Cdc24, the GDP-GTP exchange factor for Cdc42, is required for invasive hyphal growth of Candida albicans

Abstract

Candida albicans, the most common human fungal pathogen, is particularly problematic for immunocompromised individuals. The reversible transition of this fungal pathogen to a filamentous form that invades host tissue is important for its virulence. Although different signaling pathways such as a mitogen-activated protein kinase and a protein kinase A cascade are critical for this morphological transition, the function of polarity establishment proteins in this process has not been determined. We examined the role of four different polarity establishment proteins in C. albicans invasive growth and virulence by using strains in which one copy of each gene was deleted and the other copy expressed behind the regulatable promoter MET3. Strikingly, mutants with ectopic expression of either the Rho G-protein Cdc42 or its exchange factor Cdc24 are unable to form invasive hyphal filaments and germ tubes in response to serum or elevated temperature and yet grow normally as a budding yeast. Furthermore, these mutants are avirulent in a mouse model for systemic infection. This function of the Cdc42 GTPase module is not simply a general feature of polarity establishment proteins. Mutants with ectopic expression of the SH3 domain containing protein Bem1 or the Ras-like G-protein Bud1 can grow in an invasive fashion and are virulent in mice, albeit with reduced efficiency. These results indicate that a specific regulation of Cdc24/Cdc42 activity is required for invasive hyphal growth and suggest that these proteins are required for pathogenicity of C. albicans.

FIG. 1.

Comparison of fungal Cdc24 guanine nucleotide exchange factors. (A) Protein sequence comparison of fungal Cdc24s. Percentage identity and similarity of Saccharomyces cerevisiae (S.c.), Kluyveromyces lactis (K.l.), Candida albicans (C.a.), and Schizosaccharomyces pombe (S.p.) Cdc24 proteins. The GenBank accession number for C. albicans CDC24 is AY208122. The guanine nucleotide exchange factor (GEF) and plekstrin homology domains are shown in gray and black, respectively. Numbers below the schematic representation of C. albicans Cdc24 indicate the percent similarity of each region with Saccharomyces cerevisiae Cdc24. Alignments were carried out by using the BLAST algorithm (1). (B) Protein sequence alignment of fungal guanine nucleotide exchange factor domain. Residues boxed in black and in gray are identical and similar to S.c. Cdc24, respectively. The black line above the sequences represents the S. cerevisiae Cdc24 GEF domain. S.c., S. cerevisiae; K.l., K. lactis; C.a., C. albicans; S.p., S. pombe.

FIG. 2.

Construction and Southern analysis of C. albicans cdc24 mutant strains. (A) Schematic representation of CDC24 strain construction. (B) Southern blot analysis of CDC24 mutant strains. Five micrograms each of total genomic DNA from the indicated strains was digested with MfeI and analyzed by Southern blotting. Blots were probed with a radiolabeled 1.1-kb CaCDC24 MfeI fragment. The size of the fragments is indicated in kilobases.

FIG. 3.

Regulated expression of the MET3 promoter. (A) Cells carrying MET3 promoter GFP grown in SC medium lacking Met and Cys (lane 1), in SC medium containing 2.5 mM Met and Cys (lane 2), or in YEPD (lane 3) were lysed by agitation with glass beads and analyzed by SDS-PAGE, followed by immunoblotting, and then probed with anti-GFP polyclonal sera (30). (B) CDC24/pMet3xHACdc24 cells were analyzed as described for panel A with anti-HA monoclonal sera (lanes 4 to 6). (C) Cells expressing 3xHACdc24 under the control of the MET3 promoter (left panel) or CaCdc24 promoter (right panel) grown in SC medium lacking Met and Cys (lanes 7 and 9) and YEPD (lanes 8 and 10) were analyzed as described for panel A. Similar amounts of protein were loaded in each lane, as judged by Ponceau red staining of the blots.

FIG. 4.

C. albicans Cdc24 and Cdc42 are essential for normal vegetative growth. (A and B) Indicated strains were grown to exponential phase in SC medium lacking methionine and cysteine. Serial dilutions were spotted onto SC plates lacking or containing 2.5 mM of methionine and cysteine, and the plates were incubated at 30°C for 2 to 4 days.

FIG. 5.

C. albicans Cdc24 and Cdc42 are essential for invasive hyphal growth in response to serum. The indicated strains were grown in SC medium lacking methionine and cysteine, and serial dilutions were spotted onto YEPD plates containing FCS (A and B) or plates with SC medium that lacked methionine and cysteine but containing DFCS (C and D). Plates were then incubated for 5 (A, C, and D) or 3 (B) days at 30°C.

FIG. 6.

C. albicans Cdc24 and Cdc42 are required for germ tube formation. (A and B) The indicated strains were grown in YEPD, pelleted, and resuspended in YEPD, and then an equal volume of FCS was added. Cells were incubated at 37°C for 3 h, fixed with formaldehyde, and imaged. (C) The indicated strains were treated and analyzed as described for panels A and B, except that they were grown in SC medium lacking methionine and cysteine and then resuspended in the same medium containing an equal volume of DFCS. The percentages of cells with germ tubes were determined (n > 200).

FIG. 7.

C. albicans Δcdc24/pMetCDC24 and Δcdc42/pMetCDC42 strains are avirulent. (A) Δcdc24/pMetCDC24 (▵), Δcdc42/pMetCDC42 (▿), Δbud1/pMetBUD1 (⋄), and Δbem1/pMetBEM1 (○) strains (10⁶ cells) were injected into the tail vein of CD1 mice (n = 10) and monitored for survival over 40 days. A URA⁺ wild-type (□) strain was used as a control. (B) Prototrophic (HIS⁺ URA⁺ ARG⁺) versions of wild-type (⋄), Δcdc24/CDC24 (○), Δcdc24/pMetCDC24 (▵), and Δcdc24/pMetCDC24/RP10:: CDC24 (▿) strains and a URA⁺ wild-type (□) were injected in mice as described in panel A, and mouse mortality was monitored for 20 days.