Characterization of the Efflux Capability and Substrate Specificity of <named-content content-type="genus-species">Aspergillus fumigatus</named-content> PDR5-like ABC Transporters Expressed in <named-content content-type="genus-species">Saccharomyces cerevisiae</named-content>

ABSTRACT This research analyzed six Aspergillus fumigatus genes encoding putative efflux proteins for their roles as transporters. The A. fumigatus genes abcA, abcC, abcF, abcG, abcH, and abcI were cloned into plasmids and overexpressed in a Saccharomyces cerevisiae strain in which the highly active...

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Autores principales:	Brooke D. Esquivel, Jeffrey M. Rybak, Katherine S. Barker, Jarrod R. Fortwendel, P. David Rogers, Theodore C. White
Formato:	article
Lenguaje:	EN
Publicado:	American Society for Microbiology 2020
Materias:	Aspergillus fumigatus efflux drug resistance filamentous fungi ABC transporter PDR5 Microbiology QR1-502
Acceso en línea:	https://doaj.org/article/d8fc063efb164397b16c0f643c17c6f3
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spelling	oai:doaj.org-article:d8fc063efb164397b16c0f643c17c6f32021-11-15T15:57:02ZCharacterization of the Efflux Capability and Substrate Specificity of <named-content content-type="genus-species">Aspergillus fumigatus</named-content> PDR5-like ABC Transporters Expressed in <named-content content-type="genus-species">Saccharomyces cerevisiae</named-content>10.1128/mBio.00338-202150-7511https://doaj.org/article/d8fc063efb164397b16c0f643c17c6f32020-04-01T00:00:00Zhttps://journals.asm.org/doi/10.1128/mBio.00338-20https://doaj.org/toc/2150-7511ABSTRACT This research analyzed six Aspergillus fumigatus genes encoding putative efflux proteins for their roles as transporters. The A. fumigatus genes abcA, abcC, abcF, abcG, abcH, and abcI were cloned into plasmids and overexpressed in a Saccharomyces cerevisiae strain in which the highly active endogenous ABC transporter gene PDR5 was deleted. The activity of each transporter was measured by efflux of rhodamine 6G and accumulation of alanine β-naphthylamide. The transporters AbcA, AbcC, and AbcF had the strongest efflux activities of these compounds. All of the strains with plasmid-expressed transporters had more efflux activity than did the PDR5-deleted background strain. We performed broth microdilution drug susceptibility testing and agar spot assays using an array of compounds and antifungal drugs to determine the transporter specificity and drug susceptibility of the strains. The transporters AbcC and AbcF showed the broadest range of substrate specificity, while AbcG and AbcH had the narrowest range of substrates. Strains expressing the AbcA, AbcC, AbcF, or AbcI transporter were more resistant to fluconazole than was the PDR5-deleted background strain. Strains expressing AbcC and AbcF were additionally more resistant to clotrimazole, itraconazole, ketoconazole, and posaconazole than was the background strain. Finally, we analyzed the expression levels of the genes by reverse transcription-quantitative PCR (RT-qPCR) in triazole-susceptible and -resistant A. fumigatus clinical isolates. All of these transporters are expressed at a measurable level, and transporter expression varied significantly between strains, demonstrating the high degree of phenotypic variation, plasticity, and divergence of which this species is capable. IMPORTANCE One mechanism behind drug resistance is altered export out of the cell. This work is a multifaceted analysis of membrane efflux transporters in the human fungal pathogen A. fumigatus. Bioinformatics evidence infers that there is a relatively large number of genes in A. fumigatus that encode ABC efflux transporters. However, very few of these transporters have been directly characterized and analyzed for their potential role in drug resistance. Our objective was to determine if these undercharacterized proteins function as efflux transporters and then to better define whether their efflux substrates include antifungal drugs used to treat fungal infections. We chose six A. fumigatus potential plasma membrane ABC transporter genes for analysis and found that all six genes produced functional transporter proteins. We used two fungal systems to look for correlations between transporter function and drug resistance. These transporters have the potential to produce drug-resistant phenotypes in A. fumigatus. Continued characterization of these and other transporters may assist in the development of efflux inhibitor drugs.Brooke D. EsquivelJeffrey M. RybakKatherine S. BarkerJarrod R. FortwendelP. David RogersTheodore C. WhiteAmerican Society for MicrobiologyarticleAspergillus fumigatuseffluxdrug resistancefilamentous fungiABC transporterPDR5MicrobiologyQR1-502ENmBio, Vol 11, Iss 2 (2020)
institution	DOAJ
collection	DOAJ
language	EN
topic	Aspergillus fumigatus efflux drug resistance filamentous fungi ABC transporter PDR5 Microbiology QR1-502
spellingShingle	Aspergillus fumigatus efflux drug resistance filamentous fungi ABC transporter PDR5 Microbiology QR1-502 Brooke D. Esquivel Jeffrey M. Rybak Katherine S. Barker Jarrod R. Fortwendel P. David Rogers Theodore C. White Characterization of the Efflux Capability and Substrate Specificity of <named-content content-type="genus-species">Aspergillus fumigatus</named-content> PDR5-like ABC Transporters Expressed in <named-content content-type="genus-species">Saccharomyces cerevisiae</named-content>
