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Department of Molecular Biology and Microbiology, and Genetics Program, Tufts University School of Medicine, 136 Harrison Avenue, Boston, MA 02111, USA
Correspondence
Carol A. Kumamoto
carol.kumamoto{at}tufts.edu
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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Present address: Department of Pediatrics, Stanford University School of Medicine, Palo Alto, CA 94305, USA.
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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During infection, C. albicans grows invasively into the tissues of its host. C. albicans is a dimorphic fungus and, during invasive growth, typically assumes a filamentous morphology. Under other conditions, C. albicans is found in a yeast form. The ability to convert from yeast-form cells to filamentous hyphae (elongated cells lacking constrictions at the septa) and pseudohyphae (elongated cells with constrictions at the septa) is believed to be important for invasiveness and virulence (reviewed by Kumamoto & Vinces, 2005b
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To probe the mechanisms that allow C. albicans to invade host tissue, laboratory studies have made use of C. albicans invasion into agar matrices. Several proteins, including signal transduction proteins and transcription factors, which regulate invasive growth into agar have been previously described (reviewed by Kumamoto & Vinces, 2005a). The bHLH transcription factor Efg1p is a negative regulator of filamentation when cells are embedded in an agar matrix (Giusani et al., 2002), even though Efg1p has a critical positive role in filamentation under most other conditions (Stoldt et al., 1997).
In this study, a search for C. albicans genes that are important for invasive filamentation in agar was undertaken. Mutations affecting the gene, orf19.5083, were found to result in reduced invasiveness. This gene is highly homologous to the RBG1 (ribosome-interacting GTPase)/FUN11 (function unknown now) gene of Saccharomyces cerevisiae, and to the gene encoding DRG1 (developmentally regulated GTP-binding protein) of Xenopus laevis (Kumar et al., 1993) and other species. The C. albicans gene will be referred to as DRG1 in this paper.
Previous studies have suggested that the function of DRG1 may be related to growth control. For example, ectopic expression of mouse DRG1 enhances oncogenic transformation by c-myc and ras (Mahajan et al., 1996). Mouse DRG1 interacts with an oncogene for stem cell acute lymphoblastic leukaemia, TAL1/SCL1 (Mahajan et al., 1996). TAL1/SCL1 encodes a bHLH transcription factor whose HLH domain interacts with the C-terminal region of DRG1. Thus, DRG1 may influence growth control by binding and regulating transcription factors. Expression of DRG1 also correlates with growth; DRG1 is highly expressed during embryonic development in Xenopus (Kumar et al., 1993) and mice (Sazuka et al., 1992), and is expressed at higher levels in growing versus non-growing plant cells (Devitt et al., 1999). Despite these clues, the function of DRG1 is not well understood in any species.
In this study, we showed that a C. albicans drg1 mutant was attenuated in invasiveness in an agar matrix, but was capable of hyphal growth under other conditions. In addition, the drg1 null mutant exhibited delayed invasiveness in host tissue, and delayed lethality during infection. As invasive growth is a contact-dependent response, these findings suggest that Drg1p functions in contact-dependent signalling or responses.
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METHODS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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ura3 : : imm434/ura3 : : imm434) (Fonzi & Irwin, 1993) was used to construct all C. albicans strains (listed in Table 1).
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), plasmids pXC110 and pXC111, encoding 5' and 3' fragments of the DRG1 locus flanking hisG–URA3–hisG, were digested with KpnI and HindIII. Strains were characterized by Southern blotting to verify that all alleles of DRG1 were deleted. For complementation analysis, Ura– derivatives of drg1 null mutants were integratively transformed with BsaBI-digested pXC155. For cloning, Escherichia coli strains XL-1 Blue or JM109 were used.
Media and growth conditions.
For culturing C. albicans, standard media included YP (1 % yeast extract and 2 % bacto peptone) with either 2 % glucose (YPD) or 2 % sucrose (YPS), or CM-Ura (complete minimal medium lacking uridine and uracil) (Ausubel et al., 1989). For solid media, 2 % agar was added. For selection of Ura– strains, CM-Ura medium was supplemented with 60 µg uridine ml–1 and 1 mg 5-fluoroorotic acid ml–1 (Kelly et al., 1987). For the colony morphology assay, YPS medium containing 1 % agar (Difco) was used (Brown et al., 1999). Synthetic low ammonium, Lee's, proline and GlcNAc media were prepared as described by Brown et al. (1999).
