Ergothioneine Biosynthesis and Functionality in the Opportunistic Fungal Pathogen, Aspergillus fumigatus

Abstract

Ergothioneine (EGT; 2-mercaptohistidine trimethylbetaine) is a trimethylated and sulphurised histidine derivative which exhibits antioxidant properties. Here we report that deletion of Aspergillus fumigatus egtA (AFUA_2G15650), which encodes a trimodular enzyme, abrogated EGT biosynthesis in this opportunistic pathogen. EGT biosynthetic deficiency in A. fumigatus significantly reduced resistance to elevated H₂O₂ and menadione, respectively, impaired gliotoxin production and resulted in attenuated conidiation. Quantitative proteomic analysis revealed substantial proteomic remodelling in ΔegtA compared to wild-type under both basal and ROS conditions, whereby the abundance of 290 proteins was altered. Specifically, the reciprocal differential abundance of cystathionine γ-synthase and β-lyase, respectively, influenced cystathionine availability to effect EGT biosynthesis. A combined deficiency in EGT biosynthesis and the oxidative stress response regulator Yap1, which led to extreme oxidative stress susceptibility, decreased resistance to heavy metals and production of the extracellular siderophore triacetylfusarinine C and increased accumulation of the intracellular siderophore ferricrocin. EGT dissipated H₂O₂ in vitro, and elevated intracellular GSH levels accompanied abrogation of EGT biosynthesis. EGT deficiency only decreased resistance to high H₂O₂ levels which suggests functionality as an auxiliary antioxidant, required for growth at elevated oxidative stress conditions. Combined, these data reveal new interactions between cellular redox homeostasis, secondary metabolism and metal ion homeostasis.

Figure 1. Ergothioneine displayed in both thiol and thione form and EgtA structure.

(a) The structure of ergothioneine in both thiol and thione form. (b) EgtA domain architecture, 844 amino acids, domains include a methyltransferase domain at the N-terminal end, a sulfatase-modifying factor enzyme 1 at the C-terminal and an internal 5′-histidylcysteine sulfoxide synthase domain.

Figure 2. EGT detection via RP-HPLC and LC-MS in ATCC26933, ΔegtA²⁶⁹³³ and egtA^C26933.

(a) RP-HPLC Chromatograms showing the detection of 5′-IAF labelled EGT. EGT was detected at a retention time of 12.4 min. EGT was detected in the wild-type and complemented sample at 12.4 min, but is absent from ΔegtA. (b) Extracted Ion Chromatographs (m/z: 617) following LC-MS analysis of TCA precipitated 5′-IAF-labelled protein extracts from ATCC26933, ΔegtA²⁶⁹³³ and egtA^C26933, in addition to an EGT standard. A peak at 5.3 min was confirmed to be EGT. This peak was absent from the ΔegtA²⁶⁹³³ fraction, confirming the absence of EGT from the mutant. (c) Signature Ion breakdown corresponding to 5′-IAF labelled EGT.

Figure 3. Plate assays performed on AMM agar (containing 5 mM ammonium tartrate as nitrogen source) for 72 h to test for sensitivity to various ROS inducing agents.

(a) Plate assay with H₂O₂ ranging from 0 to 3 mM. ΔegtA²⁶⁹³³ shows a significant (P = 0.0081) reduction in growth compared to ATCC26933 and egtA^C at 3 mM H₂O₂. (b) Plate assay with menadione ranging from 0 to 60 μM. ΔegtA²⁶⁹³³ shows significantly reduced growth compared to ATCC26933 and egtA^C at both 40 μM (p = 0.0043) and 60 μM (P = 0.0013) menadione. (c) Plate assays with diamide ranging from 1.25 to 1.75 mM. ΔegtA²⁶⁹³³ shows no significant difference in growth compared to ATCC26933 or egtA^C.

Figure 4. Plate assays performed on AMM agar (containing 20 mM L-glutamine as nitrogen source) for 48 h to test for sensitivity to various ROS inducing agents and heavy metals.

Concentrations of stress-inducing agents (hydrogen peroxide, H₂O₂; paraquat, PQ; menadione, MD; tert-butylhydroperoxide, TBHP) and metals (iron, FeSO₄; copper, CuSO₄; zinc, ZnSO₄; cobalt, CoCl₂) are given in mM; All strains are AfS77 derivatives and 10⁴ spores were spotted. In wild-type-background EGT deficiency (strain ∆egtA) impaired resistance against H₂O₂ and MD and in a ∆yap1-background (strain ∆egtA∆yap1; Yap1 is a transcriptional activator orchestrating oxidative stress defense28) additionally against PQ and the metals copper, zinc and cobalt and iron. Complementation of EGT deficiency in the double mutant strain (egtA^c∆yap1) restored phenotype to that of a Yap1-lacking mutant.

