Transannular disulfide formation in gliotoxin biosynthesis and its role in self-resistance of the human pathogen Aspergillus fumigatus

Article abstract of DOI:10.1021/ja103262m

Gliotoxin (1), the infamous representative of the group of epipolythiodioxopiperazines (ETPs), is a virulence factor of the human pathogenic fungus Aspergillus fumigatus. The unique redox-sensitive transannular disulfide bridge is critical for deleterious effects caused by redox cycling and protein conjugation in the host. Through a combination of genetic, biochemical, and chemical analyses, we found that 1 results from GliT-mediated oxidation of the corresponding dithiol. In vitro studies using purified GliT demonstrate that the FAD-dependent, homodimeric enzyme utilizes molecular oxygen as terminal electron acceptor with concomitant formation of H₂O ₂. In analogy to the thiol-disulfide oxidoreductase superfamily, a model for dithiol-disulfide exchange involving the conserved CxxC motif is proposed. Notably, while all studied disulfide oxidases invariably form intra-or interchenar disulfide bonds in peptides, GliT is the first studied enzyme producing an epidithio bond. Furthermore, through sensitivity assays using wild type, ΔgliT mutant, and complemented strain, we found that GliT confers resistance to the producing organism. A phylogenetic study revealed that GliT falls into a clade of yet fully uncharacterized fungal gene products deduced from putative ETP biosynthesis gene loci. GliT thus not only represents the prototype of ETP-forming enzymes in eukaryotes but also delineates a novel mechanism for self-resistance.

Full text of DOI:10.1021/ja103262m

Published on Web 07/01/2010
Transannular Disulfide Formation in Gliotoxin Biosynthesis
and Its Role in Self-Resistance of the Human Pathogen
Aspergillus fumigatus
‡
‡
Daniel H. Scharf, Nicole Remme, Thorsten Heinekamp, Peter Hortschansky,
Axel A. Brakhage, and Christian Hertweck*
Leibniz Institute for Natural Product Research and Infection Biology, Hans Knoell Institute,
Beutenbergstrasse 11a, 07745 Jena, Germany, and Friedrich Schiller UniVersity,
0
7737 Jena, Germany
Received April 18, 2010; E-mail: christian.hertweck@hki-jena.de
Abstract: Gliotoxin (1), the infamous representative of the group of epipolythiodioxopiperazines (ETPs), is
a virulence factor of the human pathogenic fungus Aspergillus fumigatus. The unique redox-sensitive
transannular disulfide bridge is critical for deleterious effects caused by redox cycling and protein conjugation
in the host. Through a combination of genetic, biochemical, and chemical analyses, we found that 1 results
from GliT-mediated oxidation of the corresponding dithiol. In vitro studies using purified GliT demonstrate
that the FAD-dependent, homodimeric enzyme utilizes molecular oxygen as terminal electron acceptor
2 2
with concomitant formation of H O . In analogy to the thiol-disulfide oxidoreductase superfamily, a model
for dithiol-disulfide exchange involving the conserved CxxC motif is proposed. Notably, while all studied
disulfide oxidases invariably form intra- or interchenar disulfide bonds in peptides, GliT is the first studied
enzyme producing an epidithio bond. Furthermore, through sensitivity assays using wild type, ∆gliT mutant,
and complemented strain, we found that GliT confers resistance to the producing organism. A phylogenetic
study revealed that GliT falls into a clade of yet fully uncharacterized fungal gene products deduced from
putative ETP biosynthesis gene loci. GliT thus not only represents the prototype of ETP-forming enzymes
in eukaryotes but also delineates a novel mechanism for self-resistance.
Introduction
Pathogenic fungi have developed various chemical strategies
1
-3
to distress, weaken, or even kill their plant or animal hosts.
In invasive aspergillosis,
4
,5
the leading cause of death in
immunocompromised patients, gliotoxin (1, Figure 1A) plays
6
a critical role in virulence. Gliotoxin is the prototype of a small
family of epipolythiodioxopiperazines (ETPs) featuring unique
7
transannular di- or polysulfide bridges. Extensive molecular
studies have revealed that this rare structural motif is indispensable
6
for the deleterious effects of gliotoxin. Two individual routes
rationalize gliotoxin-mediated virulence (Figure 1). First, through
redox cycling, reactive oxygen species (ROS) may be formed,
which can severely damage the host cell. Second, gliotoxin can
target proteins that are essential for vital functions. After passage
through the membrane, the toxin enters the reductive milieu of
the cytosol and is in equilibrium with its reduced dithiol form 2,
Figure 1. Gliotoxin (1) action inside the host cell accounting for A.
fumigatus virulence: redox cycling resulting in formation of reactive oxygen
species (ROS), and protein conjugation.
readily forming thioconjugates with susceptible thiols of target
6,7
‡
proteins, such as alcohol dehydrogenase. Due to the high
These authors contributed equally.
