Proteomic dissection of the role of GliZ in gliotoxin biosynthesis in Aspergillus fumigatus

Gliotoxin (GT) biosynthesis in fungi is encoded by the gli biosynthetic gene cluster. While GT addition autoinduces biosynthesis, Zn²⁺ has been shown to attenuate cluster activity, and it was speculated that identification of Zn2Cys6 binuclear transcription factor GliZ binding partners might provide insight into this observation. Using the Tet-ON induction system, doxycycline (DOX) presence induced GliZ fusion protein expression in, and recovery of GT biosynthesis by, A. fumigatus ΔgliZ::HA-gliZ and ΔgliZ::TAP-gliZ strains, respectively. Quantitative RT-PCR confirmed that DOX induces gli cluster gene expression (n = 5) in both A. fumigatus HA-GliZ and TAP-GliZ strains. GT biosynthesis was evident in Czapek-Dox and in Sabouraud media, however tagged GliZ protein expression was more readily detected in Sabouraud media. Unexpectedly, Zn²⁺ was essential for GliZ fusion protein expression in vivo, following 3 h DOX induction. Moreover, HA-GliZ abundance was significantly higher in either DOX/GT or DOX/Zn²⁺, compared to DOX-only. This suggests that while GT induction is still intact, Zn²⁺ inhibition of HA-GliZ production in vivo is lost. Co-immunoprecipitation revealed that GT oxidoreductase GliT associates with GliZ in the presence of GT, suggesting a potential protective role. Additional putative HA-GliZ interacting proteins included cystathionine gamma lyase, ribosomal protein L15 and serine hydroxymethyltransferase (SHMT). Total mycelial quantitative proteomic data revealed that GliT and GtmA, as well as several other gli cluster proteins, are increased in abundance or uniquely expressed with GT addition. Proteins involved in sulphur metabolism are also differentially expressed with GT or Zn²⁺ presence. Overall, we disclose that under DOX induction GliZ functionality is unexpectedly evident in zinc-replete media, subject to GT induction and that GliT appears to associate with GliZ, potentially to prevent dithiol gliotoxin (DTG)-mediated GliZ inactivation by zinc ejection.