Journal of Proteomics 141 (2016) 47–56

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Journal of Proteomics

journal homepage: www.elsevier.com/locate/jprot

Proteomic analysis of an environmental isolate of Rhodotorula

mucilaginosa after arsenic and cadmium challenge: Identification of a
protein expression signature for heavy metal exposure
Sidra Ilyas a, Abdul Rehman a, Ana Varela Coelho b, David Sheehan c,⁎
a
Dept. Of Microbiology and Molecular Genetics, University of the Punjab, Quaid-e-Azam Campus, Lahore 54590, Pakistan
b
Instituto de Tecnologia Química e Biológica Antonio Xavier, Universidade Nova de Lisboa, Av. da República, 2780-157 Oeiras, Portugal
c
Environmental Research Institute and School of Biochemistry and Cell Biology, University College Cork, Ireland

a r t i c l e i n f o a b s t r a c t

Article history: A metal-resistant Rhodotorula mucilaginosa strain was isolated from an industrial wastewater. Effects on reduced/
Received 17 September 2015 oxidized glutathione (GSSG/GSH), antioxidant enzymes and proteome were assessed on metal challenge
Received in revised form 7 March 2016 (100 mg/L). Increased GSH (mM/g) was found with CdCl2 (18.43 ± 3.34), NaAsO2 (14.76 ± 2.14), CuSO4
Accepted 14 April 2016
(14.73 ± 2.49), and Pb(NO3)2 (15.74 ± 5.3) versus control (7.67 ± 0.95). GSH:GSSG ratio decreased with
Available online 16 April 2016
CdCl2, NaAsO2, and Pb(NO3)2 but not with CuSO4 and cysteine-containing protein levels increased with CdCl2
Keywords:
and NaAsO2. NaAsO2 exposure enhanced glutathione transferase activity but this decreased with CdCl2. Both
Arsenic metals significantly increased glutathione reductase and catalase activities. Metabolism-dependent uptake of
Cadmium Cd and As (12-day exposure) of approximately 65 mg/g was observed in live cells with greater cell surface inter-
Proteome action for As compared to Cd. A particular role for arsenic oxidase in As resistance was identified.
Oxidative stress One dimensional electrophoresis revealed higher oxidation of protein thiols in response to NaAsO2 than to CdCl2.
Rhodotorula Two dimensional electrophoresis showed altered abundance of some proteins on metal treatment. Selected spots
were excised for mass spectrometry and seven proteins identified. Under oxidative stress conditions, xylose re-
ductase, putative chitin deacetylase, 20S proteasome subunit, eukaryotic translation elongation factor 2, valine-
tRNA ligase and a metabolic enzyme F0F1 ATP synthase alpha subunit were all expressed as well as a unique hy-
pothetical protein. These may comprise a protein expression signature for metal-induced oxidation in this yeast.
Significance: Fungi are of widespread importance in agriculture, biodegradation and often show extensive toler-
ance to heavy metals. This makes them of interest from the perspective of bioremediation. In this study an envi-
ronmental isolate of R. mucilaginosa showing extensive tolerance of a panel of heavy metals, in particular
cadmium and arsenic, was studied. Several biochemical parameters such as activity of antioxidant enzymes, sta-
tus of reduced and oxidized glutathione and thiols associated with proteins were all found to be affected by metal
exposure. A detailed analysis with arsenic and cadmium pointed to a particular role for arsenic oxidase in arsenic
bioaccumulation and tolerance. This is the first time this has been reported in R. mucilaginosa, and suggests that
this isolate may have potential in biosorption of these metals in the environment. Proteomic analysis revealed
that seven proteins with a variety of roles - ATP synthesis, protein degradation/synthesis, and metabolism of xy-
lose and chitin - were differentially affected by metal exposure in a manner consistent with oxidative stress.
These may therefore represent a protein expression signature for exposure to cadmium and arsenic.
© 2016 Elsevier B.V. All rights reserved.

1. Introduction against metal-induced reactive oxygen species (ROS). Despite the

Rhodotorula sp. is a ubiquitous, airborne, saprophytic red yeast pres-
ent in many environmental niches [1]. Rhodotorula rubra has previously
been studied for a possible role in biodegradation of plasticisers [2].
Rhodotorula sp. also plays an important role in processes of mineral cy-
cling, can accumulate cadmium, lead, iron and other heavy metal ions
[3–5] and thus may have evolved extensive antioxidant defenses
⁎ Corresponding author.