E. coli strains were cultured in L broth or on L plates (Miller, 1972), with ampicillin added to a concentration of 100 µg ml–1.
Construction of plasmids.
Synthetic oligonucleotides are described in Table 2. pXC110 and pXC111 were constructed from p510 (http://www.sacs.ucsf.edu/home/JohnsonLab/burk/Candisrupt.html) (kind gift of B. Braun and A. Johnson, University of California, San Francisco). For pXC110, the 5' DRG1 fragment was amplified from chromosomal DNA with primers XC-01 and XC-02. The DRG 3' fragment was amplified with primers XC-03 and XC-04. pXC111, used to delete the last allele of DRG1, contained DRG1 sequences that were within the sequences deleted by pXC110. For construction of pXC111, a DRG1 5' fragment was amplified using primers XC-05 and XC-06, and a 3' fragment was amplified using primers XC-07 and XC-08.
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) as a template. These two PCR fragments were mixed and amplified with primers XC-11 and XC-14, generating a fragment that contained part of the DRG1 promoter, 13Myc, and part of the DRG1 ORF. This fragment was digested with NheI and BtgI, and ligated into NheI/BtgI-digested pXC155 (DRG1+), generating pXC156. For transformation, pXC156 was digested with BsaBI.
lexA–DRG1 was constructed by PCR amplification of the DRG1 gene with primers XC-15 and XC-16, and cloning of the PCR product into vector pEG202 (Golemis et al., 1996). DRG1–lexA was constructed by PCR amplification of the DRG1 gene with primers XC-17 and XC-16, and cloning of the PCR product into vector pNLexA (Golemis et al., 1996). GAL4AD–EFG1 was constructed by PCR amplification of EFG1 with primers XC-18 and XC-19, and cloning of the PCR product into vector pGAD-C2 (Giusani et al., 2002; James et al., 1996). plexA-Fos and pC1.1.2, encoding GAL4AD–SLK19 (control fusion proteins that are not expected to interact with either Efg1p or Drg1p), were generously provided by R. Brent (Massachusetts General Hospital, Boston) and R. Kamieniecki (Tufts University, Boston).
pXC132, containing BamHI, NsiI, SalI and EcoRV sites upstream of a promoterless yEGFP gene that was fused to the 3' UTR of ACT1 in a pNUB1 backbone, was used for insertional mutagenesis.
DNA isolation and analysis.
PCR, restriction digestion and gel electrophoresis were performed by standard methods, as described elsewhere (Sambrook et al., 1989). Automated DNA sequencing was performed by Michael Berne and coworkers at the Tufts University Core Facility. Chromosomal DNA isolation and Southern blot hybridization were performed by standard methods, as described previously (Burke et al., 2000; Sambrook et al., 1989). DNA probes were labelled with [
-32P]dATP by random priming.
To demonstrate that the DRG1 gene had been deleted, a probe corresponding to a sequence upstream of the DRG1 ORF was generated by PCR using primers XC-20 and XC-21, and was used to probe a Southern blot of MslI-digested chromosomal DNA from candidate strains. To verify that all copies of DRG1 had been deleted, a second probe, corresponding to sequences within the DRG1 ORF, was generated by PCR using primers XC-22 and XC-23, and used to probe the same blot.
For inverse PCR, genomic DNA from strain XC210 was digested with RsaI, HaeI or AluI separately. Following ligation, the circular DNA products were used as templates for PCRs using primers XC-24 and XC-25 (homologous to sequences within pXC132). The PCR products were purified from a 1 % agarose gel, and the DNA was sequenced using the primers described above.
Electroporation and restriction-enzyme-mediated integration (REMI) of C. albicans.
