Dataset Information

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

ABSTRACT: This study shows the importance of PDK1, TOR and PKC signalling pathways to the basal tolerance of Cryptococcus neoformans towards 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, pdk1?, sin1?, ypk1? and mpk1? 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.

SUBMITTER: Lee H

PROVIDER: S-EPMC3313003 | biostudies-literature | 2012 Apr

REPOSITORIES: biostudies-literature

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

Lee Hyeseung H Khanal Lamichhane Ami A Garraffo H Martin HM Kwon-Chung Kyung J KJ Chang Yun C YC

Molecular microbiology 20120315 1

This study shows the importance of PDK1, TOR and PKC signalling pathways to the basal tolerance of Cryptococcus neoformans towards 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 ...[more]

PMID: 22339665

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Project description:Resistance to fluconazole (FLC), the most widely used antifungal drug, is typically achieved by altering the azole drug target and/or drug efflux pumps. Recent reports have suggested a link between vesicular trafficking and antifungal resistance. Here, we identified novel Cryptococcus neoformans regulators of extracellular vesicle (EV) biogenesis that impact FLC resistance. In particular, the transcription factor Hap2 does not affect the expression of the drug target or efflux pumps, yet it impacts the cellular sterol profile. Subinhibitory FLC concentrations also downregulate EV production. Moreover, in vitro spontaneous FLC-resistant colonies showed altered EV production, and the acquisition of FLC resistance was associated with decreased EV production in clinical isolates. Finally, the reversion of FLC resistance was associated with increased EV production. These data suggest a model in which fungal cells can regulate EV production in place of regulating the drug target gene expression as a first line of defense against antifungal assault in this fungal pathogen. IMPORTANCE Extracellular vesicles (EVs) are membrane-enveloped particles that are released by cells into the extracellular space. Fungal EVs can mediate community interactions and biofilm formation, but their functions remain poorly understood. Here, we report the identification of the first regulators of EV production in the major fungal pathogen Cryptococcus neoformans. Surprisingly, we uncover a novel role of EVs in modulating antifungal drug resistance. Disruption of EV production was associated with altered lipid composition and changes in fluconazole susceptibility. Spontaneous azole-resistant mutants were deficient in EV production, while loss of resistance restored initial EV production levels. These findings were recapitulated in C. neoformans clinical isolates, indicating that azole resistance and EV production are coregulated in diverse strains. Our study reveals a new mechanism of drug resistance in which cells adapt to azole stress by modulating EV production.

Project description:BackgroundFluconazole is used in combination with amphotericin B for induction treatment of cryptococcal meningitis and as monotherapy for consolidation and maintenance treatment. More than 90% of isolates from first episodes of cryptococcal disease had a fluconazole minimum inhibitory concentration (MIC) ≤4 μg/ml in a Gauteng population-based surveillance study of Cryptococcus neoformans in 2007-2008. We assessed whether fluconazole resistance had emerged in clinical cryptococcal isolates over a decade.Methodology and principal findingsWe prospectively collected C. neoformans isolates from 1 January through 31 March 2017 from persons with a first episode of culture-confirmed cryptococcal disease at 37 South African hospitals. Isolates were phenotypically confirmed to C. neoformans species-complex level. We determined fluconazole MICs (range: 0.125 μg/ml to 64 μg/ml) of 229 C. neoformans isolates using custom-made broth microdilution panels prepared, inoculated and read according to Clinical and Laboratory Standards Institute M27-A3 and M60 recommendations. These MIC values were compared to MICs of 249 isolates from earlier surveillance (2007-2008). Clinical data were collected from patients during both surveillance periods. There were more males (61% vs 39%) and more participants on combination induction antifungal treatment (92% vs 32%) in 2017 compared to 2007-2008. The fluconazole MIC50, MIC90 and geometric mean MIC was 4 μg/ml, 8 μg/ml and 4.11 μg/ml in 2017 (n = 229) compared to 1 μg/ml, 2 μg/ml and 2.08 μg/ml in 2007-2008 (n = 249) respectively. Voriconazole, itraconazole and posaconazole Etests were performed on 16 of 229 (7%) C. neoformans isolates with a fluconazole MIC value of ≥16 μg/ml; only one had MIC values of >32 μg/ml for these three antifungal agents.Conclusions and significanceFluconazole MIC50 and MIC90 values were two-fold higher in 2017 compared to 2007-2008. Although there are no breakpoints, higher fluconazole doses may be required to maintain efficacy of standard treatment regimens for cryptococcal meningitis.

Dataset Information

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

Publications

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

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