Comparative transcriptome analysis reveals novel roles of the Ras and cyclic AMP signaling pathways in environmental stress response and antifungal drug sensitivity in Cryptococcus neoformans

Abstract

The cyclic AMP (cAMP) pathway plays a central role in the growth, differentiation, and virulence of pathogenic fungi, including Cryptococcus neoformans. Three upstream signaling regulators of adenylyl cyclase (Cac1), Ras, Aca1, and Gpa1, have been demonstrated to control the cAMP pathway in C. neoformans, but their functional relationship remains elusive. We performed a genome-wide transcriptome analysis with a DNA microarray using the ras1Delta, gpa1Delta, cac1Delta, aca1Delta, and pka1Delta pka2Delta mutants. The aca1Delta, gpa1Delta, cac1Delta, and pka1Delta pka2Delta mutants displayed similar transcriptome patterns, whereas the ras1Delta mutant exhibited transcriptome patterns distinct from those of the wild type and the cAMP mutants. Interestingly, a number of environmental stress response genes are modulated differentially in the ras1Delta and cAMP mutants. In fact, the Ras signaling pathway was found to be involved in osmotic and genotoxic stress responses and the maintenance of cell wall integrity via the Cdc24-dependent signaling pathway. Notably, the Ras and cAMP mutants exhibited hypersensitivity to a polyene drug, amphotericin B, without showing effects on ergosterol biosynthesis, which suggested a novel method of antifungal combination therapy. Among the cAMP-dependent gene products that we characterized, two small heat shock proteins, Hsp12 and Hsp122, were found to be involved in the polyene antifungal drug susceptibility of C. neoformans.

Fig. 1.

Genome-wide identification of genes regulated by the Ras and cAMP signaling pathways and their functional categories. (A) Schematic diagram summarizing the known signaling components of the Ras and cAMP signaling pathways in C. neoformans. (B and C) The degree of change (n-fold) in gene expression is illustrated by color coding (see the color bar scale at the bottom of the graph in panel B). (B) Relative expression levels of RAS1, ACA1, GPA1, CAC1, PKA1, and PKA2 genes in the ras1Δ (YSB51), aca1Δ (YSB6), gpa1Δ (YSB83), cac1Δ (YSB42), and pka1Δ pka2Δ (YSB200) mutant backgrounds compared to the WT strain (H99). (C) Hierarchical clustering analysis of 192 genes which are significantly up- or downregulated, by >2-fold (P < 0.05; ANOVA), in at least one of the mutant strains indicated in panel B. (D) Functional categories of genes differentially regulated by the Ras and cAMP pathways. Genes showing expression patterns in the ras1Δ (YSB51), aca1Δ (YSB6), gpa1Δ (YSB83), cac1Δ (YSB42), and pka1Δ pka2Δ (YSB200) mutants significantly different (P < 0.05; ANOVA) from those in the WT were functionally categorized based on KOG functional descriptions (http://www.ncbi.nlm.nih.gov/COG/). Bars indicate the following: yellow, percentages of cAMP-dependent genes; blue, percentages of Ras-dependent genes; white, random occurrence rates for genes in each KOG functional category in the whole C. neoformans genome.

Fig. 2.

Identification of Ras1 signaling-dependent genes in C. neoformans. (A) Relative expression profiles of Ras-dependent genes from C. neoformans, S. cerevisiae, or Schizosaccharomyces pombe which have been identified or characterized and show more than twofold induction or repression in the ras1Δ mutant compared to the WT strain. (B) Relative expression profiles of Ras-dependent genes in C. neoformans which do not have any orthologs or known functions in S. cerevisiae or S. pombe and show more than threefold induction or repression in the ras1Δ mutant compared to the WT strain. In both panels, the degree of change (n-fold) is illustrated by color coding (see the color bar scales at the bottoms of the graphs), and the exact value for each gene is indicated in the table to the right of the hierarchical clustering diagram. Abbreviations are as follows: ESR-up and ESR-down, significant up- and downregulation (more than twofold), respectively, of ESR genes; OxR, oxidative stress response; OsR, osmotic stress response; FxR, fludioxoniol response; ER, endoplasmic reticulum; and ACK, activated Cdc42-associated kinase.

Fig. 3.