The method used was that described by Brown et al. (1996), with minor modifications. Cells were grown to OD600 
0.5–0.6, harvested, and treated with 0.1 M lithium acetate, 0.01 M Tris/HCl, pH 7.5, and 1 mM EDTA for 45 min at 30 °C. DTT was added to a concentration of 2 mM, and the cells were incubated for an additional 15 min at 30 °C. After washing with cold water, and with cold 10 % (v/v) glycerol, cells were resuspended in a small volume of ice-cold 10 % glycerol. For REMI, 20 µg plasmid DNA was digested with BamHI and then precipitated using ethanol. One hundred units of BamHI mixed with the digested DNA were added to 40 µl concentrated strain CAI-4 cells kept on ice. The mixture of cells and DNA was electroporated at 1.5 kV, 200 
and 25 µF, and plated on CM-Ura. For homologous recombination, 10 µg digested plasmid DNA was added to 40 µl concentrated cells, and electroporated as above.
Analysis of filamentation.
For analysis of filamentation under embedded conditions, actively growing cells in YPD medium (400 c.f.u.) were mixed with molten YPS agar, and plated. Plates were incubated for several days. At various times, 100–200 embedded colonies were examined, and the percentage of filamentous colonies was determined (a colony that contained at least 20 filaments protruding from the periphery of the colony was arbitrarily defined as a filamentous colony). Colonies were photographed with a Nikon E400 microscope at x4 magnification.
To study filamentous growth in 10 % serum and Lee's, proline and GlcNAc media, strains were grown in CM-Ura liquid medium at room temperature, washed, starved in water, and added to each medium. After incubation at 37 °C for 6 h, cells were observed microscopically, and the percentage of filamentous cells was determined.
Protein localization.
Immunofluorescence methods were based on standard methods used for S. cerevisiae (Pringle et al., 1991), with modifications. Briefly, C. albicans cells expressing N-terminally myc-tagged Drg1p were grown in liquid medium, or were embedded in agarose. Cells were fixed in formaldehyde (4.4 %) for 2 h. After fixation, embedded cells were released from the agarose by crushing. After zymolyase treatment, cells were added to wells of polylysine-coated slides, and processed for immunofluorescence with anti-myc antibody clone 4A6 (Upstate), using standard methods. Cells were stained with 4',6-diamidino-2-phenylindole (DAPI), observed by fluorescence microscopy, and photographed using the x60 objective.
Intravenous inoculation of mice.
C. albicans cells were grown for 24 h, and harvested by centrifugation. The cells were washed three times with PBS, counted, and adjusted to a density of 5x106 cells ml–1; and 0.2 ml of the cell suspension was injected into the lateral tail vein of female CF1 mice (18–20 g body weight; Charles River Laboratories). In each experiment, four mice were inoculated per strain, and this experiment was repeated three times. Mice were observed twice daily after infection with C. albicans. In two experiments, some animals were sacrificed, and their kidneys were dissected, fixed in buffered formalin, sectioned, and stained with haematoxylin and eosin or Gomori methenamine silver.
Two-hybrid analysis.
S. cerevisiae strain EGY40 carrying the lacZ reporter plasmid pSH18-34 (Golemis et al., 1996) was transformed with the lexA plasmids pEG202, plexA-DRG1, pNLexA, pDRG1-lexA and plexA-Fos, and GAL4AD plasmids pGAD-C2, pKLEF4 and pC1.1.2, using standard methods (Ausubel et al., 1989). For assays of 
-galactosidase activity, cells grown to exponential phase in synthetic complete medium lacking uracil, histidine and leucine were harvested, and -galactosidase activity was determined using ONPG as a substrate, as described elsewhere (Clontech, 2001). Analyses were performed in triplicate.
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RESULTS |
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Following embedding in YPS agar, and growth at 25 °C, the mutant strains XC210 and XC211 were strongly defective in filamentous growth, in comparison to wild-type strain CKY101 (CAI-4, Ura+) (data not shown). Despite the strong defect in filamentous growth under matrix-embedded conditions, no obvious defect in growth rate was observed when strains XC210, XC211 and CKY101 were grown in YPD liquid medium, and the defect in filamentous growth was not observed when strains XC210 and XC211 were grown in 10 % serum, or in Lee's, proline or GlcNac medium at 37 °C (data not shown). Together, these results suggest that the gene disrupted by insertion of pXC132 in strains XC210 and XC211 plays a role in filamentous growth specifically under matrix-embedded conditions.
