Aspergillus fumigatus is the predominant pathogen of invasive
aspergillosis, a disease state credited with over 200,000 life-threatening
infections each year. The
triazole class of antifungals are clinically essential to the treatment of invasive
aspergillosis, both as frontline and as
salvage therapy. Unfortunately, resistance to the
triazoles among A. fumigatus isolates is now increasingly reported worldwide, and a large proportion of this resistance remains unexplained. In this work, we characterize the contributions of previously identified mechanisms of
triazole resistance, including mutations in the
sterol-demethylase-encoding gene cyp51A, overexpression of
sterol-demethylase genes, and overexpression of the efflux pump-encoding gene abcC, among a large collection of highly
triazole-resistant clinical A. fumigatus isolates. Upon revealing that these mechanisms alone cannot substantiate the majority of
triazole resistance exhibited by this collection, we subsequently describe the identification and characterization of a novel genetic determinant of
triazole resistance. Mutations in the 3-hydroxy-3-methyl-glutaryl-coenzyme A (
HMG-CoA) reductase-encoding gene,
hmg1, were identified in a majority of
triazole-resistant clinical isolates in our collection. Introduction of three different
hmg1 mutations, predicted to encode residue alterations in the conserved
sterol sensing domain of
Hmg1, resulted in significantly increased resistance to the
triazole class of agents. Additionally, correction of a
hmg1 mutation in a pan-
triazole-resistant clinical isolate of A. fumigatus with a novel Cas9-ribonucleoprotein-mediated system was shown to restore clinical susceptibility to all
triazole agents. Mutations in
hmg1 were also shown to lead to the accumulation of
ergosterol precursors, such as
eburicol, by
sterol profiling, while not altering the expression of
sterol-demethylase genes.IMPORTANCEAspergillus fumigatus is the predominant pathogen of invasive
aspergillosis, a disease state credited with over 200,000 life-threatening
infections annually. The
triazole class of antifungals are clinically essential to the treatment of invasive
aspergillosis. Unfortunately, resistance to the
triazoles among A. fumigatus isolates is now increasingly reported worldwide. In this work, we challenge the current paradigm of clinical
triazole resistance in A. fumigatus, by first demonstrating that previously characterized mechanisms of resistance have nominal impact on
triazole susceptibility and subsequently identifying a novel mechanism of resistance with a profound impact on clinical
triazole susceptibility. We demonstrate that mutations in the
HMG-CoA reductase gene,
hmg1, are common among resistant clinical isolates and that
hmg1 mutations confer resistance to all clinically available
triazole antifungals.