Discovery of a hapE Mutation That Causes Azole Resistance in Aspergillus fumigatus through Whole Genome Sequencing and Sexual Crossing

Simone M T Camps,Bas E Dutilh,Paul E Verweij,Maiken C Arendrup,Willem J G Melchers,Eveline Snelders,Martijn A Huynen,Antonius J M M Rijs,Matthew (Mat) Charles Fisher

doi:10.1371/journal.pone.0050034

Abstract

Azole compounds are the primary therapy for patients with diseases caused by Aspergillus fumigatus. However, prolonged treatment may cause resistance to develop, which is associated with treatment failure. The azole target cyp51A is a hotspot for mutations that confer phenotypic resistance, but in an increasing number of resistant isolates the underlying mechanism remains unknown. Here, we report the discovery of a novel resistance mechanism, caused by a mutation in the CCAAT-binding transcription factor complex subunit HapE. From one patient, four A. fumigatus isolates were serially collected. The last two isolates developed an azole resistant phenotype during prolonged azole therapy. Because the resistant isolates contained a wild type cyp51A gene and the isolates were isogenic, the complete genomes of the last susceptible isolate and the first resistant isolate (taken 17 weeks apart) were sequenced using Illumina technology to identify the resistance conferring mutation. By comparing the genome sequences to each other as well as to two A. fumigatus reference genomes, several potential non-synonymous mutations in protein-coding regions were identified, six of which could be confirmed by PCR and Sanger sequencing. Subsequent sexual crossing experiments showed that resistant progeny always contained a P88L substitution in HapE, while the presence of the other five mutations did not correlate with resistance in the progeny. Cloning the mutated hapE gene into the azole susceptible akuB KU80 strain showed that the HapE P88L mutation by itself could confer the resistant phenotype. This is the first time that whole genome sequencing and sexual crossing strategies have been used to find the genetic basis of a trait of interest in A. fumigatus. The discovery may help understand alternate pathways for azole resistance in A. fumigatus with implications for the molecular diagnosis of resistance and drug discovery.

Highlights

Aspergillus fumigatus is a ubiquitous saprophytic mold
Due to the isogenic nature of the isolates, we anticipated that the most straightforward approach to identify this resistance mechanism would be by complete genome sequencing of isolates 2 and 3
In our study the whole genome sequencing strategy was used to identify a resistance mutation that arose as a de novo mutation in a chronic granulomatous disease (CGD) patient with chronic fibrotic pulmonary aspergillosis caused by A. fumigatus

Summary

Introduction

Humans inhale at least several hundred of A. fumigatus conidia per day, it rarely causes serious medical conditions in healthy individuals. A. fumigatus is able to cause a wide range of other non-invasive diseases including allergic syndromes and aspergilloma, many of which require treatment with antifungal agents [1,2]. Current treatment options of Aspergillus diseases include three classes of antifungal agents: polyenes (amphotericin B), echinocandins (caspofungin) and azoles, the latter being the largest and most widely used class of antifungal drugs [3,4]. Itraconazole is commonly used for the treatment of chronic and allergic conditions [2] and posaconazole is effective in preventing invasive aspergillosis in patients with certain hematologic malignancies [7,8]

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Conclusion