Strain XC210 contains an insertion in the DRG1 locus
To find the location of the insertion site in strain XC210, inverse PCR and DNA sequencing were performed, as described in Methods. The inserted DNA construct was found to be located 80 bp upstream of the ORF orf19.5083. This ORF is very similar to those termed DRG1 in other eukaryotic species; therefore, we termed the C. albicans gene DRG1.
In C. albicans, DRG1 encoded a protein of 368 aa with characteristic G1–G5 motifs of GTP-binding proteins (Fig. 1). Outside of these motifs, Drg1p is not very similar to the existing subfamilies of GTP-binding proteins, such as heterotrimeric G proteins or ras-like G proteins. As suggested by Sazuka et al. (1992), Drg1p represents a novel class of GTP-binding proteins.
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). The starting strain, CAI-4, was trisomic for chromosome 1, and DRG1 mapped to chromosome 1. Therefore, the initial heterozygous mutants contained one deleted copy of DRG1 and two intact copies (e.g. XC301 and XC302; Fig. 2). The second-round heterozygous mutants, e.g. XC303 and XC304, contained two deleted copies of DRG1 and one intact copy (Fig. 2). Both types of heterozygous mutants had no obvious defect in filamentous growth when they were grown embedded in YPS agar at 25 °C (Fig. 3). Since the original insertion mutants were heterozygous for the insertion, these strains probably carried additional mutations.
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), and others had lost one copy of DRG1 (XC308; Fig. 2). To demonstrate that the phenotypes of these mutants reflected the absence of DRG1, an intact DRG1 gene under the control of its native promoter was transformed into each of the null mutants (XC414; Fig. 2). The phenotype of XC415, a drg1 null mutant carrying two copies of the drg1 locus, is shown in Fig. 3. The drg1 null mutant, carrying URA3 either at its native locus, at ADE2, or at LEU2, was defective in filamentation under embedded conditions at 30 °C (Fig. 3) and at 25 °C (data not shown). The filamentous growth defect of the drg1 null mutant could be partially complemented by reintroduction of the DRG1+ gene at its native locus, under the control of its native promoter (Fig. 3). A representative colony of each strain grown embedded in YPS agar is shown in Fig. 3(B–D). The drg1/drg1/drg1 homozygous null mutants also exhibited a delay in filamentous colony formation under matrix-embedded conditions, and these defects were partially complemented by addition of wild-type DRG1+ (data not shown). When the growth rates of the drg1 null mutants were compared with those of the wild-type strains during growth in YPS+uridine liquid medium, there were no growth defects for any of the drg1 mutants (data not shown). These results support the notion that DRG1 is important for filamentous growth when C. albicans is embedded in a matrix. Filamentous growth of drg1 null mutants containing two or three copies of chromosome 1 was further tested under different conditions known to induce C. albicans filamentation. The drg1 null mutant was defective in filamentous growth when embedded in different media (e.g. YPS, Spider or low ammonium minimal), and grown at either 25 or 30 °C. At 37 °C, drg1 null mutants exhibited defects in filamentation when embedded in low ammonium minimal medium, but not in other media. However, defects in filamentous growth were not observed at 37 °C when cells were grown on the surface of agar media (YPS, Spider, low ammonium minimal), or grown in liquid media (serum, Lee's, proline or GlcNAc). Therefore, these results indicate that DRG1 is not needed to promote filamentous growth under all conditions.
myc-tagged Drg1p was primarily localized in the cytoplasm
To study the localization of Drg1p in C. albicans, pXC156, encoding an N-terminally myc-tagged derivative of DRG1, was used. The derivatized gene was recombined into the genome of strain XC308 (drg1 null mutant) at its native locus under the control of its native promoter, as described in Methods. The derivatized mutant gene was biologically active and complemented the drg1 null mutation (Fig. 3). In contrast, a C-terminally green fluorescent protein (GFP)-tagged DRG1 derivative failed to complement the drg1/drg1 mutation (data not shown); suggesting that modification of the C terminus is not compatible with this function.
Cells expressing myc-DRG1 or untagged DRG1 were grown in liquid medium, or were embedded in agarose. These cells were fixed and processed for indirect immunofluorescence with anti-myc clone 4A6 (Upstate) and Texas red-conjugated anti-mouse IgG secondary antibody. As shown in Fig. 4, most of the cells exhibited perinuclear and cytoplasmic staining. In the majority of cells, the DAPI-stained nuclei were deficient in myc–Drg1p staining. When cells expressing untagged DRG1 were stained, no fluorescent signal was observed (data not shown). Therefore, we conclude that Drg1p exhibits a predominantly cytoplasmic localization, consistent with the results of studies of murine and human DRG1 (Sazuka et al., 1992; Sommer et al., 1994).
