TY - JOUR
T1 - 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
AU - Ballard, Eloise
AU - Weber, Jakob
AU - Melchers, Willem J. G.
AU - Tammireddy, Seshu
AU - Whitfield, Philip D.
AU - Brakhage, Axel A.
AU - Brown, Alistair J. P.
AU - Verweij, Paul E.
AU - Warris, Adilia
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.
PY - 2019/9/1
Y1 - 2019/9/1
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.
AB - 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.
KW - Aspergillus fumigatus
KW - azole resistance
KW - CRISPR-Cas9
KW - in-host adaptation
KW - ergosterol
KW - Ergosterol
KW - Azole resistance
KW - In-host adaptation
UR - https://linkinghub.elsevier.com/retrieve/pii/S1087184519300751
UR - http://www.mendeley.com/research/recreation-inhost-acquired-single-nucleotide-polymorphisms-crisprcas9-reveals-uncharacterised-gene-p
UR - http://www.scopus.com/inward/record.url?scp=85066289326&partnerID=8YFLogxK
U2 - 10.1016/j.fgb.2019.05.005
DO - 10.1016/j.fgb.2019.05.005
M3 - Article
C2 - 31128273
VL - 130
SP - 98
EP - 106
JO - Fungal Genetics and Biology
JF - Fungal Genetics and Biology
SN - 1087-1845
ER -