Use of Bulk Segregant Analysis for Determining the Genetic Basis of Azole Resistance in the Opportunistic Pathogen Aspergillus fumigatus

Abstract

A sexual cycle was described in 2009 for the opportunistic fungal pathogen Aspergillus fumigatus, opening up for the first time the possibility of using techniques reliant on sexual crossing for genetic analysis. The present study was undertaken to evaluate whether the technique ‘bulk segregant analysis’ (BSA), which involves detection of differences between pools of progeny varying in a particular trait, could be applied in conjunction with next-generation sequencing to investigate the underlying basis of monogenic traits in A. fumigatus. Resistance to the azole antifungal itraconazole was chosen as a model, with a dedicated bioinformatic pipeline developed to allow identification of SNPs that differed between the resistant progeny pool and resistant parent compared to the sensitive progeny pool and parent. A clinical isolate exhibiting monogenic resistance to itraconazole of unknown basis was crossed to a sensitive parent and F1 progeny used in BSA. In addition, the use of backcrossing and increasing the number in progeny pools was evaluated as ways to enhance the efficiency of BSA. Use of F1 pools of 40 progeny led to the identification of 123 candidate genes with SNPs distributed over several contigs when aligned to an A1163 reference genome. Successive rounds of backcrossing enhanced the ability to identify specific genes and a genomic region, with BSA of progeny (using 40 per pool) from a third backcross identifying 46 genes with SNPs, and BSA of progeny from a sixth backcross identifying 20 genes with SNPs in a single 292 kb region of the genome. The use of an increased number of 80 progeny per pool also increased the resolution of BSA, with 29 genes demonstrating SNPs between the different sensitive and resistant groupings detected using progeny from just the second backcross with the majority of variants located on the same 292 kb region. Further bioinformatic analysis of the 292 kb region identified the presence of a cyp51A gene variant resulting in a methionine to lysine (M220K) change in the CYP51A protein, which was concluded to be the causal basis of the observed resistance to itraconazole. The future use of BSA in genetic analysis of A. fumigatus is discussed.

Citizen science reveals landscape-scale exposures to multiazole-resistant Aspergillus fumigatus bioaerosols

“…Genotyping confirmed that the most common aerosolized resistanceassociated polymorphisms were TR 34 /L98H (59%) and TR 46 / Y121F/T289A (6%). We further found that 30% of TEB-resistant isolates did not contain any polymorphisms in the cyp51A promoter or coding regions suggesting that the existence of alternative resistance mechanisms as has previously been noted (table S1) (23). This is of concern in the clinical setting as only the two former mutations are picked up by commercially available polymerase chain reaction (PCR) diagnostic methods, although previously unidentified mutations leading to resistance will be identified by phenotypic testing of minimum inhibitory concentration (MIC) providing that there is an isolate available to test.…”

Landscape-scale exposure to multiazole-resistantAspergillus fumigatusbioaerosols

“…Genotyping confirmed that the most common aerosolized resistance-associated polymorphisms were TR 34 /L98H (59%) and TR 46 /Y121F/T289A (6%). We further found that 30% of TEB-resistant isolates did not contain any polymorphisms in the cyp51A promoter or coding regions suggesting the existence of alternative resistance mechanisms as have previously been noted ( 22 ) ( Table S1 ). This is of concern in the clinical setting as only the two former mutations are picked up by commercially available PCR diagnostic methods, although previously unidentified mutations leading to resistance will be picked up by phenotypic testing of minimum inhibitory concentration (MIC) providing that there is an isolate available to test.…”

Editorial: Sexual and Parasexual Reproduction of Human Fungal Pathogens

“…Finally, it was confirmed that flower bulb waste material could serve as a natural substrate for sex by the demonstration that such material could be utilized successfully to perform sexual crosses in the laboratory. Ashton et al, (2022) also demonstrate that the sexual cycle can be used for genetic analysis. They used the 'bulk segregant analysis (BSA)' technique together with next-generation sequencing and a newly developed bioinformatic pipeline to investigate the underlying basis of monogenic traits in A. fumigatus.…”