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The drg1 null mutant caused delayed lethality and exhibited reduced invasiveness following intravenous inoculation of mice
The drg1 null mutant strain XC413, containing two copies of chromosome 1, was used to test the effect of a drg1 deletion on the virulence of C. albicans. Strains XC101, XC413 (drg1 null) and XC414 (DRG1 complemented mutant) were intravenously inoculated into mice, as described in Methods. On day 7, nine of 10 mice inoculated with strain XC413 (drg1 null mutant) survived, as opposed to four of 12 mice inoculated with strain XC101 (WT), or seven of 11 mice inoculated with XC414 (DRG1+ complemented). Fisher's exact test showed that the mutant was significantly different from the wild-type strain (P=0.01). At later time points, mice inoculated with the drg1 null mutant succumbed to infection (Fig. 5A); therefore, the mutant strain exhibited delayed lethality. The location of the URA3 gene in XC414 (DRG1+ complemented strain) differed from its location in XC101 (WT), and may have influenced the virulence of the complemented strain, as observed previously (Brand et al., 2004; Lay et al., 1998).
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, in mice that survived to day 7, strain XC101 (DRG1+/DRG1+) exhibited extensive invasive growth in the renal papilla. Also, the kidney cortex showed evidence of interstitial inflammation with some damage to tubules, but organisms were not observed (data not shown). In contrast, when kidneys of mice inoculated with the drg1 null mutant were examined, organisms were not observed (Fig. 5C). Kidneys of mice inoculated with the complemented strain resembled those of mice inoculated with wild-type organisms (Fig. 5D). These results show that at an early time point following intravenous inoculation of a host, the drg1 null mutant exhibited reduced invasiveness.
In the absence of Efg1p, a repressor of embedded filamentation, the drg1 null mutant phenotype was not observed
Human and mouse DRG1 proteins have been shown to bind bHLH transcription factors via their C-terminal regions. The C-terminal region of C. albicans Drg1p contains the residues that are required for binding of murine DRG1 to TAL1/SCL1 (Fig. 1). Therefore, because the bHLH transcription factor Efg1p is an important negative regulator of filamentation during growth under embedded conditions (Giusani et al., 2002), interaction between C. albicans Drg1p and Efg1p was analysed using the two-hybrid system. A lexA–DRG1 fusion was tested for interaction with a GAL4AD–EFG1 fusion. As shown in Table 3, while control strains exhibited low levels of the reporter -galactosidase, strains containing both DRG1 and EFG1 fusions exhibited approximately two- to threefold higher activity. In contrast, C-terminally tagged DRG1–lexA failed to exhibit a two-hybrid interaction with GAL4AD–EFG1 (Table 3), consistent with the notion that modification of the C terminus of Drg1p is incompatible with its function. Therefore, Drg1p and Efg1p may interact, and the effects of Drg1p on invasive filamentation may be mediated through binding of Efg1p.
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), an efg1 null mutant was hyperfilamentous (Fig. 6; Giusani et al., 2002). For these experiments, the samples were incubated at room temperature, rather than at 30 °C. The lower temperature resulted in slower development of filamentous colonies, allowing detection of the hyperfilamentous phenotype. The efg1 drg1 double null mutant was also hyperfilamentous, and was indistinguishable from the efg1 single null mutant (Fig. 6). Reintroduction of the EFG1+ gene into either the efg1 drg1 double null mutant or the efg1 single null mutant resulted in partial complementation of the efg1 mutation, and reduced filamentation (Fig. 6; Giusani et al., 2002). Therefore, invasive filamentation can occur in the absence of Drg1p, and the effects of the drg1 mutation are not observed in the absence of Efg1p. These results suggest that Drg1p plays a role in regulating the function of Efg1p.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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).
DRG1 is a highly conserved gene with orthologues in the genomes of species from archaea to humans. The S. cerevisiae orthologue Rbg1p/Fun11p has been shown to bind to a polysome-associated protein (Fleischer et al., 2006). C. albicans Drg1p may similarly interact with polysome-associated proteins, as well as with other proteins.
