Gliotoxin effects on fungal growth: Mechanisms and exploitation

Abstract

a b s t r a c tAlthough initially investigated for its antifungal properties, little is actually known about the effect of gliotoxin on Aspergillus fumigatus and other fungi. We have observed that exposure of A. fumigatus to exogenous gliotoxin (14 lg/ml), under gliotoxin-limited growth conditions, results in significant alteration of the expression of 27 proteins (up-and down-regulated >1.9-fold; p < 0.05) including de novo expression of Cu, Zn superoxide dismutase, up-regulated allergen Asp f3 expression and down-regulated catalase and a peroxiredoxin levels. Significantly elevated glutathione GSH levels (p < 0.05), along with concomitant resistance to diamide, were evident in A. fumigatus DgliT, lacking gliotoxin oxidoreductase, a gliotoxin self-protection gene. Saccharomyces cerevisiae deletents (Dsod1 and Dyap1) were hypersensitive to exogenous gliotoxin, while Dgsh1 was resistant. Significant gliotoxin-mediated (5 lg/ml) growth inhibition (p < 0.001) of Aspergillus nidulans, Aspergillus terreus, Aspergillus niger, Cochliobolus heterostrophus and Neurospora crassa was also observed. Growth of Aspergillus flavus, Fusarium graminearum and Aspergillus oryzae was significantly inhibited (p < 0.001) at gliotoxin (10 lg/ml), indicating differential gliotoxin sensitivity amongst fungi. Re-introduction of gliT into A. fumigatus DgliT, at a different locus (ctsD; AFUA_4G07040, an aspartic protease), with selection on gliotoxin, facilitated deletion of ctsD without use of additional antibiotic selection markers. Absence of ctsD expression was accompanied by restoration of gliT expression, and resistance to gliotoxin. Thus, we propose gliT/gliotoxin as a useful selection marker system for fungal transformation. Finally, we suggest incorporation of gliotoxin sensitivity assays into all future fungal functional genomic studies.

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

“…Relevantly, it has been demonstrated that the A. fumigatus ⌬gliG strain does not exhibit increased sensitivity to gliotoxin or H 2 O 2 (11). Indeed, it has also been noted that gliotoxin may play a role in regulating the redox status of A. fumigatus (7,33). However, despite extensive analyses, no evidence of enhanced gliotoxin efflux was detectable following exposure of A. fumigatus to H 2 O 2 .…”

“…Reduction and alkylation (using 5=-iodoacetamidofluorescein; 5=-IAF) of organic extracts was performed as described previously (11,12). For intracellular metabolite analysis, mycelia were lysed as described previously (7,17) prior to reduction and alkylation (11,12). Alternatively, total mycelial thiol quantitation was undertaken using aldrithiol-4 (11 mg/ml in ethanol) analysis (35).…”

Quantitative proteomics reveals the mechanism and consequence of gliotoxin-mediated dysregulation of the methionine cycle in Aspergillus niger

“…S2) over a 4 h period. Interestingly, A. nidulans is also sensitive to exogenous GT [6], and is devoid of the gli cluster responsible for GT biosynthesis [4]. This observation shows that GT-naïve Aspergillus spp.…”

“…It has been revealed that the secretome of A. fumigatus can inhibit the growth of Candida albicans and Cryptococccus neoformans through the production of GT [5]. The anti-fungal properties of GT have also been observed in A. niger [6], although the mechanism of this growth inhibition is unclear.…”

Exploration of Sulfur Assimilation of Aspergillus fumigatus Reveals Biosynthesis of Sulfur-Containing Amino Acids as a Virulence Determinant

“…GSH and GSSG levels were measured as described previously by Carberry and colleagues (47), with the exception that 200 mg of mycelia was used for glutathione extraction and whole-cell lysates. Samples were diluted 1/10 for determination of GSSG levels and 1/50 for determination of GSH levels.…”