Invasive Filamentous Growth of Candida albicans Is Promoted by Czf1p-Dependent Relief of Efg1p-Mediated Repression

Corresponding author: Carol A. Kumamoto, Tufts University, 136 Harrison Ave., Boston, MA 02111., carol.kumamoto{at}tufts.edu (E-mail)

Communicating editor: A. P. MITCHELL

	ABSTRACT

TOP ABSTRACT Efg1p represses filamentous... In the absence of... Interaction between Czf1p and... Discussion LITERATURE CITED

Filamentation of Candida albicans occurs in response to many environmental cues. During growth within matrix, Efg1p represses filamentation and Czf1p relieves this repression. We propose that Czf1p interacts with Efg1p, altering its function. The complex regulation of filamentation may reflect the versatility of C. albicans as a pathogen.

THE opportunistic fungal pathogen, Candida albicans, grows invasively in tissues of candidiasis patients by converting from budding yeast form cells to filamentous forms. The ability to convert from one morphology to another is important for virulence (SOBEL et al. 1984 ; SHEPHERD 1985 ; RYLEY and RYLEY 1990 ; LEBERER et al. 1997 ; LO et al. 1997 ). To understand the mechanisms by which filamentous growth is stimulated during infection, regulation of hyphal development has been studied extensively (for review, see GOW 1997 ). Conditions that promote hyphal growth in the laboratory include growth at elevated temperature in medium containing special components. In the absence of these conditions, growth within a matrix also promotes hyphal growth (BROWN et al. 1999 ). The embedded condition may simulate conditions encountered by the pathogen during growth in human tissue.

Several genes whose products regulate filamentous growth have been identified (for review, see ERNST 2000 ), including CPH1 (LIU et al. 1994 ), EFG1 (STOLDT et al. 1997 ), and CZF1 (BROWN et al. 1999 ). Efg1p regulates the expression of several genes (SHARKEY et al. 1999 ; BRAUN et al. 2000 ; SCHROPPEL et al. 2000 ; LENG et al. 2001 ) and plays a major role in promoting filamentous growth (LO et al. 1997 ; STOLDT et al. 1997 ). C. albicans Cph1p, a homolog of Saccharomyces cerevisiae STE12, is important for hyphal development in some media (LIU et al. 1994 ; LANE et al. 2001 ) and nutritional signals are thought to be important in stimulation of the mitogen-activated protein kinase cascade that regulates Cph1p (KOHLER and FINK 1996 ; LEBERER et al. 1996 ; CSANK et al. 1998 ). CZF1, a zinc-finger-containing protein that has no homolog in the S. cerevisiae genome, is important for regulation of hyphal development when C. albicans cells are grown within matrix (BROWN et al. 1999 ).

Although Efg1p has been previously shown to promote filamentous growth (LO et al. 1997 ; STOLDT et al. 1997 ), this study demonstrates that Efg1p acts as a repressor of filamentous growth when cells are grown within matrix at low temperature. Genetic analysis indicates that Czf1p antagonizes the function of Efg1p. In addition, a two-hybrid interaction between Czf1p and Efg1p was observed, suggesting that Czf1p relieves repression of filamentation by binding to Efg1p.

	Efg1p represses filamentous growth under low-temperature embedded conditions

TOP ABSTRACT Efg1p represses filamentous... In the absence of... Interaction between Czf1p and... Discussion LITERATURE CITED

To determine the role of Efg1p in controlling morphology in response to growth within agar matrix at 25°, the phenotype of an efg1/efg1 null mutant, CKY136, was analyzed. Colonies of the efg1 null strain produced filaments much earlier than colonies of the wild-type strain CKY101 or the complemented mutant strain HLC74 (efg1/efg1/EFG1; Fig 1, A–C; Table 1). To demonstrate that the differences in filamentation were not caused by differences in growth rates, individual cells growing in agarose matrix at room temperature were observed. The average times required to complete one cell cycle were very similar for all three strains, 91 min for wild-type cells, 121 min for CKY136, and 114 min for HLC74. In contrast to these results, the efg1 null mutant exhibited defective filamentous growth when grown on the surface of several media at 37° (data not shown), consistent with previous reports (LO et al. 1997 ; STOLDT et al. 1997 ).