Mutants lacking Drg1p are capable of forming filaments under a wide range of conditions, demonstrating that Drg1p is not strictly needed for formation of filamentous cells. However, the mutant strain was defective in responding to the cues that normally promote filamentation when the organism is growing embedded within agar media. These results suggest that Drg1p is involved in the mechanism for sensing or responding to signals arising from growth within agar media. One of the important cues that fungal cells experience during growth within agar is contact (Kumamoto, 2005). Therefore, since G proteins typically function as molecular switches, with active and inactive forms, the activation state of Drg1p may change when cells sense contact with agar media or with host tissue, and Drg1p may participate in contact-dependent signalling or responses.
In mammalian cells, Drg1p has been previously implicated in processes related to growth control and oncogenic transformation. For example, DRG1 expression is associated with proliferating cells. In the mouse, DRG1 is relatively highly expressed in embryonic cells, and has lower expression in cells after birth (Sazuka et al., 1992). In plants, DRG is more highly expressed in growing cells as opposed to non-growing cells (Devitt et al., 1999). These findings suggest that DRG1 plays a role in a process that is necessary in proliferating cells.
In addition, ectopic expression of mouse DRG1 enhances transformation of rat embryonic fibroblasts by c-myc and ras (Mahajan et al., 1996). Since insensitivity to contact-dependent regulation of cellular proliferation is a characteristic of cancer cells, ectopic expression of DRG1 may contribute to the dysregulation of this normal control mechanism, enhancing transformation. Therefore, by analogy with the function of Drg1p in C. albicans, DRG1 in mammalian cells may play a role in contact-dependent signalling, or in the response of mammalian cells to contact-dependent cues.
Previous studies have demonstrated binding of mouse or human DRG proteins to the transcription factor oncogene TAL1/SCL1. This binding is mediated through the HLH region of TAL1 and the C-terminal region of DRG (Mahajan et al., 1996). C. albicans Drg1p exhibited a two-hybrid interaction with the bHLH transcription factor Efg1p, an important negative regulator of filamentation under embedded conditions. Since deletion of DRG1 resulted in delayed filamentation under embedded conditions, binding of Efg1p by Drg1p may be a mechanism that contributes to relief of negative regulation by Efg1p. Consistent with this possibility, deletion of EFG1 bypassed the defect in filamentation caused by deletion of DRG1.
Efg1p function appears to be tightly regulated through numerous mechanisms. At the transcriptional level, expression of the EFG1 gene is negatively autoregulated through binding of Efg1p to the TATA box region of its promoter (Tebarth et al., 2003). At the protein level, Efg1p is activated by phosphorylation by protein kinase A (Bockmuhl & Ernst, 2001). In addition, although Efg1p is required for hyphal development in liquid medium, once hypha formation is initiated, down-regulation of EFG1 expression is observed, and forced expression of EFG1 during hyphal growth inhibits hyphal development (Tebarth et al., 2003). The findings described in this communication reveal another possible mechanism for regulation of Efg1p: binding by Drg1p. Drg1p is cytoplasmically localized and, therefore, may regulate Efg1p by affecting its localization. Many transcription factors exhibit cytoplasmic localization under certain conditions, and Efg1p may undergo Drg1p-dependent cytoplasmic localization when its activity needs to be inhibited, such as during embedded filamentation. Taken together, these results suggest that tight control of Efg1p function at both the transcriptional and post-transcriptional levels is important for filamentation.
In addition to filamentation, Efg1p regulates expression of classical virulence factors (e.g. proteases and adhesins), as well as numerous genes involved in metabolism (Doedt et al., 2004; Nantel et al., 2002). Thus, the role of Efg1p in cellular physiology goes well beyond its role in controlling cellular morphology. In this context, the tight regulation of Efg1p function through multiple regulatory mechanisms allows careful control of a wide range of cellular processes that are impacted by this important transcription factor.
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ACKNOWLEDGEMENTS |
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REFERENCES |
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Bockmuhl, D. P. & Ernst, J. F. (2001). A potential phosphorylation site for an A-type kinase in the Efg1 regulator protein contributes to hyphal morphogenesis of Candida albicans. Genetics 157, 1523–1530.
