Stress-Induced Changes in the Lipid Microenvironment of β-(1,3)-<sc>d</sc>-Glucan Synthase Cause Clinically Important Echinocandin Resistance in <named-content content-type="genus-species">Aspergillus fumigatus</named-content>

ABSTRACT Aspergillus fumigatus is a leading cause of invasive fungal infections. Resistance to first-line triazole antifungals has led to therapy with echinocandin drugs. Recently, we identified several high-minimum-effective-concentration (MEC) A. fumigatus clinical isolates from patients failing e...

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Autores principales:	Shruthi Satish, Cristina Jiménez-Ortigosa, Yanan Zhao, Min Hee Lee, Enriko Dolgov, Thomas Krüger, Steven Park, David W. Denning, Olaf Kniemeyer, Axel A. Brakhage, David S. Perlin
Formato:	article
Lenguaje:	EN
Publicado:	American Society for Microbiology 2019
Materias:	Aspergillus fumigatus ROS antifungal resistance caspofungin echinocandins glucan synthase Microbiology QR1-502
Acceso en línea:	https://doaj.org/article/2dcc72bc71854ea8b9eab4d51ffbbb11
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spelling	oai:doaj.org-article:2dcc72bc71854ea8b9eab4d51ffbbb112021-11-15T15:55:25ZStress-Induced Changes in the Lipid Microenvironment of β-(1,3)-<sc>d</sc>-Glucan Synthase Cause Clinically Important Echinocandin Resistance in <named-content content-type="genus-species">Aspergillus fumigatus</named-content>10.1128/mBio.00779-192150-7511https://doaj.org/article/2dcc72bc71854ea8b9eab4d51ffbbb112019-06-01T00:00:00Zhttps://journals.asm.org/doi/10.1128/mBio.00779-19https://doaj.org/toc/2150-7511ABSTRACT Aspergillus fumigatus is a leading cause of invasive fungal infections. Resistance to first-line triazole antifungals has led to therapy with echinocandin drugs. Recently, we identified several high-minimum-effective-concentration (MEC) A. fumigatus clinical isolates from patients failing echinocandin therapy. Echinocandin resistance is known to arise from amino acid substitutions in β-(1,3)-d-glucan synthase encoded by the fks1 gene. Yet these clinical isolates did not contain mutations in fks1, indicating an undefined resistance mechanism. To explore this new mechanism, we used a laboratory-derived strain, RG101, with a nearly identical caspofungin (CAS) susceptibility phenotype that also does not contain fks1 mutations. Glucan synthase isolated from RG101 was fully sensitive to echinocandins. Yet exposure of RG101 to CAS during growth yielded a modified enzyme that was drug insensitive (4 log orders) in kinetic inhibition assays, and this insensitivity was also observed for enzymes isolated from clinical isolates. To understand this alteration, we analyzed whole-enzyme posttranslational modifications (PTMs) but found none linked to resistance. However, analysis of the lipid microenvironment of the enzyme with resistance induced by CAS revealed a prominent increase in the abundances of dihydrosphingosine (DhSph) and phytosphingosine (PhSph). Exogenous addition of DhSph and PhSph to the sensitive enzyme recapitulated the drug insensitivity of the CAS-derived enzyme. Further analysis demonstrated that CAS induces mitochondrion-derived reactive oxygen species (ROS) and that dampening ROS formation by antimycin A or thiourea eliminated drug-induced resistance. We conclude that CAS induces cellular stress, promoting formation of ROS and triggering an alteration in the composition of plasma membrane lipids surrounding glucan synthase, rendering it insensitive to echinocandins. IMPORTANCE Resistance to first-line triazole antifungal agents among Aspergillus species has prompted the use of second-line therapy with echinocandins. As the number of Aspergillus-infected patients treated with echinocandins is rising, clinical observations of drug resistance are also increasing, indicating an emerging global health threat. Our knowledge regarding the development of clinical echinocandin resistance is largely derived from Candida spp., while little is known about resistance in Aspergillus. Therefore, it is important to understand the specific cellular responses raised by A. fumigatus against echinocandins. We discovered a new mechanism of resistance in A. fumigatus that is independent of the well-characterized FKS mutation mechanism observed in Candida. This study identified an off-target effect of CAS, i.e., ROS production, and integrated oxidative stress and sphingolipid alterations into a novel mechanism of resistance. This stress-induced response has implications for drug resistance and/or tolerance mechanisms in other fungal pathogens.Shruthi SatishCristina Jiménez-OrtigosaYanan ZhaoMin Hee LeeEnriko DolgovThomas KrügerSteven ParkDavid W. DenningOlaf KniemeyerAxel A. BrakhageDavid S. PerlinAmerican Society for MicrobiologyarticleAspergillus fumigatusROSantifungal resistancecaspofunginechinocandinsglucan synthaseMicrobiologyQR1-502ENmBio, Vol 10, Iss 3 (2019)
institution	DOAJ
collection	DOAJ
language	EN
topic	Aspergillus fumigatus ROS antifungal resistance caspofungin echinocandins glucan synthase Microbiology QR1-502
spellingShingle	Aspergillus fumigatus ROS antifungal resistance caspofungin echinocandins glucan synthase Microbiology QR1-502 Shruthi Satish Cristina Jiménez-Ortigosa Yanan Zhao Min Hee Lee Enriko Dolgov Thomas Krüger Steven Park David W. Denning Olaf Kniemeyer Axel A. Brakhage David S. Perlin Stress-Induced Changes in the Lipid Microenvironment of β-(1,3)-<sc>d</sc>-Glucan Synthase Cause Clinically Important Echinocandin Resistance in <named-content content-type="genus-species">Aspergillus fumigatus</named-content>
