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A Proteomic Approach to Investigating Gene Cluster
Expression and Secondary Metabolite Functionality in
Aspergillus fumigatus
Rebecca A. Owens, Stephen Hammel, Kevin J. Sheridan, Gary W. Jones, Sean Doyle*
Department of Biology, National University of Ireland Maynooth, Maynooth, Co. Kildare, Ireland
Abstract
A combined proteomics and metabolomics approach was utilised to advance the identification and characterisation of
secondary metabolites in Aspergillus fumigatus. Here, implementation of a shotgun proteomic strategy led to the
identification of non-redundant mycelial proteins (n= 414) from A. fumigatus including proteins typically under-represented
in 2-D proteome maps: proteins with multiple transmembrane regions, hydrophobic proteins and proteins with extremes of
molecular mass and pI. Indirect identification of secondary metabolite cluster expression was also achieved, with proteins
(n= 18) from LaeA-regulated clusters detected, including GliT encoded within the gliotoxin biosynthetic cluster. Biochemical
analysis then revealed that gliotoxin significantly attenuates H
2
O
2
-induced oxidative stress in A. fumigatus (p.0.0001),
confirming observations from proteomics data. A complementary 2-D/LC-MS/MS approach further elucidated significantly
increased abundance (p,0.05) of proliferating cell nuclear antigen (PCNA), NADH-quinone oxidoreductase and the gliotoxin
oxidoreductase GliT, along with significantly attenuated abundance (p,0.05) of a heat shock protein, an oxidative stress
protein and an autolysis-associated chitinase, when gliotoxin and H
2
O
2
were present, compared to H
2
O
2
alone. Moreover,
gliotoxin exposure significantly reduced the abundance of selected proteins (p,0.05) involved in de novo purine
biosynthesis. Significantly elevated abundance (p,0.05) of a key enzyme, xanthine-guanine phosphoribosyl transferase
Xpt1, utilised in purine salvage, was observed in the presence of H
2
O
2
and gliotoxin. This work provides new insights into
the A. fumigatus proteome and experimental strategies, plus mechanistic data pertaining to gliotoxin functionality in the
organism.
Citation: Owens RA, Hammel S, Sheridan KJ, Jones GW, Doyle S (2014) A Proteomic Approach to Investigating Gene Cluster Expression and Secondary Metabolite
Functionality in Aspergillus fumigatus. PLoS ONE 9(9): e106942. doi:10.1371/journal.pone.0106942
Editor: Kap-Hoon Han, Woosuk University, Republic of Korea
Received May 29, 2014; Accepted August 11, 2014; Published September 8, 2014
Copyright: ß2014 Owens et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits
unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Data Availability: The authors confirm that all data underlying the findings are fully available without restriction. All relevant data are within the paper and its
Supporting Information files.
Funding: This work was funded in part by a Science Foundation Ireland Principal Investigator Award to SD (PI/11/1188). RAO and SH were recipients of Irish
Research Council for Science Engineering and Technology Embark PhD Fellowships. LC-MS facilities were funded by a competitive award from the Irish Higher
Education Authority. Funding from the 3U Partnership (DCU/NUIM/RCSI) is also acknowledged. The funders had no role in study design, data collection and
analysis, decision to publish, or preparation of the manuscript.
Competing Interests: The authors have declared that no competing interests exist.
* Email: sean.doyle@nuim.ie
Introduction
Following the publication of A. fumigatus Af293 [1] genomic
sequence and the sequencing of a second A. fumigatus strain,
A1163 [2], extensive efforts have been undertaken to characterise
the proteome of this opportunistic human pathogen [3–10].
Traditional proteomic strategies have utilised 2-D separation with
subsequent protein identification by MS. Shotgun MS-based
proteomics has developed more recently and provides a comple-
mentary method to 2-D for proteome profiling [9,10], since 2-D
can occasionally be limiting for the identification of particular
subsets of proteins, especially hydrophobic proteins, membrane
proteins, and proteins with large molecular mass or extreme pI
[11].
MS-based or shotgun proteomics can adopt multiple approach-
es including, (i) direct LC-MS/MS, (ii) indirect LC-MS/MS and
(iii) 2-D-LC-MS/MS (multidimensional protein identification
technology, MudPIT) [12,13]. Direct LC-MS/MS involves the
on-line separation of complex peptide mixtures using reversed
phase nano-LC columns with extended acetonitrile gradients to
effect peptide separation [14]. Indirect LC-MS/MS is where
complex peptide or protein mixtures are pre-fractionated off-line
(e.g. by SDS-PAGE) before LC-MS/MS analysis [15]. Sub-
proteome strategies have also been implemented to investigate
glutathione binding [4] and mitochondrial proteins [6]. Indeed,
the recent emergence of MS-based proteomics studies of A.
fumigatus has been undertaken whereby 530 plasma membrane
associated proteins were identified by utilising a combination of
SDS-PAGE fractionation of total protein followed by peptide
separation and identification by 2-D-LC-MS/MS [16]. This study
would have been difficult to perform using 2-D due to the
incompatibility of hydrophobic proteins, and proteins with
transmembrane (TM) regions, with detergents used in isoelectric
focusing, the first separation stage of 2-D [17]. Quantitative MS-
based proteomics, both label-free and using isobaric tagging for
relative and absolute quantitation (iTRAQ), have been used to
comparatively profile the stages of A. fumigatus germination
[9,10]. Activity-based MS proteomics has also recently been
PLOS ONE | www.plosone.org 1 September 2014 | Volume 9 | Issue 9 | e106942
developed to investigate A. fumigatus following incubation with
human sera [18]. The application of MS-based proteomics to
dissect the proteome of A. fumigatus has the potential to provide a
global overview of the pathways and biological processes active
under a set of conditions. In addition, (i) bioinformatic analysis can
expand the characterisation of large datasets generated by MS-
based proteomics, and (ii) shotgun proteomics offers the possibility
of identifying the presence of either hypothetical proteins or
proteins of unknown function, whose existence may either be
unclear, or only previously demonstrated at the transcript level.
Furthermore, (iii) shotgun MS-based proteomics has the potential
to be used for the non-targeted identification of secondary
metabolite (SM) cluster expression, which, coupled with subse-
quent metabolomics, could result in the identification of novel
cluster products [19].