8
(
(
1) Fung, F.; Clark, R. F. J. Toxicol. Clin. Toxicol. 2004, 42, 217–234.
importance of this group of ETP toxins, much effort has been
2) Moebius, N.; Hertweck, C. Curr. Opin. Plant Biol. 2009, 12, 390–
9
devoted to the elucidation of their genetic basis. Several work
3
98.
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55.
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groups have independently demonstrated that a mutant with a
(
(
(
3
disruption of a nonribosomal peptide synthetase (NRPS) gene (gliP,
10-13
Figure 2A) stalls the production of 1 in Aspergillus fumigatus.
4
Yet, apart from the finding that the diketopiperazine core of
4
65.
(
(
6) Kwon-Chung, K. J.; Sugui, J. A. Med. Mycol. 2009, 47, S97–S103.
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1
021–1032.
1
0136 9 J. AM. CHEM. SOC. 2010, 132, 10136–10141
10.1021/ja103262m  2010 American Chemical Society

Transannular Disulfide Formation in Gliotoxin Biosynthesis
A R T I C L E S
Figure 3. Phylogenetic analysis of GliT, its homologues from other fungal
ETP clusters (clade III), (putative) bacterial natural product oxidizing
proteins (clade II), and thioredoxin reductase-like proteins from various
fungi (clade I). Homologue from E. coli as outgroup. Amino acid sequences
were obtained from GenBank database and aligned with ClustalW.
Figure 2. Functional analysis of gliT. (A) Organization of the gliotoxin
biosynthesis gene cluster. The predicted function of the encoded proteins
is as follows: zinc finger transcription factor, GliZ; aminocyclopropane
carboxylate synthase, GliI; dipeptidase, GliJ; nonribosomal peptide syn-
thetase, GliP; cytochrome P450 mono-oxygenases, GliC and GliF; methyl
transferases, GliM and GliN; glutathione S-transferase, GliG; major
facilitator superfamily transporter, GliA; hypothetical protein, GliK; and
gliotoxin sulfhydryl oxidase, GliT. (B) Southern blot analysis of A. fumigatus
wild type (WT), the ∆gliT mutant, and the gliT-complemented mutant strain
in bacterial pathways. We thus postulated that GliT could
function as an oxidase in the gliotoxin pathway. To investigate
the role of gliT in gliotoxin biosynthesis, we first constructed a
targeted A. fumigatus mutant lacking gliT (∆gliT) and verified
the successful gene deletion by polymerase chain reaction (PCR)
(
gliTc). (C) Metabolic profiling of A. fumigatus strains by HPLC: WT (a),
gliTc (b), ∆gliT mutant strain (c), and standard of 1 (d).
1
4
(
data not shown) and Southern blot analysis (Figure 2B). After
gliotoxin is assembled by a bimodular NRPS thiotemplate,
digesting chromosomal DNA with NdeI, we detected a specific
signal for gliT at 5.6 kb in the wild type (WT). The band at 6.0
kb indicates the partial deletion of gliT in the ∆gliT mutant.
According to high-performance liquid chromatography (HPLC)
analyses of the mutant broth, the loss of gliT results in a
complete abrogation of gliotoxin production (Figure 2C). To
exclude the possibility of polar effects in the mutant, we
monitored gene expression by reverse transcription (RT)-PCR
and found that the absence of gliT does not have any impact
on the expression of other genes from the gli locus (Figure S1,
Supporting Information). Furthermore, we successfully comple-
mented the ∆gliT mutant through ectopic integration of a gliT
expression cassette and yielded a strain (gliTc) producing 1 at
the wild-type level (WT, 4.5 mg L ; gliTc, 5.5 mg L ). We
confirmed the complementation at the genetic level by detecting
the 3.5 kb band characteristic of a DNA fragment comprising
the gliT gene with flanking promoter and terminator sequences
in the Southern blot (Figure 2B). While these experiments
clearly show that GliT plays a crucial role in gliotoxin
biosynthesis, HPLC-MS profiles of the ∆gliT mutant broth did
not provide any clue about potential late-pathway intermediates.
