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 ras1⌬, gpa1⌬, cac1⌬, aca1⌬, and pka1⌬ pka2⌬ mutants. The aca1⌬, gpa1⌬, cac1⌬, and pka1⌬ pka2⌬ mutants displayed similar transcriptome patterns, whereas the ras1⌬ 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 ras1⌬ 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.

Regulatory Circuitry Governing Fungal Development, Drug Resistance, and Disease

“…Ras1-dependent stress control was mediated primarily by Cdc24-dependent signaling, as cdc24 mutants tightly phenocopied the stress response phenotypes exhibited by a ras1 mutant (353). Furthermore, a significant proportion of Ras/cAMP-dependent genes were also controlled by the environmental stress response (353).…”

“…Furthermore, in serotype A strains, the loss of the PKA regulatory subunit Pkr1 results in the overproduction of capsule, hypervirulence, and the suppression of phenotypes observed for a gpa1 mutant (160). Recently, it was shown that serotype A pka2 mutants are hypersensitive to osmotic shock under conditions of glucose starvation (353); hypersensitivity to osmotic shock is abolished by the additional disruption of PKA1, suggesting that Pka1 and Pka2 play opposite roles in the osmostress response (353). One of the key transcription factors that functions downstream of Pka1 in C. neoformans is Nrg1 (121).…”

“…Recently, a comparative transcriptome analysis was conducted with C. neoformans ras1, aca1, gpa1, cac1, and pka1 pka2 mutants to evaluate the functional connections between these signaling components in the cAMP-PKA cascade (353). That study identified numerous Ras1 and cAMP-dependent genes and illustrated that Ras1-mediated signaling was largely independent of the cAMP-PKA pathway.…”

“…That study identified numerous Ras1 and cAMP-dependent genes and illustrated that Ras1-mediated signaling was largely independent of the cAMP-PKA pathway. Ras1 was shown to be a regulator of the osmotic stress response and the oxidative stress response and was required for the maintenance of cell wall integrity (353). Ras1-dependent stress control was mediated primarily by Cdc24-dependent signaling, as cdc24 mutants tightly phenocopied the stress response phenotypes exhibited by a ras1 mutant (353).…”

“…Ras1 was shown to be a regulator of the osmotic stress response and the oxidative stress response and was required for the maintenance of cell wall integrity (353). Ras1-dependent stress control was mediated primarily by Cdc24-dependent signaling, as cdc24 mutants tightly phenocopied the stress response phenotypes exhibited by a ras1 mutant (353). Furthermore, a significant proportion of Ras/cAMP-dependent genes were also controlled by the environmental stress response (353).…”