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Novel ABC Transporter Associated with Fluconazole Resistance in Aging of Cryptococcus neoformans.

ABSTRACT: Cryptococcus neoformans causes meningoencephalitis in immunocompromised individuals, which is treated with fluconazole (FLC) monotherapy when resources are limited. This can lead to azole resistance, which can be mediated by overexpression of ABC transporters, a class of efflux pumps. ABC pump-mediated efflux of FLC is also augmented in 10-generation old C. neoformans cells. Here, we describe a new ABC transporter Afr3 (CNAG_06909), which is overexpressed in C. neoformans cells of advanced generational age that accumulate during chronic infection. The Δafr3 mutant strain showed higher FLC susceptibility by FLC E-Test strip testing and also by a killing test that measured survival after 3 h FLC exposure. Furthermore, Δafr3 cells exhibited lower Rhodamine 6G efflux compared to the H99 wild-type cells. Afr3 was expressed in the Saccharomyces cerevisiae ADΔ strain, which lacks several drug transporters, thus reducing background transport. The ADΔ + Afr3 strain demonstrated a higher efflux with both Rhodamine 6G and Nile red, and a higher FLC resistance. Afr3-GFP localized in the plasma membrane of the ADΔ + Afr3 strain, further highlighting its importance as an efflux pump. Characterization of the Δafr3 mutant revealed unattenuated growth but a prolongation (29%) of the replicative life span. In addition, Δafr3 exhibited decreased resistance to macrophage killing and attenuated virulence in the Galleria mellonella infection model. In summary, our data indicate that a novel ABC pump Afr3, which is upregulated in C. neoformans cells of advanced age, may contribute to their enhanced FLC tolerance, by promoting drug efflux. Lastly, its role in macrophage resistance may also contribute to the selection of older C. neoformans cells during chronic infection.

SUBMITTER: Oliveira NK

PROVIDER: S-EPMC9320417 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description: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.

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.

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Novel ABC Transporter Associated with Fluconazole Resistance in Aging of Cryptococcus neoformans.

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