Extensive proteomic remodeling is induced by eukaryotic translation elongation factor 1Bγ deletion in Aspergillus fumigatus

Abstract

The opportunistic pathogen Aspergillus fumigatus is ubiquitous in the environment and predominantly infects immunocompromised patients. The functions of many genes remain unknown despite sequencing of the fungal genome. A putative translation elongation factor 1Bγ (eEF1Bγ, termed elfA; 750 bp) is expressed, and exhibits glutathione S-transferase activity, in A. fumigatus. Here, we demonstrate the role of ElfA in the oxidative stress response, as well as a possible involvement in translation and actin cytoskeleton organization, respectively. Comparative proteomics, in addition to phenotypic analysis, under basal and oxidative stress conditions, demonstrated a role for A. fumigatus elfA in the oxidative stress response. An elfA-deficient strain (A. fumigatus ΔelfA) was significantly more sensitive to the oxidants H2O2, diamide, and 4,4'-dipyridyl disulfide (DPS) than the wild-type. This was further supported with the identification of differentially expressed proteins of the oxidative stress response, including; mitochondrial peroxiredoxin Prx1, molecular chaperone Hsp70 and mitochondrial glycerol-3-phosphate dehydrogenase. Phenotypic analysis also revealed that A. fumigatus ΔelfA was significantly more tolerant to voriconazole than the wild-type. The differential expression of two aminoacyl-tRNA synthetases suggests a role for A. fumigatus elfA in translation, while the identification of actin-bundling protein Sac6 and vacuolar dynamin-like GTPase VpsA link A. fumigatus elfA to the actin cytoskeleton. Overall, this work highlights the diverse roles of A. fumigatus elfA, with respect to translation, oxidative stress and actin cytoskeleton organization. In addition to this, the strategy of combining targeted gene deletion with comparative proteomics for elucidating the role of proteins of unknown function is further revealed.

Keywords: GST; elongation factor; glutathione; proteomics; redox; translation.

Figure 1

(A) H₂O₂-induced oxidative stress increases elfA expression, particularly following 15 min exposure (**P = 0.0009). (B) Impact of H₂O₂ (0–4 mM) on the growth of A. fumigatus ATCC46645, ΔelfA, and elfA^C, respectively, after 72 h. (C) Growth of A. fumigatus ATCC46645, ΔelfA, and elfA^C in response to diamide (0–4 mM) exposure. (D) The impact of 4,4′-dipyridyl disulfide (0–7.5 µM) on the growth of A. fumigatus ATCC46645, ΔelfA, and elfA^C. (E) GSH and GSSG determination in A. fumigatus ATCC46645, ΔelfA, and elfA^C under normal growth conditions. (F) Relative amounts of GSH and GSSG in A. fumigatus ATCC46645, ΔelfA, and elfA^C in the absence and presence of 2 mM H₂O₂ for 15 min. (G) GSH and GSSG amounts in A. fumigatus ATCC46645, ΔelfA, and elfA^C in the absence and presence of 2 mM H₂O₂ for 30 min. (H) Exposure to diamide causes a reduction in GSH levels in A. fumigatus ΔelfA.

Figure 2

Proteomic remodeling in A. fumigatus ΔelfA in the absence and presence of oxidative stress. 2DE analysis of A. fumigatus ATCC46645 (A) and ΔelfA (B) under normal growth conditions. 2DE analysis of A. fumigatus ATCC46645 (C) and ΔelfA (D) following exposure to 2 mM H₂O₂ for 1 h. The proteins found to be differentially expressed after analysis using Progenesis™ SameSpot software are numbered.

Figure 3

Western blot analysis of protein glutathionylation in A. fumigatus ATCC46645 and ΔelfA. (A) SDS-PAGE of A. fumigatus ATCC46645 (Lanes 1, 3, 5, 7) and ΔelfA (Lanes 2, 4, 6, 8) whole cell lysates (50 µg) under reducing (Lanes 1–4) and nonreducing conditions (Lanes 5–8). The samples in Lanes 1, 2, 5, and 6 were incubated with biotin-GSSG, while the samples in Lanes 3, 4, 7, and 8 were incubated with water as a control. (B) Western blot probed with streptavidin-HRP. Proteins glutathionylated upon addition of biotin-GSSG were detected in A. fumigatus ATCC46645 (Lane 5) and ΔelfA (Lane 6). There were more glutathionylated proteins detected in A. fumigatus ΔelfA (Lane 6) than in A. fumigatus ATCC46645 (Lane 5). No glutathionylated proteins were detected on the reduced blot (Lanes 1–4) or in the samples treated with water (Lanes 7–8). (C) Image analysis indicates a 24% increase in chemiluminescent signal intensity in A. fumigatus ΔelfA compared to A. fumigatus ATCC46645 supporting the observation that there was a higher level of protein glutathionylation in A. fumigatus ΔelfA.