Brand, A., MacCallum, D. M., Brown, A. J., Gow, N. A. & Odds, F. C. (2004). Ectopic expression of URA3 can influence the virulence phenotypes and proteome of Candida albicans but can be overcome by targeted reintegration of URA3 at the RPS10 locus. Eukaryot Cell 3, 900–909.
Brown, D. H., Jr, Slobodkin, I. V. & Kumamoto, C. A. (1996). Stable transformation and regulated expression of an inducible reporter construct in Candida albicans using restriction enzyme-mediated integration. Mol Gen Genet 251, 75–80.[Medline]
Brown, D. H., Jr, Giusani, A. D., Chen, X. & Kumamoto, C. A. (1999). Filamentous growth of Candida albicans in response to physical environmental cues and its regulation by the unique CZF1 gene. Mol Microbiol 34, 651–662.[CrossRef][Medline]
Burke, D., Dawson, D. & Stearns, T. (2000). Methods in Yeast Genetics. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory.
Chen, X., Magee, B. B., Dawson, D., Magee, P. T. & Kumamoto, C. A. (2004). Chromosome 1 trisomy compromises the virulence of Candida albicans. Mol Microbiol 51, 551–565.[CrossRef][Medline]
Clontech (2001). Clontech Yeast Protocols Handbook. Manual no. PT3024-1. Palo Alto, CA: Clontech Laboratories.
Devitt, M. L., Maas, K. J. & Stafstrom, J. P. (1999). Characterization of DRGs, developmentally regulated GTP-binding proteins, from pea and Arabidopsis. Plant Mol Biol 39, 75–82.[CrossRef][Medline]
Doedt, T., Krishnamurthy, S., Bockmuhl, D. P., Tebarth, B., Stempel, C., Russell, C. L., Brown, A. J. & Ernst, J. F. (2004). APSES proteins regulate morphogenesis and metabolism in Candida albicans. Mol Biol Cell 15, 3167–3180.
Felk, A., Kretschmar, M., Albrecht, A. & 7 other authors (2002). Candida albicans hyphal formation and the expression of the Efg1-regulated proteinases Sap4 to Sap6 are required for the invasion of parenchymal organs. Infect Immun 70, 3689–3700.
Fleischer, T. C., Weaver, C. M., McAfee, K. J., Jennings, J. L. & Link, A. J. (2006). Systematic identification and functional screens of uncharacterized proteins associated with eukaryotic ribosomal complexes. Genes Dev 20, 1294–1307.
Fonzi, W. A. & Irwin, M. Y. (1993). Isogenic strain construction and gene mapping in Candida albicans. Genetics 134, 717–728.[Abstract]
Giusani, A. D., Vinces, M. & Kumamoto, C. A. (2002). Invasive filamentous growth of Candida albicans is promoted by Czf1p-dependent relief of Efg1p-mediated repression. Genetics 160, 1749–1753.
Golemis, E. A., Gyuris, J. & Brent, R. (1996). Interaction trap/two-hybrid system to identify interacting proteins. In Current Protocols in Molecular Biology. Edited by F. Ausubel, R. Brent, R. Kingston, D. Moore, J. Seidman, J. Smith & K. Struhl. New York: Wiley.
James, P., Halladay, J. & Craig, E. A. (1996). Genomic libraries and a host strain designed for highly efficient two-hybrid selection in yeast. Genetics 144, 1425–1436.[Abstract]
Kelly, R., Miller, S. M., Kurtz, M. B. & Kirsch, D. R. (1987). Directed mutagenesis in Candida albicans: one-step gene disruption to isolate ura3 mutants. Mol Cell Biol 7, 199–208.
Kumamoto, C. A. (2005). A contact-activated kinase signals Candida albicans invasive growth and biofilm development. Proc Natl Acad Sci U S A 102, 5576–5581.