description	ABSTRACT Aspergillus fumigatus is a leading cause of invasive fungal infections. Resistance to first-line triazole antifungals has led to therapy with echinocandin drugs. Recently, we identified several high-minimum-effective-concentration (MEC) A. fumigatus clinical isolates from patients failing echinocandin therapy. Echinocandin resistance is known to arise from amino acid substitutions in β-(1,3)-d-glucan synthase encoded by the fks1 gene. Yet these clinical isolates did not contain mutations in fks1, indicating an undefined resistance mechanism. To explore this new mechanism, we used a laboratory-derived strain, RG101, with a nearly identical caspofungin (CAS) susceptibility phenotype that also does not contain fks1 mutations. Glucan synthase isolated from RG101 was fully sensitive to echinocandins. Yet exposure of RG101 to CAS during growth yielded a modified enzyme that was drug insensitive (4 log orders) in kinetic inhibition assays, and this insensitivity was also observed for enzymes isolated from clinical isolates. To understand this alteration, we analyzed whole-enzyme posttranslational modifications (PTMs) but found none linked to resistance. However, analysis of the lipid microenvironment of the enzyme with resistance induced by CAS revealed a prominent increase in the abundances of dihydrosphingosine (DhSph) and phytosphingosine (PhSph). Exogenous addition of DhSph and PhSph to the sensitive enzyme recapitulated the drug insensitivity of the CAS-derived enzyme. Further analysis demonstrated that CAS induces mitochondrion-derived reactive oxygen species (ROS) and that dampening ROS formation by antimycin A or thiourea eliminated drug-induced resistance. We conclude that CAS induces cellular stress, promoting formation of ROS and triggering an alteration in the composition of plasma membrane lipids surrounding glucan synthase, rendering it insensitive to echinocandins. IMPORTANCE Resistance to first-line triazole antifungal agents among Aspergillus species has prompted the use of second-line therapy with echinocandins. As the number of Aspergillus-infected patients treated with echinocandins is rising, clinical observations of drug resistance are also increasing, indicating an emerging global health threat. Our knowledge regarding the development of clinical echinocandin resistance is largely derived from Candida spp., while little is known about resistance in Aspergillus. Therefore, it is important to understand the specific cellular responses raised by A. fumigatus against echinocandins. We discovered a new mechanism of resistance in A. fumigatus that is independent of the well-characterized FKS mutation mechanism observed in Candida. This study identified an off-target effect of CAS, i.e., ROS production, and integrated oxidative stress and sphingolipid alterations into a novel mechanism of resistance. This stress-induced response has implications for drug resistance and/or tolerance mechanisms in other fungal pathogens.
format	article
author	Shruthi Satish Cristina Jiménez-Ortigosa Yanan Zhao Min Hee Lee Enriko Dolgov Thomas Krüger Steven Park David W. Denning Olaf Kniemeyer Axel A. Brakhage David S. Perlin
author_facet	Shruthi Satish Cristina Jiménez-Ortigosa Yanan Zhao Min Hee Lee Enriko Dolgov Thomas Krüger Steven Park David W. Denning Olaf Kniemeyer Axel A. Brakhage David S. Perlin
author_sort	Shruthi Satish
title	Stress-Induced Changes in the Lipid Microenvironment of β-(1,3)-<sc>d</sc>-Glucan Synthase Cause Clinically Important Echinocandin Resistance in <named-content content-type="genus-species">Aspergillus fumigatus</named-content>
title_short	Stress-Induced Changes in the Lipid Microenvironment of β-(1,3)-<sc>d</sc>-Glucan Synthase Cause Clinically Important Echinocandin Resistance in <named-content content-type="genus-species">Aspergillus fumigatus</named-content>
title_full	Stress-Induced Changes in the Lipid Microenvironment of β-(1,3)-<sc>d</sc>-Glucan Synthase Cause Clinically Important Echinocandin Resistance in <named-content content-type="genus-species">Aspergillus fumigatus</named-content>
title_fullStr	Stress-Induced Changes in the Lipid Microenvironment of β-(1,3)-<sc>d</sc>-Glucan Synthase Cause Clinically Important Echinocandin Resistance in <named-content content-type="genus-species">Aspergillus fumigatus</named-content>
title_full_unstemmed	Stress-Induced Changes in the Lipid Microenvironment of β-(1,3)-<sc>d</sc>-Glucan Synthase Cause Clinically Important Echinocandin Resistance in <named-content content-type="genus-species">Aspergillus fumigatus</named-content>
title_sort	stress-induced changes in the lipid microenvironment of β-(1,3)-<sc>d</sc>-glucan synthase cause clinically important echinocandin resistance in <named-content content-type="genus-species">aspergillus fumigatus</named-content>
publisher	American Society for Microbiology
publishDate	2019
url	https://doaj.org/article/2dcc72bc71854ea8b9eab4d51ffbbb11
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Stress-Induced Changes in the Lipid Microenvironment of β-(1,3)-<sc>d</sc>-Glucan Synthase Cause Clinically Important Echinocandin Resistance in <named-content content-type="genus-species">Aspergillus fumigatus</named-content>

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