E-mail address: d.sheehan@ucc.ie (D. Sheehan).
known potential of yeasts for the removal of heavy metal cations from
aqueous solution [6], little information on the detailed effects of cad-
mium and arsenic on Rhodotorula mucilaginosa is available. In the pres-
ent study, this carotenoid-rich red yeast was isolated from water
effluent from a synthetic mill in Lahore, Pakistan. It was found that
this isolate tolerated high levels of heavy metals in culture and grew
well under these conditions.
ROS can be generated by a variety of intracellular processes or as a
result of xenobiotic intoxication [7]. Pollutants such as heavy metals
(e.g. Cd) and metalloids (e.g. As) have been linked with generation

http://dx.doi.org/10.1016/j.jprot.2016.04.012
1874-3919/© 2016 Elsevier B.V. All rights reserved.
48 S. Ilyas et al. / Journal of Proteomics 141 (2016) 47–56
and accumulation of ROS in yeasts [8,9]. Unlike As, Cd is a non-redox
metal widespread in aquatic sediments. It does not participate in Fenton
reactions, but nonetheless enhances ROS production [10]. CdCl2 and
NaAsO2 are considered highly toxic as they have high affinity for pro-
teins and can induce synthesis of cysteine-rich binding peptides and
metallothioneins (MT) [11]. These metals can also bind directly to pro-
tein -SH groups, indirectly generate oxidative modifications in yeast
cells and can alter protein properties [12]. Cadmium also has recently
been reported as influencing fungal community composition in soil
fungi [13]. Enzymes such as glutathione transferase (GST), glutathione
reductase (GR), catalase (CAT) and superoxide dismutase (SOD) are
key components of enzymatic detoxification of ROS and their by-
products. Amongst these, GSTs are important phase II detoxification en-
zymes which are widespread in fungi [14,15].
Redox proteomics enables comparative analysis of protein profiles of
control and oxidatively-stressed cells [16]. Proteins absorb ∼70% of ROS
and this causes covalent changes to amino acid side-chains of proteins
with the potential to complicate the proteome [17].\\SH groups can be-
come reversibly oxidized to sulfenic acid (SOH), disulfides (S\\S) or
mixed disulfides (P\\S\\S\\G) with reduced glutathione (GSH) and ir-
reversibly modified to sulfinic (SO2H) or sulfonic (SO3H) acids [17,18].
The aim of this study was to characterize an environmental isolate of
R. mucilaginosa displaying resistance to heavy metals and to study
metal-induced toxicity responses to oxidative stress. We aimed, in par-
ticular, to investigate effects on key antioxidant factors necessary for
ROS detoxification and to identify a protein expression signature for ex-
posure to metal ions.
2. Materials and methods
2.1. Culture and maintenance conditions
Whilst screening for metal-resistant environmental fungi,
R. mucilaginosa was serendipitously isolated from wastewater effluents
of a synthetic mill in Lahore, Pakistan. Screening and selection were per-
formed in the presence of a panel of heavy metal ions (Cd, As, Cu, Cr, Pb)
in YPD medium containing 2% (w/v) glucose, 1% (w/v) yeast extract, 2%
(w/v) peptone and 2% (w/v) agar per litre (pH 6.5). Stock solutions of
heavy metal salts were prepared in deionized water. All experiments
were carried out in chemically defined MSM containing 0.1% (w/v)
(NH4)2SO4, 0.015% (w/v) KH2PO4, 0.01% (w/v) K2HPO4, 0.01% (w/v)
MgSO4·7H2O, 0.0026% (w/v) FeSO4, 0.0086% (w/v) CaCl2 (pH 7.0–7.2)
fortified with 1% (w/v) glucose as a carbon source. Biomass grown to
log phase was used to inoculate MSM and heavy metals were incorpo-
rated in cultures and incubated at 30 °C for 2 days. Control cultures
were treated identically but did not contain heavy metals.
2.2. Metal tolerance analysis
The minimum inhibitory concentration (MIC) was assessed by in-
creasing concentrations of each metal ion (Cd, As, Cu, Cr, and Pb) on
YPD agar medium plates. MIC is defined as the lowest concentration
of metal at which a single colony-derived streak could not grow. The ini-
tial concentration range of metals tested was 0.1–30 mM. Yeast cultures
were subsequently transferred at a given concentration to the next
highest concentration and a maximum resistance value was evaluated
when the yeast was unable to produce colonies on agar plates.
