The <named-content content-type="genus-species">Aspergillus fumigatus</named-content> Damage Resistance Protein Family Coordinately Regulates Ergosterol Biosynthesis and Azole Susceptibility

ABSTRACT Ergosterol is a major and specific component of the fungal plasma membrane, and thus, the cytochrome P450 enzymes (Erg proteins) that catalyze ergosterol synthesis have been selected as valuable targets of azole antifungals. However, the opportunistic pathogen Aspergillus fumigatus has deve...

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Autores principales:	Jinxing Song, Pengfei Zhai, Yuanwei Zhang, Caiyun Zhang, Hong Sang, Guanzhu Han, Nancy P. Keller, Ling Lu
Formato:	article
Lenguaje:	EN
Publicado:	American Society for Microbiology 2016
Materias:	Microbiology QR1-502
Acceso en línea:	https://doaj.org/article/ed51baa5307d48b4b1e2858ac2179a2e
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spelling	oai:doaj.org-article:ed51baa5307d48b4b1e2858ac2179a2e2021-11-15T15:49:40ZThe <named-content content-type="genus-species">Aspergillus fumigatus</named-content> Damage Resistance Protein Family Coordinately Regulates Ergosterol Biosynthesis and Azole Susceptibility10.1128/mBio.01919-152150-7511https://doaj.org/article/ed51baa5307d48b4b1e2858ac2179a2e2016-03-01T00:00:00Zhttps://journals.asm.org/doi/10.1128/mBio.01919-15https://doaj.org/toc/2150-7511ABSTRACT Ergosterol is a major and specific component of the fungal plasma membrane, and thus, the cytochrome P450 enzymes (Erg proteins) that catalyze ergosterol synthesis have been selected as valuable targets of azole antifungals. However, the opportunistic pathogen Aspergillus fumigatus has developed worldwide resistance to azoles largely through mutations in the cytochrome P450 enzyme Cyp51 (Erg11). In this study, we demonstrate that a cytochrome b5-like heme-binding damage resistance protein (Dap) family, comprised of DapA, DapB, and DapC, coordinately regulates the functionality of cytochrome P450 enzymes Erg5 and Erg11 and oppositely affects susceptibility to azoles. The expression of all three genes is induced in an azole concentration-dependent way, and the decreased susceptibility to azoles requires DapA stabilization of cytochrome P450 protein activity. In contrast, overexpression of DapB and DapC causes dysfunction of Erg5 and Erg11, resulting in abnormal accumulation of sterol intermediates and further accentuating the sensitivity of ΔdapA strains to azoles. The results of exogenous-hemin rescue and heme-binding-site mutagenesis experiments demonstrate that the heme binding of DapA contributes the decreased azole susceptibility, while DapB and -C are capable of reducing the activities of Erg5 and Erg11 through depletion of heme. In vivo data demonstrate that inactivated DapA combined with activated DapB yields an A. fumigatus mutant that is easily treatable with azoles in an immunocompromised mouse model of invasive pulmonary aspergillosis. Compared to the single Dap proteins found in Saccharomyces cerevisiae and Schizosaccharomyces pombe, we suggest that this complex Dap family regulatory system emerged during the evolution of fungi as an adaptive means to regulate ergosterol synthesis in response to environmental stimuli. IMPORTANCE Knowledge of the ergosterol biosynthesis route in fungal pathogens is useful in the design of new antifungal drugs and could aid in the study of antifungal-drug resistance mechanisms. In this study, we demonstrate that three cytochrome b5-like Dap proteins coordinately regulate the azole resistance and ergosterol biosynthesis catalyzed by cytochrome P450 proteins. Our new insights into the Dap regulatory system in fungal pathogens may have broad therapeutic ramifications beyond their usefulness for classic azole antifungals. Moreover, our elucidation of the molecular mechanism of Dap regulation of cytochrome P450 protein functionality through heme-binding activity may extend beyond the Kingdom Fungi with applicability toward Dap protein regulation of mammalian sterol synthesis.Jinxing SongPengfei ZhaiYuanwei ZhangCaiyun ZhangHong SangGuanzhu HanNancy P. KellerLing LuAmerican Society for MicrobiologyarticleMicrobiologyQR1-502ENmBio, Vol 7, Iss 1 (2016)
institution	DOAJ
collection	DOAJ
language	EN
topic	Microbiology QR1-502
spellingShingle	Microbiology QR1-502 Jinxing Song Pengfei Zhai Yuanwei Zhang Caiyun Zhang Hong Sang Guanzhu Han Nancy P. Keller Ling Lu The <named-content content-type="genus-species">Aspergillus fumigatus</named-content> Damage Resistance Protein Family Coordinately Regulates Ergosterol Biosynthesis and Azole Susceptibility
