Repression of N-glycosylation triggers the unfolded protein response (UPR) and overexpression of cell wall protein and chitin in Aspergillus fumigatus

Microbiology ◽  
2011 ◽  
Vol 157 (7) ◽  
pp. 1968-1979 ◽  
Author(s):  
Kai Li ◽  
Haomiao Ouyang ◽  
Yang Lü ◽  
Jingnan Liang ◽  
Iain B. H. Wilson ◽  
...  
Keyword(s):  
Cell Wall ◽  
Aspergillus Fumigatus ◽  
Unfolded Protein Response ◽  
Cell Wall Protein ◽  
Signalling Pathway ◽  
Cell Wall Biosynthesis ◽  
Unfolded Protein ◽  
Cell Wall Biogenesis ◽  
The Unfolded Protein Response ◽  
Protein Response

Aspergillus fumigatus is the most common airborne fungal pathogen, causing fatal invasive aspergillosis in immunocompromised patients. The crude mortality is 60–90 % and remains around 29–42 % even with treatment. The main reason for patient death is the low efficiency of the drug therapies. As protein N-glycosylation is involved in cell wall biogenesis in A. fumigatus, a deeper understanding of its role in cell wall biogenesis will help to develop new drug targets. The Afstt3 gene encodes the essential catalytic subunit of oligosaccharyltransferase, an enzyme complex responsible for the transfer of the N-glycan to nascent polypeptides. To evaluate the role of N-glycosylation in cell wall biosynthesis, we constructed the conditional mutant strain CPR-stt3 by replacing the endogenous promoter of Afstt3 with the nitrogen-dependent niiA promoter. Repression of the Afstt3 gene in the CPR-stt3 strain led to a severe retardation of growth and a slight defect in cell wall integrity (CWI). One of the most interesting findings was that upregulation of the cell wall-related genes was not accompanied by an activation of the MpkA kinase, which has been shown to be a central element in the CWI signalling pathway in both Saccharomyces cerevisiae and A. fumigatus. Considering that the unfolded protein response (UPR) was found to be activated, which might upregulate the expression of cell wall protein and chitin, our data suggest that the UPR, instead of the MpkA-dependent CWI signalling pathway, is the major compensatory mechanism induced by repression but not abolition of N-glycosylation in A. fumigatus. Our finding is a key to understanding the complex compensatory mechanisms of cell wall biosynthesis and may provide a new strategy for drug development.

scholarly journals Functional Coupling between the Unfolded Protein Response and Endoplasmic Reticulum/Golgi Ca2+-ATPases Promotes Stress Tolerance, Cell Wall Biosynthesis, and Virulence of Aspergillus fumigatus

mBio ◽  
2020 ◽  
Vol 11 (3) ◽  
Author(s):  
Martin Weichert ◽  
José Guirao-Abad ◽  
Vishukumar Aimanianda ◽  
Karthik Krishnan ◽  
Christina Grisham ◽  
...  
Keyword(s):  
Endoplasmic Reticulum ◽  
Cell Wall ◽  
Er Stress ◽  
Aspergillus Fumigatus ◽  
Secretory Pathway ◽  
Functional Coupling ◽  
Content Type ◽  
Unfolded Protein ◽  
The Unfolded Protein Response ◽  
Protein Response