Thus, in order to corroborate the function of GliT, in Vitro
experiments were needed.
the enzymology of the downstream reactions including dithiol
formation has remained fully enigmatic.
Here we report that gliotoxin is generated from the corresponding
dithiol by a novel FAD-dependent dithiol oxidase, GliT, and reveal
that this enzyme also confers resistance against the toxin.
Results and Discussion
GliT Plays a Key Role in Gliotoxin Biosynthesis. Analysis
1
5
of the gliotoxin biosynthesis gene cluster from A. fumigatus
revealed a candidate gene (gliT) that might code for an
oxidoreductase (Figure 2A). The deduced gene product, GliT,
shares amino acid sequence similarities with thioredoxin-like
oxidoreductases. To evaluate the phylogenetic relationship of
GliT, we constructed a phylogenetic tree (neighbor-joining)
using homologous amino acid sequences and found that GliT
falls into a clade (III) of not yet fully characterized fungal gene
products deduced from putative ETP biosynthesis gene loci
-
1
-1
(Figure 3, and Figure S2, Supporting Information). Surprisingly,
clade III is rather distant from clade I, comprising putative fungal
thioredoxin reductases, and more closely related to clade II,
which comprises gene products implicated as oxidizing enzymes
(
10) Cramer, R. A.; Gamcsik, M. P.; Brooking, R. M.; Najvar, L. K.;
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GliT Is a Homodimeric, FAD-Dependent Oxidase Employ-
ing Molecular Oxygen as Terminal Electron Acceptor. To
investigate the function of GliT in Vitro, we amplified the gliT
ORF (1002 bp) and cloned the generated PCR fragment into
an Escherichia coli expression vector. The resulting plasmid
was propagated in E. coli DH5R cells, and after sequencing of
the insert, the vector was introduced into E. coli BL21(DE3)
cells for protein overproduction. After the cells were harvested,
5
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His
Figure 4A). Size exclusion chromatography revealed the
homodimeric status of GliT (Figure 4B).
6
-tagged GliT was purified using a NiSepharose column
(
14) Balibar, C. J.; Walsh, C. T. Biochemistry 2006, 45, 15029–15038.
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115.
J. AM. CHEM. SOC. 9 VOL. 132, NO. 29, 2010 10137

A R T I C L E S
Scharf et al.
Figure 4. Purification and biochemical characterization of heterologously
produced GliT. (A) Molecular mass of His -tagged GliT analyzed by
6
SDS-PAGE. (B) Native molecular mass determination by gel filtration.
(
C) UV-visible absorbance spectra of FAD-saturated GliT.
To identify the cofactor(s) of the putative oxidoreductase,
we searched for consensus motifs in the amino acid alignment
and identified a conserved FAD binding motif within the GliT
sequence (Figure S3, Supporting Information). This was con-
firmed by the spectrophotometric characterization of GliT
(Figure 4C), which indicated a 1:1 stoichiometry of GliT-FAD.
Notably, GliT lacks a canonical NADPH binding domain, which
16
is typically found in thioredoxin reductases. Nevertheless, we
found a conserved CxxC motif in the amino acid sequence of
GliT and homologous proteins (Figure S3). This motif is a
hallmark for various oxidoreductases, such as the quiescin-
1
7
sulfhydryl oxidase family of flavoenzymes.
Our next goal was to elucidate whether GliT functions as an
oxidase or a reductase by various methods. First, we chemically
reduced 1 to 2 using tris(2-carboxyethyl)phosphine hydrochlo-
ride (TCEP) to provide the reference and appropriate substrate
for the enzymatic assays (Figure 5C, i). The potential reductase
function was probed by standardized thioredoxin reductase
Figure 5. In Vitro analysis of GliT action. (A) HPLC profile of GliT-
catalyzed transformation of 2 to 1 after (a) 5, (b) 20, (c) 30, and (d) 40
min; (e) control with heat-inactivated GliT; (f) control with O
buffer; (g) reference of 2; and (h) reference of 1. (B) Monitoring O
2
-saturated
depletion
2
in GliT assay. (C) Model for GliT-mediated transformation of 2 to 1 and
preparation of 2 through chemical reduction of 1: (i) 1.5 equiv of TCEP,
MeOH/buffer, rt, pH ) 7.0, 3 h.
