Dataset Information

Structural basis of HapEP88L-linked antifungal triazole resistance in Aspergillus fumigatus.

ABSTRACT: Azoles are first-line therapeutics for human and plant fungal infections, but their broad use has promoted the development of resistances. Recently, a pan-azole-resistant clinical Aspergillus fumigatus isolate was identified to carry the mutation P88L in subunit HapE of the CCAAT-binding complex (CBC), a conserved eukaryotic transcription factor. Here, we define the mechanistic basis for resistance in this isolate by showing that the HapEP88L mutation interferes with the CBC's ability to bend and sense CCAAT motifs. This failure leads to transcriptional derepression of the cyp51A gene, which encodes the target of azoles, the 14-? sterol demethylase Cyp51A, and ultimately causes drug resistance. In addition, we demonstrate that the CBC-associated transcriptional regulator HapX assists cyp51A repression in low-iron environments and that this iron-dependent effect is lost in the HapEP88L mutant. Altogether, these results indicate that the mutation HapEP88L confers increased resistance to azoles compared with wt A. fumigatus, particularly in low-iron clinical niches such as the lung.

SUBMITTER: Hortschansky P

PROVIDER: S-EPMC7266990 | biostudies-literature | 2020 Jul

REPOSITORIES: biostudies-literature

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Structural basis of HapEP88L-linked antifungal triazole resistance in Aspergillus fumigatus.

Hortschansky Peter P Misslinger Matthias M Mörl Jasmin J Gsaller Fabio F Bromley Michael J MJ Brakhage Axel A AA Groll Michael M Haas Hubertus H Huber Eva M EM

Life science alliance 20200528 7

Azoles are first-line therapeutics for human and plant fungal infections, but their broad use has promoted the development of resistances. Recently, a pan-azole-resistant clinical Aspergillus fumigatus isolate was identified to carry the mutation P88L in subunit HapE of the CCAAT-binding complex (CBC), a conserved eukaryotic transcription factor. Here, we define the mechanistic basis for resistance in this isolate by showing that the HapEP88L mutation interferes with the CBC's ...[more]

PMID: 32467317

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Project description:BACKGROUND:Azoles play an important role in the management of Aspergillus diseases. Azole resistance is an emerging global problem in Aspergillus fumigatus, and may develop through patient therapy. In addition, an environmental route of resistance development has been suggested through exposure to 14?-demethylase inhibitors (DMIs). The main resistance mechanism associated with this putative fungicide-driven route is a combination of alterations in the Cyp51A-gene (TR(34)/L98H). We investigated if TR(34)/L98H could have developed through exposure to DMIs. METHODS AND FINDINGS:Thirty-one compounds that have been authorized for use as fungicides, herbicides, herbicide safeners and plant growth regulators in The Netherlands between 1970 and 2005, were investigated for cross-resistance to medical triazoles. Furthermore, CYP51-protein homology modeling and molecule alignment studies were performed to identify similarity in molecule structure and docking modes. Five triazole DMIs, propiconazole, bromuconazole, tebuconazole, epoxiconazole and difenoconazole, showed very similar molecule structures to the medical triazoles and adopted similar poses while docking the protein. These DMIs also showed the greatest cross-resistance and, importantly, were authorized for use between 1990 and 1996, directly preceding the recovery of the first clinical TR(34)/L98H isolate in 1998. Through microsatellite genotyping of TR(34)/L98H isolates we were able to calculate that the first isolate would have arisen in 1997, confirming the results of the abovementioned experiments. Finally, we performed induction experiments to investigate if TR(34)/L98H could be induced under laboratory conditions. One isolate evolved from two copies of the tandem repeat to three, indicating that fungicide pressure can indeed result in these genomic changes. CONCLUSIONS:Our findings support a fungicide-driven route of TR(34)/L98H development in A. fumigatus. Similar molecule structure characteristics of five triazole DMIs and the three medical triazoles appear the underlying mechanism of cross resistance development. Our findings have major implications for the assessment of health risks associated with the use of triazole DMIs.

Project description:Aspergillus fumigatus is the predominant pathogen of invasive aspergillosis, a disease state credited with over 200,000 life-threatening infections each year. The triazole class of antifungals are clinically essential to the treatment of invasive aspergillosis, both as frontline and as salvage therapy. Unfortunately, resistance to the triazoles among A. fumigatus isolates is now increasingly reported worldwide, and a large proportion of this resistance remains unexplained. In this work, we characterize the contributions of previously identified mechanisms of triazole resistance, including mutations in the sterol-demethylase-encoding gene cyp51A, overexpression of sterol-demethylase genes, and overexpression of the efflux pump-encoding gene abcC, among a large collection of highly triazole-resistant clinical A. fumigatus isolates. Upon revealing that these mechanisms alone cannot substantiate the majority of triazole resistance exhibited by this collection, we subsequently describe the identification and characterization of a novel genetic determinant of triazole resistance. Mutations in the 3-hydroxy-3-methyl-glutaryl-coenzyme A (HMG-CoA) reductase-encoding gene, hmg1, were identified in a majority of triazole-resistant clinical isolates in our collection. Introduction of three different hmg1 mutations, predicted to encode residue alterations in the conserved sterol sensing domain of Hmg1, resulted in significantly increased resistance to the triazole class of agents. Additionally, correction of a hmg1 mutation in a pan-triazole-resistant clinical isolate of A. fumigatus with a novel Cas9-ribonucleoprotein-mediated system was shown to restore clinical susceptibility to all triazole agents. Mutations in hmg1 were also shown to lead to the accumulation of ergosterol precursors, such as eburicol, by sterol profiling, while not altering the expression of sterol-demethylase genes.IMPORTANCEAspergillus fumigatus is the predominant pathogen of invasive aspergillosis, a disease state credited with over 200,000 life-threatening infections annually. The triazole class of antifungals are clinically essential to the treatment of invasive aspergillosis. Unfortunately, resistance to the triazoles among A. fumigatus isolates is now increasingly reported worldwide. In this work, we challenge the current paradigm of clinical triazole resistance in A. fumigatus, by first demonstrating that previously characterized mechanisms of resistance have nominal impact on triazole susceptibility and subsequently identifying a novel mechanism of resistance with a profound impact on clinical triazole susceptibility. We demonstrate that mutations in the HMG-CoA reductase gene, hmg1, are common among resistant clinical isolates and that hmg1 mutations confer resistance to all clinically available triazole antifungals.

Dataset Information

Structural basis of HapEP88L-linked antifungal triazole resistance in Aspergillus fumigatus.

Publications

Structural basis of HapE<sup>P88L</sup>-linked antifungal triazole resistance in <i>Aspergillus fumigatus</i>.

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