The Ras1 signaling pathway controls the osmotic stress response and is required for maintaining cell wall integrity in C. neoformans. Each C. neoformans strain indicated below was grown overnight at 30°C in liquid YPD medium, serially diluted 10-fold (yielding 1- to 10⁴-fold dilutions), and spotted (in 3-μl volumes) onto yeast extract-peptone (YP) or YPD agar containing the indicated concentrations of either NaCl or KCl (A and B), fludioxonil (C and D), and Congo red and SDS (E and F). Cells were incubated at 30°C for 72 h and photographed. (A, C, and E) The WT H99 strain (MATα) and the ras1Δ (YSB53), aca1Δ (YSB6), gpa1Δ (YSB83), cac1Δ (YSB42), pka1Δ (YSB188), pka2Δ (YSB194), and pka1Δ pka2Δ (YSB200) mutant strains were used. In addition, the mpk1Δ (KK3), cna1Δ (KK1), and hog1Δ (YSB64) mutants were used for the analysis in panel E. (B, D, and F) The WT KN99 strain (MATa) and the ras1Δ (YSB73), cac1Δ (YSB183), aca1Δ (YSB176), hog1Δ (YSB81), aca1Δ ras1Δ (YSB175), and cac1Δ ras1Δ (YSB187) mutant stains were used.

Fig. 4.

The Ras1 signaling pathway is involved in oxidative and genotoxic stress responses in C. neoformans. Each C. neoformans strain indicated below was grown overnight at 30°C in liquid YPD medium, serially diluted 10-fold (yielding 1- to 10⁴-fold dilutions), and spotted (in 3-μl volumes) onto YPD agar containing the indicated concentrations of hydrogen peroxide (H₂O₂), the superoxide generator menadione, diamide, and tBOOH (A), cadmium sulfate (B and C), and HU and MMS (D and E). Cells were incubated at 30°C for 72 h and photographed. (A, B, and D) The WT H99 strain (MATα) and the ras1Δ (YSB53), aca1Δ (YSB6), gpa1Δ (YSB83), cac1Δ (YSB42), pka1Δ (YSB188), pka2Δ (YSB194), and pka1Δ pka2Δ (YSB200) mutant strains were used. (C and E) The WT KN99 strain (MATa) and the ras1Δ (YSB73), cac1Δ (YSB183), aca1Δ (YSB176), hog1Δ (YSB81), aca1Δ ras1Δ (YSB175), and cac1Δ ras1Δ (YSB187) mutant stains were used.

Fig. 5.

The Ras1-mediated stress response is controlled by the Cdc24-dependent signaling pathway in C. neoformans. C. neoformans strains (the WT H99 strain, the ras1Δ mutant [CBN45], the ras1Δ RAS1 complemented strain [CBN64], the ras2Δ mutant [MWC12], the cdc24Δ mutant [CBN32], and the cdc24Δ CDC24 complemented strain [CBN33]) were grown overnight at 30°C in liquid YPD medium, serially diluted 10-fold (yielding 1- to 10⁴-fold dilutions), and spotted (in 3-μl volumes) onto YPD agar containing the indicated concentrations of either NaCl or KCl, fludioxonil, Congo red and SDS, H₂O₂, menadione, tBOOH, cadmium sulfate, HU, and MMS. Cells were incubated at 30°C for 72 h and photographed.

Fig. 6.

Identification of cAMP signaling-dependent genes in C. neoformans. (A) Relative expression profiles of cAMP-dependent genes which show more than twofold induction or repression in the aca1Δ, gpa1Δ, cac1Δ, or pka1Δ pka2Δ mutant compared to the WT strain. The degree of change (n-fold) is illustrated by color coding (see the color bar scale at the bottom of the graph), and the exact value for each gene is indicated in the table to the right of the hierarchical clustering diagram. Sce, S. cerevisiae; Cne, C. neoformans; NMDA, N-methyl-d-aspartate. (B and C) C. neoformans strains (the WT H99 strain and the corresponding ras1Δ [YSB53], aca1Δ [YSB6], gpa1Δ [YSB83], cac1Δ [YSB42], pka1Δ [YSB188], pka2Δ [YSB194], and pka1Δ pka2Δ [YSB200] mutant strains [B] and the WT KN99 strain and the corresponding ras1Δ [YSB73], cac1Δ [YSB183], aca1Δ [YSB176], hog1Δ [YSB81], aca1Δ ras1Δ [YSB175], and cac1Δ ras1Δ [YSB187] mutant strains [C]) were grown overnight at 30°C in liquid YPD medium, serially diluted 10-fold (yielding 1- to 10⁴-fold dilutions), and spotted (in 3-μl volumes) onto YPD agar containing the indicated concentrations of MG. Cells were incubated at 30°C for 72 h and photographed.

