Structural basis of HapE^P88L-linked antifungal triazole resistance in Aspergillus fumigatus

Abstract

Azoles are first-line therapeutics for human and plant fungal infections, but their broad use has promoted the development of resistances. Recently, a pan-azole–resistant clinical Aspergillus fumigatus isolate was identified to carry the mutation P88L in subunit HapE of the CCAAT-binding complex (CBC), a conserved eukaryotic transcription factor. Here, we define the mechanistic basis for resistance in this isolate by showing that the HapEP88L mutation interferes with the CBC’s ability to bend and sense CCAAT motifs. This failure leads to transcriptional derepression of the cyp51A gene, which encodes the target of azoles, the 14-α sterol demethylase Cyp51A, and ultimately causes drug resistance. In addition, we demonstrate that the CBC-associated transcriptional regulator HapX assists cyp51A repression in low-iron environments and that this iron-dependent effect is lost in the HapEP88L mutant. Altogether, these results indicate that the mutation HapEP88L confers increased resistance to azoles compared with wt A. fumigatus, particularly in low-iron clinical niches such as the lung.

Azole resistance associated regulatory motifs within the promoter of cyp51A in Aspergillus fumigatus

“…We identified iron-independent, cyp51A -activating functions of AtrR and SrbA binding sites during steady-state cultivation and found the HapX binding motif to be crucial for the induction of the gene during short-term iron exposure. Moreover, despite the known -Fe specific repressing role of HapX on iron consuming genes such as cyp51A (18, 19, 23), our results suggest the requirement of its functional binding motif for the repression of cyp51A during steady-state growth, regardless of the iron availability. Hampering this repression by disrupting the HapX binding site elevates resistance to voriconazole and, thus, unveils its binding motif as potential azole resistance hotspot.…”

“…HapX, together with the CBC, was previously shown to mediate repression of cyp51A during iron starvation and upregulation during short-term iron exposure (19). This is in accordance with the cyp51A transcript levels found in wt and mutant strains lacking HapX (Δ hapX ) or CBC (Δ hapC ) as well as in the strains carrying the unmutated ( cyp51A WT ) and mutated ( cyp51A HapX* ) HapX binding motif, respectively (Fig.…”

Investigational Agents for the Treatment of Resistant Yeasts and Molds

“…The most prevalent azole resistance mechanisms are the overexpression and alteration of the target enzyme gene, Cyp51A, and its promoter region (TR 46 /Tyr121Phe/Thr289Ala; TR 34 / Leu98His) [8]. Other mechanisms include biofilm formation, drug efflux, and mutations in transcription factor, HapE [8,13]. Polyene resistance amongst Aspergillus species typically involves selection for inherently resistant species including A. terreus, A. flavus, and A. nidulans [8].…”

Aspergillosis, Avian Species and the One Health Perspective: The Possible Importance of Birds in Azole Resistance

“…Despite the great importance of cyp51A mutations conferring azole resistance, a survey of Manchester isolates showed that 43% of resistant isolates were not cyp51A mutants [122]. Thus other mechanisms than cyp51A mutation may have an important role in azole resistance, such as point mutation in the subunit HapE, of the CCAAT-biding complex (CBC) [123,124], in cox10 gene [125], and in hmg1 gene [126]. In this sense, genome-wide sequencing is a good tool to identify possible mutations conferring azole resistance in cyp51-unrelated resistant strains [125,126].…”