Genome-wide transcriptional profiling of the cyclic AMP-dependent signaling pathway during morphogenic transitions of Candida albicans

Abstract

Candida albicans is an opportunistic human fungal pathogen that causes systemic candidiasis as well as superficial mucosal candidiasis. In response to the host environment, C. albicans transitions between yeast and hyphal forms. In particular, hyphal growth is important in facilitating adhesion and invasion of host tissues, concomitant with the expression of various hypha-specific virulence factors. In previous work, we showed that the cyclic AMP (cAMP) signaling pathway plays a crucial role in morphogenic transitions and virulence of C. albicans by studying genes encoding adenylate cyclase-associated protein (CAP1) and high-affinity phosphodiesterase (PDE2) (Y. S. Bahn, J. Staab, and P. Sundstrom, Mol. Microbiol. 50:391-409, 2003; and Y. S. Bahn and P. Sundstrom, J. Bacteriol. 183:3211-3223, 2001). However, little is known about the downstream targets of the cAMP signaling pathway that are responsible for morphological transitions and the expression of virulence factors. Here, microarrays were probed with RNA from strains with hypoactive (cap1/cap1 null mutant), hyperactive (pde2/pde2 null mutant), and wild-type cAMP signaling pathways to provide insight into the molecular mechanisms of virulence that are regulated by cAMP and that are related to the morphogenesis of C. albicans. Genes controlling metabolic specialization, cell wall structure, ergosterol/lipid biosynthesis, and stress responses were modulated by cAMP during hypha formation. Phenotypic traits predicted to be regulated by cAMP from the profiling results correlated with the relative strengths of the mutants when tested for resistance to azoles and subjected to heat shock stress and oxidative/nitrosative stress. The results from this study provide important insights into the role of the cAMP signaling pathway not only in morphogenic transitions of C. albicans but also for adaptation to stress and for survival during host infections.

FIG. 1.

Morphologies of the WT, hyperfilamentous pde2/pde2 mutant, and budding cap1/cap1 mutant used to prepare RNA in profiling experiments. Cells were grown at 25°C in YNB to mid-logarithmic phase, reinoculated at a concentration of 5 × 10⁶ cells/ml into M199, and further incubated for 5 h at 25°C for yeast growth or at 37°C for germ tube induction. At each time point, cells were sampled and their morphologies were observed by microscopy. Total RNAs were isolated from a 1.5-h culture of each strain in M199 at 25 or 37°C (black-outlined boxes). True hyphal formation of the WT and pde2/pde2 mutant was confirmed by an indirect immunofluorescence assay with hypha wall protein Hwp1-specific antibodies as described before (5). The reference RNA was pooled from RNA prepared from each strain at 25°C (denoted as R). The proportion of cells with germ tubes after 1 h of incubation in M199 at 37°C was nearly 100% for the pde2/pde2 (Δpde2; strain BPS15) mutant, 20 to 30% for the WT strain (UnoPP-1), and 0% for the cap1/cap1 (Δcap1; CAC1-1A1E1) mutant.

FIG. 2.

Expression patterns of known hypha-specific genes. (A) Microarray data showing the fold induction of ECE1, HWP1, and RBT1 in the WT (UnoPP-1), the pde2/pde2 (Δpde2; BPS15), and the cap1/cap1 (Δcap1; CAC1-1A1E1) strains under hypha-inducing (M199 at 37°C for 1.5 h) conditions relative to induction levels under yeast growth conditions (M199 at 25°C for 1.5 h), as described in Materials and Methods. (B) Northern blot showing ECE1 and HWP1 expression. Total RNA samples that also were used for the microarray experiments were separated by electrophoresis in a formaldehyde agarose gel, transferred to a nitrocellulose membrane, and probed with ³²P-labeled DNA probes for ECE1, HWP1, and 18S rRNA as a loading control. The membrane was exposed to X-ray film for 1 and 8 h for the detection of HWP1 and ECE1 mRNA, respectively, and for 4 h for the detection of 18S rRNA. exp., experiment. Y, yeast growth conditions; H, hyphal growth conditions.

FIG. 3.

Verification of expression patterns of genes modulated by the cAMP signaling pathway during bud-hypha transitions by Northern blot analysis. Northern blot analysis was performed with gene-specific PCR probes (genes positively regulated [A] and negatively regulated [B]) as described in the legend to Fig. 2B and Materials and Methods. Several replicated membranes were prepared using the same sample of total RNA and were used repeatedly for multiple probes after stripping the membrane as previously described (63). In general, the membrane was exposed to X-ray film for 1 to 2 days for detection of the mRNA of each probe, except for ERG1, ERG11, UCF1, and MCR1 probes, which required only 6 to 7 h of exposure, and for the XJP15 probe, which required only 1 h of exposure. The membrane probed with the control 18S rRNA was exposed for 4 h.

FIG. 4.

Increased azole resistance by hyperactivation of the cAMP pathway of C. albicans. Susceptibilities to fluconazole were determined by the Etest strip method for the following isogenic strains: WT (UnoPP-1), pde2/pde2 (Δpde2; BPS15), pde2/PDE2 reintegrants (Δpde2+PDE2; BPS9), cap1/cap1 (Δcap1; CAC1-1A1E1), and cap1/CAP1 reintegrants (Δcap1+CAP1; CACRE1). The plates were photographed, and the fluconazole MICs for each strain were determined after 48 h of incubation at 35°C.

FIG. 5.

Effect of heat shock, high salt concentrations, and osmotic shock on the viability of the WT and the cAMP signaling mutants. The WT (Unopp-1) strain and the mutants cap1/cap1 (Δcap1; CAC1-1A1E1), pde2/pde2 (Δpde2; BPS15), and cap1/cap1 pde2/pde2 (Δcap1 Δpde2; BPS27) were tested for (A) heat shock sensitivity (55°C) or (B) high salt concentrations (1.5 M NaCl or KCl) and high osmolarity (2 M sorbitol).

FIG. 6.

Effect of hydrogen peroxide, superoxide, nitric oxide, and peroxynitrite on the viability of the WT and cAMP signaling mutants. (A) Volumes of 2 μl each of 10⁶ cells/ml of the C. albicans strains described in the legend to Fig. 5 were spotted on YNB plates containing the indicated concentrations of H₂O₂ or menadione, a O₂⁻ generator, were incubated for 2 days, and were photographed. (B) Each strain was incubated at 37°C for 3 h with the indicated concentrations of SNP, SIN-1 (an ONOO⁻ generator), or ONOO⁻ itself. Error bars indicate the standard deviations from two independent experiments. The significant difference in percent survival between the pde2/pde2 mutant and the other strains was denoted with an asterisk. The significant difference in percent survival between the cap1/cap1 mutant and the other strains was denoted with a pound sign (P < 0.05 using Bonferroni's multiple comparison test [Prism 2.0b; GraphPad Software]).

FIG. 7.

Northern blot analysis of oxidative/nitrosative stress defense genes in response to peroxynitrite. Total RNAs isolated from C. albicans strains (WT [UnoPP-1], cap1/cap1 mutant [Δcap1; CAC1-1A1E1], and pde2/pde2 mutant [Δpde2; BPS15]) exposed to 50 μM of peroxynitrite for each indicated time of incubation were used for Northern blot analysis. A single membrane was stripped and repeatedly probed with stress-related genes (MCR1, CCP1, SOD2, and HSP12) as previously described. The membrane was exposed to X-ray film for 1 day (MCR1) or 2 days (SOD2, HSP12, and CCP1). The membrane probed with the control 18S rRNA was exposed for 4 h.