HacA-Independent Functions of the ER Stress Sensor IreA Synergize with the Canonical UPR to Influence Virulence Traits in Aspergillus fumigatus

Abstract

Endoplasmic reticulum (ER) stress is a condition in which the protein folding capacity of the ER becomes overwhelmed by an increased demand for secretion or by exposure to compounds that disrupt ER homeostasis. In yeast and other fungi, the accumulation of unfolded proteins is detected by the ER-transmembrane sensor IreA/Ire1, which responds by cleaving an intron from the downstream cytoplasmic mRNA HacA/Hac1, allowing for the translation of a transcription factor that coordinates a series of adaptive responses that are collectively known as the unfolded protein response (UPR). Here, we examined the contribution of IreA to growth and virulence in the human fungal pathogen Aspergillus fumigatus. Gene expression profiling revealed that A. fumigatus IreA signals predominantly through the canonical IreA-HacA pathway under conditions of severe ER stress. However, in the absence of ER stress IreA controls dual signaling circuits that are both HacA-dependent and HacA-independent. We found that a ΔireA mutant was avirulent in a mouse model of invasive aspergillosis, which contrasts the partial virulence of a ΔhacA mutant, suggesting that IreA contributes to pathogenesis independently of HacA. In support of this conclusion, we found that the ΔireA mutant had more severe defects in the expression of multiple virulence-related traits relative to ΔhacA, including reduced thermotolerance, decreased nutritional versatility, impaired growth under hypoxia, altered cell wall and membrane composition, and increased susceptibility to azole antifungals. In addition, full or partial virulence could be restored to the ΔireA mutant by complementation with either the induced form of the hacA mRNA, hacA i, or an ireA deletion mutant that was incapable of processing the hacA mRNA, ireA Δ10. Together, these findings demonstrate that IreA has both HacA-dependent and HacA-independent functions that contribute to the expression of traits that are essential for virulence in A. fumigatus.

Effects of a Defective Endoplasmic Reticulum-Associated Degradation Pathway on the Stress Response, Virulence, and Antifungal Drug Susceptibility of the Mold Pathogen Aspergillus fumigatus

“…An emerging body of evidence suggests that the secretory pathway of filamentous fungi is a point of vulnerability that could be accessible to therapeutic intervention (6,7). The genome of A. fumigatus, like those of many other environmental saprophytic fungi, is enriched for secreted hydrolytic enzymes.…”

“…This is accomplished by remodeling gene expression to expand the protein-folding capacity of the ER in proportion to the accumulation of unfolded nascent proteins in the ER lumen. The A. fumigatus UPR is regulated by IreA (Ire1 in yeast and higher eukaryotes), a molecular sensor embedded in the ER membrane that is comprised of a lumenal sensing domain and a cytosolic effector domain (6). Studies in Saccharomyces cerevisiae have shown that unfolded proteins trigger oligomerization of this protein in the ER membrane, which activates the cytosolic domain and induces the expression of a bZIP transcription factor known as HacA in filamentous fungi (10).…”

The unfolded protein response (UPR) pathway inCryptococcus

“…48 The expression levels of some ergosterol biosynthesis genes, including ERG2, ERG11, ERG24, and ERG3, decrease in both ireAΔ and hacAΔ mutants in A. fumigatus and subsequent ergosterol levels in both stains are reduced. In Cryptococcus, ire1Δ and hxl1Δ mutants also show significantly enhanced susceptibility to azole drugs, including fluconazole, ketoconazole, and itraconazole, although the mechanism appears to be different from that of A. fumigatus based on several observations.…”

“…Comprehensive gene expression analysis of the UPR pathways in Caenorhabditis elegans and A. fumigatus strongly support the divergent regulation of Ire1 and Hac1 orthologs. 47,48 Moreover, in metazoans, the endoribonuclease activity of Ire1 was directly involved in controlling the expression of a subset of genes encoding ER proteins by degrading mRNA to reduce the unfolded protein load in the ER lumen. 49,50 It was thus proposed that Ire1 selectively degrades groups of ER-bound mRNAs to relieve the burden of unfolded proteins in the ER lumen via the regulated Ire1-dependent decay (RIDD) pathway.…”

“…57 Recent studies suggest that the UPR pathway could potentially be exploited as a novel antifungal drug target, since fungal UPR pathways play a critical role in antifungal drug resistance. 33,38,41,48 A. fumigatus ireAΔ and hacAΔ mutants exhibit increased susceptibility to azole drugs, such as itraconazole and voriconazole, which function through the inhibition of ergosterol Figure 3. The eR stress response and UPR pathways in C. neoformans.…”