CAP1, an adenylate cyclase-associated protein gene, regulates bud-hypha transitions, filamentous growth, and cyclic AMP levels and is required for virulence of Candida albicans

Abstract

In response to a wide variety of environmental stimuli, the opportunistic fungal pathogen Candida albicans exits the budding cycle, producing germ tubes and hyphae concomitant with expression of virulence genes, such as that encoding hyphal wall protein 1 (HWP1). Biochemical studies implicate cyclic AMP (cAMP) increases in promoting bud-hypha transitions, but genetic evidence relating genes that control cAMP levels to bud-hypha transitions has not been reported. Adenylate cyclase-associated proteins (CAPs) of nonpathogenic fungi interact with Ras and adenylate cyclase to increase cAMP levels under specific environmental conditions. To initiate studies on the relationship between cAMP signaling and bud-hypha transitions in C. albicans, we identified, cloned, characterized, and disrupted the C. albicans CAP1 gene. C. albicans strains with inactivated CAP1 budded in conditions that led to germ tube formation in isogenic strains with CAP1. The addition of 10 mM cAMP and dibutyryl cAMP promoted bud-hypha transitions and filamentous growth in the cap1/cap1 mutant in liquid and solid media, respectively, showing clearly that cAMP promotes hypha formation in C. albicans. Increases in cytoplasmic cAMP preceding germ tube emergence in strains having CAP1 were markedly diminished in the budding cap1/cap1 mutant. C. albicans strains with deletions of both alleles of CAP1 were avirulent in a mouse model of systemic candidiasis. The avirulence of a germ tube-deficient cap1/cap1 mutant coupled with the role of Cap1 in regulating cAMP levels shows that the Cap1-mediated cAMP signaling pathway is required for bud-hypha transitions, filamentous growth, and the pathogenesis of candidiasis.

FIG. 1

Disruption of C. albicans CAP1. (A) Genetic organization of the CAP1 locus. The CAP1 open reading frame (shaded bar) and PCR products (solid line) (PCR-1.2 and PCR-1.6) are indicated. Each arrowhead indicates primers used for RT-PCR to confirm the disruption of CAP1 (1, CAP-NRT1; 2, CAP-F1; 3, CAP-R3; 4, CAP-3F1). (B) Southern blot analysis of HindIII-digested C. albicans genomic DNA probed with PCR-1.2 as described in Materials and Methods. Lanes: 1, parental strain CAI4; 2 and 3, CAP1/cap1 strains CAC1 and CAC1-1, Ura⁺ and Ura⁻, respectively; 4 and 5, homozygous cap1/cap1 strains CAC1-1A and CAC1-1A1, Ura⁺ and Ura⁻, respectively; 6, CAP1-complemented strain CACRE1.

FIG. 2

Primary structure alignment of C. albicans Cap1 with CAPs of other organisms. Multiple sequence alignments of CAPs from C. albicans (CaCAP1), S. cerevisiae (ScCAP), S. pombe (SpCAP), mouse (MouseCAP1), and human (HumanCAP1) were performed with ClustalW (79) and illustrated with MacVector 6.5.3 (Oxford Molecular Company). Solid lines indicate residues for the conserved RLE/RLE motif (21 to 30), the polyproline region (289 to 297), and two consensus SH3-binding motifs (358 to 361 and 364 to 367) in C. albicans.

FIG. 3

Northern blot and RT-PCR analysis of cap1/cap1 mutants. CAP1 mRNA is absent in the cap1/cap1 strain and present at equivalent low levels in other strains during yeast growth (A) or germ tube induction (B). Total RNA (7 μg/lane), isolated as described in Materials and Methods, was separated in a formaldehyde agarose gel transferred to a nitrocellulose membrane and probed with radiolabeled PCR-1.2 to detect CAP1 mRNA and 18S rRNA as a control. The membrane was exposed to X-ray film for 7 days for detection of CAP1 mRNA and for 4 h for detection of 18S rRNA. (C) Amplification of 5′ (605 bp, 1 to 605) and 3′ (713 bp, 922 to 1634) portions of CAP1 mRNA using RT-PCR followed by Southern blotting using radiolabeled PCR-1.6 as probe. ACT1 mRNA (304 bp) was amplified as a positive control. Lanes 1 to 4, strains UnoPP-1, CAC1, CAC1-1A, and CACRE1.

