Discovery of a HapE mutation that causes azole resistance in Aspergillus fumigatus through whole genome sequencing and sexual crossing

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.

Figure 1. Relapsing infection with A. fumigatus in a CGD patient.

During the course of the disease, four A. fumigatus isolates were obtained. Sampling dates of isolates 1 through 4 are indicated. S indicates susceptibility to azoles and R indicates resistance. Minimum inhibitory concentrations (MICs) for itraconazole, voriconazole, and posaconazole are also shown. The patient was treated with voriconazole monotherapy (week −67 to +12), caspofungin+voriconazole combination therapy (week +12 to +90) and caspofungin+posaconazole combination therapy (week +90 to +134). Plasma concentrations at week 86 (voriconazole) and weeks 99, 105, and 123 (posaconazole) are also indicated. The patient died from the pulmonary infection at week 134 .

Figure 2. In vitro growth curves of susceptible and resistant progeny.

A. In vitro growth curves of susceptible (n = 12) and resistant (n = 4) progeny. B. The maximum growth per hour during the exponential phase of the growth curve (6–10 hours, between dashed lines of Figure 2A) was averaged (±standard deviation) for susceptible and resistant progeny. The difference is significant according to a Student's T-test (one-tailed, heteroscedastic).

Figure 3. Cyp51A mRNA levels in the parental isolates (47–169, isolate 3, isolate 4) and three of the progeny (v122-12, v121-64, v121-48).

Isolate v122-12 contains only the HapE substitution and not the other five gene mutations. As a control, we selected two other progeny: v121-64 containing only the erg25 mutation and v121-48 containing the erg6/erg25 double mutation. S: susceptible, R: resistant.