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 ΔhrdA 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 ΔhrdA ΔderA 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.

Fig 1

Deletion of A. fumigatus hrdA and derA. The hrdA gene was replaced by a phleomycin resistance cassette (ble) and the derA gene by a hygromycin resistance cassette (hph), as shown. Homologous recombination with the hrdA gene was demonstrated by Southern blotting of XbaI-digested genomic DNA using a probe in the right arm of the deletion cassette (P1). Homologous reconstitution of the ΔhrdA mutant to create a ΔhrdA::hrdA strain was demonstrated using the same probe. Homologous recombination with the derA gene was shown by hybridizing NsiI-digested genomic DNA with a probe in the left arm of the deletion cassette (P2). A ΔhrdA ΔderA strain, lacking both genes, was constructed by deleting the derA gene in the background of the ΔhrdA mutant using the same derA deletion strategy. The ΔhrdA::hrdA strain has the hrdA deletion homologously reconstituted, as detailed in Materials and Methods. X, XbaI; N, NsiI.

Fig 2

Loss of hrdA reduces ERAD efficiency. A CHX chase assay was used to monitor the degradation efficiency of a folding-defective mutant of A. fumigatus CPY (Af-CPY*). Overnight cultures of the indicated strains were treated with CHX to arrest protein synthesis, and total protein was extracted from the crushed mycelium after 6 h of incubation at 37°C. Equal amounts of protein were fractionated by SDS-PAGE and transferred to a PVDF membrane. The expected 62-kDa Af-CPY* protein was detected by immunoblot analysis using an anti-FLAG tag antibody, and actin was detected with an anti-actin antibody, as described in Materials and Methods. Quantitation of Af-CPY* levels was accomplished by measuring the relative band intensity normalized to actin levels. The values shown are relative to the wt in the absence of CHX.

Fig 3

Loss of hrdA induces the UPR. Total RNA was extracted from overnight cultures in the absence of ER stress-inducing conditions, and the expression of the UPR target gene bipA was determined by Northern blotting. Lane 1, wt; lane 2, ΔhrdA; lane 3, ΔhrdA ΔderA. Equivalent RNA loading was confirmed by staining rRNA with SYBR Green.

Fig 4

Loss of ERAD increases sensitivity to acute ER stress. Conidia from the indicated strains were inoculated onto solid medium in a multiwell plate containing AMM and the indicated concentrations of BFA or TM. The plates were incubated at 37°C for 2 days. Dimethyl sulfoxide (DMSO) was used as the vehicle control for TM.

Fig 5

HrdA promotes thermotolerant growth. Conidia from the indicated strains were spotted onto the center of a plate containing rich medium (IMA), and colony diameter was measured after 3 days of growth at the indicated temperatures. The experiment was performed in triplicate, and the values represent the average ± SD. *, statistically significant by Student's t test (P < 0.001).

Fig 6

ERAD protects against cell wall stress. A total of 2,000 conidia were inoculated into the center of each well of a 24-well plate containing 2 ml of IMA agar supplemented with the indicated concentrations of CFW or CR and incubated for 2 days at 37°C.

Fig 7

Loss of ERAD impairs the secretion of collagenolytic activity. Conidia from the indicated strains were inoculated in liquid cultures of AMM containing fetal bovine serum as the sole carbon/nitrogen source. After incubation for 72 h at 37°C, the collagenolytic activity of culture supernatants was quantified using the Azocoll assay, as described in Materials and Methods. The experiment was performed in triplicate, and the values represent the mean A₅₂₀/g (dry weight) plus SD. *, statistically significant by Student's t test (P < 0.001).

Fig 8

Loss of HrdA reduces voriconazole sensitivity. Conidia from the indicated strains were spread onto the surface of a plate of IMA, and an Etest strip containing voriconazole was applied to the inoculated agar surface. The plates were incubated at 37°C for 24 h. The two strains here are shown for clarity. The voriconazole sensitivities of the additional strains in this study are shown in Fig. S2 in the supplemental material.

Fig 9

The ΔhrdA mutant accumulates higher levels of sterol intermediates in response to voriconazole treatment. Analysis of sterol content (ergosterol and two unidentified intermediates) was performed in the presence (+) or absence (−) of voriconazole (VCZ) (0.125 μg/ml) by gas chromatography. The values represent the averages of three replicates, expressed as μg sterol per mg dry fungal weight plus SD (*, P < 0.005; **, P < 0.0005).

Fig 10

HrdA is dispensable for virulence. Groups of 12 CF-1 outbred mice were immunosuppressed with triamcinolone on day −1 and infected intranasally with conidia from the indicated strains on day 0. Mortality was monitored for 7 days. The virulence levels of all mutant strains were statistically indistinguishable from that of the wt.