18
activity assays. However, in a series of tested conditions, GliT
did not show any reductase activity, neither with gliotoxin (1)
nor with other substrates, e.g. oxidized glutathione. In contrast,
we observed that 2 (0.36 µmol, 1.8 mM) is readily converted
into 1 in the presence of GliT (9.6 pmol, 0.32 µmol). The time
course of the reaction was monitored by HPLC (Figure 5A,
a-d). Notably, no transformation of 2 could be observed with
reoxidized by molecular oxygen with formation of H
2 2
O , as
shown by the use of O electrodes in our O depletion assay,
2
2
respectively. As has been demonstrated for thiol-disulfide
oxidoreductase model systems, there are several possible
scenarios for the exact mechanisms of electron/hydrogen transfer
heat-inactivated GliT or with O
Gliotoxin oxidase activity was further corroborated by measuring
the decrease of c(O ) from an assay after adding reduced
2
alone (Figure 5A, e, f).
2
0-22
and thiol-disulfide exchange,
and in particular oxygen
23
reactivity of flavoenzymes is still a matter of intense research.
2
gliotoxin (2) using an oxygen monitor (Figure 5B). Furthermore,
oxygen depletion could be reversed by the addition of catalase
GliT Confers Gliotoxin Resistance to the Producing Organ-
ism. Our in Vitro studies unequivocally show that GliT catalyzes
disulfide formation in gliotoxin biosynthesis. Accordingly, one
may conclude that the dithiol 2 is the immediate biosynthetic
precursor of 1. In light of this, the baffling phenotype of the
gliT knockout mutant appears more plausible: In the absence
of GliT, 2 would accumulate in the fungal cytosol, hardly
detectable because of protein conjugation. This assumption is
supported by some important further observations. Using higher
doses of 1, we were able to detect the dithiol in the cell lysate,
supporting the model for cytosolic disulfide reduction (see
Supporting Information). Furthermore, we noted that growth
of the ∆gliT mutant is dramatically impaired compared to that
of the WT upon addition of 1. To quantify this effect, we probed
the sensitivity of A. fumigatus strains toward 1 in an inhibition
zone assay (Figure 6).
(
data not shown), which implied the formation of hydrogen
peroxide as a side product of gliotoxin oxidation.
In sum, several lines of evidence support a model according
to which GliT functions as a sulfhydryl oxidase using FAD as
cofactor and oxygen as terminal electron acceptor (Figure 5C).
On the basis of our biochemical data and analogies to well-
1
9
17
studied flavoproteins and sulfhydryl oxidases, we propose
a model for electron/proton flow in gliotoxin biosynthesis
(
Figure 5C): As in thioredoxin reductases, two redox centers
are present, the flavin and the disulfide-dithiol (CxxC) redox
rheostat. Accordingly, the sulfhydryl hydrogens of 2 are first
transferred through the CxxC redox switch to the oxidized form
of GliT (disulfide), yielding the reduced form of GliT (dithiol).
Subsequently, FADox receives the hydrogens from the active
site of GliT with concomitant reduction. FADred is then
(
20) Wouters, M. A.; Fan, S. W.; Haworth, N. L. Antioxidants Redox
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0138 J. AM. CHEM. SOC. 9 VOL. 132, NO. 29, 2010

Transannular Disulfide Formation in Gliotoxin Biosynthesis
A R T I C L E S
+
+
classical NADP /NADPH binding site. Instead of NADP , GliT
employs molecular oxygen as terminal electron acceptor.
Another novelty reported in this study is the proven role of a
disulfide-forming enzyme for self-resistance. In this context it
is intriguing that our phylogenetic analysis revealed that GliT
clusters with a number of yet unidentified putative gene products
likely involved in fungal ETP pathways. Since GliT represents
the prototype of this novel enzyme clade, we hypothesize that
these enzymes may also confer resistance to unwanted ETP
redox cycling in the requisite producer strains. Finally, consider-
ing the crucial role of GliT for self-resistance and virulence,
one may consider an evaluation of this key enzyme as a novel
target for antifungal therapy.
Figure 6. Sensitivity of A. fumigatus strains toward gliotoxin (1) (c ) 3
mM) determined in an inhibition zone assay.