Proteomic approaches may also have an application in
characterizing the effect of exogenous SMs on A. fumigatus
[20]. Indeed, despite the many advantages of shotgun proteomics,
2-D has been successfully deployed to inform on proteomic
alterations in A. fumigatus under various conditions [20–27].
Thus, a complementary strategy of shotgun and 2-D proteomics
offers much in terms of the ability to reveal the nature of the
proteome in pathogenic microorganisms, provide further insight
into SM biosynthesis- and explore how apparently synergistic
stressors may interact in unexpected ways. Interestingly, both
gliotoxin and H
2
O
2
, separately, have been shown to result in
numerous, growth inhibitory-associated, alterations to the pro-
teome of A. fumigatus [20,23,28]. Indeed, exposure of mamma-
lian cells to gliotoxin has been shown to increase the production of
ROS, while H
2
O
2
induces oxidative stress [29]. Paradoxically
however, it has been revealed [30] that gliotoxin actually relieved
H
2
O
2
–induced growth inhibition of A. fumigatus in a concentra-
tion-dependent manner, although the basis for this phenomenon
was not investigated.
The aim of the work presented here was to investigate the
potential for shotgun MS to dissect the mycelial proteome,
particularly with respect to identifying SM cluster expression,
allied to cognate metabolite biosynthesis. Moreover, dissection of
the molecular basis of SM (gliotoxin)-mediated relief of H
2
O
2
-
induced oxidative stress in A. fumigatus was explored by 2-D and
LC-MS/MS analysis. Overall, these combinatorial approaches
reveal new insights into the expression, functionality and dynamic
nature of the A. fumigatus proteome during normal growth and
consequent to attenuated oxidative stress conditions.
Materials and Methods
Mycelial proteomics
For shotgun proteomics, mycelia from A. fumigatus
ATCC26933 shaking cultures were harvested after 48 h,
200 rpm, 37uCinAspergillus minimal media (AMM) and snap-
frozen in liquid nitrogen. A. fumigatus ATCC26933 mycelia were
also harvested from shaking cultures grown for 72 h in Czapek-
Dox media, 37uC, 200 rpm. Protein was extracted and subjected
to trypsin digestion as described [31]. Briefly, frozen mycelia (1 g)
were ground in liquid nitrogen and resuspended in 6 ml of 25 mM
Tris-HCl, 6 M Guanidine-HCl, 10 mM DTT pH 8.6. Extracts
were sonicated five times at 10% power, cycle 6 for 10 sec
intervals, followed by centrifugation at 10000 g for 10 min at 4uC.
DTT (1 M; 10 ml per ml lysate) was added to the supernatants and
incubated at 56uC for 30 min. Iodoacetamide (1 M; 55 ml per ml
lysate) was added and incubated in the dark for 20 min. Whole cell
lysates were dialysed twice against 100 mM ammonium bicar-
bonate. Aliquots of denatured protein solutions (100 ml) were
digested with trypsin (5 ml; 0.4 mg/ml in 10% (v/v) acetonitrile,
10 mM ammonium bicarbonate), overnight at 37uC. Tryptic
peptide mixtures were spin-filtered (Agilent Technologies,
0.22 mm cellulose acetate), separated on extended liquid chroma-
tography gradients on a nanoflow Agilent 1200 LC system and
subjected to tandem mass spectrometry using an Agilent 6340 Ion
Trap LC-MS System (Agilent Technologies, Santa Clara, CA).
Database searches for identification of proteins were carried out
using Spectrum Mill MS Proteomics Workbench (Revision
B.04.00.127). Validation criteria were set to (i) maximum of two
missed cleavages by trypsin, (ii) fixed modification: carbamido-
methylation of cysteines, (iii) variable modifications: oxidation of
methionine, (iv) mass tolerance of precursor ions 62.5 Da and
product ions 60.7 Da were employed and searches were carried
out against a protein database of Aspergillus fumigatus strains
Af293 (reference strain) and A1163, acquired from [32]. Protein
grouping was carried out based on the presence of $1 shared
peptide. Protein identifications were validated based on fixed
thresholds (minimum protein score set to 20), with single peptide
identifications requiring a Spectrum Mill score $17.0 and SPI .
70%. In order to determine the relative hydrophobicity of the
identified proteins, the grand average of hydropathy (GRAVY)
index was calculated using GRAVY calculator (www.gravy-
calculator.de). Using Phobius (http://phobius.cbr.su.se), the num-
ber of putative transmembrane regions present in each identified
protein was determined. Identified proteins were grouped into
functional categories based on the FunCat (Functional Catalogue),
GO (Gene Ontology) and KEGG (Kyoto Encyclopedia of Genes
and Genomes) annotations, using the FungiFun application
(https://www.omnifung.hki-jena.de/FungiFun/) [33].
Detection of Secreted Secondary Metabolites in A.
fumigatus
Culture supernatants were harvested from A. fumigatus
ATCC26933 grown for 48 h in AMM or 72 h in Czapek-Dox,
as described above. Organic extractions were carried out using a
1:1 mixture of chloroform to culture supernatant [34]. Chloroform
extracts were dried to completion using a rotary evaporator and
resuspended in methanol for LC-MS analysis using an Agilent
6340 Ion Trap LC-MS System (Agilent Technologies, Santa
Clara, CA). Settings were adjusted to include the detection of
singly charged molecules and molecules were separated on an
acetonitrile gradient over a 15 min runtime.
2-D and LC-MS/MS
The mechanisms involved in gliotoxin-mediated relief of H
2
O
2
-
induced stress in A. fumigatus ATCC26933 were investigated by
comparative 2-D and LC-MS/MS. A. fumigatus ATCC26933
was grown in Sabouraud dextrose media at 200 rpm, 37uC for
24 h prior to addition of one of the following four treatments: (i)
Solvent control (500 ml methanol added per 50 ml culture), (ii)
Gliotoxin alone (gliotoxin, dissolved in methanol, added to a final
concentration of 10 mg/ml), (iii) H
2
O
2
alone (H
2
O
2
added to a
final concentration of 2 mM; 500 ml methanol added per 50 ml
culture), (iv) Gliotoxin and H
2
O
2
combined (gliotoxin added to a
final 10 mg/ml and H
2
O
2
added to final 2 mM). Mycelia (n=5
biological replicates/treatment) were harvested after 4 h and
ground in liquid nitrogen. Crushed mycelia were resuspended in
10% (w/v) TCA and sonicated three times at 10% power, cycle 6,
10 sec. Samples were incubated on ice for 20 min and centrifuged
at 10000 g, 4uC for 10 min. Pellets were washed with ice-cold
acetone to remove excess TCA and resuspended in IEF buffer [4].