description	ABSTRACT Ergosterol is a major and specific component of the fungal plasma membrane, and thus, the cytochrome P450 enzymes (Erg proteins) that catalyze ergosterol synthesis have been selected as valuable targets of azole antifungals. However, the opportunistic pathogen Aspergillus fumigatus has developed worldwide resistance to azoles largely through mutations in the cytochrome P450 enzyme Cyp51 (Erg11). In this study, we demonstrate that a cytochrome b5-like heme-binding damage resistance protein (Dap) family, comprised of DapA, DapB, and DapC, coordinately regulates the functionality of cytochrome P450 enzymes Erg5 and Erg11 and oppositely affects susceptibility to azoles. The expression of all three genes is induced in an azole concentration-dependent way, and the decreased susceptibility to azoles requires DapA stabilization of cytochrome P450 protein activity. In contrast, overexpression of DapB and DapC causes dysfunction of Erg5 and Erg11, resulting in abnormal accumulation of sterol intermediates and further accentuating the sensitivity of ΔdapA strains to azoles. The results of exogenous-hemin rescue and heme-binding-site mutagenesis experiments demonstrate that the heme binding of DapA contributes the decreased azole susceptibility, while DapB and -C are capable of reducing the activities of Erg5 and Erg11 through depletion of heme. In vivo data demonstrate that inactivated DapA combined with activated DapB yields an A. fumigatus mutant that is easily treatable with azoles in an immunocompromised mouse model of invasive pulmonary aspergillosis. Compared to the single Dap proteins found in Saccharomyces cerevisiae and Schizosaccharomyces pombe, we suggest that this complex Dap family regulatory system emerged during the evolution of fungi as an adaptive means to regulate ergosterol synthesis in response to environmental stimuli. IMPORTANCE Knowledge of the ergosterol biosynthesis route in fungal pathogens is useful in the design of new antifungal drugs and could aid in the study of antifungal-drug resistance mechanisms. In this study, we demonstrate that three cytochrome b5-like Dap proteins coordinately regulate the azole resistance and ergosterol biosynthesis catalyzed by cytochrome P450 proteins. Our new insights into the Dap regulatory system in fungal pathogens may have broad therapeutic ramifications beyond their usefulness for classic azole antifungals. Moreover, our elucidation of the molecular mechanism of Dap regulation of cytochrome P450 protein functionality through heme-binding activity may extend beyond the Kingdom Fungi with applicability toward Dap protein regulation of mammalian sterol synthesis.
format	article
author	Jinxing Song Pengfei Zhai Yuanwei Zhang Caiyun Zhang Hong Sang Guanzhu Han Nancy P. Keller Ling Lu
author_facet	Jinxing Song Pengfei Zhai Yuanwei Zhang Caiyun Zhang Hong Sang Guanzhu Han Nancy P. Keller Ling Lu
author_sort	Jinxing Song
title	The <named-content content-type="genus-species">Aspergillus fumigatus</named-content> Damage Resistance Protein Family Coordinately Regulates Ergosterol Biosynthesis and Azole Susceptibility
title_short	The <named-content content-type="genus-species">Aspergillus fumigatus</named-content> Damage Resistance Protein Family Coordinately Regulates Ergosterol Biosynthesis and Azole Susceptibility
title_full	The <named-content content-type="genus-species">Aspergillus fumigatus</named-content> Damage Resistance Protein Family Coordinately Regulates Ergosterol Biosynthesis and Azole Susceptibility
title_fullStr	The <named-content content-type="genus-species">Aspergillus fumigatus</named-content> Damage Resistance Protein Family Coordinately Regulates Ergosterol Biosynthesis and Azole Susceptibility
title_full_unstemmed	The <named-content content-type="genus-species">Aspergillus fumigatus</named-content> Damage Resistance Protein Family Coordinately Regulates Ergosterol Biosynthesis and Azole Susceptibility
title_sort	<named-content content-type="genus-species">aspergillus fumigatus</named-content> damage resistance protein family coordinately regulates ergosterol biosynthesis and azole susceptibility
publisher	American Society for Microbiology
publishDate	2016
url	https://doaj.org/article/ed51baa5307d48b4b1e2858ac2179a2e
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The <named-content content-type="genus-species">Aspergillus fumigatus</named-content> Damage Resistance Protein Family Coordinately Regulates Ergosterol Biosynthesis and Azole Susceptibility

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