The Aspergillus fumigatus Protein GliK Protects against Oxidative Stress and Is Essential for Gliotoxin Biosynthesis

Abstract

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 H 2 O 2 (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 H 2 O 2 (P < 0.01), unexpectedly, exogenous gliotoxin relieved H 2 O 2 -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 ⌬gliK 26933 deletion mutant. The A. fumigatus ⌬gliK 26933 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 ⌬gliK 26933 mutant than in those of the wild type (P ‫؍‬ 0.0024 [fold difference, 24] and P ‫؍‬ 0.0003 [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 ⌬gliK 26933 mutant compared to the wild type (P < 0.001). Determination of the gliotoxin uptake rate revealed a significant difference (P ‫؍‬ 0.0045) between that of A. fumigatus ATCC 46645 (9.3 pg/mg mycelium/min) and the ⌬gliK 46645 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. Gliotoxin was discovered in 1936, and its structure was elucidated in 1943 (19, 38). Much initial interest in the molecule was directed toward exploiting its antifungal activities; however, after observations that gliotoxin exhibited growth-inhibitory effects against animal cells, it was posited as a putative anti-cancer or organ transplant rejection agent, and a large number of subsequent studies investigated the mechanism of action of gliotoxin against mammalian cell types (3, 24, 37).Until recently, the mechanism of gliotoxin biosynthesis had proven to be especially refractory to analysis, despite multiple studies between 1960 and 1985 involving radiolabeled precursor feeding and analysis of proteins produced during gliotoxin biosynthesis (2, 18). Since the discovery of the gliotoxin gene cluster gli (14), now known to contain 13 genes, the function of a number of cluster genes has been established by a combination of targeted gene deletion and phenotypic and biochemical analyses (4, 10, 11, 13, 22, 31-33, 34, 36). gliZ [a Zn(II) 2 -Cys(6) binuclear cluster domain transcription factor] deletio...

Interplay between Gliotoxin Resistance, Secretion, and the Methyl/Methionine Cycle in Aspergillus fumigatus

“…This scenario is in contrast to that observed for wild-type A. fumigatus, whereby downregulation of MTHFR/MtrA was noted in response to gliotoxin and H 2 O 2 combined, relative to the solvent control (11). Although an altered GliT abundance was not detectable in the A. fumigatus ⌬gliK strain during the above-mentioned 2-DE-LC-MS/MS analysis, it has been reported that elevated gliT expression occurs in response to gliotoxin exposure in this mutant (7). Here, parallel LFQ proteomic investigations of the A. fumigatus ⌬gliK strain, in the presence and absence of gliotoxin, revealed a 14-to 15-fold (log 2 -fold change ϭ 3.837)-increased abundance of GliT, compared to the 250-fold increase in wild-type A. fumigatus following gliotoxin exposure.…”

“…BmGT formation in the A. fumigatus ⌬gliT strain was identically evaluated. Culture supernatants were subjected to organic extraction and liquid chromatography-mass spectrometry (LC-MS) analysis to detect gliotoxin and BmGT, as previously described (7,10). GtmA activity in the A. fumigatus ⌬gliK strain was determined as described previously for the A. fumigatus ⌬gliT strain (10).…”

“…Wild-type A. terreus did not exhibit acetylaranotin sensitivity; however, no data were presented regarding the sensitivity of the ⌬ataTC strain to exogenous acetylaranotin. Deletion of a major facilitator superfamily (MFS) transporter, gliA, and gliK, a ␥-glutamyl cyclotransferase, also sensitizes A. fumigatus to exogenous gliotoxin albeit to a lesser extent than in the absence of GliT (7,15). While gliK deletion prevents gliotoxin biosynthesis (5, 7), interestingly, Wang et al (15) noted only a reduction, not an abolition, of gliotoxin secretion by the A. fumigatus ⌬gliA strain.…”

“…Although bisdethiobis(methylthio)gliotoxin (BmGT) (Fig. 1) has not been reported in most studies relating to gliT, gliA, gliK, or gliG (a glutathione S-transferase responsible for biosynthetic intermediate sulfurization via bis-glutathionylation) gene cluster deletion (4,5,7,13,15,18,19), this metabolite is readily detectable in culture supernatants of A. fumigatus (8)(9)(10). Moreover, endogenous dithiol gliotoxin [GT-(SH) 2 ] and exogenous gliotoxin can be converted to BmGT via a novel, S-adenosylmethionine (SAM)-dependent gliotoxin bis-thiomethyltransferase (GtmA) (10).…”

“…Indeed, existing paradigms of gliotoxin ( Fig. 1) as a toxin and the perspective of the disulfide bridge-containing (oxidized) form as the final, or only, product are undergoing significant reconsideration (6)(7)(8)(9)(10)(11).…”