Protein was separated on pH 4–7 IEF strips, followed by
resolution by SDS-PAGE [35,36]. Colloidal Coomassie staining
A. fumigatus Proteomics
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was carried out on gels, with subsequent protein spot analysis using
Progenesis SameSpot software (Nonlinear Dynamics, Newcastle
upon Tyne, United Kingdom). Gels (n= 20; 5 replicates/
treatment) from all four treatments were aligned and subsets of
treatments were compared. Spots demonstrating significant
changes in abundance (p,0.05, fold change $1.5) were excised
from gels and trypsin digested according to the method described
by Shevchenko (2007) [37]. Digested peptides were analysed by
LC-MS/MS as described previously, with SpectrumMill used for
database searching.
Fluorescent Detection of ROS in A. fumigatus
A. fumigatus ATCC26933 conidia (4610
6
per well, in 6 well
plates) were added to Sabouraud liquid media (4 ml/well) and
incubated for 24 h at 37uC, static. Each well contained a glass
microscope slide. After removal of the top layer of mycelia, H
2
O
2
(2 mM final) only, or with gliotoxin (10 mg/ml final) was added.
H
2
O
2
alone acts as a positive control as it is a known ROS
inducing agent. Gliotoxin (10 mg/ml final) and an equivalent
volume of MeOH were also added individually to separate wells as
negative control. Plates were re-incubated 37uC, static, for 30 min,
culture supernatants removed and mycelial mats washed once with
4 ml PBS for 5 min. After PBS removal, Sabouraud medium
(3 ml) containing 29,79-dichlorodihydrofluorescein diacetate
(2.5 mg/ml; H
2
DCFDA; Life Technologies) was added to each
well followed by incubation 37uC for 40 min, static. After washing
twice with PBS (2620 min each), mycelia were visualised using a
fluorescent microscope (GFP filter: Ex/Em: 492–495/517–
527 nm). Fluorescence was quantified by measuring Integrated
Density Value (IDV) of selected areas from each image (n=5/
treatment) representing subsequent data as means 6standard
error. Differences were evaluated using ANOVA, and statistical
significance was accepted at p,0.05.
Results
Shotgun mass spectrometry identifies 414 proteins from
A. fumigatus mycelia grown in AMM
Utilising a direct shotgun proteomics approach, a total of 1826
unique peptides were identified in A. fumigatus mycelial lysates,
corresponding to 414 distinct A. fumigatus proteins from 405
protein groups (Table S1). A false discovery rate (FDR) of 1.97%
was determined for the distinct peptides identified in this study,
using the validation criteria outlined (Table S1). These proteins
(n= 414) spanned a theoretical pIrange of 3.9 to 11.8 and a M
r
range of 7.8 to 444 kDa (Figure 1; Table S1). All peptides
identified contributed to a sequence coverage range of between 1
and 64% of the respective proteins, with Spectrum Mill scores
ranging between 17 and 1327.
The GRAVY index for identified proteins ranged from 21.632
to 0.483, with positive scores indicating hydrophobicity (Figure 1).
A number of hydrophobic proteins were identified (n= 33; 7.97%
of total identified proteins), based on positive GRAVY scores.
Additionally, GRAVY scores were computed for the entire
predicted proteome of A. fumigatus and it was observed that
15.6% of the total proteome possess positive GRAVY scores
(Figure 1). The majority of proteins identified by shotgun mass
spectrometry (70.05%) were slightly hydrophilic, with GRAVY
scores ranging from 20.5 to 0. This is in accordance with the total
predicted proteome of A. fumigatus, where 55.1% of all predicted
proteins fall within this range. Proteins with transmembrane
helices (n= 44; 10.62% of total identified proteins) were detected.
Several proteins were detected with 10 or more putative TM
regions, including a plasma membrane H
+
-ATPase
(AFUA_3G07640), an amino acid permease (Gap1)
(AFUA_7G04290) and two ABC transporters (AFUA_1G14330
and AFUA_5G06070). One protein, a small oligopeptide trans-
porter (OPT family) (AFUA_2G15240) was detected with 14
putative transmembrane regions and a GRAVY score of 0.276.
Annotations were available for 89.37%, 86.47% and 49.27% of
identified proteins using the FunCat, GO and KEGG schemes,
respectively (Figure 1). Based on the FunCat classification,
functional categories that were significantly over-represented were
protein synthesis (n= 86, p= 4.68610
223
), energy (n= 91,
p= 4.22610
217
), protein with binding function or cofactor
requirement (n= 254, p= 4.43610
214
) and transcription (n= 32,
p= 2.79610
29
). Proteins (n= 23; 5.5%) were identified which
have no functional classifications using the aforementioned
methods.
Identification of A. fumigatus secondary metabolite
cluster expression at protein level
Proteins identified by shotgun mass spectrometry were mapped
based on their relative loci on each of the eight A. fumigatus
chromosomes, using their gene locus identifiers (Figure S1). A
number of proteins (n= 15) that comprise a secondary metabolite
supercluster, involved in the production of pseurotin A, fumi-
tremorgins and fumagillin [38–42], were identified (Table 1). In
addition, proteins were identified from the gliotoxin biosynthetic
cluster on chromosome 6 [43], including GliT, the gliotoxin
oxidoreductase responsible for self-protection against gliotoxin
[28], and two clusters responsible for the production of unknown
metabolites on chromosomes 3 and 4 respectively (Table 1). A
phosphoglycerate kinase PgkA protein (AFUA_1G10350) was also
identified, which is predicted to be part of the Afpes1 NRPS
cluster on chromosome 1 [1]. The identification of these proteins is
indicative of the respective cluster activity under the growth
conditions used. To confirm whether this detection of secondary
metabolism-associated proteins correlated with the production of
the respective molecules, LC-MS analysis was carried out on
culture supernatants. This analysis revealed the presence of
fumagillin, tryprostatin B and pseurotin A, along with an array
of other, as yet unidentified, molecules (Figure 1F). These
secondary metabolites are all products of the ‘supercluster’ on
Chromosone 8 [44–46], demonstrating correlation between the
proteomic and metabolomic profiles. Interestingly, expression of
the clusters identified here is partially or completely regulated by
the transcription regulator LaeA [38]. Two additional proteins,
encoded by AFUA_3G03280 and AFUA_3G03330, were also
detected, from a cluster with predicted involvement in the
production of a siderophore and a toxin [38] (Table 1). Following
on from this observation of SM cluster protein detection, mycelia
from cultures grown in Czapek-Dox media for 72 h were also
analysed. Shotgun proteomic analysis revealed the presence of
four proteins from the gliotoxin biosynthetic cluster under the
conditions used. The identification of peptides from GliN, GliF,
GliH and GliT (Table S2), correlated with the presence of
gliotoxin in the culture supernatants of these cultures (Figure 1G).
