Cryptococcus neoformans Overcomes Stress of Azole Drugs by Formation of Disomy in Specific Multiple Chromosomes

Abstract

Cryptococcus neoformans is a haploid environmental organism and the major cause of fungal meningoencephalitis in AIDS patients. Fluconazole (FLC), a triazole, is widely used for the maintenance therapy of cryptococcosis. Heteroresistance to FLC, an adaptive mode of azole resistance, was associated with FLC therapy failure cases but the mechanism underlying the resistance was unknown. We used comparative genome hybridization and quantitative real-time PCR in order to show that C. neoformans adapts to high concentrations of FLC by duplication of multiple chromosomes. Formation of disomic chromosomes in response to FLC stress was observed in both serotype A and D strains. Strains that adapted to FLC concentrations higher than their minimal inhibitory concentration (MIC) contained disomies of chromosome 1 and stepwise exposure to even higher drug concentrations induced additional duplications of several other specific chromosomes. The number of disomic chromosomes in each resistant strain directly correlated with the concentration of FLC tolerated by each strain. Upon removal of the drug pressure, strains that had adapted to high concentrations of FLC returned to their original level of susceptibility by initially losing the extra copy of chromosome 1 followed by loss of the extra copies of the remaining disomic chromosomes. The duplication of chromosome 1 was closely associated with two of its resident genes: ERG11, the target of FLC and AFR1, the major transporter of azoles in C. neoformans. This adaptive mechanism in C. neoformans may play an important role in FLC therapy failure of cryptococcosis leading to relapse during azole maintenance therapy.

Aneuploidy underlies a multicellular phenotypic switch

“…In either case, the fact that aneuploidy provides cells multiple routes to the same phenotypic endpoint has fundamental implications for understanding how organisms respond to environmental perturbations. Because aneuploidy is strongly correlated with drug resistance in pathogenic microorganisms (40)(41)(42)(43), rampant in cancer (44), and associated with high levels of cancer relapse after drug treatment (45), aneuploidy itself could be an important target for the development of novel antifungal and cancer therapeutics (44). The complexity of the problem is compounded by the fact that the genome sequence (genetic background) of an individual has the potential to affect not only the traits displayed by aneuploid cells, but also how well specific aneuploidies are tolerated.…”

Regulatory Circuitry Governing Fungal Development, Drug Resistance, and Disease

“…In that study it was discovered that adaptive resistance to the azoles was achieved by the duplication of multiple chromosomes in response to fluconazole (538). A duplication of chromosome 1 was common, resulting in an increased copy number of the gene encoding the azole target Erg11, which resides on chromosome 1 (538).…”

Mechanisms of Antifungal Drug Resistance

“…For C. neoformans, azole resistance is associated with specific chromosome alterations, especially disomies of chromosomes 1 and 4. Chromosome 1 contains two azole-resistance determinants, the azole target gene ERG11 and ABC transporter gene AFR1 (Sionov et al 2010). Alteration in copy number of these chromosomes also relates to the phenomenon of heteroresistance, in which subpopulations within the same clone vary in resistance based on the frequent loss and gain of chromosomes in response to selection (Sionov et al 2010(Sionov et al , 2013.…”