ABSTRACT Many species of pathogenic fungi deploy the unfolded protein response (UPR) to expand the folding capacity of the endoplasmic reticulum (ER) in proportion to the demand for virulence-related proteins that traffic through the secretory pathway. Although Ca2+ plays a pivotal role in ER function, the mechanism by which transcriptional upregulation of the protein folding machinery is coordinated with Ca2+ homeostasis is incompletely understood. In this study, we investigated the link between the UPR and genes encoding P-type Ca2+-ATPases in the human-pathogenic mold Aspergillus fumigatus. We demonstrate that acute ER stress increases transcription of the srcA gene, encoding a member of the sarco/endoplasmic reticulum Ca2+-ATPase (SERCA) family, as well as that of pmrA, encoding a secretory pathway Ca2+-ATPase (SPCA) in the Golgi membrane. Loss of the UPR transcription factor HacA prevented the induction of srcA and pmrA transcription during ER stress, defining these ER/Golgi Ca2+ pumps as novel downstream targets of this pathway. While deletion of srcA alone caused no major deficiencies, a ΔsrcA/ΔpmrA mutant displayed a severe polarity defect, was hypersensitive to ER stress, and showed attenuated virulence. In addition, cell wall analyses revealed a striking reduction in mannose levels in the absence of both Ca2+ pumps. The ΔhacA mutant was hypersensitive to agents that block calcineurin-dependent signaling, consistent with a functional coupling between the UPR and Ca2+ homeostasis. Together, these findings demonstrate that the UPR integrates the need for increased levels of chaperone and folding enzymes with an influx of Ca2+ into the secretory pathway to support fungal growth, stress adaptation, and pathogenicity. IMPORTANCE The UPR is an intracellular signal transduction pathway that maintains homeostasis of the ER. The pathway is also tightly linked to the expression of virulence-related traits in diverse species of human-pathogenic and plant-pathogenic fungal species, including the predominant mold pathogen infecting humans, Aspergillus fumigatus. Despite advances in the understanding of UPR signaling, the linkages and networks that are governed by this pathway are not well defined. In this study, we revealed that the UPR is a major driving force for stimulating Ca2+ influx at the ER and Golgi membranes and that the coupling between the UPR and Ca2+ import is important for virulence, cell wall biosynthesis, and resistance to antifungal compounds that inhibit Ca2+ signaling.

Presenilin-1 mutations downregulate the signalling pathway of the unfolded-protein response

10.1038/70265 ◽  
1999 ◽  
Vol 1 (8) ◽  
pp. 479-485 ◽  
Author(s):  
Taiichi Katayama ◽  
Kazunori Imaizumi ◽  
Naoya Sato ◽  
Ko Miyoshi ◽  
Takashi Kudo ◽  
...  
Keyword(s):  
Unfolded Protein Response ◽  
Signalling Pathway ◽  
Presenilin 1 ◽  
Unfolded Protein ◽  
The Unfolded Protein Response ◽  
Protein Response

scholarly journals A Human IRE1 Inhibitor Blocks the Unfolded Protein Response in the Pathogenic Fungus Aspergillus fumigatus and Suggests Noncanonical Functions within the Pathway

mSphere ◽  
2020 ◽  
Vol 5 (5) ◽  
Author(s):  
José P. Guirao-Abad ◽  
Martin Weichert ◽  
Aaron Albee ◽  
Katie Deck ◽  
David S. Askew
Keyword(s):  
Endoplasmic Reticulum ◽  
Aspergillus Fumigatus ◽  
Unfolded Protein Response ◽  
Pathogenic Fungus ◽  
Transcriptional Regulator ◽  
Content Type ◽  
Unfolded Protein ◽  
Er Stress Response ◽  
The Unfolded Protein Response ◽  
Protein Response

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.

scholarly journals A Role for the Unfolded Protein Response (UPR) in Virulence and Antifungal Susceptibility in Aspergillus fumigatus

2009 ◽  
Vol 5 (1) ◽  
pp. e1000258 ◽  
Author(s):  
Daryl L. Richie ◽  
Lukas Hartl ◽  
Vishukumar Aimanianda ◽  
Michael S. Winters ◽  
Kevin K. Fuller ◽  
...  
Keyword(s):  
Aspergillus Fumigatus ◽  
Unfolded Protein Response ◽  
Antifungal Susceptibility ◽  
Unfolded Protein ◽  
The Unfolded Protein Response ◽  
Protein Response

scholarly journals The Unfolded Protein Response Is Induced by the Cell Wall Integrity Mitogen-activated Protein Kinase Signaling Cascade and Is Required for Cell Wall Integrity in Saccharomyces cerevisiae

2009 ◽  
Vol 20 (1) ◽  
pp. 164-175 ◽  
Author(s):  
Thomas Scrimale ◽  
Louis Didone ◽  
Karen L. de Mesy Bentley ◽  
Damian J. Krysan
Keyword(s):  
Cell Wall ◽  
Unfolded Protein Response ◽  
Wall Stress ◽  
Cell Wall Integrity ◽  
Double Mutation ◽  
Unfolded Protein ◽  
Cell Wall Stress ◽  
Mitogen Activated Protein ◽  
The Unfolded Protein Response ◽  
Protein Response