2.3. Growth curves
Growth rates in the presence of heavy metals were measured at
30 °C on a shaker (120 rpm). Aliquots (5 mL) were removed at intervals
of 4 h (from 4 to 32 h) and growth patterns of the yeast in the presence
and absence of metals were determined.
2.4. Protein extraction and estimation
Approximately 1 g yeast cells (wet weight) was ground in a mortar
and pestle to a fine powder in liquid nitrogen and suspended in extrac-
tion buffer (2 mL) containing 10 mM Tris-HCl (pH 7.2), 0.5 M sucrose,
0.15 M KCl, 1 mM EDTA and 1 mM PMSF. Homogenates were centri-
fuged at 11,000g for 10 min (Sorvall RC 5C plus) at 4 °C and extracts
were stored at −80 °C until used further for biochemical analysis. The
concentration of protein in the extracts was quantified by the method
of Bradford [19] using bovine serum albumin (BSA) as a standard.
2.5. Quantification of glutathione and cysteine levels
GSSG, GSH and non-protein thiol contents of cell-free lysates pre-
pared from metal-exposed and control yeast cultures were estimated
[20]. MSM (100 mL) was inoculated with 5 × 106/mL of fresh pre-
culture yeast cells and incubated at 30 °C with constant agitation
(120 rpm). GSH and GSSG levels were determined following 48 h treat-
ment with oxidants. Cells were weighed, and suspended in 5% (w/v)
sulfosalicylic acid prior to sonication with 60 s interval (5 cycles).
Quantification of GSH and GSSG was performed by incubating the
reaction mixture containing reaction buffer (0.1 M phosphate buffer
[pH 7]; 0.5 mM EDTA), 3 mM of 5-dithio-bis-(2 nitrobenzoic acid) and
enzyme extract at 30 °C for 5 min followed by addition of NADPH
(0.4 mM) and 2 μL GR. Samples were maintained at 30 °C for 20 min
and absorbance at 412 nm was measured. GSH was expressed as mM
of GSH per gram of cells. Extracts prepared from oxidant free salt me-
dium were taken as control. Cysteine and non-protein thiols were quan-
tified by incubating 0.1 M reaction buffer, 1 mM 5-dithio-bis-(2-
nitrobenzoic acid), and enzyme extract at 30 °C for 10 min.
2.6. Metal processing ability of R. mucilaginosa
Atomic absorption spectroscopy was used to estimate the amount of
metal ions present in the medium or processed by yeast [21]. Metal-
treated and control cultures were incubated at 30 °C under shaking con-
dition (120 rpm) and aliquots (5 mL) were removed under sterile con-
ditions every two days from two to twelve days of growth. Cultures
were centrifuged at 4000g for 10 min for estimation of metal ions pres-
ent in the supernatants. To determine metal adsorption and uptake, col-
lected culture pellets were washed three times with distilled water and
divided into two parts. One part was washed three times with 0.1 M
EDTA for 10 min and the amount of metal associated with the cell sur-
face was removed as a soluble fraction. The second part was treated
with 0.2 N HNO3 (1:1) for acid digestion and left on a hot plate for
half an hour to allow complete acid digestion for estimation of intracel-
lular metal content. The total metal adsorbed and absorbed by the or-
ganism was estimated. Metals in the prepared samples were
estimated by flame and furnace atomic absorption spectrometry (Zee-
man atomic absorption spectrophotometer, model Z-5000, Hitachi Ltd.
Japan) using an air-acetylene flame at wavelengths of 228.3 and
193.7 nm, respectively.
2.7. Enzyme assays
2.7.1. Superoxide dismutase assay
SOD activity was assessed by the method of Ewing and Janero [22].
The reaction mixture contained 62.5 mM sodium phosphate buffer
(pH 7.4), 0.125 mM EDTA, 83.3 μM NBT, 150 μM NADH and 16.5 μM
phenazine methosulfate (PMS). NBT reduction was monitored by in-
crease in absorbance at 560 nm wavelength for 5 min (every 20 or
30 s) at 25 °C. One unit of SOD is defined as the amount of enzyme caus-
ing 50% inhibition of NBT reduction.
