Recreation of in-host acquired single nucleotide polymorphisms by CRISPR-Cas9 reveals an uncharacterised gene playing a role in Aspergillus fumigatus azole resistance via a non-cyp51A mediated resistance mechanism

Eloise Ballard,Jakob Weber,Willem J.G Melchers,Seshu Tammireddy,Phillip D Whitfield,Axel A Brakhage,Alistair J.P Brown,Paul E Verweij,Adilia Warris

doi:10.1016/j.fgb.2019.05.005

Abstract

The human host comprises a range of specific niche environments. In order to successfully persist, pathogens such as Aspergillus fumigatus must adapt to these environments. One key example of in-host adaptation is the development of resistance to azole antifungals. Azole resistance in A. fumigatus is increasingly reported worldwide and the most commonly reported mechanisms are cyp51A mediated. Using a unique series of A. fumigatus isolates, obtained from a patient suffering from persistent and recurrent invasive aspergillosis over 2 years, this study aimed to gain insight into the genetic basis of in-host adaptation. Single nucleotide polymorphisms (SNPs) unique to a single isolate in this series, which had developed multi-azole resistance in-host, were identified. Two nonsense SNPs were recreated using CRISPR-Cas9; these were 213* in svf1 and 167* in uncharacterised gene AFUA_7G01960. Phenotypic analyses including antifungal susceptibility testing, mycelial growth rate assessment, lipidomics analysis and statin susceptibility testing were performed to associate genotypes to phenotypes. This revealed a role for svf1 in A. fumigatus oxidative stress sensitivity. In contrast, recapitulation of 167* in AFUA_7G01960 resulted in increased itraconazole resistance. Comprehensive lipidomics analysis revealed decreased ergosterol levels in strains containing this SNP, providing insight to the observed itraconazole resistance. Decreases in ergosterol levels were reflected in increased resistance to lovastatin and nystatin. Importantly, this study has identified a SNP in an uncharacterised gene playing a role in azole resistance via a non-cyp51A mediated resistance mechanism. This mechanism is of clinical importance, as this SNP was identified in a clinical isolate, which acquired azole resistance in-host.

Highlights

In order to survive in-host, the human fungal pathogen Aspergillus fumigatus must adapt to specific niche environments (Verweij et al, 2016)
We wanted to establish that CRISPR-Cas9 can be used to recapitulate Single nucleotide polymorphisms (SNPs) that affect antifungal drug resistance in clinical isolates of A. fumigatus
Clinical isolates are exposed to numerous known and unknown stress factors, underscoring the diversity and complexity of the adaptation process involving various pathways. Genome editing systems such as CRISPR-Cas9 will help to gain detailed insights into the complex interplay of SNPs involved in in-host adaptation in A. fumigatus

Summary

Introduction

In order to survive in-host, the human fungal pathogen Aspergillus fumigatus must adapt to specific niche environments (Verweij et al, 2016). A range of mutations in the cyp51A gene, which encodes the ergosterol biosynthetic enzyme lanosterol 14-alphademethylase, have been shown to cause azole resistance by altering the structure of ligand access channels. Amino acid changes in these channels are thought to disturb docking of azole molecules by altering interactions (Snelders et al, 2010). These include mutations such as P216, G138, M220 and G54 (Albarrag et al, 2011; Garcia-Effron et al, 2005; Mann et al, 2003; Mellado et al, 2004; Snelders et al, 2010). One example is P88L in the HapE subunit of the CCAAT-binding complex, which is a multimeric transcription factor acting as a negative regulator of ergosterol biosynthesis

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