A Glutathione S-Transferase, GliG, M ay M ediate Thiol Incorporation in Gliotoxin Biosynthesis And Is Not Involved in Auto-Protection Against Gliotoxin

Carol Davis 1*, Stephen Carberry 1, Markus Schrettl1, Dermot Brougham 3, Kevin Kavanagh 1, John Stephens 2 and Sean Doyle 1.

Author address: 

Department of Biology and National Institute for Cellular Biotechnology, National University of Ireland Maynooth, Co. Kildare, Ireland 1. Department of Chemistry, National University of Ireland Maynooth, Co. Kildare, Ireland 2. School of Chemical Sc

Abstract: 

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 ( )aku80 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 )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 (GT P). 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 )gliG 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
2010

abstract No: 

1.

Full conference title: 

The 7th International Aspergillus Meeting
    • Asperfest 7 (2010)