Figure 5. Metabolic pathways linking cystathionine, glutathione and methionine metabolism.

(a) Comparison of ΔegtA²⁶⁹³³ and ATCC26933 under basal conditions showing an absence of cystathionine γ-synthase, which converts cysteine to cystathionine. This could be due to a switch towards increased glutathione production. (b) Comparison of ΔegtA²⁶⁹³³ and ATCC26933 upon addition of 3 mM H₂O₂ shows an increased abundance of cystathionine β-synthase, which converts cystathionine to homocysteine. This would result in increased production of SAM, which is required for EGT biosynthesis.

Figure 6. egtA expression and EGT detection following 3 mM H₂O₂ exposure.

(a) Quantitative RT-PCR data showing the relative expression of egtA in ATCC26933 following exposure to 3 mM H₂O₂. egtA levels are significantly (P = 0.0002) increased in ATCC26933 when exposed to H₂O₂ compared to control levels. (b) RP-HPLC analysis of 5′-IAF labelled EGT following ATCC26933 exposure to 3 mM H₂O₂. No significant variation in EGT levels was observed in H₂O₂ samples compared to control. (c) RP-HPLC data showing peak area of 5′-IAF labelled EGT following purified EGT incubation with 3 mM in H₂O₂. EGT levels drop significantly (P = 0.029) following 3 h reaction with 3 mM H₂O₂.

Figure 7. GSH detection via LC-MS in ATCC26933, ΔegtA²⁶⁹³³ and egtA^C26933.

(a) Extracted Ion Chromatographs (m/z: 695) following LC-MS analysis of TCA precipitated 5′-IAF-labelled mycelial extracts from ATCC26933, ΔegtA²⁶⁹³³ and egtA^C26933, in addition to a GSH standard. A peak at 5.9 min was confirmed to be GSH. (b) Peak height data from LC-MS analysis comparing GSH levels in ATCC26933, ΔegtA²⁶⁹³³ and egtA^C26933. GSH levels are significantly (P = 0.0016) increased in ΔegtA²⁶⁹³³ compared to the wild-type and complemented samples. (c) Signature Ion breakdown corresponding to 5-IAF labelled GSH.

Figure 8. Biomass levels and siderophore production of AfS77, ∆egtA^AfS77, ∆yap1 and ∆egtA∆yap1 under iron-deplete conditions (containing 20 mM L-glutamine as nitrogen source).

(a) Biomass production of A. fumigatus strains cultivated for 24 h at 37 °C during iron starvation (-Fe) and iron sufficiency (+Fe) show no remarkable differences. (b) EGT-deficiency (strain ∆egtA) caused higher levels of FC. (c) Combined with Yap1 deficiency, EGT deficiency increased FsC and decreased TAFC production (d) Exemplar RP-HPLC chromatogram (detection at 435 nm to detect red siderophores after iron saturation of the culture supernatants) confirming increased FsC and decreased TAFC production in ∆egtA∆yap1 (red line) in comparison to ∆yap1 (black line).

Figure 9. Northern analysis revealing downregulation of sidG expression in ∆egtA∆yap1.

Total RNA (10 μg) from AfS77 (wt), ∆egtA^AfS77 (∆e), ∆yap1 (∆y) and ∆egtA∆yap1 (∆e∆y) was isolated from submersed AMM cultures (containing 20 mM L-glutamine as nitrogen source) grown under iron starvation (−Fe) or sufficiency (+Fe) at 37 °C for 16 h plus 45 min with or without addition of H₂O₂ to a final concentration of 1 mM. See Supplementary Figure S8.

Figure 10. Gliotoxin detection via RP-HPLC and LC-MS in ATCC26933 and ΔegtA²⁶⁹³³.

(a) RP-HPLC analysis of organic extracts from the supernatants of 72 h cultures of ATCC26933 and ΔegtA²⁶⁹³³. Gliotoxin is present in all chromatograms for both samples at 14.9 min. (b) Comparison of gliotoxin peak area from RP-HPLC analysis for ATCC26933 and ΔegtA²⁶⁹³³ performed in triplicate. The peak area was found to be significantly (P = 0.0003) lowered in ΔegtA²⁶⁹³³. (c) Extracted Ion Chromatographs (m/z: 327) following LC-MS analysis of organic extracts of supernatants from ATCC26933 and ΔegtA²⁶⁹³³, in addition to a gliotoxin standard. A peak at 6.3 min was confirmed to be gliotoxin. (d) Signature ion breakdown corresponding to gliotoxin.

Figure 11. Conidiation in ATCC26933 and ΔegtA²⁶⁹³³.

(a) Comparison of conidial colour and appearance in colonies from ATCC26933 and egtA²⁶⁹³³. (b) Comparison of conidiation levels from ATCC26933 and egtA²⁶⁹³³ (5 mM ammonium tartrate as nitrogen source).