Overexpression of cyp51A and cyp51B contribute to triazole resistance in clinical isolates of Aspergillus fumigatus from the United States

Objectives:
In Candida albicans, mutations leading to the activation of Upc2, and upregulation genes of the ergosterol biosynthesis pathway including ERG11, represents an important mechanism of resistance to the triazole antifungals. In order to determine if activation of the ergosterol biosynthesis pathway contributes to triazole resistance among clinical isolates of Aspergillus fumigatus, we examined the transcriptional profiles of triazole resistant isolates from the United States using RNAseq.

Methods:
21 triazole-resistant clinical isolates of A. fumigatus and 5 triazole-susceptible control isolates of A. fumigatus were included in this study. Genomic DNA and RNA were isolated from mature hyphal cells, sequenced on the Ion Torrent platform, and reads were aligned to the Af293 reference genome. Transcript counts among the control isolates were averaged to create a susceptible composite. This composite was then compared to the transcript count measured in individual resistant isolates to create fold- change values. In vitro assembled Cas9 ribonucleoproteins coupled with microhomology repair templates were utilized to replace 1000bp of the cyp51A or cyp51B promoter with that of the strong hspA promoter in the akuBKU80 background. Voriconazole minimum inhibitory concentrations (MIC) for each isolate were determined using CLSI methods in duplicate.

Results:
Overexpression of numerous ergosterol biosynthesis associated genes, most notably cyp51A, cyp51B, erg3A, erg3B, erg13A, erg13B, erg24, erg25A, erg25B, hmg1, hapX, and srbA was observed in 6 of the triazole-resistant clinical isolates. This overexpression was observed among both isolates with previously characterized cyp51A mutations, and isolates without cyp51A mutations. Overexpression of cyp51A and cyp51B was observed to range from 3- to 67-fold and 3- to 7-fold that of the susceptible composite, respectively. Whole genome sequencing revealed no unique mutations in srbA in these 6 isolates, however mutations in genes involved in srbA activation (dscA, insA, rbdB, and sppA) were identified. Constitutive overexpression of both cyp51A and cyp51B in the akuBKU80 background using the hspA promoter led to increases in voriconazole MIC by 2 and 4-fold respectively.

Conclusion:
While tandem repeat elements in the promoter of region of cyp51A have been described and contribute to both cyp51A overexpression and triazole resistance, coordinate upregulation of multiple genes of the ergosterol biosynthesis pathway in resistant clinical isolates have not been reported. Overexpression of a multitude of genes associated with ergosterol biosynthesis, including both cyp51A and cyp51B, was observed in 29% (6/21) of the triazole-resistant A. fumigatus clinical isolates in this collection. Triazole MIC did not clearly correlate with the extent of either cyp51A or cyp51B overexpression, suggesting the concomitant involvement of additional resistance mechanisms. Consistent with findings reported in previous studies, overexpression of cyp51A mediated by promoter replacement in the akuBKU80 background led to a 2-fold increase in voriconazole MIC. Intriguingly, overexpression of cyp51B, which has not previously been demonstrated to have a direct impact on triazole MIC, was observed to increase voriconazole MIC above the previously reported ECV (1mg/L) in the akuBKU80 background. Further research is needed to reveal the direct contribution of overexpression of genes associated with ergosterol biosynthesis on triazole resistance in A. fumigatus.

7 / Overexpression of cyp51A and cyp51b contribute to triazole resistance in clinical isolates of Aspergillus fumigatus from the United States