 S. Ilyas et al. / Journal of Proteomics 141 (2016) 47–56
2.7.2. 2. Glutathione reductase assay
GR activity was determined by the method of Carlberg and
Mannervik [23]. The assay contained 0.48 M potassium phosphate
buffer, pH 7.2, extract and 1.1 mM EDTA with 8.5 mM GSSG. The tem-
perature of the reaction mixture was equilibrated at 25 °C before adding
freshly-prepared 0.65 mM β-NADPH and absorbance was read at
340 nm for 5 min.
2.7.3. Glutathione transferase assay
GST activity was measured as described by Habig et al. [24]. The re-
action mixture contained; 1 mM 1-chloro-2, 4-dinitrobenzene (CNDB),
1 mM GSH in 150 mM phosphate buffer (pH 6.5) and enzyme extract.
The formation of S-2, 4-dinitrophenyl-GSH conjugate was followed by
measuring increase in absorbance at 340 nm for 5 min (every 20 or
30 s) at 25 °C.
2.7.4. Catalase assay
H2O2 (0.059 M) in phosphate buffer, pH 7.0 (1 mL), and 1.9 mL of re-
agent grade water were added to a cuvette [25]. Protein extract (50 μL)
in 0.05 M phosphate buffer (pH 7.0) was added to initiate the reaction
and a decrease in optical density due to H2O2 consumption was re-
corded at 240 nm for 2–3 min in a temperature-controlled spectropho-
tometer. A blank solution containing H2O2-phosphate buffer and water
but no extract served as a control. One unit of catalase decomposes one
micromole of H2O2 per min at 25 °C (pH 7.0) under specific conditions.
2.7.5. Arsenite oxidase assay
ARO activity was determined according to Anderson et al. [26]. Sam-
ples containing supernatant as a soluble fraction and pellet as a mem-
brane fraction were suspended in 50 mM phosphate buffer (pH 7).
The reduction of 2, 4-dichlorophenolindophenol catalyzed by ARO was
measured with 2 mM PMS at 600 nm.
2.8. AgNO3 and KMnO4 assay
Arsenite conversion to arsenate was judged by the AgNO3 test [27].
Arsenite consumption and arsenate production were also assessed qual-
itatively by a KMnO4 screening technique [28].
2.9. Quantification of arsenite and arsenate
Arsenite was quantified by the method of Pasha and Narayana [29].
The reaction mixture contained 2% (w/v) potassium iodate, 1 M hydro-
chloric acid (HCl) 0.02% (w/v) safranine O and enzyme extract and op-
tical densities were measured spectrophotometrically against reagent
blank at 532 nm wavelength. For arsenate, the reaction mixture con-
taining enzyme extract, ascorbic acid and ammonium molybdenum
blue reagent in 9 M H2SO4 were incubated at 30 °C for 30 min and absor-
bance was read at 870 nm [30].
2.10. Measurement of protein thiols
Protein thiols in cellular extracts were tagged with IAF, and incu-
bated at 4 °C for 2 h in the dark [31]. Protein samples were precipitated
with an equal volume of 10% (w/v) chilled TCA and centrifuged at
11,000g for 3 min at 4 °C. Labeled protein pellets (15 μg) were solubi-
lized in sample buffer (3.55 mL deionized water; 0.5 M Tris-HCl,
pH 6.8; 2.5 mL glycerol; 10% (w/v) SDS and 0.5% (w/v) bromophenol
blue) and separated by one dimensional electrophoresis (1-DE) in the
presence of SDS at a constant voltage (120 V) [32]. Fluorescence protein
images were captured with a typhoon scanner (model 9410, Amersham
Biosciences) using 450 PMT and the fluorescence intensity value for
each lane was calculated using ImageQuant software. Gels were then
stained for total protein by colloidal Coomassie staining. The total thiols
in the extracts were quantified as a ratio of fluorescence protein signal
to Coomassie protein stain intensity for each electrophoresis track [9].
49
Two dimensional electrophoresis (2-DE) was performed by
dissolving IAF-labeled protein pellets (150 μg) in rehydration buffer
containing 8 M urea, 2 M thiourea, 2% (w/v) CHAPS, 4% (w/v) carrier
ampholyte (Pharmalyte 3–10), 1% (w/v) DeStreak reagent (GE
Healthcare) and a trace amount of bromophenol blue. Proteins
(125 μg) were loaded on 7-cm immobilised pH gradient (IPG) dry
strips (pH 3–10, GE Healthcare) and incubated at room temperature
in rehydration buffer for at least 18 h covered with 2 mL of mineral
oil. Proteins impregnated on IPG strips were focused in a Protean
IEF Cell (BioRad) and subjected to reduction by incubating IPG strips
on a shaker at room temperature for 20 min containing equilibration
buffer (0.375 M Tris-HCl, pH 8.8; 6 M urea; 2% (w/v) SDS; 20% (w/v)
glycerol) and 2% (w/v) DTT followed by alkylation with 2.5% (w/v)
iodoacetamide added to the equilibration buffer.