While the WT is insensitive toward 1, we observed a clear
inhibition zone (32.8 mm) on the ∆gliT mutant lawn. Notably,
the gliT-complemented mutant fully restored resistance. To
exclude any secondary effects, we employed a gliotoxin-negative
strain lacking the NRPS gene gliP (∆gliP) as control. The
finding that the presence of the gliT gene directly correlates
with gliotoxin tolerance provides strong evidence that GliT
confers host self-resistance toward gliotoxin. In light of our
biochemical analysis, we reason that the activity of GliT would
prevent unwanted redox cycling as well as conjugation of
gliotoxin with susceptible proteins. Since reduced gliotoxin is
not found in the broth of the ∆gliT mutant, one may also
speculate that formation of the internal sulfur bridge is a
requirement for the export of gliotoxin. To the best of our
knowledge, enzyme-catalyzed epidisulfide formation represents
an unprecedented antibiotic self-resistance mechanism.
Experimental Section
General Analytical Procedures. Samples were measured on a
JASCO HPLC with DAD and on an LC-Q electrospray MS from
Thermo Electron, respectively, using a C18 Nucleosil column (250
×
4.6 mm, 5 µm) from JASCO. HRESIMS were recorded on a
Finnigan TSQ Quantum Ultra AM instrument from Thermo
Electron. IR spectra were measured on a Bruker FT-IR (IFS 55)
spectrometer using ATR technique. All solvents used were spectral
grade or distilled prior to use. For HPLC measurements, 20 µL of
the concentrated sample was injected. Column, Nucleosil 100 (250
-1
×
2
4.6 mm, C18, 5 µm); eluent, 1 mL min flow; gradient A, H O,
0.1% TFA, B, acetonitrile, start 20% B, in 20 min 65% B, after 28
min 100% B for 10 min. For LC-MS measurements, 25 µL of the
concentrated sample was injected. Column, Nucleosil 100 (250 ×
GliT Is the Prototype of Fungal Enzymes Forming Epidithio
Bridges. Enzyme-mediated disulfide formation is a universal
reaction found in all kingdoms of life. However, to date virtually
all investigated disulfide oxidoreductases are involved in protein
folding and cross-linking of ribosomally synthesized peptide
1
4
0
.6 mm, C18, 5 µm); eluent, 0.6 mL min- flow; gradient A, H
2
O,
.1% HCOOH, B, acetonitrile, start 20% B, in 18 min 65% B,
after 28 min 95% B for 10 min. Gliotoxin standard elutes on HPLC
system after 12.1 min and from the LC-MS system after 17.3 min.
Reduced gliotoxin elutes on HPLC system after 10.2 min and from
the LC-MS system after 14.8 min.
2
4-26
strands.
In contrast to the ubiquitous occurrence of disul-
fides in proteins, disulfide-containing secondary metabolites are
scarce, and there is only very limited knowledge on disulfide
formation in such small molecules. Although various gene
clusters coding for the biosynthesis of di- and trisulfide-
containing secondary metabolites have been cloned and
Fungal and Bacterial Strains, Media, and Growth Conditions.
3
3
A. fumigatus strain CEA17∆akuB was used as wild type and for
generation of gliT mutant strains. Strains were grown in Aspergillus
3
4
minimal medium (AMM) as described previously. AMM agar
was prepared by addition of 1.6% (w/v) Select Agar (Invitrogen,
Germany). For transformation of Escherichia coli, TOP10F’ cells
9,27-31
sequenced,
requisite biochemical functions have remained
(
°
Invitrogen, Germany) were used. E. coli cells were grown at 37
elusive. Only recently, an oxidase (DepH) has been identified
and characterized that mediates the formation of a disulfide
-
1
C in LB medium supplemented with ampicillin (100 µg mL ).
For production of A. fumigatus conidia, the fungus was cultivated
32
bridge of a bacterial depsipeptide. To date, all studied disulfide
oxidases invariably form intra- or interchenar disulfide bonds
in peptides. In stark contrast, GliT is the first studied enzyme
producing an epidithio bond. While GliT shares the FAD
binding motif and the CxxC box with thiol-disulfide oxi-
doreductases such as thioredoxin reductases, GliT exhibits no
for 5 days on AMM agar plates. The spores were harvested in 0.9%
w/v) NaCl/0.1% (v/v) Tween 80 and counted using a CASY cell
counter (model TT, Innovatis AG, Germany). For isolation of DNA,
the fungus was grown in liquid AMM for 24 h.