Comparative 2-D analysis of A. fumigatus ATCC26933
following exposure to a combination of gliotoxin and
H
2
O
2
Proteins (n= 13) were found to be significantly differentially
abundant (p,0.05) when A. fumigatus was co-exposed to
gliotoxin/H
2
O
2
compared to H
2
O
2
alone (Figure 2; Table 2).
These comprised six proteins with an abundance increase, and
seven proteins with a decrease, of at least 1.5 fold. Furthermore,
A. fumigatus Proteomics
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Figure 1. Overview of
A. fumigatus
shotgun proteomic data. (a) Proteome map showing distribution of A. fumigatus proteins based on
theoretical M
r
and pIwhere proteins identified by shotgun mass spectrometry (n= 414; red) are shown overlaid on the total A. fumigatus proteome
(black). tM
r
, theoretical molecular mass, axis drawn on logarithmic scale; tpI, theoretical isoelectric point, axis drawn on linear scale. (b, c) Distribution
of proteins identified by shotgun mass spectrometry (MS) according to their relative hydrophobicity and the number of putative transmembrane
regions per protein. The number of putative transmembrane regions on each protein identified by shotgun MS is shown. (d) Distribution of
functional annotations of A. fumigatus proteins identified using shotgun proteomics strategy. GO, KEGG and FunCat classification schemes were used
for functional annotation utilizing the FungiFun application. A number of proteins (n= 23) were identified that possessed no functional classification
A. fumigatus Proteomics
PLOS ONE | www.plosone.org 4 September 2014 | Volume 9 | Issue 9 | e106942
comparative analyses were carried out on all comparitor sets, to
identify alterations in protein abundance between individual
treatments (i.e., gliotoxin alone, H
2
O
2
alone, co-addition and
solvent control). Redundancy was noted, with some proteins
included in multiple comparison sets, resulting in the net
differential expression of 27 unique proteins (Figure 2). These
proteins were excised and subjected to in-gel trypsin digestion,
followed by LC-MS/MS analysis for protein identification.
using this system. (e) The functional categorization of the proteins identified here, based on the FunCat annotation scheme, are shown. Note: GRAVY,
grand average of hydropathy; TM, transmembrane; MS, mass spectrometry. (f) LC-MS detection of SM in A. fumigatus organic extracts from AMM
cultures and (g) Czapek-Dox cultures (BPC: Base Peak Chromatogram; EIC: Extracted Ion Chromatogram).
doi:10.1371/journal.pone.0106942.g001
Table 1. A. fumigatus proteins, involved in secondary metabolism, and identified by shotgun mass spectrometry.
Cluster No
a
CADRE ID.
(AFUA_) Protein name Chromosome No LaeA regulation
a
Product(s)
11G10350 Phosphoglycerate
kinase PgkA (EC 2.7.2.3)
1 Yes Fumigaclavine C
73G03280 FAD binding
monooxygenase
3 No Putatively two products: a siderophore and a toxin
73G03330 Mitochondrial enoyl
reductase
3No
10 3G14680 Lysophospholipase 3
(EC 3.1.1.5)
(Phospholipase B 3)
3 Partial Unknown
13 4G14380 Glutathione S-transferase,
putative
4 Partial Unknown
18 6G09740 GliT (Thioredoxin
reductase GliT)
(EC 1.-.-.-)
6 Yes Gliotoxin
22 8G00230 Phytanoyl-CoA
dioxygenase family
protein
8 Yes ‘Supercluster’ producing Fumitremorgin B, Pseurotin A
and Fumagillin
22 8G00370 Polyketide synthase,
putative
8Yes
22 8G00380 DltD N-terminal
domain protein
8Yes
22 8G00390 O-methyltransferase,
putative
8Yes
22 8G00400 Unknown function
protein
8Yes
22 8G00430 Unknown function
protein
8Yes
22 8G00440 Steroid monooxygenase,
putative (EC 1.-.-.-)
8Yes
22 8G00480 Phytanoyl-CoA
dioxygenase family
protein
8Yes
22 8G00500 Acetate-CoA ligase,
putative (EC 6.2.1.1)
8Yes
22 8G00510 Cytochrome P450
oxidoreductase
OrdA-like, putative
8 Yes ‘Supercluster’ producing Fumitremorgin B, Pseurotin A
and Fumagillin
22 8G00530 Alpha/beta hydrolase,
putative
8Yes
22 8G00540 Hybrid PKS-NRPS
enzyme, putative
8Yes
22 8G00550 Methyltransferase
SirN-like, putative
8Yes
22 8G00580 Glutathione
S-transferase, putative
8Yes
CADRE ID., A. fumigatus gene annotation nomenclature according to [1] and [71].
a
Cluster numbers and LaeA regulation as denoted in [38].
doi:10.1371/journal.pone.0106942.t001
A. fumigatus Proteomics
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Identification of differentially abundant proteins by LC-
MS/MS
LC-MS/MS analysis was used to identify the 27 proteins which
were differentially abundant following challenges with gliotoxin
and H
2
O
2
, individually or in combination (Tables S3 and S4,
Table 2). Protein abundance was assessed for all conditions
relative to the solvent control (Tables S3 and S4). The abundance
of proteins after gliotoxin/H
2
O
2
co-exposure was also assessed
relative to the individual treatments of gliotoxin alone or H
2
O
2
alone (Table 2). Proteins (n= 13) were significantly altered in
abundance following co-addition relative to H
2
O
2
alone (p,0.05)
(Table 2, Figure 2). Proteins exhibiting increased abundance in
the co-addition included those with oxidation-reduction activity.