Fig. 7.

Perturbation of the Ras and cAMP signaling pathways increases polyene drug sensitivity independently of ergosterol biosynthesis. (A, B, C, E, F, G, and H) Each C. neoformans strain indicated below was grown overnight at 30°C in liquid YPD medium, serially diluted 10-fold (yielding 1- to 10⁴-fold dilutions), and spotted (in 3-μl volumes) onto YPD agar containing the indicated concentrations of AmpB, fluconazole, ketoconazole, or itraconazole. Cells were incubated at 30°C for 72 h and photographed. (D) Verification of transcriptional activation of ERG5, ERG25, and ERG11 in the Ras and cAMP pathway mutants by Northern blot analysis. (A, E, F, and G) The WT H99 strain and the ras1Δ (YSB53), aca1Δ (YSB6), gpa1Δ (YSB83), cac1Δ (YSB42), pka1Δ (YSB188), pka2Δ (YSB194), pka1Δ pka2Δ (YSB200), hog1Δ (YSB64), hog1Δ cac1Δ (YSB156), and hog1Δ pka1Δ (YSB112) mutant strains were used. (B) The WT KN99 strain (MATa) and the ras1Δ (YSB73), cac1Δ (YSB183), aca1Δ (YSB176), hog1Δ (YSB81), aca1Δ ras1Δ (YSB175), and cac1Δ ras1Δ (YSB187) mutant stains were used. (C and H) The WT H99 strain, the ras1Δ mutant (CBN45), the ras1Δ RAS1 complemented strain (CBN64), the ras2Δ mutant (MWC12), the cdc24Δ mutant (CBN32), and the cdc24Δ CDC24 complemented strain (CBN33) were used.

Fig. 8.

The heat shock proteins, Hsp12 and Hsp122, are partly involved in polyene sensitivity and is coregulated by the cAMP and HOG signaling pathways. (A) Northern blot analysis showing basal expression levels of GRE2, PKP1, HSP12, and HSP122 in the WT H99 strain and the ras1Δ (YSB51), aca1Δ (YSB6), gpa1Δ (YSB83), cac1Δ (YSB42), and pka1Δ pka2Δ (YSB200) mutants. (B) C. neoformans strains (the WT H99 strain and the ras1Δ [YSB53], cac1Δ [YSB42], hog1Δ [YSB64], hsp12Δ [YSB599 and YSB600], hsp122Δ [YSB603 and YSB604], and hsp12Δ hsp122Δ [YSB757 and YSB758] mutant strains) were grown overnight at 30°C in liquid YPD medium, serially diluted 10-fold (yielding 1- to 10⁴-fold dilutions), spotted (in 3-μl volumes) onto YPD agar containing the indicated concentration of AmpB with or without osmotic stress (0.5 M NaCl), incubated at 30°C for 72 h, and photographed. (C) Northern blot analysis showed basal expression levels of HSP12, HSP122, and GRE2 in the WT H99 strain and the hog1Δ (YSB64), ssk1Δ (YSB261), and skn7Δ (YSB349) mutants.

Fig. 9.

Proposed model for the regulatory mechanisms of the Ras and cAMP signaling pathways in stress response and antifungal drug susceptibility in C. neoformans. The Ras1-dependent signaling pathway promotes cell survival under diverse environmental stresses, such as cell wall destabilization, osmotic shock, oxidative and genotoxic stresses, and high temperature, in a manner mediated by Cdc24 and Cdc42. Ras1 appears to affect resistance to antifungal drugs by maintaining cell wall and membrane integrity. Along with Ras1, Ras2 plays some minor role in controlling antifungal and oxidative stress sensitivity. The cAMP signaling pathway is involved in responses to stress from heavy metals and toxic metabolites. Notably, small heat shock proteins, Hsp12 and Hsp122, are positively regulated by the cAMP signaling pathway and appears to affect resistance against AmpB treatment. However, expression of HSP12 and HSP122 is much more significantly regulated by the HOG pathway.