At the metal–metabolite interface in Aspergillus fumigatus: towards untangling the intersecting roles of zinc and gliotoxin

Cryptic links between apparently unrelated metabolic systems represent potential new drug targets in fungi. Evidence of such a link between zinc and gliotoxin (GT) biosynthesis in Aspergillus fumigatus is emerging. Expression of some genes of the GT bio-synthetic gene cluster gli is influenced by the zinc-dependent transcription activator ZafA, zinc may relieve GT-mediated fungal growth inhibition and, surprisingly, GT biosynthesis is influenced by zinc availability. In A. fumigatus, dithiol gliotoxin (DTG), which has zinc-chelating properties, is converted to either GT or bis-dethiobis(methylthio)gliotoxin (BmGT) by oxidoreductase GliT and methyltransferase GtmA, respectively. A double deletion mutant lacking both GliT and GtmA was previously observed to be hypersensitive to exogenous GT exposure. Here we show that compared to wild-type exposure, exogenous GT and the zinc cheator N,N,N′,N′-tetrakis(2-pyridinylmethyl)−1,2-ethanediamine (TPEN) inhibit A. fumigatus ΔgliTΔgtmA growth, specifically under zinc-limiting conditions, which can be reversed by zinc addition. While GT biosynthesis is evident in zinc- depleted medium, addition of zinc (1μM) suppressed GT and activated BmGT production. In addition, secretion of the unferrated siderophore, tria- cetylfusarinine C (TAFC), was evident by A. fumigatus wild-type (at >5 μM zinc) and ΔgtmA (at >1 μM zinc) in a low-iron medium. TAFC secretion suggests that differential zinc-sensing between both strains may influence fungal Fe3+ requirement. Label-free quantitative proteomic analysis of both strains under equivalent differential zinc conditions revealed protein abundance alterations in accordance with altered metabolomic observations, in addition to increased GliT abundance in ΔgtmA at 5 μM zinc, compared to wild-type, supporting a zinc-sensing deficiency in the mutant strain. The relative abundance of a range of oxidore-ductase- and secondary metabolism- related enzymes was also evident in a zinc- and strain- dependent manner. Overall, we elaborate new linkages between zinc availability, natural product biosynthesis and oxidative stress homeostasis in A. fumigatuA.M.T. is funded by a John and Pat Hume PhD Scholarship from Maynooth University. This work was in part supported by the Ministerio de Ciencia e Innovación (Spain) through grant PID2019-110994RB-I00 to J.A.C. LC-MS and HPLC facilities were funded by competitive awards from Science Foundation Ireland [12/RI/2346 (3)], to S.D., and the Irish Higher Education Authority, respectively