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

Abstract

Proteins that are destined for release outside the eukaryotic cell, insertion into the plasma membrane, or delivery to intracellular organelles are processed and folded in the endoplasmic reticulum (ER). An imbalance between the level of nascent proteins entering the ER and the organelle's ability to manage that load results in the accumulation of unfolded proteins. Terminally unfolded proteins are disposed of by ER-associated degradation (ERAD), a pathway that transports the aberrant proteins across the ER membrane into the cytosol for proteasomal degradation. The ERAD pathway was targeted in the mold pathogen Aspergillus fumigatus by deleting the hrdA gene, encoding the A. fumigatus ortholog of Hrd1, the E3 ubiquitin ligase previously shown to contribute to ERAD in other species. Loss of HrdA was associated with impaired degradation of a folding-defective ERAD substrate, CPY*, as well as activation of the unfolded-protein response (UPR). The ⌬hrdA mutant showed resistance to voriconazole and reduced thermotolerance but was otherwise unaffected by a variety of environmental stressors. A double-deletion mutant deficient in both HrdA and another component of the same ERAD complex, DerA, was defective in secretion and showed hypersensitivity to ER, thermal, and cell wall stress. However, the ⌬hrdA ⌬derA mutant remained virulent in mouse and insect infection models. These data demonstrate that HrdA and DerA support complementary ERAD functions that promote survival under conditions of ER stress but are dispensable for virulence in the host environment.

The fungal UPR

“…Moreover, the Δ derA /Δ hacA mutant was avirulent, which contrasted the wt virulence of Δ derA and the partial virulence of Δ hacA 50 . Similar findings were observed by deleting the hrdA gene, encoding a ubiquitin ligase that is part of the same ERAD complex 51 . Together, these findings established that ERAD cooperates with the UPR to support functions of the secretory pathway that are necessary for virulence.…”

Autophagy is dispensable to overcome ER stress in the filamentous fungus Aspergillus niger

“…Yet another possibility is proteasomal degradation independent of DerA. In A. fumigatus it has been demonstrated that deletion of the ERAD gene hrdA in addition to the deletion of derA highly increased the susceptibility to ER stressing agents such as tunicamycin (Krishnan et al 2013), indicating that DerA and HrdA have synergistic functions. Further studies are required to explore the proteasomal pathway either by making double knockout mutants of the ERAD system or by applying proteasome inhibitors like MG132 (Kautto et al 2013) and the fluorescent mtGlaA::GFP protein could be used as a reporter to monitor protein accumulations under these conditions.…”

“…; Krishnan et al. ). Deletion of the der1 homolog derA in a heterologous protein overexpression strain of Aspergillus niger resulted in a sixfold increase in the intracellular accumulation of the protein accompanied by the induction of UPR target genes, but had no effect on growth and conidiation (Carvalho et al.…”

“…Studies in yeast and mammalian cells indicate that recognition, retrograde transport, and ubiquitination of misfolded proteins that accumulate in the ER lumen are mediated by the Hrd1 membrane complex, which includes the E3-ubiquitin ligase Hrd1 itself as well as Hrd3, Der1, and other regulatory and scaffold proteins (Vembar and Brodsky 2008;Ruggiano et al 2014). Homologs of ERAD genes were also identified in filamentous fungi and studying deletion mutants of a number of those in Aspergilli species showed that a functional ERAD pathway is not required for growth both under normal and ER stress conditions (Carvalho et al 2011a;Krishnan et al 2013). Deletion of the der1 homolog derA in a heterologous protein overexpression strain of Aspergillus niger resulted in a sixfold increase in the intracellular accumulation of the protein accompanied by the induction of UPR target genes, but had no effect on growth and conidiation (Carvalho et al 2011a).…”

Endoplasmic reticulum stress and fungal pathogenesis

“…The disposal of any proteins that ultimately fail to achieve an appropriate conformation is accomplished by ERAD, a pathway that is regulated in part by the UPR (Travers et al, 2000). The deletion of the ERAD genes derA or hrdA had no effect on the virulence of A. fumigatus (Krishnan et al, 2013; Richie et al, 2011). However, the combined loss of derA and hacA caused a more severe reduction in hyphal growth, antifungal drug resistance and protease secretion than the loss of either gene alone.…”