GliT, the gliotoxin oxidoreductase [28,47], was more abundant in
the co-addition condition relative to H
2
O
2
alone (3.5 fold) but was
unaltered by H
2
O
2
addition alone (Figure 2, Table 2 and Table
S4). An increase in abundance of the Ran-specific GTPase and the
proliferating cell nuclear antigen (PCNA), involved in cell-cycle
regulation and DNA-repair [48,49], respectively, was also
observed in the presence of gliotoxin/H
2
O
2
together. The HAD
family hydrolase, also exhibited increased abundance when both
gliotoxin and H
2
O
2
were present, relative to any of the control
conditions. Proteins involved in amino acid and nucleic acid
metabolism [50,51], glutamine amidotransferase: cyclase and
methylenetetrahydrofolate reductase, also showed differential
abundance. The class V chitinase, associated with cell autolysis
[52], was significantly less abundant (p= 5.2610
25
) upon co-
addition relative to H
2
O
2
alone. A decrease in abundance of
proteins associated with response to stress was observed following
gliotoxin/H
2
O
2
co-exposure, relative to H
2
O
2
alone. Hsp90 and
the oxidative stress protein Svf1 were of lower abundance in the
co-addition (2.7 and 1.6 fold, respectively), reflective of the relief of
H
2
O
2
-induced stress (Table 2, Figure 2). Additionally two un-
known function proteins were detected, which underwent a 4.9
Figure 2. 2-D analysis reveals differential proteomic response
of A. fumigatus
to a combination of gliotoxin and H
2
O
2
.2-D proteome
maps of A. fumigatus ATCC26933 (a) solvent control, (b) following exposure to gliotoxin (10 mg/ml) for 4 h, (c) following exposure to 2 mM H
2
O
2
for
4 h, (d) following exposure to a combination of gliotoxin (10 mg/ml) and H
2
O
2
(2 mM) for 4 h. The proteins were first separated on pH 4–7 strips
followed by SDS-PAGE. Proteins found to be significantly differentially expressed (p,0.05), after analysis using Progenesis SameSpot software, are
numbered. (e) Increased expression of the gliotoxin oxidoreductase GliT in response to gliotoxin but not H
2
O
2
. GliT expression was increased
following exposure to exogenous gliotoxin alone (5.1 fold) and in combination with H
2
O
2
(4.8 fold), relative to the solvent control. No significant
difference in expression of GliT was detected upon exposure of A. fumigatus to H
2
O
2
alone, relative to the control (p.0.05), indicating GliT expression
is mediated by gliotoxin only.
doi:10.1371/journal.pone.0106942.g002
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fold increase (AFUA_2G11120) and a 2.9 fold decrease
(AFUA_6G03460) in abundance in the co-addition, relative to
H
2
O
2
alone. This latter observation underpins the necessity to
undertake both shotgun and 2-D based approaches to identify
novel proteins.
Gliotoxin inhibits H
2
O
2
-induced ROS formation in A.
fumigatus
Previous work has demonstrated that GliT is essential to protect
A. fumigatus against exogenous gliotoxin [28] but contra-
intuitively, that gliotoxin reverses H
2
O
2
-mediated growth inhibi-
tion of A. fumigatus ATCC26933 and DgliK
26933
[30], yet, no
mechanistic explanation was forthcoming. Results from the
comparative proteomics analysis indicated that gliotoxin may
effect an alleviation of H
2
O
2
induced-oxidative stress. Data in
Figure 3 show ROS formation consequent to H
2
O
2
exposure in A.
fumigatus. However, co-addition of gliotoxin results in a
significant reduction (p.0.0001) in the production of reactive
metabolites, as judged by decreased 29,79-dichlorofluorescein
fluorescence. This suggests that gliotoxin acts as an anti-oxidant
and functions to impede H
2
O
2
-mediated growth inhibition.
Discussion
A comprehensive mycelial proteome reference map, produced
by Vo¨disch et al. [53], identified proteins with a GRAVY score up
to 0.158 and fourteen proteins with 1–2 putative TM regions,
which computes to 4.2% of the identified proteins possessing TM
regions. Here, by comparison, 44 proteins possessing predicted
TM domains were identified from A. fumigatus mycelia,
corresponding to 10.62% of the proteins identified using shotgun
mass spectrometry alone (Table S1). This represents a substantial
increase (2.5 fold) in the identification of proteins with TM regions,
compared to previous 2-D based studies with similar targets [6].
Using the shotgun proteomics approach, 33 hydrophobic proteins,
corresponding to 7.97% of total identified proteins, were detected,
including a protein transport protein SEC61 alpha subunit
(AFUA_5G08130) with a GRAVY score of 0.4828, compared to
3.4% hydrophobic protein content by previous 2-D coupled MS
analysis [53]. The standard molecular mass resolution of A.
fumigatus mycelial proteins, using 2-D, ranges from 10 to
142 kDa [3,4,6]. The constraint of high molecular mass did not
apply to the shotgun proteomic approach used in this study, with
the identification of 12 proteins possessing a molecular mass
greater than 142 kDa. The largest protein detected was encoded
by AFUA_5G02570, with predicted histone acetyltransferase
activity and a theoretical mass of 444 kDa. PesO [54], a hybrid
polyketide synthase/ non-ribosomal peptide synthetase (PKS/
NRPS) (AFUA_8G00540), with a theoretical molecular mass of
434 kDa was also identified. Here, 73 unique peptides were
identified from this protein, contributing to sequence coverage of
28%. PesO is involved in the production of pseurotin A [40] and
its identification provides evidence of expression of this secondary
metabolite cluster. A 267 kDa polyketide synthase
(AFUA_8G00370) was also identified by 10 unique peptides,
contributing to 10% sequence coverage. These findings represent
some of the largest A. fumigatus proteins to be identified by mass
spectrometry to date. Cagas et al. [9] utilised iTRAQ in order to
profile the early development proteome of A. fumigatus. This gel-
free method of large scale proteomic identification extended the
Table 2. Proteins (n = 13) exhibiting significant differential abundance1 in A. fumigatus ATCC26933 following the co-addition of
gliotoxin and H2O2, relative to H2O2 alone.
Protein Name
Co-addition v
Control (iii)
Co-addition v
Gliotoxin (iv)
Co-addition v
H
2
O
2
(v)
H
2
O
2
v
Control
CADRE ID.
(AFUA_) Spot No.