FIG. 4

cap1/cap1 strains are defective in bud-hypha transitions. Germ tubes were induced at cell concentrations of 5 × 10⁶ cells/ml (A and B, first four rows) or 10⁶ cells/ml (A and B, bottom rows) in prewarmed Lee's medium, saliva, M199, or M199+serum for 5 h (A) and 20 h (B). cap1/cap1 mutant cells formed buds (arrows 1) or pseudohyphae at low frequency (arrows 2), whereas strains having CAP1 (UnoPP-1, CAC1, and CACRE1) produced typical germ tubes (A and B, first two and fourth columns). At 20 h a few cap1/cap1 mutant yeasts (<10%) produced germ tubes in saliva or M199 (arrows 3). In the presence of serum the frequency of germ tube formation was higher (20 to 30%) (arrow 4). Reducing the inoculum concentration in the presence of serum led to production of germ tubes by 40% of cap1/cap1 mutant yeasts at 5 h (arrow 5), and at 20 h the majority of yeasts had formed germ tubes that were shorter than those of the other strains (arrow 6). Bars, 5 μm.

FIG. 5

cap1/cap1 strains are defective in filamentous growth. Colonial appearances (A) and cellular morphologies at colony rims (B), respectively, in each agar medium condition are shown. (A) Colonies of the cap1/cap1 mutant consisted of budding yeasts (A and B, third columns), whereas strains with CAP1 (UnoPP-1, CAC1, and CACRE1) produced filamentous growths of differing characteristics depending on the media. The asymmetric colonies formed by strains with CAP1 in serum contained infrequent thick plumes composed of filaments covered with buds radiating from the colony center (arrow). (B) Strains with CAP1 produced uniform hyphae with short branches in M199 and Spider plates (arrows 1) or hyphae with thick-walled terminal buds in SLAD medium (arrow 2). In media with serum, colonies of strains with CAP1 were composed primarily of hyphae bereft of buds (arrow 3). M199 plates were incubated first at 30°C for 48 h and transferred to 37°C for another 48 h, whereas the other plates were incubated for 6 days at 37°C. Black (A) and white (B) bars, 1 mm and 50 μm, respectively.

FIG. 6

Reduced cAMP levels of the C. albicans cap1/cap1 mutant in germ tube-inducing conditions compared to those of strains with CAP1. Intracellular cAMP levels for each strain (UnoPP-1 [CAP1/CAP1] [□], CAC1 [CAP1/cap1] [■], CACRE1 [CAP1/cap1] [●], and CAC1-1A [cap1/cap1] [○]) were measured as described in Materials and Methods. Each value in the y axis indicates the fold increase in cAMP over the basal level in each strain at time zero. Error bars indicate the standard deviation of each value from three independent experiments performed in triplicate. (A) Germ tube-inducing conditions (M199 at 37°C). cAMP levels (picomoles per milligram of protein) at time zero for UnoPP-1, CAC1, CAC1-1A, and CACRE1 were 45.3 ± 4.6, 55.1 ± 6.9, 61.8 ± 6.5, and 51.4 ± 6.7 (mean value ± standard deviation), respectively. The decreased cAMP level in the CAP1/cap1 mutant compared to results for strains with CAP1 at 1 h was statistically significant (asterisk, P < 0.01 [UnoPP-1 or CAC1 versus CAC1-1A] and P < 0.05 [CACRE1 versus CAC1-1A] using Bonferroni's multiple comparison test performed with Prism 2.0b [GraphPad Software]). (B) Budding growth in M199 at 27°C. cAMP levels (picomoles per milligram of protein) at time zero for UnoPP-1, CAC1, CAC1-1A, and CACRE1 were 50.9 ± 22.4, 58.1 ± 8.4, 37.4 ± 2.9, and 52.6 ± 6.6, respectively. (C) Morphological changes of UnoPP-1 (CAP1/CAP1), CAP1/cap1 strain (CAC1 and CACRE1), and cap1/cap1 strain (CAC1-1A) were monitored during germ tube induction. Bars, 5 μm.

FIG. 7

Suppression of defective bud-hypha transitions and filamentous growth in the cap1/cap1 mutant by exogenous cAMP or its derivative, dbcAMP. (A) The wild-type CAP1/CAP1 strain, UnoPP-1, and the cap1/cap1 mutant strain, CAC1-1A, were grown in SLAD medium with or without 10 mM cAMP or dbcAMP for 5 days at 37°C. Bars, 1 mm. (B) Bud-hypha transitions were induced at cell concentrations of 10⁶ cells/ml in prewarmed M199+serum with or without 10 mM dbcAMP for 13 h (first [UnoPP-1] and second [CAC1-1A] columns, 20× objective; third [CAC1-1A] column, 40× objective). Bars, 30 μm.

FIG. 8

Survival curves of mice (CBA/J, 5 to 6 weeks old, six mice per group) infected with 2 × 10⁵ cells of C. albicans strains SC5314 (CAP1/CAP1), CAC1 (CAP1/cap1), CAC1-1A (CAP1/cap1), and CACRE1 (CAP1/cap1, revertant). Similar results were obtained in two independent experiments. Survival curves were illustrated according to the Kaplan-Meier method using the PRISM program and compared using the log-rank test. A P value of <0.05 was considered significant.