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, Philip D. Whitfield, Axel A. Brakhage, Alistair J. P. Brown, Paul E. Verweij, Adilia Warris (Corresponding Author)

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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.
Original languageEnglish
Pages (from-to)98-106
Number of pages9
JournalFungal Genetics and Biology
Volume130
Early online date23 May 2019
DOIs
Publication statusPublished - 1 Sep 2019

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Clustered Regularly Interspaced Short Palindromic Repeats
Recreation
Azoles
Aspergillus fumigatus
Single Nucleotide Polymorphism
Ergosterol
Itraconazole
Genes
Hydroxymethylglutaryl-CoA Reductase Inhibitors
Nystatin
Lovastatin
Aspergillosis
Oxidative Stress
Genotype
Phenotype
Growth

Keywords

  • Aspergillus fumigatus
  • azole resistance
  • CRISPR-Cas9
  • in-host adaptation
  • ergosterol
  • Ergosterol
  • Azole resistance
  • In-host adaptation

ASJC Scopus subject areas

  • Genetics
  • Microbiology

Cite this

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. / Ballard, Eloise; Weber, Jakob; Melchers, Willem J. G. ; Tammireddy, Seshu; Whitfield, Philip D.; Brakhage, Axel A.; Brown, Alistair J. P.; Verweij, Paul E.; Warris, Adilia (Corresponding Author).

In: Fungal Genetics and Biology, Vol. 130, 01.09.2019, p. 98-106.

Research output: Contribution to journalArticle

Ballard, Eloise ; Weber, Jakob ; Melchers, Willem J. G. ; Tammireddy, Seshu ; Whitfield, Philip D. ; Brakhage, Axel A. ; Brown, Alistair J. P. ; Verweij, Paul E. ; Warris, Adilia. / 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. In: Fungal Genetics and Biology. 2019 ; Vol. 130. pp. 98-106.
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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.",
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AU - Weber, Jakob

AU - Melchers, Willem J. G.

AU - Tammireddy, Seshu

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N1 - EB, AB and AW are supported by the Wellcome Trust Strategic Award (grant 097377) and the MRC Centre for Medical Mycology (grant MR/N006364/1) at the University of Aberdeen. AB was also supported by the Biotechnology and Biological Sciences Research Council (BBSRC) (BB/K017365/1) and the Medical Research Council (MR/M026663/1). JW and AAB were supported by the BMBF-funded project DrugBioTune in the frame of InfectControl2020 and the excellence graduate school Jena School of Microbial Communication. ST and PW acknowledge the support of the European Regional Development Fund, Scottish Funding Council and Highlands and Islands Enterprise. The work in this paper is funded by a BBSRC EASTBIO grant (BB/M010996/1) awarded to AW. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

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N2 - 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.

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