Increased Abundance Proteins in Co-addition
vH
2
O
2
Thioredoxin reductase GliT q4.8 - q3.5 - 6G09740 738
Unknown function protein q4.4 - q4.9 - 2G11120 803
Proliferating cell nuclear antigen (PCNA) q5.9 q7.4 q2.4 - 1G04900 980
Ran-specific GTPase-activating protein 1 - - q1.8 - 5G12180 850
NADH-quinone oxidoreductase (23 kDa subunit) - - q1.9 - 1G06610 897
HAD family hydrolase q1.8 q2.1 q1.5 - 5G08270 989
Decreased Abundance Proteins in Co-
addition v H
2
O
2
Unknown function protein - - Q2.9 q2.3 6G03460 964
Molecular chaperone and allergen
Mod-E/Hsp90/Hsp1
Q1.5 - Q2.7 q2.0 5G04170 966
Oxidative stress protein Svf1 - - Q1.6 - 5G11820 975
Glutamine amidotransferase: cyclase Q1.9 - Q1.7 - 2G06230 968
Glycyl-tRNA synthetase Q1.9 - Q1.5 - 5G05920 305
Methylenetetrahydrofolate reductase Q1.9 - Q1.6 - 2G11300 379
Class V chitinase - - Q1.8 - 3G11280 662
Data extracted from Tables S3 and S4 and re-charted for clarity. Proteins detected with a significant change in abundance in H
2
O
2
compared to the control are also
reported.
1
p,0.05; Fold increase (q) or decrease (Q) of protein in the co-additive condition, relative to the solvent control, gliotoxin alone or H
2
O
2
alone. Co-addition: incubation
with both gliotoxin and H
2
O
2
. CADRE ID., A. fumigatus gene annotation nomenclature according to [1] and [71]; Spot No, according to Figure 2. Numbers in bold
indicate fold change of proteins (n= 13) differentially regulated in the co-addition, relative to H
2
O
2
alone.
doi:10.1371/journal.pone.0106942.t002
A. fumigatus Proteomics
PLOS ONE | www.plosone.org 7 September 2014 | Volume 9 | Issue 9 | e106942
molecular mass limits of detection to 9 to 255 kDa, thereby
confirming the value of alternative methods for proteomic
investigation. Supplementary information (Text S1) provides
further discussion of shotgun MS data.
Verification of proteins encoded by genes within secondary
metabolite clusters was achieved in this study using shotgun mass
spectrometry. Proteins identified from AMM cultures are puta-
tively encoded by six clusters involved in the production of up to
nine secondary metabolites (SM) [38]. Products of these SM
clusters include up to 3 unknown metabolites, fumigaclavine C
[55], fumitremorgins [39,44,45], pseurotin A [40], fumagillin
[41,46], gliotoxin [43] and a putative siderophore [38]. Perrin et
al. [38] annotated a ‘supercluster’ on Chromosome 8
(AFUA_8G00100-AFUA_8G00720) that is involved in the pro-
duction of fumitremorgins, pseurotin A and fumagillin [39,40,46].
Fifteen proteins identified by shotgun mass spectrometry are
annotated members of this ‘supercluster’, with one identified
protein involved in the production of fumitremorgins and
tryprostatins, four proteins involved in the pseurotin A biosyn-
thetic portion of the cluster and the remaining nine proteins
associated with fumagillin biosynthesis. Metabolomic investigation
confirmed the presence of fumagillin, tryprostatin B and pseurotin
A in culture supernatants, confirming the activity of these clusters,
as indicated by shotgun proteomics. Phytanoyl-CoA dioxygenase
family protein (FtmF) (AFUA_8G00230) was identified by 3
unique peptides and a sequence coverage of 14%. FtmF, a non-
heme Fe(II) and a-ketoglutarate-dependent dioxygenase, catalyses
the conversion of fumitremorgin B to verruculogen via endoper-
oxide bond formation [56]. This enzyme is also capable of
converting fumitremorgin B to 12a,13a-dihydroxyfumitremorgin
C and 13-oxo-verruculogen, by deprenylation and oxidation
mechanisms respectively [57]. Verruculogen, like fumitremorgin
B, is a tremorgenic mycotoxin and has been shown to produce
deleterious effects on respiratory epithelial cells [58]. A second
protein (AFUA_8G00280) was also identified from the border
region of this cluster, with putative oxidoreductase activity, and
along with the detection of FtmF, suggests the production of
fumitremorgins or tryprostatins by A. fumigatus under the
conditions of culture. Pseurotin A production is also encoded by
the ‘supercluster’ on Chromosome 8 [40], and four enzymes, that
form part of the pseurotin biosynthetic cluster, were detected here;
an alpha/beta hydrolase (AFUA_8G00530), a hybrid PKS-NRPS
enzyme PesO (AFUA_8G00540), a methyltransferase SirN-like
(AFUA_8G00550) and a putative glutathione S-transferase
(AFUA_8G00580) [4]. This cluster has demonstrated increased
expression at both the transcript and protein level under hypoxic
conditions [21]. Furthermore, up-regulation of the methyltrans-
ferase and PesO transcripts were also shown in the mouse lung
during infection by A. fumigatus [21]. Identification of nine
proteins from the portion of the supercluster associated with
fumagillin biosynthesis, represents significant coverage of this
fifteen member cluster by shotgun mass spectrometry. Fumagillin
is a meroterpenoid, with demonstrated anti-angiogenic activity
through interaction with methionine aminopeptidase II (MetAP2)
[59]. Fumagillin has also been associated with disruption of
NADPH oxidase function could represent a putative virulence
Figure 3. Gliotoxin attenuates H
2
O
2
-induced ROS formation. (a) Neither methanol (solvent control) or gliotoxin induce significant ROS
formation in A. fumigatus, however H
2
O
2
exposure leads to clear formation of ROS. Co-addition of gliotoxin dissipates ROS as judged by reduced
fluorescence. (b) Gliotoxin significantly reduces H
2
O
2
-induced ROS levels during co-incubation with H
2
O
2
(p.0.0001).
doi:10.1371/journal.pone.0106942.g003
A. fumigatus Proteomics
PLOS ONE | www.plosone.org 8 September 2014 | Volume 9 | Issue 9 | e106942
factor [60]. Indeed, cognate transcript expression of six of the
proteins identified from this cluster was up-regulated in A.
fumigatus Af293 during the initiation of murine infection [61].
