Dataset Information

Deletion of Cryptococcus neoformans AIF ortholog promotes chromosome aneuploidy and fluconazole-resistance in a metacaspase-independent manner.

ABSTRACT: Apoptosis is a form of programmed cell death critical for development and homeostasis in multicellular organisms. Apoptosis-like cell death (ALCD) has been described in several fungi, including the opportunistic human pathogen Cryptococcus neoformans. In addition, capsular polysaccharides of C. neoformans are known to induce apoptosis in host immune cells, thereby contributing to its virulence. Our goals were to characterize the apoptotic signaling cascade in C. neoformans as well as its unique features compared to the host machinery to exploit the endogenous fungal apoptotic pathways as a novel antifungal strategy in the future. The dissection of apoptotic pathways revealed that apoptosis-inducing factor (Aif1) and metacaspases (Mca1 and Mca2) are independently required for ALCD in C. neoformans. We show that the apoptotic pathways are required for cell fusion and sporulation during mating, indicating that apoptosis may occur during sexual development. Previous studies showed that antifungal drugs induce ALCD in fungi and that C. neoformans adapts to high concentrations of the antifungal fluconazole (FLC) by acquisition of aneuploidy, especially duplication of chromosome 1 (Chr1). Disruption of aif1, but not the metacaspases, stimulates the emergence of aneuploid subpopulations with Chr1 disomy that are resistant to fluconazole (FLC(R)) in vitro and in vivo. FLC(R) isolates in the aif1 background are stable in the absence of the drug, while those in the wild-type background readily revert to FLC sensitivity. We propose that apoptosis orchestrated by Aif1 might eliminate aneuploid cells from the population and defects in this pathway contribute to the selection of aneuploid FLC(R) subpopulations during treatment. Aneuploid clinical isolates with disomies for chromosomes other than Chr1 exhibit reduced AIF1 expression, suggesting that inactivation of Aif1 might be a novel aneuploidy-tolerating mechanism in fungi that facilitates the selection of antifungal drug resistance.

SUBMITTER: Semighini CP

PROVIDER: S-EPMC3219705 | biostudies-literature | 2011 Nov

REPOSITORIES: biostudies-literature

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Deletion of Cryptococcus neoformans AIF ortholog promotes chromosome aneuploidy and fluconazole-resistance in a metacaspase-independent manner.

Semighini Camile P CP Averette Anna F AF Perfect John R JR Heitman Joseph J

PLoS pathogens 20111117 11

Apoptosis is a form of programmed cell death critical for development and homeostasis in multicellular organisms. Apoptosis-like cell death (ALCD) has been described in several fungi, including the opportunistic human pathogen Cryptococcus neoformans. In addition, capsular polysaccharides of C. neoformans are known to induce apoptosis in host immune cells, thereby contributing to its virulence. Our goals were to characterize the apoptotic signaling cascade in C. neoformans as well as its unique ...[more]

PMID: 22114551

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Project description:The high morbidity and mortality of cryptococcal meningitis is due to the limited range of therapeutic options: only three classes of antifungal drugs are available (polyenes [amphotericin B], azoles [fluconazole], and pyrimidine analogues [flucytosine]). Fluconazole is the most widely used antifungal drug in sub-Saharan Africa, where cryptococcal meningitis is a major cause of death in patients infected with HIV. In this study, we found that exposure to fluconazole, even for short times (48 h) at subinhibitory concentrations, drove rapid adaptation of Cryptococcus neoformans serotype A strain H99 via the acquisition of different aneuploid chromosomes. These aneuploidies conferred heteroresistance to fluconazole. Importantly, most of the adaptors were cross-tolerant to flucytosine. Some of the aneuploid adaptors were not heteroresistant to fluconazole but were tolerant to amphotericin B. Thus, exposure to one antifungal drug class can promote adaptation to two antifungal drug classes, highlighting the plasticity of the C. neoformans genome and raising concerns about the rapid reduction in the range of treatment options for cryptococcal infections. IMPORTANCE Cryptococcosis is a globally distributed invasive fungal infection caused by infections with Cryptococcus neoformans or Cryptococcus gattii. Only three classes of therapeutic drugs are clinically available for treating cryptococcosis: polyenes (amphotericin B), azoles (fluconazole), and pyrimidine analogues (flucytosine). Fluconazole is the primary drug available in resource-limited countries. Aneuploidy is a genomic state due to the gain or loss of chromosomes. We found that C. neoformans rapidly adapted to fluconazole by acquiring diverse aneuploidies and that specific aneuploidies enabled improved growth of isolates susceptible (tolerance) to amphotericin B and/or cross-tolerance to both fluconazole and flucytosine. Therefore, aneuploidy is an underlying mechanism of drug tolerance that not only arises rapidly during growth in fluconazole but can also confer tolerance to other antifungal drugs without prior exposure to those drugs. Resistant isolates have high MICs, and all cells grow similarly in medium with the drug, while tolerant isolates test as susceptible and grow slowly at drug concentrations above the MIC.

Project description:Heteroresistance in Cryptococcus neoformans is an intrinsic adaptive resistance to azoles and the heteroresistant phenotype is associated with disomic chromosomes. Two chromosome 1 (Chr1) genes, ERG11, the fluconazole target, and AFR1, a drug transporter, were reported as major factors in the emergence of Chr1 disomy. In the present study, we show Chr4 to be the second most frequently formed disomy at high concentrations of fluconazole (FLC) and characterize the importance of resident genes contributing to disomy formation. We deleted nine Chr4 genes presumed to have functions in ergosterol biosynthesis, membrane composition/integrity or drug transportation that could influence Chr4 disomy under FLC stress. Of these nine, disruption of three genes homologous to Sey1 (a GTPase), Glo3 and Gcs2 (the ADP-ribosylation factor GTPase activating proteins) significantly reduced the frequency of Chr4 disomy in heteroresistant clones. Furthermore, FLC resistant clones derived from sey1?glo3? did not show disomy of either Chr4 or Chr1 but instead had increased the copy number of the genes proximal to ERG11 locus on Chr1. Since the three genes are critical for the integrity of endoplasmic reticulum (ER) in Saccharomyces cerevisiae, we used Sec61ß-GFP fusion as a marker to study the ER in the mutants. The cytoplasmic ER was found to be elongated in sey1? but without any discernable alteration in gcs2? and glo3? under fluorescence microscopy. The aberrant ER morphology of all three mutant strains, however, was discernable by transmission electron microscopy. A 3D reconstruction using Focused Ion Beam Scanning Electron Microscopy (FIB-SEM) revealed considerably reduced reticulation in the ER of glo3? and gcs2? strains. In sey1?, ER reticulation was barely detectable and cisternae were expanded extensively compared to the wild type strains. These data suggest that the genes required for maintenance of ER integrity are important for the formation of disomic chromosomes in C. neoformans under azole stress.

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

Deletion of Cryptococcus neoformans AIF ortholog promotes chromosome aneuploidy and fluconazole-resistance in a metacaspase-independent manner.

Publications

Deletion of Cryptococcus neoformans AIF ortholog promotes chromosome aneuploidy and fluconazole-resistance in a metacaspase-independent manner.

Similar Datasets

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