Mutations in Aspergillus fumigatus Resulting in Reduced Susceptibility to Posaconazole Appear To Be Restricted to a Single Amino Acid in the Cytochrome P450 14α-Demethylase

Abstract

To better understand the molecular basis of posaconazole (POS) resistance in Aspergillus fumigatus, resistant laboratory isolates were selected. Spontaneous mutants arose at a frequency of 1 in 10 8 and fell into two susceptibility groups, moderately resistant and highly resistant. Azole resistance in A. fumigatus was previously associated with decreased drug accumulation. We therefore analyzed the mutants for changes in levels of transcripts of genes encoding efflux pumps (mdr1 and mdr2) and/or alterations in accumulation of [ 14 C]POS. No changes in either pump expression or drug accumulation were detected. Similarly, there was no change in expression of cyp51A or cyp51B, which encode the presumed target site for POS, cytochrome P450 14␣-demethylase. DNA sequencing revealed that each resistant isolate carried a single point mutation in residue 54 of cyp51A. Mutations at the same locus were identified in three clinical A. fumigatus isolates exhibiting reduced POS susceptibility but not in susceptible clinical strains. To verify that these mutations were responsible for the resistance phenotype, we introduced them into the chromosome of a POS-susceptible A. fumigatus strain under the control of the glyceraldehyde phosphate dehydrogenase promoter. The transformants exhibited reductions in susceptibility to POS comparable to those exhibited by the original mutants, confirming that point mutations in the cyp51A gene in A. fumigatus can confer reduced susceptibility to POS.

Regulatory Circuitry Governing Fungal Development, Drug Resistance, and Disease

“…The first mutation identified to have a role in A. fumigatus azole resistance was the cyp51A glycine 54 (G54) point mutation, which was detected in both laboratory-evolved posaconazole-resistant strains as well as clinical isolates with reduced posaconazole susceptibility (362). This same mutation was also found to contribute to itraconazole resistance in clinical isolates (152).…”

Structural Insights into Inhibition of Sterol 14α-Demethylase in the Human Pathogen Trypanosoma cruzi

“…Repositioning the rigid I helix that is observed in all inhibitor bound trypanosomal 14DMs is marked with a blue arrow 3. the available shape within the enzyme binding cleft and potentially contributing to its inhibitory potency. Location of posaconazole in the TC14DM structure provides a possible explanation to posaconazole resistance observed in some strains (clinical isolates) of a pathogenic fungus Aspergillus fumigatus (27), where single point mutations resulted in substitutions of Gly 54 (aligns with Gly 49 in TC14DM) to arginine or tryptophan have been detected. Assuming that the fungal 14DM structure is similar to that of TC14DM, the introduction of a bulky and less flexible residue in this position might narrow the channel entrance and affect the surface interactions (Fig.…”

“…Moreover, easily induced resistance to fluconazole was demonstrated in the laboratory strains of TC (26). Posaconazole has been reported not to induce the efflux pump mechanism (24); however, sparse cases of resistance associated with point mutations in the fungal 14DM have been observed (27).…”

Current Concepts in Antifungal Pharmacology

“…20 For molecules derived from ketoconazole (ie, itraconazole, posaconazole), extension of the nonpolar side chains enhances azole binding to the 14α-demethylase apoprotein, resulting in an enhanced spectrum of activity against molds ( Figure 3). 21 Voriconazole, a derivative of fluconazole, possesses an α-o-methyl group that confers activity against Aspergillus species and other filamentous fungi. 21,22 Resistance to triazole antifungal agents is most commonly the result of mutations in the azole binding pocket of 14α-demethylase 21,22 and/or the overexpression of MDR1 efflux pumps that expel fluconazole or the multidrug adenosine triphosphate-dependent efflux pumps CDR1 and CDR2, which expel all triazoles, thereby leading to cross-resistance.…”

“…21 Voriconazole, a derivative of fluconazole, possesses an α-o-methyl group that confers activity against Aspergillus species and other filamentous fungi. 21,22 Resistance to triazole antifungal agents is most commonly the result of mutations in the azole binding pocket of 14α-demethylase 21,22 and/or the overexpression of MDR1 efflux pumps that expel fluconazole or the multidrug adenosine triphosphate-dependent efflux pumps CDR1 and CDR2, which expel all triazoles, thereby leading to cross-resistance. 3 Because intrinsic resistance in C krusei is a result of impaired binding of fluconazole to 14α-demethylase, newer triazoles with enhanced binding to the enzyme retain activity against fluconazoleresistant strains such as C krusei.…”