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 R.; Whitfield, Phillip D.; Brakhage, Axel A.; Brown, Alistair J. P.; Verweij, Paul E.; Warris, Adilia

doi:10.1016/j.fgb.2019.05.005

Cited by 26 publications

(25 citation statements)

References 56 publications

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“…A series of 13 isogenic strains was also included, isolated from a single chronic granulomatous disease patient over a period of 2 years. We have previously characterised these isolates phenotypically and genotypically, revealing which have developed azole resistance and conidiation defects in-host throughout the course of infection [2,[37][38][39]. We also included a range of A. fumigatus strains isolated from the soil in Scotland.…”

Section: A Fumigatus Strainsmentioning

confidence: 99%

Antifungal Activity of Antimicrobial Peptides and Proteins against Aspergillus fumigatus

Ballard

Yucel

Melchers

et al. 2020

JoF

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Antimicrobial peptides and proteins (AMPs) provide an important line of defence against invading microorganisms. However, the activity of AMPs against the human fungal pathogen Aspergillus fumigatus remains poorly understood. Therefore, the aim of this study was to characterise the anti-Aspergillus activity of specific human AMPs, and to determine whether A. fumigatus can possess resistance to specific AMPs, as a result of in-host adaptation. AMPs were tested against a wide range of clinical isolates of various origins (including cystic fibrosis patients, as well as patients with chronic and acute aspergillosis). We also tested a series of isogenic A. fumigatus isolates obtained from a single patient over a period of 2 years. A range of environmental isolates, obtained from soil in Scotland, was also included. Firstly, the activity of specific peptides was assessed against hyphae using a measure of fungal metabolic activity. Secondly, the activity of specific peptides was assessed against germinating conidia, using imaging flow cytometry as a measure of hyphal growth. We showed that lysozyme and histones inhibited hyphal metabolic activity in all the A. fumigatus isolates tested in a dose-dependent fashion. In addition, imaging flow cytometry revealed that histones, β-defensin-1 and lactoferrin inhibited the germination of A. fumigatus conidia.

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Section: A Fumigatus Strainsmentioning

confidence: 99%

Antifungal Activity of Antimicrobial Peptides and Proteins against Aspergillus fumigatus

Ballard

Yucel

Melchers

et al. 2020

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“…Therefore, the emergence of azole resistance in A. fumigatus has raised global concern. As demonstrated in earlier studies (4)(5)(6)(7)(8)(9)(10), azole-resistant strains often appear during treatment. Resistant-responsible mutations were found mainly in Cyp51A (Erg11), which is the target protein of azole antifungals (11,12).…”

Section: Introductionmentioning

confidence: 56%

Identification of novel mutations contributing to azole tolerance ofAspergillus fumigatus throughin vitroexposure to tebuconazole

Toyotome

Onishi

Sato

et al. 2020

Preprint

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Azole resistance of Aspergillus fumigatus is a global problem. The major resistant mechanism is a cyp51A alteration such as mutation(s) in the gene and the acquisition of a tandem repeat in the promoter. Although other azole tolerances and resistant mechanisms such as hmg1 mutation are known, few reports describe studies elucidating non-cyp51A resistance mechanisms. This study explored genes contributing to azole tolerance in A. fumigatus by in vitro mutant selection with tebuconazole, an azole fungicide. After three-round selection, we obtained four isolates with low susceptibility to tebuconazole. These isolates also showed low susceptibility to itraconazole and voriconazole. Comparison of the genome sequences of the obtained isolates and the parental strain revealed a non-synonymous mutation in MfsD (Afu1g11820, R337L mutation) in all isolates. Furthermore, non-synonymous mutations in AgcA (Afu7g05220, E535Stop mutation), UbcD (Afu3g06030, T98K mutation), AbcJ (Afu3g12220, G297E mutation), and RttA (Afu7g04740, A83T mutation), a protein responsible for tebuconazole tolerance, were found in at least one isolate. Clarification by constructing the MfsD R337L mutant suggests that the mutation contributes to azole tolerance. Disruption of the agcA gene and reconstruction of the A83T point mutation in RttA led to decreased susceptibility to azoles. The reversion of T98K mutation to wild type in UbcD led to the level of azole susceptibility comparable to the parental strain. These results suggest that these mutations contribute to lowered susceptibility to medical azoles and to agricultural azole fungicides.

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“…The current A. fumigatus PCR-based resistance assays do not reveal the less common or potential mutations involved in azole resistance. In Manchester, 43% of the azole-resistant patient isolates were reported to be non-cyp51A related [26] and numerous studies have revealed that azole resistance is most certainly due to other genes [46][47][48][49][50][51][131][132][133][134]. Besides gene mutations in the ergosterol biosynthesis pathway, other non-cyp51A-mediated resistance mechanisms involve the overexpression of drug efflux transporters such as ABC-and MFS-transporters [96,[135][136][137].…”

Section: The Pyrosequencing Methodsmentioning

confidence: 99%

Detecting Azole-Antifungal Resistance in Aspergillus fumigatus by Pyrosequencing

Torre

Frazer

Rautemaa‐Richardson

2020

JoF

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Guidelines on the diagnosis and management of Aspergillus disease recommend a multi-test approach including CT scans, culture, fungal biomarker tests, microscopy and fungal PCR. The first-line treatment of confirmed invasive aspergillosis (IA) consists of drugs in the azole family; however, the emergence of azole-resistant isolates has negatively impacted the management of IA. Failure to detect azole-resistance dramatically increases the mortality rates of azole-treated patients. Despite drug susceptibility tests not being routinely performed currently, we suggest including resistance testing whilst diagnosing Aspergillus disease. Multiple tools, including DNA sequencing, are available to screen for drug-resistant Aspergillus in clinical samples. This is particularly beneficial as a large proportion of IA samples are culture negative, consequently impeding susceptibility testing through conventional methods. Pyrosequencing is a promising in-house DNA sequencing method that can rapidly screen for genetic hotspots associated with antifungal resistance. Pyrosequencing outperforms other susceptibility testing methods due to its fast turnaround time, accurate detection of polymorphisms within critical genes, including simultaneous detection of wild type and mutated sequences, and—most importantly—it is not limited to specific genes nor fungal species. Here we review current diagnostic methods and highlight the potential of pyrosequencing to aid in a diagnosis complete with a resistance profile to improve clinical outcomes.

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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

Abstract: HighlightsNew in-host acquired non-cyp51-mediated azole resistance mechanism in A. fumigatus.167* mutation in AFUA_7G01960 leads to azole resistance and thermotolerance.Azole resistance associated with decreased ergosterol content in fungal membrane.

Cited by 26 publications

References 56 publications

Antifungal Activity of Antimicrobial Peptides and Proteins against Aspergillus fumigatus

Antifungal Activity of Antimicrobial Peptides and Proteins against Aspergillus fumigatus

Identification of novel mutations contributing to azole tolerance ofAspergillus fumigatus throughin vitroexposure to tebuconazole

Detecting Azole-Antifungal Resistance in Aspergillus fumigatus by Pyrosequencing

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