A Human IRE1 Inhibitor Blocks the Unfolded Protein Response in the Pathogenic Fungus <named-content content-type="genus-species">Aspergillus fumigatus</named-content> and Suggests Noncanonical Functions within the Pathway

ABSTRACT The unfolded protein response (UPR) is a signaling network that maintains homeostasis of the endoplasmic reticulum (ER). In the human-pathogenic fungus Aspergillus fumigatus, the UPR is initiated by activation of an endoribonuclease (RNase) domain in the ER transmembrane stress sensor IreA,...

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Autores principales:	José P. Guirao-Abad, Martin Weichert, Aaron Albee, Katie Deck, David S. Askew
Formato:	article
Lenguaje:	EN
Publicado:	American Society for Microbiology 2020
Materias:	A. fumigatus UPR 4μ8C ER stress IRE1 IreA Microbiology QR1-502
Acceso en línea:	https://doaj.org/article/de64c7c49fec4315af7cd50de0ba93da
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spelling	oai:doaj.org-article:de64c7c49fec4315af7cd50de0ba93da2021-11-15T15:30:58ZA Human IRE1 Inhibitor Blocks the Unfolded Protein Response in the Pathogenic Fungus <named-content content-type="genus-species">Aspergillus fumigatus</named-content> and Suggests Noncanonical Functions within the Pathway10.1128/mSphere.00879-202379-5042https://doaj.org/article/de64c7c49fec4315af7cd50de0ba93da2020-10-01T00:00:00Zhttps://journals.asm.org/doi/10.1128/mSphere.00879-20https://doaj.org/toc/2379-5042ABSTRACT The unfolded protein response (UPR) is a signaling network that maintains homeostasis of the endoplasmic reticulum (ER). In the human-pathogenic fungus Aspergillus fumigatus, the UPR is initiated by activation of an endoribonuclease (RNase) domain in the ER transmembrane stress sensor IreA, which splices the downstream mRNA hacAu into its active form, hacAi, encoding the master transcriptional regulator of the pathway. Small-molecule inhibitors against IRE1, the human ortholog of IreA, have been developed for anticancer therapy, but their effects on the fungal UPR are unexplored. Here, we demonstrate that the IRE1 RNase inhibitor 4μ8C prevented A. fumigatus from increasing the levels of hacAi mRNA, thereby blocking induction of downstream UPR target gene expression. Treatment with 4μ8C had minimal effects on growth in minimal medium but severely impaired growth on a collagen substrate that requires high levels of hydrolytic enzyme secretion, mirroring the phenotype of other fungal UPR mutants. 4μ8C also increased sensitivity to carvacrol, a natural compound that disrupts ER integrity in fungi, and hygromycin B, which correlated with reduced expression of glycosylation-related genes. Interestingly, treatment with 4μ8C was unable to induce all of the phenotypes attributed to the loss of the canonical UPR in a ΔhacA mutant but showed remarkable similarity to the phenotype of an RNase-deficient IreA mutant that is also unable to generate the hacAi mRNA. These results establish proof of principle that pharmacological inhibition of the canonical UPR pathway is feasible in A. fumigatus and support a noncanonical role for the hacAu mRNA in ER stress response. IMPORTANCE The unfolded protein response (UPR) is a signaling pathway that maintains endoplasmic reticulum (ER) homeostasis, with functions that overlap virulence mechanisms in the human-pathogenic mold Aspergillus fumigatus. The canonical pathway centers on HacA, its master transcriptional regulator. Translation of this protein requires the removal of an unconventional intron from the cytoplasmic mRNA of the hacA gene, which is achieved by an RNase domain located in the ER-transmembrane stress sensor IreA. Here, we show that targeting this RNase activity with a small-molecule inhibitor effectively blocked UPR activation, resulting in effects that mirror the consequences of genetic deletion of the RNase domain. However, these phenotypes were surprisingly narrow in scope relative to those associated with a complete deletion of the hacA gene. These findings expand the understanding of UPR signaling in this species by supporting the existence of noncanonical functions for the unspliced hacA mRNA in ER stress response.José P. Guirao-AbadMartin WeichertAaron AlbeeKatie DeckDavid S. AskewAmerican Society for MicrobiologyarticleA. fumigatusUPR4μ8CER stressIRE1IreAMicrobiologyQR1-502ENmSphere, Vol 5, Iss 5 (2020)
institution	DOAJ
collection	DOAJ
language	EN
topic	A. fumigatus UPR 4μ8C ER stress IRE1 IreA Microbiology QR1-502
spellingShingle	A. fumigatus UPR 4μ8C ER stress IRE1 IreA Microbiology QR1-502 José P. Guirao-Abad Martin Weichert Aaron Albee Katie Deck David S. Askew A Human IRE1 Inhibitor Blocks the Unfolded Protein Response in the Pathogenic Fungus <named-content content-type="genus-species">Aspergillus fumigatus</named-content> and Suggests Noncanonical Functions within the Pathway