View larger version (143K): In this window In a new window Download PPT slide   Figure 1. Repressive effects of EFG1 on filamentous growth within agar matrix. Strains (Table 1) were grown in YPD (AUSUBEL 1989, no. 20) overnight, diluted and grown in YPD at 30° for 4 hr, mixed with molten YPS (yeast extract, peptone, 2% sucrose) agar medium, and plated. Plates were incubated at either 25° (A, B, and D) or room temperature (C). At various times, 100–200 colonies were examined microscopically as described previously (BROWN 1999, no. 45), and the percentage of filamentous colonies was plotted as a function of time. To measure the doubling times of cells embedded in agar, cells were mixed with molten YPS containing 0.7% low-melt agarose and poured onto a glass microscope slide. During growth at room temperature, individual cells on the glass slide were observed and the time required for emergence of a bud was measured for two cell cycles. Emergence of 14 buds was monitored for strains CKY101 (EFG1/EFG1) and CKY136 (efg1/efg1) and emergence of 7 buds was monitored for strain HLC74 (efg1/efg1/EFG1). (A) Colony of CKY101 (EFG1/EFG1) grown for 43 hr at 25°. (B) Colony of CKY136 (efg1/efg1) grown for 43 hr at 25°. (C) Formation of filamentous colonies by strains CKY101 (EFG1/EFG1; diamonds), CKY136 (efg1/efg1; squares) and HLC74 (efg1/efg1/EFG1; triangles). (D) Formation of filamentous colonies by strains CKY101 (EFG1/EFG1; diamonds), CKY138 (efg1/efg1 cph1/cph1; triangles), CKY169 (cph1/cph1 czf1/czf1; squares), and CKY248 (efg1/efg1 cph1/cph1 czf1/czf1; circles).

The repressive effect of Efg1p on filamentation during growth within matrix was also observed in strains lacking Czf1p. czf1 null mutants and czf1 cph1 double null mutants are defective in production of filamentous colonies under low-temperature embedded conditions (Fig 1D; BROWN et al. 1999 ). However, a triple null mutant lacking Efg1p, Cph1p, and Czf1p exhibited close to wild-type production of filamentous colonies under low-temperature embedded conditions (Fig 1D).

	In the absence of Efg1p, changes in the expression of Czf1p had no effect on development of filamentous colonies

TOP ABSTRACT Efg1p represses filamentous... In the absence of... Interaction between Czf1p and... Discussion LITERATURE CITED

In wild-type cells incubated under low-temperature embedded conditions, changing the expression of Czf1p altered the kinetics of filamentous colony production (BROWN et al. 1999 ). When Czf1p was ectopically expressed using the maltase promoter, filamentous colonies were observed precociously. When Czf1p was absent, production of filamentous colonies was delayed. We propose that ectopically expressed Czf1p relieves repression by Efg1p precociously and that, in the absence of Czf1p, repression by Efg1p is not relieved normally. A prediction of this model is that in the absence of Efg1p, changes in the expression of Czf1p will have no effect.

To test this model, we first showed that double null mutants, lacking Efg1p and Czf1p, exhibited precocious filamentation indistinguishable from the efg1 null mutant (data not shown). This observation is consistent with the hypothesis that Czf1p acts on Efg1p-dependent repression.

Second, we ectopically expressed Czf1p in strains lacking Efg1p. An efg1 null strain ectopically expressing Czf1p produced filamentous colonies with the same kinetics as an efg1 null mutant alone (data not shown). However, due to the precociously filamentous phenotype of this mutant strain, it might be difficult to detect an effect of Czf1p ectopic expression. During growth within matrix at low temperature, the efg1 cph1 double null mutant is precociously filamentous in many media but produces filamentous colonies with close to wild-type kinetics in YPS media (RIGGLE et al. 1999 ). Therefore, we examined the effect of ectopic expression of Czf1p in the efg1 cph1 double null mutant. While ectopic expression of Czf1p in a cph1 single null mutant did accelerate production of filamentous colonies (Fig 2), ectopic expression of Czf1p in the efg1 cph1 double null mutant did not accelerate production of filamentous colonies (Fig 2). These results demonstrate that in the absence of Efg1p ectopic expression of Czf1p did not alter the rate of production of filamentous colonies and are consistent with the model that Czf1p promotes filamentous growth by relieving repression due to Efg1p.

View larger version (18K): In this window In a new window Download PPT slide   Figure 2. Filamentous growth in strains lacking Efg1p is not affected by changing the expression of Czf1p. Cells were grown and plated as in Fig 1 and the percentage of filamentous colonies was plotted as a function of time. Results of duplicate samples are shown and the average value is plotted. Strains were as follows: CKY138 (efg1/efg1 cph1/cph1; circles) and CKY139 (efg1/efg1 cph1/cph1 PMAL-CZF1; triangles), CKY196 (cph1/cph1; diamonds), and CKY197 (cph1/cph1 PMAL-CZF1; squares).

