CYP51 Paralogue Structure Is Associated with Intrinsic Azole Resistance in Fungi

Abstract

Azoles are the most commonly used clinical antifungal therapy and also play an important role in control of plant pathogens. Intrinsic resistance to the azole class of fungicides, which target lanosterol demethylase (CYP51), is observed in many fungal species; however, the mechanisms underpinning this phenomenon are unknown. In this study, 5 azole-resistant Penicillium isolates from patients attending the UK National Aspergillosis Centre that could not be morphologically identified to species level were analyzed by genome sequencing. The genomes and CYP51 paralogue structure from these isolates were compared with those of 46 representative fungal isolates to identify to species level and examine possible mechanisms of drug resistance. Analysis of CYP51 paralogues showed that azole-resistant isolates from this study (n = 2) and from public databases (n = 6) contained a new CYP51 paralogue, CYP51D, which was associated with azole resistance in 6/8 cases and never occurred in azole-sensitive species (46/46 tested). Furthermore, one isolate from this study and an azole-resistant Aspergillus fumigatiaffinis isolate were shown to encode a CYP51A paralogue, CYP51A2. Introduction of CYP51A2 to the closely related but azole-sensitive Aspergillus fumigatus resulted in azole resistance. The identification of novel CYP51A and CYP51D paralogues in resistant fungi and the observation that resistance to azoles can be conferred by introducing a CYP51A paralogue from a resistant species into an azole-sensitive species are a potentially important new azole resistance mechanism. IMPORTANCE Azole antifungals are the main treatment for fungal disease in humans. Many species are intrinsically resistant to azoles-in other words all members of the species are resistant without prior exposure-and we do not understand why. In this study, we serendipitously discovered that many intrinsically resistant species have alternative or extra copies of the azole target gene, CYP51. Transfer of one of these genes from a resistant species to a sensitive one resulted in drug resistance, showing that the extra copies of CYP51 can confer drug resistance. Understanding how clinically important species are resistant to therapy allows us to predict whether a species could be resistant from genome sequence.

Keywords: Aspergillus; CYP51; Penicillium; antifungal agents; antifungal resistance; cryptic; paralogy.

FIG 1

Illustrative phylogeny of CYP51 paralogues in this study. Typical CYP51 paralogues were selected. Complete protein sequences were aligned using MUSCLE, and phylogenies were calculated using FastTree with -gamma -spr 4 -mlacc 2 -slownni parameters.

FIG 2

A. fumigatiaffinis cyp51a2 knock-in results in resistance. (a) Overview of replacing the nonfunctional aft4 transposon locus with the A. fumigatiaffinis cyp51a2 gene including 1.5 kb upstream and 500 bp downstream. Microhomology arms of 50 bp are included to allow CRISPR-Cas9-mediated transformation. (b) A phenotypic test on Aspergillus complete medium, grown for 48 h at 37°C. (c) MIC assay for itraconazole for both the parental isolate (A1160p+) and A1160p+^cyp51a2. Growth was normalized to nondrug control (1% dimethyl sulfoxide [DMSO]).