description	ABSTRACT The unfolded protein response (UPR) is a signaling network that maintains homeostasis of the endoplasmic reticulum (ER). In the human-pathogenic fungus Aspergillus fumigatus, the UPR is initiated by activation of an endoribonuclease (RNase) domain in the ER transmembrane stress sensor IreA, which splices the downstream mRNA hacAu into its active form, hacAi, encoding the master transcriptional regulator of the pathway. Small-molecule inhibitors against IRE1, the human ortholog of IreA, have been developed for anticancer therapy, but their effects on the fungal UPR are unexplored. Here, we demonstrate that the IRE1 RNase inhibitor 4μ8C prevented A. fumigatus from increasing the levels of hacAi mRNA, thereby blocking induction of downstream UPR target gene expression. Treatment with 4μ8C had minimal effects on growth in minimal medium but severely impaired growth on a collagen substrate that requires high levels of hydrolytic enzyme secretion, mirroring the phenotype of other fungal UPR mutants. 4μ8C also increased sensitivity to carvacrol, a natural compound that disrupts ER integrity in fungi, and hygromycin B, which correlated with reduced expression of glycosylation-related genes. Interestingly, treatment with 4μ8C was unable to induce all of the phenotypes attributed to the loss of the canonical UPR in a ΔhacA mutant but showed remarkable similarity to the phenotype of an RNase-deficient IreA mutant that is also unable to generate the hacAi mRNA. These results establish proof of principle that pharmacological inhibition of the canonical UPR pathway is feasible in A. fumigatus and support a noncanonical role for the hacAu mRNA in ER stress response. IMPORTANCE The unfolded protein response (UPR) is a signaling pathway that maintains endoplasmic reticulum (ER) homeostasis, with functions that overlap virulence mechanisms in the human-pathogenic mold Aspergillus fumigatus. The canonical pathway centers on HacA, its master transcriptional regulator. Translation of this protein requires the removal of an unconventional intron from the cytoplasmic mRNA of the hacA gene, which is achieved by an RNase domain located in the ER-transmembrane stress sensor IreA. Here, we show that targeting this RNase activity with a small-molecule inhibitor effectively blocked UPR activation, resulting in effects that mirror the consequences of genetic deletion of the RNase domain. However, these phenotypes were surprisingly narrow in scope relative to those associated with a complete deletion of the hacA gene. These findings expand the understanding of UPR signaling in this species by supporting the existence of noncanonical functions for the unspliced hacA mRNA in ER stress response.
format	article
author	José P. Guirao-Abad Martin Weichert Aaron Albee Katie Deck David S. Askew
author_facet	José P. Guirao-Abad Martin Weichert Aaron Albee Katie Deck David S. Askew
author_sort	José P. Guirao-Abad
title	A Human IRE1 Inhibitor Blocks the Unfolded Protein Response in the Pathogenic Fungus <named-content content-type="genus-species">Aspergillus fumigatus</named-content> and Suggests Noncanonical Functions within the Pathway
title_short	A Human IRE1 Inhibitor Blocks the Unfolded Protein Response in the Pathogenic Fungus <named-content content-type="genus-species">Aspergillus fumigatus</named-content> and Suggests Noncanonical Functions within the Pathway
title_full	A Human IRE1 Inhibitor Blocks the Unfolded Protein Response in the Pathogenic Fungus <named-content content-type="genus-species">Aspergillus fumigatus</named-content> and Suggests Noncanonical Functions within the Pathway
title_fullStr	A Human IRE1 Inhibitor Blocks the Unfolded Protein Response in the Pathogenic Fungus <named-content content-type="genus-species">Aspergillus fumigatus</named-content> and Suggests Noncanonical Functions within the Pathway
title_full_unstemmed	A Human IRE1 Inhibitor Blocks the Unfolded Protein Response in the Pathogenic Fungus <named-content content-type="genus-species">Aspergillus fumigatus</named-content> and Suggests Noncanonical Functions within the Pathway
title_sort	human ire1 inhibitor blocks the unfolded protein response in the pathogenic fungus <named-content content-type="genus-species">aspergillus fumigatus</named-content> and suggests noncanonical functions within the pathway
publisher	American Society for Microbiology
publishDate	2020
url	https://doaj.org/article/de64c7c49fec4315af7cd50de0ba93da
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A Human IRE1 Inhibitor Blocks the Unfolded Protein Response in the Pathogenic Fungus <named-content content-type="genus-species">Aspergillus fumigatus</named-content> and Suggests Noncanonical Functions within the Pathway

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