	Interaction between Czf1p and Efg1p

TOP ABSTRACT Efg1p represses filamentous... In the absence of... Interaction between Czf1p and... Discussion LITERATURE CITED

Czf1p might antagonize repression by Efg1p through physical interaction. To test this possibility, a two-hybrid experiment was performed. As shown in Table 2, the ability of CZF1-lexA or GAL4AD-EFG1 to activate lacZ reporter gene expression in the presence of unfused GAL4AD or unfused lexA, respectively, or in the presence of irrelevant protein fusions was poor. However, when CZF1-lexA and GAL4AD-EFG1 were both present in the strain, lacZ reporter gene expression was observed. These results indicate that Czf1p and Efg1p are capable of interaction and suggest that relief of Efg1p-mediated repression by Czf1p occurs via protein-protein interaction.

	Discussion

TOP ABSTRACT Efg1p represses filamentous... In the absence of... Interaction between Czf1p and... Discussion LITERATURE CITED

Efg1p regulates filamentous growth under several conditions but performs different functions. At 37° in many media, strains lacking Efg1p are defective in filamentation. In contrast, during growth within matrix at low temperature, Efg1p acts as a repressor of filamentous growth. The negative effects of Efg1p on filamentous growth under these conditions are observed in strains containing or lacking Czf1p and Cph1p, indicating that the repressive effects do not require either protein. However, the efg1 null mutant is not hyperfilamentous when grown on the surface of agar media. Therefore, cells also possess an Efg1p-independent mechanism for repression of filamentous growth under nonembedded conditions.

Ectopic expression of Czf1p accelerates production of filamentous colonies under low-temperature embedded conditions and ectopic expression of Cph1p has a similar, though weaker, effect (D. H. BROWN, JR. and C. KUMAMOTO, unpublished observations). Ectopic expression of Czf1p also accelerates production of filamentous colonies in the absence of Cph1p. Similarly, deletion of czf1 results in defective production of filamentous colonies during growth within matrix at low temperature in both CPH1 and cph1 strains. These results support the model that Czf1p and Cph1p have independent functions. In contrast, when efg1 is deleted, the effects of changes in CZF1 expression are eliminated. This epistatic effect is inconsistent with a model in which Czf1p and Efg1p function independently and supports the model that Czf1p acts on Efg1p. We propose that Czf1p acts to antagonize repression mediated by Efg1p, by binding and altering the function of Efg1p. As a result, expression of critical genes needed for formation of highly elongated, filamentous cells occurs, leading to filamentous growth.

At least two mechanisms for repression of transcription by Efg1p could be imagined. As a bHLH protein with homology to the Myc family of transcription factors, Efg1p may bind a partner that mediates transcriptional repression (FACCHINI and PENN 1998 ), analogous to Mad. A second possibility is that Efg1p may interact with components of the basal transcription machinery or with other transcription factors. Myc represses expression of its own gene in the absence of DNA binding (FACCHINI et al. 1997 ), possibly through interaction with the basal transcription machinery. v- and c-Myc also interact with and repress the transcription factors C/EBPß and MIZ-1 (MINK et al. 1996 ; PEUKERT et al. 1997 ).

Several studies, including this one, demonstrate that numerous mechanisms for promotion of filamentous growth exist in C. albicans (RIGGLE et al. 1999 ; BRAUN and JOHNSON 2000 ). The presence of these multiple pathways presumably allows the cell to produce hyphae in a variety of environments and in the presence of different stimuli. For example, the mutant lacking Efg1p and Cph1p fails to form hyphae in serum and other media at 37° but forms filaments well under low-temperature matrix embedded conditions (RIGGLE et al. 1999 ). This mutant forms filaments in the tongue during experimental infection (RIGGLE et al. 1999 ) but fails to develop filaments following ingestion by macrophages (LO et al. 1997 ). The complex regulation of hyphal growth in C. albicans may reflect the high versatility of C. albicans as a pathogen and the wide variety of environmental cues that may be encountered by the organism during infection in different tissue sites.

	ACKNOWLEDGMENTS

We thank Ralph Isberg, Dean Dawson, Claire Moore, Andrew Wright, Linc Sonenshein, Perry Riggle, and Xi Chen for helpful discussions and comments on the manuscript. We are grateful to Dr. G. Fink for kindly providing strains JKC18 (cph1/cph1), Can35 (efg1/efg1), and Can36 (efg1/efg1 cph1/cph1). The contributions of Katherine Lew to this project are gratefully acknowledged. This work was supported by grant AI38591 from the National Institutes of Health (to C.A.K.)

Manuscript received January 17, 2001; Accepted for publication January 14, 2002.

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