Biosynthesis of gliotoxin is directed by the multi-gene (gli) cluster in the opportunistic fungal pathogen, Aspergillus fumigatus. Minimal functional cluster annotation is available. The gene gliG, located in the gli cluster, is classified as a glutathione s-transferase by in silico analysis and recombinant GliG exhibits GST and glutathione reductase activity. Two overlapping constructs, each containing part of a marker gene (ptrA) and with homology to gliG flanking regions, were used to disrupt gliG in A. fumigatus (Daku80 and Af293 strains). The generation of a gliG mutant was confirmed using Southern Blot analysis using a digoxigenin-labelled probe specific for an XbaI digested fragment size of 2124 bp in the wild-type and 1668 bp in the gliG mutant. Absence of gliG expression in the mutant was confirmed by Northern analysis. RP-HPLC-DAD and LC-MS analysis of extracts from A. fumigatus wild-type and 916;gliG revealed that gliotoxin (Rt= 14.4 min) was absent from the mutant strains, strongly indicating that gliG is involved in gliotoxin biosynthesis. Interestingly, an additional metabolite (Rt = 12.3 min) was present in mutant culture supernatants which may represent a precursor of gliotoxin (GTP). LC-ToF analysis determined that the metabolic intermediate had a mass of 263 Da and targeted alkylation demonstrated the lack both free thiol residues and an intact disulphide bridge. Reconstitution of gliG into A. fumigatus DgliG restored gliotoxin biosynthesis. Unlike another component of the gli cluster, gliA, it appears that gliG is not involved in the auto-protection of A. fumigatus against exogenous gliotoxin. In conclusion, we confirm a key role for the glutathione s-transferase, GliG, in the biosynthesis of, and not auto-protection against, gliotoxin- which, to our knowledge, is the first time this enzyme has been shown to play a pivotal function in ETP biosynthesis.
Full conference title:
10th EUROPEAN CONFERENCE ON FUNGAL GENETICS
- ECFG 10th (2010)