description	ABSTRACT This research analyzed six Aspergillus fumigatus genes encoding putative efflux proteins for their roles as transporters. The A. fumigatus genes abcA, abcC, abcF, abcG, abcH, and abcI were cloned into plasmids and overexpressed in a Saccharomyces cerevisiae strain in which the highly active endogenous ABC transporter gene PDR5 was deleted. The activity of each transporter was measured by efflux of rhodamine 6G and accumulation of alanine β-naphthylamide. The transporters AbcA, AbcC, and AbcF had the strongest efflux activities of these compounds. All of the strains with plasmid-expressed transporters had more efflux activity than did the PDR5-deleted background strain. We performed broth microdilution drug susceptibility testing and agar spot assays using an array of compounds and antifungal drugs to determine the transporter specificity and drug susceptibility of the strains. The transporters AbcC and AbcF showed the broadest range of substrate specificity, while AbcG and AbcH had the narrowest range of substrates. Strains expressing the AbcA, AbcC, AbcF, or AbcI transporter were more resistant to fluconazole than was the PDR5-deleted background strain. Strains expressing AbcC and AbcF were additionally more resistant to clotrimazole, itraconazole, ketoconazole, and posaconazole than was the background strain. Finally, we analyzed the expression levels of the genes by reverse transcription-quantitative PCR (RT-qPCR) in triazole-susceptible and -resistant A. fumigatus clinical isolates. All of these transporters are expressed at a measurable level, and transporter expression varied significantly between strains, demonstrating the high degree of phenotypic variation, plasticity, and divergence of which this species is capable. IMPORTANCE One mechanism behind drug resistance is altered export out of the cell. This work is a multifaceted analysis of membrane efflux transporters in the human fungal pathogen A. fumigatus. Bioinformatics evidence infers that there is a relatively large number of genes in A. fumigatus that encode ABC efflux transporters. However, very few of these transporters have been directly characterized and analyzed for their potential role in drug resistance. Our objective was to determine if these undercharacterized proteins function as efflux transporters and then to better define whether their efflux substrates include antifungal drugs used to treat fungal infections. We chose six A. fumigatus potential plasma membrane ABC transporter genes for analysis and found that all six genes produced functional transporter proteins. We used two fungal systems to look for correlations between transporter function and drug resistance. These transporters have the potential to produce drug-resistant phenotypes in A. fumigatus. Continued characterization of these and other transporters may assist in the development of efflux inhibitor drugs.
format	article
author	Brooke D. Esquivel Jeffrey M. Rybak Katherine S. Barker Jarrod R. Fortwendel P. David Rogers Theodore C. White
author_facet	Brooke D. Esquivel Jeffrey M. Rybak Katherine S. Barker Jarrod R. Fortwendel P. David Rogers Theodore C. White
author_sort	Brooke D. Esquivel
title	Characterization of the Efflux Capability and Substrate Specificity of <named-content content-type="genus-species">Aspergillus fumigatus</named-content> PDR5-like ABC Transporters Expressed in <named-content content-type="genus-species">Saccharomyces cerevisiae</named-content>
title_short	Characterization of the Efflux Capability and Substrate Specificity of <named-content content-type="genus-species">Aspergillus fumigatus</named-content> PDR5-like ABC Transporters Expressed in <named-content content-type="genus-species">Saccharomyces cerevisiae</named-content>
title_full	Characterization of the Efflux Capability and Substrate Specificity of <named-content content-type="genus-species">Aspergillus fumigatus</named-content> PDR5-like ABC Transporters Expressed in <named-content content-type="genus-species">Saccharomyces cerevisiae</named-content>
title_fullStr	Characterization of the Efflux Capability and Substrate Specificity of <named-content content-type="genus-species">Aspergillus fumigatus</named-content> PDR5-like ABC Transporters Expressed in <named-content content-type="genus-species">Saccharomyces cerevisiae</named-content>
title_full_unstemmed	Characterization of the Efflux Capability and Substrate Specificity of <named-content content-type="genus-species">Aspergillus fumigatus</named-content> PDR5-like ABC Transporters Expressed in <named-content content-type="genus-species">Saccharomyces cerevisiae</named-content>
title_sort	characterization of the efflux capability and substrate specificity of <named-content content-type="genus-species">aspergillus fumigatus</named-content> pdr5-like abc transporters expressed in <named-content content-type="genus-species">saccharomyces cerevisiae</named-content>
publisher	American Society for Microbiology
publishDate	2020
url	https://doaj.org/article/d8fc063efb164397b16c0f643c17c6f3
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Characterization of the Efflux Capability and Substrate Specificity of <named-content content-type="genus-species">Aspergillus fumigatus</named-content> PDR5-like ABC Transporters Expressed in <named-content content-type="genus-species">Saccharomyces cerevisiae</named-content>

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