Isolation and Manipulation of Nucleic Acids. Standard tech-
(
niques for manipulation of DNA were carried out using standard
3
5
procedures. Chromosomal DNA of A. fumigatus was prepared
using the Master Pure Yeast DNA purification kit (Epicentre
Biotechnologies, USA). For Southern blot analysis, DNA fragments
(
(
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were separated on an agarose gel and blotted onto Hybond N nylon
membranes (GE Healthcare Bio-Sciences, Germany). Labeling of
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were performed as described previously. For RNA isolation, A.
fumigatus was cultivated under gliotoxin-producing conditions for
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8 h. For RNA extraction, 100 mg of mycelium was used employing
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A R T I C L E S
Scharf et al.
the RNeasy Plant Mini Kit (Qiagen, Germany). After DNase
treatment with the TURBO DNA-free Kit (Ambion, Applied
Biosystems, Germany), 10 µg of total RNA was used for first-
strand cDNA synthesis employing SuperScript III reverse tran-
scriptase and anchored oligo(dT)20 primers (Invitrogen, Germany).
Reverse transcription was conducted at 50 °C for 3 h.
RT-PCR. Reverse transcription PCR analysis was used to
determine RNA transcription levels. Specific oligonucleotide
combinations (Table S1, Supporting Information) allowed the
amplification of short fragments of selected genes of the gliotoxin
biosynthesis gene cluster. As control, mRNA steady-state levels
of the constitutively transcribed actin gene were determined.
Transcript amplification was carried out using GoTaq DNA
polymerase (Promega, Germany) with 5 ng of A. fumigatus cDNA
as template.
at 160 rpm for 6 days. After 2 days, 5 mg of gliotoxin, dissolved
in 1 mL of DMSO, was added. After 4 days, 10 g of Amberlites
XAD-4 (SERVA), suspended in 50 mL of water, was added to the
culture. The mycelium and amberlites were filtered off and washed
with 200 mL of degassed MeOH. The solvent was removed under
reduced pressure. The residue was dissolved in 500 µL of degassed
water and measured immediately by LC-MS. Reduced gliotoxin
eluted after 14.8 min and could be detected only in the ∆gliT mutant
when gliotoxin was added.
Chemical Reduction of Gliotoxin. Gliotoxin (1) was isolated
from A. fumigatus and purified on silica gel (cyclohexane/EtOAc,
1:1), Sephadex LH-20 (MeOH), and RP-18 preparative HPLC. The
3
9
physicochemical properties were identical with reported data.
Gliotoxin was reduced using tris(2-carboxyethyl)phosphine hydro-
chloride (TCEP) as reducing agent. To a solution of 2.4 mg (7.4 ×
-3
Generation of A. fumigatus Mutant Strains and Complemen-
tation. Partial deletion of gliT was done by using a PCR-based
strategy. Upstream and downstream flanking regions of gene gliT
10 mmol) of gliotoxin, dissolved in 1 mL of degassed MeOH,
3.2 mg (0.01 mmol) of TCEP was added, dissolved in 1 mL of
phosphate buffer (pH 7.0, 0.1 M). The reaction was complete after
stirring for 3 h under an argon atmosphere. The solvent was
removed under vacuum, and the residue was dissolved in 4 mL of
phosphate buffer (pH 7.0, 0.1 M). Reduced gliotoxin eluted after
10.2 min, and gliotoxin eluted as the standard after 12.1 min.
Reduced gliotoxin was purified by preparative HPLC (column,
Nucleosil Macherey-Nagel; eluent, 10 mL min- flow; gradient A,
(
AFUA_6G09740) were amplified by PCR using primer pairs gliT5-
for and gliT-ptrA-rev and gliT3-rev and gliT-ptrA-for, respectively.
By this reaction, overlapping ends of the pyrithiamine resistance
3
7
cassette were introduced at the 3′-end of the upstream flanking
region and at the 5′-end of the downstream flanking region of the
gliT gene. The ptrA resistance cassette was amplified from plasmid
pSK275 (kind gift from Sven Krappmann) with primers ptrA-for
and ptrA-rev. All PCR fragments were purified by gel extraction.
