Text].

MARTINEZ, E., V. B. PALHAN, A. TJERNBERG, E. S. LYMAR, and A. M. GAMPER et al., 2001  Human STAGA complex is a chromatin-acetylating transcription coactivator that interacts with pre-mRNA splicing and DNA damage-binding factors in vivo. Mol. Cell. Biol. 21:6782-6795[Abstract/Free Full Text].

MITCHELL, A. P., 1998  Dimorphism and virulence in Candida albicans.. Curr. Opin. Microbiol. 1:687-692[Medline].

MURAD, A. M., P. LENG, M. STRAFFON, J. WISHART, and S. MACASKILL et al., 2001  NRG1 represses yeast-hypha morphogenesis and hypha-specific gene expression in Candida albicans.. EMBO J. 20:4742-4752[Medline].

NAVARRO-GARCIA, F., R. M. PEREZ-DIAZ, A. I. NEGREDO, J. PLA, and C. NOMBELA, 1998  Cloning and sequence of a 3.835 kbp DNA fragment containing the HIS4 gene and a fragment of a PEX5-like gene from Candida albicans.. Yeast 14:1147-1157[Medline].

NAVARRO-GARCIA, F., M. SANCHEZ, C. NOMBELA, and J. PLA, 2001  Virulence genes in the pathogenic yeast Candida albicans. FEMS Microbiol. Rev. 25:245-268[Medline].

ODDS, F. C., 1988 Candida and Candidosis. Bailliere Tindall, London.

OGRYZKO, V. V., T. KOTANI, X. ZHANG, R. L. SCHLITZ, and T. HOWARD et al., 1998  Histone-like TAFs within the PCAF histone acetylase complex. Cell 94:35-44[Medline].

PAN, X., T. HARASHIMA, and J. HEITMAN, 2000  Signal transduction cascades regulating pseudohyphal differentiation of Saccharomyces cerevisiae.. Curr. Opin. Microbiol. 3:567-572[Medline].

ROBERTS, R. L. and G. R. FINK, 1994  Elements of a single MAP kinase cascade in Saccharomyces cerevisiae mediate two developmental programs in the same cell type: mating and invasive growth. Genes Dev. 8:2974-2985[Abstract/Free Full Text].

ROBERTS, S. M. and F. WINSTON, 1996  SPT20/ADA5 encodes a novel protein functionally related to the TATA-binding protein and important for transcription in Saccharomyces cerevisiae.. Mol. Cell. Biol. 16:3206-3213[Abstract].

ROBERTS, S. M. and F. WINSTON, 1997  Essential functional interactions of SAGA, a Saccharomyces cerevisiae complex of Spt, Ada, and Gcn5 proteins, with the Snf/Swi and Srb/mediator complexes. Genetics 147:451-465[Abstract].

ROSE, M. D., F. WINSTON and P. HIETER