Equilibrated strips were quickly rinsed with running buffer and
loaded on 14% polyacrylamide gels along with 4 μL of marker on
chromatography paper and covered with 0.5% (w/v) molten
agarose-containing trace amounts of bromophenol blue. Electropho-
resis was performed at 90 V for 30 min followed by a constant
voltage (120 V) using a mini PAGE system (Atto, Tokyo, Japan)
until the dye front reached the end of the gel cassette. Gels were
washed three times with milliQ water and protein spots in analytical
gels were visualized by colloidal Coomassie G-250 (Bio-Rad). Gel
images were imported into Progenesis SameSpots software used
for identification and analysis of protein spots of interest [9].
2.11. Identification of proteins by MALDI-TOF/MS
Selected spots evident in colloidal Coomassie stained 2-DE gels
were excised manually. Following in-gel tryptic digestion, extracted
peptides were loaded onto an R2 micro-column (RP-C18 equivalent)
where they were desalted, concentrated and eluted directly onto a
MALDI plate using α-cyano-4-hydroxycinnamic acid (CHCA) as the
matrix solution in 50% (v/v) acetonitrile and 5% (v/v) formic acid.
Mass spectra of the peptides were acquired in positive reflectron
MS and MS/MS modes using a MALDI-TOF/TOF MS instrument
(4800plus MALDI TOF/TOF analyzer) with an exclusion list of the
trypsin autolysis peaks (842.51, 1045.56, 2211.11 and 2225.12).
The collected MS and MS/MS spectra were analyzed in combined
mode by the Mascot search engine (version 2.2; Matrix Science,
Boston, MA) and the NCBI database restricted to 50 ppm peptide
mass tolerance for the parent ions, an error of ± 0.3 Da for fragments,
one missed cleavage in peptide masses, carbamidomethylation of
Cys and oxidation of Met were selected as fixed and variable amino
acid modifications, respectively. No taxonomy restrictions were
applied. The identified proteins were only considered if a MASCOT
score above 95% confidence was obtained (p b 0.05) and at least
one peptide was identified with a score above 95% confidence
(p b 0.05). This analysis was conducted at the Analytical Services
Unit, Instituto de Tecnologia Química e Biológica (ITQB), New
University of Lisbon, Lisbon, Portugal.
2.12. Statistical analyses
All experiments were performed in triplicate and results reported
as means and standard deviation (S.D.). Statistical analysis for
enzymatic assays was carried out by paired Student's t-test. Data
obtained from proteome-based assays were tested for normality
prior to any analysis and significance testing level was set at 0.05.
p-Values of b0.05 were considered to be statistically significant.
Principal component analysis (PCA) and analysis of variance (one way
ANOVA) were performed for comparison and assessment of statistically
significant expression and redox variation between treatments and
control.
50 S. Ilyas et al. / Journal of Proteomics 141 (2016) 47–56
3. Results and discussion
3.1. Identification and culture conditions
A yeast isolated from an industrial effluent grew well at 30 °C (pH 7)
with agitation and nucleotide sequence similarities were determined
using the basic local alignment search tool (http://www.ncbi.nlm.nih.
gov/BLAST). Yeast identity was confirmed as R. mucilaginosa under ac-
cession number JQ 966756 and cells were maintained and stored on
YPD agar medium plates.
3.2. Determination of minimum inhibitory concentrations for metals
An ability to withstand high levels of heavy metals was investigated
in R. mucilaginosa and the maximum metal resistance values observed
were 29 mM (Pb), 27 mM (As), 21 mM (Cu), and 14 mM (Cd).
R. mucilaginosa has previously been reported as showing tolerance for
Ni, Cd, Cu, Pb and Cr [5,33]. Rhodotorula glutinis, apparently uses several
mechanisms to balance or reduce intracellular heavy metal toxicity
which may include sequestration of heavy metals by metallothioneins
and adsorption of heavy metal cations by the cell wall [34]. Biomass
content was also found to be reduced in the presence of As and Cd
(Table 1).
