The function of a number of genes in the gliotoxin biosynthetic cluster (gli) in Aspergillus fumigatus remains unknown. Here, we
demonstrate that gliK deletion from two strains of A. fumigatus completely abolished gliotoxin biosynthesis. Furthermore,
exogenous H2O2 (1 mM), but not gliotoxin, significantly induced A. fumigatus gliK expression (P 0.0101). While both mutants
exhibited significant sensitivity to both exogenous gliotoxin (P<0.001) and H2O2 (P<0.01), unexpectedly, exogenous
gliotoxin relieved H2O2-induced growth inhibition in a dose-dependent manner (0 to 10 g/ml). Gliotoxin-containing organic
extracts derived from A. fumigatus ATCC 26933 significantly inhibited (P<0.05) the growth of the gliK26933 deletion mutant.
The A. fumigatus gliK26933 mutant secreted metabolites, devoid of disulfide linkages or free thiols, that were detectable by reverse-
phase high-performance liquid chromatography and liquid chromatography-mass spectrometry with m/z 394 to 396.
These metabolites (m/z 394 to 396) were present at significantly higher levels in the culture supernatants of the A. fumigatus
gliK26933 mutant than in those of the wild type (P0.0024 [fold difference, 24] and P0.0003 [fold difference, 9.6], respectively)
and were absent from A. fumigatus gliG. Significantly elevated levels of ergothioneine were present in aqueous mycelial
extracts of the A. fumigatus gliK26933 mutant compared to the wild type (P<0.001). Determination of the gliotoxin uptake rate
revealed a significant difference (P0.0045) between that of A. fumigatus ATCC 46645 (9.3 pg/mg mycelium/min) and the
gliK46645 mutant (31.4 pg/mg mycelium/min), strongly suggesting that gliK absence and the presence of elevated ergothioneine
levels impede exogenously added gliotoxin efflux. Our results confirm a role for gliK in gliotoxin biosynthesis and reveal new
insights into gliotoxin functionality in A. fumigatus
