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

Molecular Microbiology Section, Laboratory of Clinical Infectious Diseases, National Institute of Allergy and Infectious Diseases, NIH, Bethesda, Maryland, United States of America.
PLoS Pathogens (Impact Factor: 7.56). 04/2010; 6(4):e1000848. DOI: 10.1371/journal.ppat.1000848
Source: PubMed


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.

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Available from: Kyung J Kwon-Chung, Feb 17, 2015
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    • "Twelve of the 34 lines were aneuploid, and each of these 12 lines contained either chrVIII aneuploidy (5 lines) or chrII aneuploidy (8 lines). Chromosomal aneuploidy seems to be a common route to adaptation for fungal species reproducing asexually in a diverse array of environmental stressors such as drug resistance (Selmecki et al. 2009; Sionov et al. 2010), high temperature (Yona et al. 2012), and salt (Dhar et al. 2011). Aneuploidy is an intriguing beneficial mutation, as it has the potential to affect many genes simultaneously , yet has a much higher reversion rate than other types of mutations. "
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    ABSTRACT: Copper is a micronutrient essential for growth due to its role as a co-factor in enzymes involved in respiration, defense against oxidative damage, and iron uptake. Yet too much of a good thing can be lethal, and yeast cells typically do not have tolerance to copper levels much beyond the concentration in their ancestral environment. Here, we report a short-term evolutionary study of Saccharomyces cerevisiae exposed to levels of copper sulfate that are inhibitory to the initial strain. We isolated and identified adaptive mutations soon after they arose, reducing the number of neutral mutations, to determine the first genetic steps that yeast take when adapting to copper. We analyzed 34 such strains through whole-genome sequencing and by assaying fitness within different environments; we also isolated a subset of mutations through tetrad analysis of four lines. We identified a multi-layered evolutionary response. In total, 57 single base-pair mutations were identified across the 34 lines. In addition, gene amplification of the copper metallothionein protein, CUP1, was rampant, as was chromosomal aneuploidy. Four other genes received multiple, independent mutations in different lines (the vacuolar transporter genes VTC1 and VTC4; the plasma membrane H+-ATPase PMA1; and MAM3, a protein required for normal mitochondrial morphology). Analyses indicated that mutations in all four genes, as well as CUP1 copy number, contributed significantly to explaining variation in copper tolerance. Our study thus finds that evolution takes both common and less trodden pathways toward evolving tolerance to an essential, but highly toxic, micronutrient. Copyright © 2014, The Genetics Society of America.
    Genetics 12/2014; 199(2). DOI:10.1534/genetics.114.171124 · 5.96 Impact Factor
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    • "Treatment of haploid C. neoformans cells with fluconazole resulted in disomy of up to four chromosomes (Chrs 1, 4, 10, and 14) (Sionov et al. 2009, 2010). Chr1 disomy was common to all resistant isolates and included the genes encoding Erg11 and the major azole transporter, Afr1 (Sionov et al. 2010). Genes on Chr4 also were linked to drug resistance. "
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    ABSTRACT: Human fungal pathogens can exist in a variety of ploidy states, including euploid and aneuploid forms. Ploidy change has a major impact on phenotypic properties, including the regulation of interactions with the human host. In addition, the rapid emergence of drug-resistant isolates is often associated with the formation of specific supernumerary chromosomes. Pathogens such as Candida albicans and Cryptococcus neoformans appear particularly well adapted for propagation in multiple ploidy states with novel pathways driving ploidy variation. In both species, heterozygous cells also readily undergo loss of heterozygosity (LOH), leading to additional phenotypic changes such as altered drug resistance. Here, we examine the sexual and parasexual cycles that drive ploidy variation in human fungal pathogens and discuss ploidy and LOH events with respect to their far-reaching roles in fungal adaptation and pathogenesis.
    Cold Spring Harbor Perspectives in Medicine 07/2014; 4(10). DOI:10.1101/cshperspect.a019604 · 9.47 Impact Factor
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    • "The molecular mechanism that lies behind the recent emergence of drug-resistant phenotype among Sporothrix species is currently unknown. However, judging from other fungi, the increased and prolonged use of triazoles has raised concerns about resistant infections by Cryptococcus neoformans [38], Candida albicans [39] and Aspergillus fumigatus [40]. Azole resistance may occur through a diversity of mechanisms including the upregulation of multidrug transporter genes that leads to enhanced efflux of azoles and therefore reduce drug accumulation [41]; multiple genetic alterations of the target enzyme that can affect the affinity of the enzyme and therefore prevents azole binding [42], and alteration of metabolism, usually sterol synthesis [43]. "
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    BMC Infectious Diseases 04/2014; 14(1):219. DOI:10.1186/1471-2334-14-219 · 2.61 Impact Factor
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