Identification of a number of proteins from both the pseurotin A
and fumagillin clusters is in-line with the recent identification of a
transcription factor, FapR, which co-regulates expression of genes
in these two clusters [46]. Further investigation of a second
minimal media culture condition (Czapek-Dox, 72 h) revealed a
similar observation. Proteins (n= 4) from the gliotoxin biosynthetic
cluster were identified (Table S2), in line with the detection of
gliotoxin in culture supernatants (Figure 1G). Enlisting a shotgun
proteomic approach provides a non-targeted method to detect the
expression of proteins involved in secondary metabolism, in any
given growth condition, and could prove useful as a tool for the
identification of novel metabolites.
Proteomics also revealed changes in protein abundance
associated with SM (gliotoxin)-mediated relief of H
2
O
2
-induced
stress. Proteins (n= 13) were differentially abundant following
exposure to a combination of H
2
O
2
(2 mM) and gliotoxin (10 mg/
ml), relative to H
2
O
2
alone (2 mM), which facilitates dissection of
the mechanisms involved in gliotoxin-mediated relief of H
2
O
2
-
induced stress (Table 2). Increased abundance of two proteins, in
response to gliotoxin and H
2
O
2
in combination, relative to H
2
O
2
alone, with predicted or demonstrated oxidoreductase activity
included the gliotoxin oxidoreductase GliT and the NADH-
quinone oxidoreductase (23 kDa subunit), with 3.5 and 1.9 fold
increase in abundance, respectively. In addition to a key role in the
gliotoxin biosynthetic process, GliT also mediates self-protection
against the harmful effects of gliotoxin [28,47]. Increased
expression of GliT was detected following exposure to gliotoxin
alone (5.1 fold), as previously noted [20,28] and combined with
H
2
O
2
(4.8 fold) relative to the solvent control (Figure 2). There
was no significant alteration to abundance of GliT in response to
H
2
O
2
alone (p= 0.297) and this demonstrates that GliT
abundance is not regulated by H
2
O
2
and increased levels in the
co-addition condition is solely a result of gliotoxin presence. Choi
Figure 4. Overview of the regulation of the purine metabolic pathway by gliotoxin and H
2
O
2
, either alone or in combination. (a)
Purine-related enzymes and pathways undergoing an increase in expression, relative to the solvent control, are indicated in red and decreased
expression is indicated in green. Metabolites are indicated in black. Ade1, bifunctional purine biosynthetic protein; Xpt1, xanthine-guanine
phosphoribosyltransferase; Apt1, adenine phosphoribosyltransferase. Enzymes of the histidine and de novo purine biosythesis converging pathways,
glutamine amidotransferase: cyclase and Ade1, are down-regulated in response to gliotoxin. Expression of enzymes involved in the purine salvage
pathways, Xpt1 and Apt1, is up-regulated in the presence of H
2
O
2
and gliotoxin, repsectively, relative to a solvent control [20,23]. Figure adapted
from pathway.yeastgenome.org. (b) Structures of intermediate molecules in the purine and histidine biosynthesis pathway; 5-aminoimidazole
ribonucleotide (AIR), N-succinyl-5-aminoimidazole-4-carboxamide ribonucleotide (SAICAR), 5-aminoimidazole-4-carboxamide ribonucleotide (AICAR)
and inosine monophosphate (IMP). R5P = ribulose-5-phosphate.
doi:10.1371/journal.pone.0106942.g004
A. fumigatus Proteomics
PLOS ONE | www.plosone.org 9 September 2014 | Volume 9 | Issue 9 | e106942
et al. [62] noted that gliotoxin catalysed H
2
O
2
reduction,
mediated by the mammalian thioredoxin redox system and
proposed that gliotoxin replaces 2-cys peroxiredoxin as an electron
acceptor, in the reduction of H
2
O
2
to H
2
O in mammalian cells.
The proliferating cell nuclear antigen (PCNA) exhibited a 2.4
fold increase in abundance following incubation with a combina-
tion of gliotoxin and H
2
O
2
, relative to H
2
O
2
alone. Moreover,
abundance of this protein was also further increased in the co-
addition condition, relative to gliotoxin alone (7.4 fold) and the
solvent control (5.9 fold; Table 2). Thus, PCNA abundance is
induced by H
2
O
2
(approximately 3.5 fold) and not by exposure to
gliotoxin alone. Clearly, a combination of H
2
O
2
with gliotoxin
leads to further induction of this protein. PCNA is involved in the
process of DNA-repair following H
2
O
2
-mediated damage [48]
and acts as an anchor to the DNA template for binding partners
[63]. The increase in PCNA abundance observed in response to
H
2
O
2
, alone or coupled with gliotoxin, may therefore be indicative
of H
2
O
2
-induced DNA damage in these conditions. Furthermore,
additional induction of PCNA abundance in the co-addition
condition relative to H
2
O
2
, alone, may account for the recovery of
growth, due to enhanced DNA repair capacity.
Proteins involved in the response to cellular stress underwent
decreased abundance in the co-addition condition, relative to
H
2
O
2
alone. A decrease in abundance of Hsp90 (2.7 fold) and the
oxidative stress protein Svf1 (1.6 fold) was noted. Hsp90 displayed
increased abundance in the presence of H
2
O
2
alone, relative to the
solvent control (2.0 fold), with this response reversing upon co-
incubation with gliotoxin. In accordance with these observations at
the proteomic level, the transcript of Hsp90 was also reported to
be up-regulated in A. fumigatus in response to exogenous H
2
O
2
[64]. Hsp90 is a stress-induced protein involved in the refolding of
denatured proteins and signal transduction [64,65]. The decrease
in abundance of Hsp90 is indicative of the decrease in oxidative
stress, correlating with the relief of growth inhibition observed.
The decreased abundance of Svf1, with a nuclear localisation and
a role in the response to oxidative stress [7,66], is also diagnostic
for the attenuation of oxidative stress in the co-addition condition
relative to H
2
O
2
alone. Indeed, a significant reduction (p,0.0001)
in ROS levels was detected following co-application of gliotoxin
and H
2
O
2
, relative to H
2
O
2
alone (Figure 3), thus providing
biochemical verification of the proteomics data. A decrease in
abundance (1.8 fold) of the class V chitinase in the presence of a
combination of gliotoxin and H
2
O
2
was observed, relative H
2
O
2
alone. This protein belongs to subgroup A of fungal/bacterial
chitinases which are associated with fungal growth and autolysis
[67,68]. The orthologous A. nidulans protein, ChiB, has
demonstrated involvement in the autolysis of fungal mycelia in
response to stress [52]. A higher abundance of this protein, in the
presence of H
2
O
2
alone, may indicate the occurrence of mycelial
autolysis, which could have been stimulated by the presence of
oxidative stress. This autolysis could also account for the growth-
inhibited phenotype observed in the presence of H
2
O
2
alone [30].
