Mutations in <i>hmg1</i> , Challenging the Paradigm of Clinical Triazole Resistance in Aspergillus fumigatus

Abstract

<i>Aspergillus fumigatus</i> 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 <i>A. fumigatus</i> 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 <i>cyp51A</i>, overexpression of sterol-demethylase genes, and overexpression of the efflux pump-encoding gene <i>abcC</i>, among a large collection of highly triazole-resistant clinical <i>A. fumigatus</i> 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, <i>hmg1</i>, were identified in a majority of triazole-resistant clinical isolates in our collection. Introduction of three different <i>hmg1</i> 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 <i>hmg1</i> mutation in a pan-triazole-resistant clinical isolate of <i>A. fumigatus</i> with a novel Cas9-ribonucleoprotein-mediated system was shown to restore clinical susceptibility to all triazole agents. Mutations in <i>hmg1</i> 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.<b>IMPORTANCE</b><i>Aspergillus fumigatus</i> 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 <i>A. fumigatus</i> isolates is now increasingly reported worldwide. In this work, we challenge the current paradigm of clinical triazole resistance in <i>A. fumigatus</i>, 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, <i>hmg1</i>, are common among resistant clinical isolates and that <i>hmg1</i> mutations confer resistance to all clinically available triazole antifungals.