The final deletion construct was generated by a three-fragment PCR
employing primers gliT-for and gliT-rev. All PCRs were performed
with Phusion High-Fidelity DNA polymerase (Finnzymes) accord-
ing to the manufacturer’s recommendations. The resulting 3.7 kb
PCR product was purified and used for transformation of A.
1
H
2
O, B, acetonitrile, start 20% B, in 20 min 65% B, after 28 min
100% B for 10 min) and analyzed by mass spectrometry. The
4
0
physicochemical properties were identical with reported data.
Heterologous Production and Purification of GliT. The gliT
ORF (1002 bp) was amplified by PCR using primer gliT forward
and gliT reverse. The generated PCR fragment was cloned
into the BamHI/HindIII sites of pET43.1H6HapX to create
34
41
fumigatus wild-type protoplasts as described previously. Pyrithia-
mine (Sigma-Aldrich, Germany)-resistant transformants were ana-
lyzed for partial deletion of gliT by Southern blot analysis. One
positive transformant, designated ∆gliT, was chosen for further
analysis and for generation of a gliT-complemented strain.
To re-introduce the gliT gene in the ∆gliT mutant, i.e. to generate
a gliT-complemented strain, the gliT gene was amplified from wild-
type genomic DNA by PCR using Phusion High-Fidelity DNA
polymerase and primers gliT-HindIII-F and gliT-XhoI-R. The
obtained 1.7 kb PCR fragment comprised the intergenic region
between gliF (AFUA_6G09730) and gliT and 0.3 kb of gliT
terminator sequences. This fragment was inserted via XhoI and
pET43.1H6GliT. The resulting plasmid was propagated in E. coli
DH5R cells. The DNA sequence was verified by sequencing, and
the vector was introduced into E. coli BL21(DE3) cells for protein
overproduction. Recombinant GliT was produced by auto-induction
in Overnight Express Instant TB medium (Novagen). Cells were
harvested after 24 h incubation at 30 °C. His6-GliT-containing
2 4
cleared cellular extracts in 50 mM NaH PO , 300 mM NaCl, 10%
glycerol, 1 mM DTT, 1 mM PMSF, pH 8.0, were applied to a 25
mL NiSepharose 6 FF column (all columns purchased from GE
Healthcare Bio-Sciences, Freiburg, Germany). GliT was eluted with
200 mM imidazole and incubated with an excess of FAD at 4 °C
2 4
for 2 h. GliT was transferred to storage buffer (100 mM KH PO ,
3
8
HindIII restriction into plasmid pUChph, mediating hygromycin
resistance. The resulting plasmid, pUChph-gliT, was used for
transformation of strain ∆gliT. Transformants were selected for
resistance against hygromycin (Roche Applied Science, Germany)
and further analyzed by PCR and Southern blot.
pH 7.0) using a HiPrep desalting column. GliT-containing fraction
was identified by SDS-PAGE and stored in 50% glycerol at -20
°C. Protein concentrations were determined according to Bradford
using the Coomassie Plus protein assay reagent (Pierce).
Determination of Physical and Biochemical Properties of
GliT. The oligomeric status of GliT was determined by size
exclusion chromatography using a HiLoad 16/60 Superdex 200 pg
column. Running buffer used was 20 mM Tris/HCl (pH 7.5) and
Cultivation of A. fumigatus Wild-Type and Mutant Strains.
For formation of conidia, A. fumigatus strains CEA17∆akuB, ∆gliT,
and gliT complemented were grown on Aspergillus minimal
medium at 37 °C for 5 days. In the case of ∆gliT mutant cultivation,
-1
150 mM NaCl, flow rate 1 mL min . UV/vis spectra of GliT were
obtained with a spectrophotometer (V-630, Jasco). The stoichiom-
etry of GliT-FAD was determined in duplicate as follows. The
spectrum of FAD-saturated GliT was recorded from 250 to 600
nm. GliT was subsequently denatured by boiling for 5 min to release
bound FAD, the sample was centrifuged for 5 min, and the spectrum
of the supernatant from 250 to 600 nm was recorded. The protein
concentration was determined from the absorbance at 280 nm before
heat inactivation using a molar extinction coefficient of A ) 30.0
-1
pyrithiamine (1 mg mL ) was added to the media as selection
marker. A. fumigatus strains CEA17∆akuB, ∆gliT, and gliT
complemented were cultivated at 28 °C in 250 mL of Czapek Dox
medium at 160 rpm for 6 days. The cultures were extracted three
times with 100 mL of ethyl acetate. The combined organic phases
were dried over sodium sulfate and concentrated to dryness. For
gliotoxin determination, the residues were dissolved in 3 mL of
MeOH and measured with HPLC. For quantification of gliotoxin,
a calibration curve was obtained from 16 µg to 1 mg of gliotoxin
standard (Sigma-Aldrich). Gliotoxin eluted after 12.1 min as the
standard.