Proteins involved in amino acid and nucleotide metabolism also
decreased in abundance in the presence of gliotoxin and H
2
O
2
combined, relative to H
2
O
2
alone. Glutamine amidotransferase:
cyclase and methylenetetrahydrofolate reductase underwent a 1.7
and 1.6 fold decrease in abundance, respectively, relative to H
2
O
2
alone. Additionally, abundance of both of these proteins was
decreased 1.9 fold in the co-addition condition, relative to the
solvent control. Considering these observations, H
2
O
2
does not
appear to be involved in the controlling the levels of these proteins.
Instead, gliotoxin, either independently or in combination with
H
2
O
2
, is responsible for triggering the decrease in abundance of
these proteins. Glutamine amidotransferase: cyclase catalyses two
Table 3. Summary of abundance changes of proteins involved in purine biosynthesis pathways.
Protein Name
Gliotoxin v
Control (i)
H
2
O
2
v
Control (ii)
Co-addition v
Control (iii)
Co-addition v
Gliotoxin (iv)
Co-addition
vH
2
O
2
(v) CADRE ID. (AFUA_) Spot No.
Proteins involved in purine
salvage pathway
Adenine phosphoribosyl transferase Apt1 q20.5* 7G02310 N/A
Xanthine-guanine phosphoribosyl
transferase Xpt1
q3.4* q1.8 q1.6 4G04550 992
Proteins involved in de-novo
purine biosynthesis
Glutamine amidotransferase: cyclase Q1.9 Q1.7 2G06230 968
Bifunctional purine biosynthesis protein Ade1 Q1.8 6G04730 959
Fold increase (q) or decrease (Q) of protein, relative to the respective control. CADRE ID., A. fumigatus gene annotation nomenclature according to [1] and [71]; Spot No, according to Figure 2.
*Change in protein abundance was reported previously [20,23].
doi:10.1371/journal.pone.0106942.t003
A. fumigatus Proteomics
PLOS ONE | www.plosone.org 10 September 2014 | Volume 9 | Issue 9 | e106942
steps in the biosynthesis of histidine, producing both a histidine
precursor and 5-aminoimidazole-4-carboxamide ribonucleotide
(AICAR), an intermediate of the purine biosynthetic process, thus
linking these pathways [50]. Interestingly, the bifunctional purine
biosynthetic protein, Ade1, was reduced in abundance in the
presence of gliotoxin relative to the solvent control (1.8 fold)
(Figure 2). Conversely, xanthine-guanine phosphoribosyl transfer-
ase Xpt1, was more abundant in the presence of gliotoxin and
H
2
O
2
combined, relative to the solvent control (1.8 fold) and
gliotoxin alone (1.6 fold), indicating that levels of this protein are
influenced by H
2
O
2
. Indeed, Lessing et al. [23] observed an
increase in abundance of Xpt1 following exposure to H
2
O
2
for
45 min. Xpt1 is involved in the purine nucleotide salvage
pathway, whereby XMP and GMP are formed from precursors,
xanthine and guanine, respectively [69] (Figure 4; Table 3).
Additionally, the increased abundance of another component of
the purine salvage pathway, adenine phosphoribosyltransferase,
has been noted in response to exogenous gliotoxin [20]. These
observations reveal a diminution of de novo purine biosynthesis in
the presence of gliotoxin and that the alternative salvage pathway
is utilised in its place (Figure 4; Table 3). Together, these
observations underline the influence of gliotoxin and H
2
O
2
, either
alone or in combination, on nucleotide biosynthesis in A.
fumigatus.
Furthermore, while no definitive functions have been demon-
strated for the unknown function proteins encoded by
AFUA_6G03460 and AFUA_2G11120, computational analysis
has assigned the function of D-alanine-D-alanine ligase and
methyltransferase to these proteins, respectively [32]. An ortho-
logue of this methyltransferase (MT-II) was found to be up-
regulated in A. niger in response to reductive stress from DTT
[70], which may resemble the stress induced by gliotoxin and
presents an interesting target for future investigations.
Conclusions
In summary, shotgun proteomics has revealed expression of
multiple proteins involved in secondary metabolite biosynthesis
coincident with detection of the cognate metabolites, and provides
strong evidence for the activation of multiple clusters under the
control of the transcriptional regulator LaeA, in the conditions
tested. Our findings also demonstrate how proteomics can inform
how the SM, gliotoxin, effects attenuation of H
2
O
2
-mediated
oxidative stress.
Supporting Information
Figure S1 Distribution of proteins identified using
shotgun mass spectrometry (n= 414) based on gene
locus (blue lines). Identification of proteins (n= 15) from
a supercluster on chromosome 8, involved in the
production of fumitremorgin B, pseurotin A and fuma-
gillin (red circle).
(DOC)
Table S1 Proteins identified by shotgun MS analysis.
(XLS)
Table S2 Peptides detected from gliotoxin cluster
proteins following growth in Czapek-Dox media for
72 h at 376C.
(XLS)
Table S3 Proteins undergoing significant differential
abundance
1
in A. fumigatus ATCC26933 following
exposure to gliotoxin and H
2
O
2
, separately or combined,
relative to the solvent control. Protein identification was
achieved by 2D-PAGE and LC-MS/MS.
(DOC)
Table S4 Proteins undergoing significant differential
abundance
1
in A. fumigatus ATCC26933 following
exposure to a combination of gliotoxin and H
2
O
2
(co-
addition), relative to the control, gliotoxin alone or H
2
O
2
alone. Protein identification was achieved by 2D-PAGE and LC-
MS/MS.
(DOC)
Text S1 Supplementary Discussion.
(DOC)
Author Contributions
Performed the experiments: RAO SH KJS. Analyzed the data: RAO SH
KJS GWJ SD. Contributed to the writing of the manuscript: RAO GWJ
SD.
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