Involvement of PDK1, PKC and TOR signalling pathways in basal fluconazole tolerance in Cryptococcus neoformans

Abstract

Summary This study shows the importance of PDK1, TOR and PKC signaling pathways to the basal tolerance of Cryptococcus neoformans toward fluconazole, the widely used drug for treatment of cryptococcosis. Mutations in genes integral to these pathway resulted in hypersensitivity to the drug. Upon fluconazole treatment, Mpk1, the downstream target of PKC was phosphorylated and its phosphorylation required Pdk1. We show genetically that the PDK1 and TOR phosphorylation sites in Ypk1 as well as the kinase activity of Ypk1 are required for the fluconazole basal tolerance. The involvement of these pathways in fluconazole basal tolerance was associated with sphingolipid homeostasis. Deletion of PDK1, SIN1, or YPK1 but not MPK1 affected cell viability in the presence of sphingolipid biosynthesis inhibitors. Concurrently, pdk1Δ, sinΔ1, ypk1Δ, and mpk1Δ exhibited altered sphingolipid content and elevated fluconazole accumulation compared with the wild-type. The fluconazole hypersensitivity phenotype of these mutants, therefore, appears to be the result of malfunction of the influx/efflux systems due to modifications of membrane sphingolipid content. Interestingly, the reduced virulence of these strains in mice suggests that the cryptococcal PDK1, PKC, and likely the TOR pathways play an important role in managing stress exerted either by fluconazole or by the host environment.

Cryptococcus neoformans Phosphoinositide-Dependent Kinase 1 (PDK1) Ortholog Is Required for Stress Tolerance and Survival in Murine Phagocytes

“…In the absence of cell wall stress, Mpk1 phosphorylation is mark- edly reduced in the pkh2-02⌬ mutant compared to the WT and the revertant (Fig. 2C), a finding consistent with the data reported by Lee et al (32). The addition of CFW to the cultures increased Mpk1 phosphorylation in both the WT and the pkh2-02⌬:: PKH2-02 revertant mutant (37,44), but only a barely detectable amount of phosphorylated Mpk1 was observed in the pkh2-02⌬ mutant (Fig.…”

“…2A) compared to congenic wild-type strains. This is also consistent with the observations of Lee et al (32) and with the fact that the PDK1/Pkh kinases have been linked to the TOR pathway in both mammalian and S. cerevisiae systems.…”

“…1A), while the pkh2-01⌬ mutant showed no growth defect at elevated temperature (data not shown). Lee et al also observed identical phenotypes for the CM108-derived pkh2-01⌬ and pkh2-02⌬ mutants (32). In the KN99␣ background, the pkh2-02⌬ mutant was also sensitive to high-temperature growth, but the phenotype did not manifest until 39°C (data not shown).…”

“…In addition, Lee et al found that activation of the cell wall integrity (CWI) mitogen-activated protein kinase (MAPK) cascade by fluconazole was dependent on PKH2-02 and that pkh2-02⌬ mutants have altered sphingolipid profiles. They also provided data that suggest that PKH2-02-dependent fluconazole tolerance is mediated by phosphorylation of Ypk1 (32).…”

“…As our work was in progress, Lee et al reported a series of experiments investigating the role of PKHs in fluconazole susceptibility and renamed PKH2-02 as PDK1; they also suggested that because the PKH2-01 product does not have a clearly definable catalytic domain, it may not be a true PDK1/Pkh kinase (32). Deletion of C. neoformans PKH2-02 (CnPKH2-02) led to decreased fluconazole tolerance as well as hypersensitivity to high salt, rapamycin, aureobasidin A, myriocin, and phytosphingosine.…”