Kumamoto, C. A. & Vinces, M. D. (2005a). Alternative Candida albicans lifestyles: growth on surfaces. Annu Rev Microbiol 59, 113–133.[CrossRef][Medline]
Kumamoto, C. A. & Vinces, M. D. (2005b). Contributions of hyphae and hypha-co-regulated genes to Candida albicans virulence. Cell Microbiol 7, 1546–1554.[CrossRef][Medline]
Kumar, S., Iwao, M., Yamagishi, T., Noda, M. & Asashima, M. (1993). Expression of GTP-binding protein gene drg during Xenopus laevis development. Int J Dev Biol 37, 539–546.[Medline]
Lay, J., Henry, L. K., Clifford, J., Koltin, Y., Bulawa, C. E. & Becker, J. M. (1998). Altered expression of selectable marker URA3 in gene-disrupted Candida albicans strains complicates interpretation of virulence studies. Infect Immun 66, 5301–5306.
Lo, H. J., Kohler, J. R., DiDomenico, B., Loebenberg, D., Cacciapuoti, A. & Fink, G. R. (1997). Nonfilamentous C. albicans mutants are avirulent. Cell 90, 939–949.[CrossRef][Medline]
Longtine, M. S., McKenzie, A., 3rd, Demarini, D. J., Shah, N. G., Wach, A., Brachat, A., Philippsen, P. & Pringle, J. R. (1998). Additional modules for versatile and economical PCR-based gene deletion and modification in Saccharomyces cerevisiae. Yeast 14, 953–961.[CrossRef][Medline]
Mahajan, M. A., Park, S. T. & Sun, X. H. (1996). Association of a novel GTP binding protein, DRG, with TAL oncogenic proteins. Oncogene 12, 2343–2350.[Medline]
Miller, J. H. (1972). Experiments in Molecular Genetics. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory.
Nantel, A., Dignard, D., Bachewich, C. & 12 other authors (2002). Transcription profiling of Candida albicans cells undergoing the yeast-to-hyphal transition. Mol Biol Cell 13, 3452–3465.
Pringle, J. R., Adams, A. E., Drubin, D. G. & Haarer, B. K. (1991). Immunofluorescence methods for yeast. Methods Enzymol 194, 565–602.[Medline]
Sambrook, J., Fritsch, E. F. & Maniatis, T. (1989). Molecular Cloning: a Laboratory Manual, 2nd edn. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory
Sazuka, T., Tomooka, Y., Ikawa, Y., Noda, M. & Kumar, S. (1992). DRG: a novel developmentally regulated GTP-binding protein. Biochem Biophys Res Commun 189, 363–370.[CrossRef][Medline]
Schenker, T., Lach, C., Kessler, B., Calderara, S. & Trueb, B. (1994). A novel GTP-binding protein which is selectively repressed in SV40 transformed fibroblasts. J Biol Chem 269, 25447–25453.
Sommer, K. A., Petersen, G. & Bautz, E. K. (1994). The gene upstream of DmRP128 codes for a novel GTP-binding protein of Drosophila melanogaster. Mol Gen Genet 242, 391–398.[Medline]
Stoldt, V. R., Sonneborn, A., Leuker, C. E. & Ernst, J. F. (1997). Efg1p, an essential regulator of morphogenesis of the human pathogen Candida albicans, is a member of a conserved class of bHLH proteins regulating morphogenetic processes in fungi. EMBO J 16, 1982–1991.[CrossRef][Medline]
Tebarth, B., Doedt, T., Krishnamurthy, S., Weide, M., Monterola, F., Dominguez, A. & Ernst, J. F. (2003). Adaptation of the Efg1p morphogenetic pathway in Candida albicans by negative autoregulation and PKA-dependent repression of the EFG1 gene. J Mol Biol 329, 949–962.[CrossRef][Medline]
Received 25 June 2006;
accepted 5 August 2006.
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, WT), XC413 (drg1
, drg1 null) and XC414 (drg1
, Comp, dotted line) were inoculated intravenously into 10–12 mice, and survival as a function of time post-inoculation was monitored. Some animals were sacrificed for histological analysis of kidneys. All mice from three experiments are shown in the graph. (B–D) Mice surviving at day 7 were sacrificed, and kidneys were fixed, sectioned and stained with Gomori methenamine silver staining. Sections were photographed with the x40 objective. (B) Extensive growth of strain XC101 (WT) in the renal papilla is shown. (C) Organisms were not detected in the renal papilla of mice inoculated with drg1 null strain XC413. (D) Complemented mutant XC414 exhibiting growth in the renal papilla.