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 The PGRS Domain of Mycobacterium tuberculosis PE_PGRS Protein Rv0297 Is Involved in Endoplasmic Reticulum Stress-Mediated Apoptosis through Toll-Like Receptor 4

mBio ◽  
2018 ◽  
Vol 9 (3) ◽  
Author(s):  
Sonam Grover ◽  
Tarina Sharma ◽  
Yadvir Singh ◽  
Sakshi Kohli ◽  
Manjunath P. ◽  
...  
Keyword(s):  
Endoplasmic Reticulum ◽  
Mycobacterium Tuberculosis ◽  
Er Stress ◽  
Unfolded Protein Response ◽  
Toll Like Receptor ◽  
Toll Like Receptor 4 ◽  
Content Type ◽  
Unfolded Protein ◽  
The Unfolded Protein Response ◽  
Protein Response

ABSTRACT The genome of Mycobacterium tuberculosis , the causal organism of tuberculosis (TB), encodes a unique protein family known as the PE/PPE/PGRS family, present exclusively in the genus Mycobacterium and nowhere else in the living kingdom, with largely unexplored functions. We describe the functional significance of the PGRS domain of Rv0297, a member of this family. In silico analyses revealed the presence of intrinsically disordered stretches and putative endoplasmic reticulum (ER) localization signals in the PGRS domain of Rv0297 (Rv0297PGRS). The PGRS domain aids in ER localization, which was shown by infecting macrophage cells with M. tuberculosis and by overexpressing the protein by transfection in macrophage cells followed by activation of the unfolded protein response, as evident from increased expression of GRP78/GRP94 and CHOP/ATF4, leading to disruption of intracellular Ca 2+ homeostasis and increased nitric oxide (NO) and reactive oxygen species (ROS) production. The consequent activation of the effector caspase-8 resulted in apoptosis of macrophages, which was Toll-like receptor 4 (TLR4) dependent. Administration of recombinant Rv0297PGRS (rRv0297PGRS) also exhibited similar effects. These results implicate a hitherto-unknown role of the PGRS domain of the PE_PGRS protein family in ER stress-mediated cell death through TLR4. Since this protein is already known to be present at later stages of infection in human granulomas it points to the possibility of it being employed by M. tuberculosis for its dissemination via an apoptotic mechanism. IMPORTANCE Apoptosis is generally thought to be a defense mechanism in protecting the host against Mycobacterium tuberculosis in early stages of infection. However, apoptosis during later stages in lung granulomas may favor the bacterium in disseminating the disease. ER stress has been found to induce apoptosis in TB granulomas, in zones where apoptotic macrophages accumulate in mice and humans. In this study, we report ER stress-mediated apoptosis of host cells by the Rv0297-encoded PE_PGRS5 protein of M. tuberculosis exceptionally present in the pathogenic Mycobacterium genus. The PGRS domain of Rv0297 aids the protein in localizing to the ER and induces the unfolded protein response followed by apoptosis of macrophages. The effect of the Rv0297PGRS domain was found to be TLR4 dependent. This study presents novel insights on the strategies employed by M. tuberculosis to disseminate the disease.

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.

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.

scholarly journals The unfolded protein response coordinates the production of endoplasmic reticulum protein and endoplasmic reticulum membrane.

1997 ◽  
Vol 8 (9) ◽  
pp. 1805-1814 ◽  
Author(s):  
J S Cox ◽  
R E Chapman ◽  
P Walter
Keyword(s):  
Endoplasmic Reticulum ◽  
Unfolded Protein Response ◽  
Secretory Pathway ◽  
Lipid Synthesis ◽  
Secretory Proteins ◽  
Endoplasmic Reticulum Membrane ◽  
Yeast Saccharomyces Cerevisiae ◽  
Unfolded Protein ◽  
The Unfolded Protein Response ◽  
Protein Response

The endoplasmic reticulum (ER) is a multifunctional organelle responsible for production of both lumenal and membrane components of secretory pathway compartments. Secretory proteins are folded, processed, and sorted in the ER lumen and lipid synthesis occurs on the ER membrane itself. In the yeast Saccharomyces cerevisiae, synthesis of ER components is highly regulated: the ER-resident proteins by the unfolded protein response and membrane lipid synthesis by the inositol response. We demonstrate that these two responses are intimately linked, forming different branches of the same pathway. Furthermore, we present evidence indicating that this coordinate regulation plays a role in ER biogenesis.

scholarly journals Class I histone deacetylases localize to the endoplasmic reticulum and modulate the unfolded protein response

2012 ◽  
Vol 26 (6) ◽  
pp. 2437-2445 ◽  
Author(s):  
Soumen Kahali ◽  
Bhaswati Sarcar ◽  
Antony Prabhu ◽  
Edward Seto ◽  
Prakash Chinnaiyan
Keyword(s):  
Endoplasmic Reticulum ◽  
Unfolded Protein Response ◽  
Histone Deacetylases ◽  
Class I ◽  
Unfolded Protein ◽  
The Unfolded Protein Response ◽  
Protein Response
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