In Vitro Evolution of Itraconazole Resistance in Aspergillus fumigatus Involves Multiple Mechanisms of Resistance

Abstract

We investigated the evolution of resistance to the antifungal drug itraconazole in replicate populations of Aspergillus fumigatus that were founded from a strain with a genotype of sensitivity to a single drug and then propagated under uniform conditions. For each population, conidia were serially transferred 10 times to agar medium either with or without itraconazole. After 10 transfers in medium supplemented with itraconazole, 10 itraconazole-resistant mutant strains were isolated from two populations. These mutant strains had different growth rates and different levels of itraconazole resistance. Analysis of the ergosterol contents of these mutants showed that they accumulate ergosterol when they are grown in the presence of itraconazole. The replacement of the CYP51A gene of the wild-type strain changed the susceptibility pattern of this strain to one of itraconazole resistance only when CYP51A genes with N22D and M220I mutations were used as selectable marker genes. Real-time quantitative reverse transcription-PCR was used to assess the levels of expression of the Afumdr1, Afumdr2, Afumdr3, Afumdr4, AtrF transporter, CYP51A, and CYP51B genes in these mutant strains. Most mutants showed either constitutive high-level expression or induction upon exposure of Afumdr3, Afumdr4, and AtrF to itraconazole. Our results suggest that overexpression of drug efflux pumps and/or selection of drug target site mutations are at least partially responsible for itraconazole resistance and could be considered mechanisms for the emergence of clinical resistance to this drug.

Stress, Drugs, and Evolution: the Role of Cellular Signaling in Fungal Drug Resistance

“…Most mutants showed constitutive overexpression of mdr3 and mdr4, and some showed overexpression of atrF. In the presence of itraconazole, gene expression increased in most resistant strains, but not all, again suggesting multiple mechanisms (47).…”

Regulatory Circuitry Governing Fungal Development, Drug Resistance, and Disease

“…The MDR pumps are also involved in A. fumigatus azole resistance. In itraconazole-resistant mutants selected in vitro, mdr3, mdr4, and atrF showed pronounced changes in expression in many evolved mutants, with either constitutively high levels of expression of these transcripts or an induction of expression upon exposure to itraconazole (132,411). Some resistant mutants also showed increased expression levels of mdr1 or mdr2, although this expression change is very uncommon (132).…”

“…Furthermore, ⌬cnaA mutants lacking the calcineurin catalytic subunit display defective hyphal morphology, including improper polarized growth and overall decreased filamentation (132,557). These mutants have altered sporulation and abnormal conidial morphology as well as decreased virulence in several independent models of A. fumigatus infection (132,557).…”

An Invisible Threat: Mutation-Mediated Resistance to Triazole Drugs in Aspergillus

“…Slaven et al demonstrated that increased expression of atrF, an A. fumigatus ABC transporter gene, conferred resistance to itraconazole [82]. Moreover, da Silva Ferriera et al demonstrated that mRNA levels of A. fumigatus ABC genes Afumdr1 Afumdr2, Afumdr4, and AtrF as well as the MFS gene Afumdr3 increased from 0.2 to 23-fold, depending on the gene, when ITZ was included in the growth medium [83]. Using restriction-mediated plasmid integration (REMI) Bowyer et al demonstrated that disruption of an ABC transporter gene led to the hyper-sensitization to triazoles in the affected A. fumigatus strain [84].…”

“…In addition to the above clinical observations, triazole-mediated cyp51A mutations have also been induced in vitro. To demonstrate this point, da Silva Ferreira et al propagated Cyp51A wild type A. fumigatus on medium containing 1μg/ml of ITZ [71]. After 10 transfers, G54R…”