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Adaptation to Fluconazole via Aneuploidy Enables Cross-Adaptation to Amphotericin B and Flucytosine in Cryptococcus
Feng Yang1,2, Vladimir Gritsenko2, Hui Lu1
1Department of Pharmacy, Shanghai Tenth People's Hospital, School of Medicine, Tongji University, Shanghai, China.
Abstract:
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.
Insights
Exposure to fluconazole rapidly adapted Cryptococcus neoformans through aneuploidy, conferring tolerance to other antifungal drugs. This genomic plasticity raises concerns for cryptococcal meningitis treatment options.
Area of Science:
- Medical Mycology
- Antimicrobial Resistance
- Genomics
Background:
- Cryptococcal meningitis has high mortality due to limited antifungal drug options.
- Fluconazole is a key treatment in resource-limited settings, particularly for HIV-infected individuals.
- Cryptococcus neoformans adaptation to antifungals is a significant clinical challenge.
Purpose of the Study:
- To investigate the rapid adaptation mechanisms of Cryptococcus neoformans to fluconazole.
- To determine if aneuploidy confers tolerance to other antifungal drug classes.
- To assess the implications of these findings for cryptococcosis treatment.
Main Methods:
- Exposing Cryptococcus neoformans serotype A strain H99 to subinhibitory concentrations of fluconazole.
- Analyzing genomic changes, specifically aneuploidies, in adapted isolates.
- Testing the drug susceptibility and cross-tolerance of adapted strains to fluconazole, flucytosine, and amphotericin B.
Main Results:
- Short-term fluconazole exposure induced rapid aneuploid adaptation in C. neoformans.
- Aneuploidies conferred heteroresistance to fluconazole and cross-tolerance to flucytosine.
- Some aneuploid strains showed tolerance to amphotericin B without prior exposure.
Conclusions:
- Aneuploidy is a key mechanism for rapid antifungal drug tolerance in C. neoformans.
- Adaptation to one drug can confer tolerance to multiple antifungal classes, limiting therapeutic options.
- The genomic plasticity of C. neoformans underscores the urgent need for novel therapeutic strategies.
