Helmholtz Centre for Infection Research, Structure and Function of Proteins, Inhoffenstraße 7, 38124 Braunschweig, GermanyInstitute of Biochemistry, Biotechnology and Bioinformatics, Technische Universität Braunschweig, Spielmannstrasse 7, 38106 Braunschweig, Germany
Conversion of gliotoxin between the reduced (dithiol gliotoxin), oxidized (gliotoxin) and bisthiomethyl forms. In A. fumigatus, the gli-cluster that encodes gliotoxin biosynthesis consists of 13 genes (in colour and labelled with their last letter) and is located on chromosome 6 [4–6]. gtmA is encoded outside the cluster and is on chromosome 2.
Comparison of apo-GtmA and the SAH/SAM complexes. (a) apo- and SAH-bound GtmA. The missing secondary structure elements of apo GtmA are labelled. (b) SAH- and SAM-bound complex. The dramatic movement of the lower domain and helix α1 is indicated by arrows. (c) Co-factor binding: comparison of SAH (magenta) and SAM (yellow) binding mode. Thr27, Ala54, Asp82 and Asn109 are similar in position, only Tyr20 is flipped out in the SAM structure owing to the movement of the lower domain and helix α1. (d) Comparison of apo- (cyan) and SAH-complexed (magenta) GtmA. The disulfide bridge found between Cys55 and Cys80 of the apo structure has to be reduced to allow SAH/SAM binding. (e) Asn159 is involved in a hydrogen bonding network with Asn161, Glu182, a Na+ cation, a water molecule and Trp162. (f) Disruption of the GT binding pocket in the SAM complex. The SAH complex is coloured magenta and the SAM complex yellow. Also see electronic supplementary material, figure S5.
(a) RP-HPLC chromatograms of GtmA wild-type, GtmA W157V, GtmA W162V, GtmA N159V, GtmA F185G, GtmA F127V and a ‘no GtmA’ control incubated with dithiol gliotoxin and SAM. Dithiol gliotoxin (green), mono(methylthio)gliotoxin (orange), bis(methyl)gliotoxin (red). (b) GtmA Km determination for dithiol gliotoxin (rGT; 38.62 µM) was almost fivefold lower than that for purified MmGT (184.5 µM). (c) GtmA-mediated bis-thiomethylation occurs sequentially. The addition of increasing amounts of SAM (100–400 µM) results in the increased formation of bis(methyl)gliotoxin (red) compared with monomethylgliotoxin (orange). Dithiol gliotoxin is shown in green.
(a) Deletion of gtmA does not result in gliotoxin sensitivity when compared with A. fumigatus wild-type. Deletion of gtmA in the gliotoxin-sensitive strains ΔgliT and ΔgliA resulted in double mutants with an increased sensitivity to gliotoxin exposure. Aspergillus fumigatus ΔgliT::ΔgtmA was shown to be particularly sensitive to exogenous gliotoxin whereby the radial growth of this mutant was inhibited at 2.5 µg ml−1 gliotoxin.
(a) SAM detection in A. fumigatus wild-type and selected mutants after 21 h growth in Czapek Dox liquid medium followed by 3 h exposure to control (MeOH) or gliotoxin exposure (5 µg ml−1 final). Compared with the wild-type, significantly higher cellular SAM was detectable in ΔgliT (p = 0.0093), ΔgliA (p = 0.0003), ΔgliT::ΔgtmA (p = 0.0003) and ΔgliA::ΔgtmA (p = 0.0021). Gliotoxin exposure results in highly significant SAM depletion in ΔgliT (p = 0.0001). This SAM depletion does not occur in the ΔgliT::ΔgtmA mutant. SAM levels were also significantly reduced in ΔgliA upon gliotoxin exposure (p = 0.0100). In comparison, this reduction was not significant in the ΔgliA::ΔgtmA double mutant. SAM depletion did not occur in ΔgtmA to the same degree as in the wild-type or complemented strain (p = 0.0021). (b) Exposure of wild-type and mutant strains to 2.5 µg ml−1 gliotoxin for 3 h and quantification of the remaining GT/BmGT in the organically extracted supernatants. The bars represent the combined intensity of GT/BmGT signal on the RP-HPLC 280 nm chromatogram. Aspergillus fumigatus ΔgliT::ΔgtmA has significantly lower extracellular GT/BmGT than ΔgliT after 3 h (p = 0.0118).
(a) Aspergillus fumigatus ΔgliT responds to exogenous gliotoxin by effecting excessive bis-thiomethylation on this metabolite, resulting in cellular SAM depletion. (b) The highly sensitive A. fumigatus ΔgliT::ΔgtmA cannot remove intracellular accumulated dithiol gliotoxin by S-methylation [6,35] or oxidation. This results in cell death as a result of the combined effect of gliotoxin toxicity and gli-cluster over-activation owing to the sustained presence of dithiol gliotoxin.
Proteomic analysis of selected A. fumigatus mutants exposed to gliotoxin (2.5 µg ml−1) or a solvent (MeOH) control. The quantity of proteins deregulated in abundance upon gliotoxin exposure correlates with the sensitivity of these mutants to gliotoxin.
total proteins detected
higher abundance (+)
lower abundance (−)
below detection limit
Wild-type GT versus MeOH
ΔgtmA GT versus MeOH
ΔgliT GT versus MeOH
ΔgliT::ΔgtmA GT versus MeOH
ΔgliT versus ΔgliT::ΔgtmA GT
ΔgliT versus ΔgliT::ΔgtmA MeOH
Top five proteins with increased abundance in A. fumigatus ΔgliT::ΔgtmA compared with ΔgliT following a 3 h exposure to gliotoxin (2.5 µg ml−1). Data sorted by fold change, in descending order.
sequence coverage (%)
glutathione S-transferase encoded in the gliotoxin biosynthetic gene cluster, GliG
N methyltransferase, encoded in the putative gliotoxin biosynthetic gene cluster, GliN
conserved hypothetical protein, encoded in the putative gliotoxin biosynthetic gene cluster, GliH
predicted O-methyltransferase, encoded in the putative gliotoxin biosynthetic gene cluster, GliM
glutamyl-tRNA(Gln) amidotransferase, subunit A
D-3-phosphoglycerate dehydrogenase, role in l-serine biosynthetic process