2
80
-1
-1
mM cm and the absorbance after boiling as blank. The
(39) Kaouadji, M.; Steiman, R.; Seigle-Murandi, F.; Krivobok, S.; Sage-
Steiman, L. J. Nat. Prod. 1990, 53, 717-719.
(40) Bernardo, P. H.; Chai, C. L.; Deeble, G. J.; Liu, X. M.; Waring, P.
Bioorg. Med. Chem. Lett. 2001, 11, 483-485.
Detection of Reduced Gliotoxin in ∆gliT Mutant. A. fumigatus
strains were cultivated at 28 °C in 250 mL of Czapek Dox medium
(
(
37) Kubodera, T.; Yamashita, N.; Nishimura, A. Biosci. Biotechnol.
Biochem. 2002, 66, 404.
(41) Hortschansky, P.; Eisendle, M.; Al-Abdallah, Q.; Schmidt, A. D.;
Bergmann, S.; Th o¨ n, M.; Kniemeyer, O.; Abt, B.; Seeber, B.; Werner,
E. R.; Kato, M.; Brakhage, A. A.; Haas, H. EMBO J. 2007, 26, 3157-
3168.
38) Liebmann, B.; Muller, M.; Braun, A.; Brakhage, A. A. Infect. Immunol.
2
004, 72, 5193-5203.
1
0140 J. AM. CHEM. SOC. 9 VOL. 132, NO. 29, 2010

Transannular Disulfide Formation in Gliotoxin Biosynthesis
A R T I C L E S
concentration of free FAD was determined from the absorbance at
Inhibition Zone Assays for Gliotoxin Sensitivity. The sensitiv-
ity of various A. fumigatus strains to gliotoxin was determined in
50 nm using a molar extinction coefficient of A450 ) 11.0 mM-
-1
1
4
7
cm .
Enzyme Assay. Enzymatic activity was proven by conversion
an inhibition zone assay. Of the test strains, 1.5 × 10 conidia were
mixed with 15 mL of YAG agar (0.5 % (w/v) yeast extract, 2 %
(w/v) agar, 2 % (w/v) glucose, trace elements). Next, 100 µL of 3
mM gliotoxin was added in a hole in the center of the plate, and
the diameter of the inhibition zone was determined after 24 h
incubation at 37 °C. The experiment was repeated in quadruplicate.
of reduced gliotoxin (2) with GliT into gliotoxin (1). To GliT
solution (0.32 µmol, 9.6 pmol in 30 µL of phosphate buffer, pH
6
.5, 0.1 M) reduced gliotoxin (2) was added (1.8 mM, in 0.2 mL
of phosphate buffer, pH 6.5, 0.1 M). The reaction was stopped after
, 20, 30, and 40 min with TFA (10 µL), filtered, and measured
5
Acknowledgment. We thank A. Perner, S. Fricke, N. Hann-
wacker, and C. Schult for technical assistance and Dr. E. Shelest
for phylogenetic analysis. This research was financially supported
by the JSMC and the ILRS.
directly with HPLC. The reaction was complete after 40 min. As
a blind value, the same experiment was examined with heat-
inactivated GliT and stopped after 40 min. Reduced gliotoxin (2)
eluted after 10.2 min, and gliotoxin (1) eluted as the standard after
1
2.1 min.
Gliotoxin Oxidase Activity. Determination of gliotoxin oxidase
Supporting Information Available: RT-PCR, biochemical
assays, bioinformatics studies, chemical analyses. This material
is available free of charge via the Internet at http://pubs.acs.org.
2
activity was based upon depletion of O from assay containing
reduced gliotoxin (2), as measured with an oxygen monitor (Yellow
Springs Instruments 5300) equipped with polarographic Clark-type
electrodes.
JA103262M
J. AM. CHEM. SOC. 9 VOL. 132, NO. 29, 2010 10141