The yeast cell wall is an extracellular structure that is dependent on secretory and membrane proteins for its construction. We investigated the role of protein quality control mechanisms in cell wall integrity and found that the unfolded protein response (UPR) and, to a lesser extent, endoplasmic reticulum (ER)-associated degradation (ERAD) pathways are required for proper cell wall construction. Null mutation of IRE1, double mutation of ERAD components (hrd1Δ and ubc7Δ) and ire1Δ, or expression of misfolded proteins show phenotypes similar to mutation of cell wall proteins, including hypersensitivity to cell wall-targeted molecules, alterations to cell wall protein layer, decreased cell wall thickness by electron microscopy, and increased cellular aggregation. Consistent with its important role in cell wall integrity, UPR is activated by signaling through the cell wall integrity mitogen-activated protein (MAP) kinase pathway during cell wall stress and unstressed vegetative growth. Both cell wall stress and basal UPR activity is mediated by Swi6p, a regulator of cell cycle and cell wall stress gene transcription, in a manner that is independent of its known coregulatory molecules. We propose that the cellular responses to ER and cell wall stress are coordinated to buffer the cell against these two related cellular stresses.

scholarly journals Identification of an Exceptionally Long Intron in theHAC1Gene ofCandida parapsilosis

mSphere ◽  
2018 ◽  
Vol 3 (6) ◽  
Author(s):  
Elise Iracane ◽  
Paul D. Donovan ◽  
Mihaela Ola ◽  
Geraldine Butler ◽  
Linda M. Holland
Keyword(s):  
Endoplasmic Reticulum ◽  
Cell Wall ◽  
Transcription Factor ◽  
Unfolded Protein Response ◽  
Candida Parapsilosis ◽  
Calcofluor White ◽  
Content Type ◽  
Unfolded Protein ◽  
The Unfolded Protein Response ◽  
Protein Response

ABSTRACTThe unfolded protein response (UPR) in the endoplasmic reticulum (ER) is well conserved in eukaryotes from metazoa to yeast. The transcription factorHAC1is a major regulator of the UPR in many eukaryotes. DeletingHAC1in the yeastCandida parapsilosisrendered cells more sensitive to DTT, a known inducer of the UPR. The deletion strain was also sensitive to Congo red, calcofluor white, and the antifungal drug ketoconazole, indicating thatHAC1has a role in cell wall maintenance. Transcriptomic analysis revealed that treatment of the wild type with DTT resulted in the increased expression of 368 genes. Comparison with mutant cells treated with DTT reveals that expression of 137 of these genes requiresHAC1. Enriched GO term analysis includes response to ER stress, cell wall biogenesis and glycosylation. Orthologs of many of these are associated with UPR inSaccharomyces cerevisiaeandCandida albicans. Unconventional splicing of an intron fromHAC1mRNA is required to produce a functional transcription factor. The spliced intron varies in length from 19 bases inC. albicansto 379 bases inCandida glabrata, but has not been previously identified inCandida parapsilosisand related species. We used RNA-seq data andin silicoanalysis to identify theHAC1intron in 12 species in the CTG-Ser1 clade. We show that the intron has undergone major contractions and expansions in this clade, reaching up to 848 bases. Exposure to DTT induced splicing of the long intron inC. parapsilosisHAC1, inducing the UPR.IMPORTANCEThe unfolded protein response (UPR) responds to the build-up of misfolded proteins in the endoplasmic reticulum. The UPR has wide-ranging functions from fungal pathogenesis to applications in biotechnology. The UPR is regulated through the splicing of an unconventional intron in theHAC1gene. This intron has been described in many fungal species and is of variable length. Until now it was believed that some members of the CTG-Ser1 clade such asC. parapsilosisdid not contain an intron inHAC1, suggesting that the UPR was regulated in a different manner. Here we demonstrate thatHAC1plays an important role in regulating the UPR inC. parapsilosis. We also identified an unusually long intron (626 bp) inC. parapsilosisHAC1. Further analysis showed thatHAC1orthologs in several species in the CTG-Ser1 clade contain long introns.

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