3.3. Growth curve analysis
Growth patterns were determined in cells grown in minimal salt
medium (MSM) in the presence and absence of 100 mg/L of metals
(Fig. 1). Control cells showed the shortest lag phase and accelerated
growth during the log phase with stationary phase achieved by approx-
imately 32 h. Metals inhibited cell growth, by contrast, especially in log
phase and a stationary phase comparable to controls was never
achieved. The order of growth inhibition was Cd N Cu N As N Pb.
3.4. Measurement of GSH, GSSG and NPSH
Effects of heavy metals on the GSH/oxidized glutathione (GSSG) sys-
tem were studied as changes in cellular GSH and GSSG content may
alter the cell's redox status. An approximately 2.5-fold increase in total
glutathione was observed in CdCl2-treated cells and this was almost
doubled also in cells grown in NaAsO2, CuSO4, and Pb(NO3)2 (Table 2).
Fauchon et al. previously reported that Cd strongly increases intracellu-
lar GSH content which optimizes Cd detoxification [35]. Table 2 suggests
this effect is triggered by multiple metals.
GSH was strongly elevated by Cd, Pb, As and Cu (Table 2). Thorsen
et al. reported that arsenite challenge increased GSH biosynthesis in
Saccharomyces cerevisiae [36]. Previous studies have revealed that in-
creasing GSH synthesis through overexpression of GSH1 can augment
the GSH pool by 50–66% [37] and various metabolic processes lead to in-
duction of enzymes involved in the GSH biosynthetic pathway [35,38].
In this study, elevated GSSG levels were also detected in cells cultured
in CdCl2, and Pb(NO3)2 which resulted in a decreased overall GSH/
GSSG ratio as compared to controls or the rest of the metal panel
(Fig. 2A). This dramatic variation in the GSSG/GSH ratio suggested a
Table 1
Biomass of environmental isolate R. mucilaginosa grown
in MSM supplemented with (100 mg/L) and without
heavy metals. Data are expressed as percentages (mean
of triplicate determinations ± S.D.) relevant to controls.
Sample Biomass (%)
Control 100
CdCl2 86 ± 4.7
NaAsO2 96 ± 3.59
Pb(NO3)2 85 ± 7.15
CuSO4 83 ± 12.5
Fig. 1. Effect of heavy metals (100 mg/L) on the growth pattern of R. mucilaginosa grown in
MSM.
significant alteration to cellular redox balance in response to metal ex-
posure. In contrast, this ratio increased significantly on CuSO4 treatment
in agreement with Peña-Llopis et al. [39] with only modest effects ob-
served on exposure to NaAsO2 (Fig. 2A). S. cerevisiae mutants for yap1
have previously been reported to have a decreased GSH:GSSG ratio on
exposure to As [8]. Cysteine and non-protein thiols were elevated in
CdCl2-exposed cells followed by NaAsO2, CuSO4 and Pb(NO3)2 as com-
pared to controls (Fig. 2B). These data suggest that the redox status of
R. mucilaginosa is significantly altered by exposure to metals. To explore
these findings further, more detailed experiments were performed on
cells grown in the presence of 100 mg/L CdCl2 and NaAsO2for two
days at 30 °C (with agitation at 120 rpm).
In addition to thiol-based redox-buffering, yeasts also possess a bat-
tery of enzymatic antioxidant defenses to deal with ROS and their by-
products. GST, GR, CAT and SOD are crucial components of enzymatic
antioxidant defense. CdCl2 and NaAsO2both altered the profile of these
enzymes in R. mucilaginosa (Fig. 3). ROS production can be transient
and variable for different chemicals, species and enzymes [17] and
amount, nature and subcellular localization of ROS can influence antiox-
idant activity patterns [40]. As was found to stimulate generally higher
levels of antioxidant defense enzymes [41] whilst Cd treatment reduced
GST activity (Fig. 3A). It is possible that increased production of ROS
may inactivate GST in CdCl2-treated samples. Enhanced GR activity
was evident on exposure to both CdCl2 and NaAsO2 (Fig. 3B) implying
that more GSH may be required to cope with these metals since GR re-
cycles GSSG back to GSH [23]. Alteration in GR activity is likely to affect
cellular antioxidant capacity and the ability to withstand oxidative
stress.
CdCl2 and NaAsO2 increased CAT activity by 140 and 47%, respec-
tively (Fig. 3C). It was previously reported that Cd exposure (1 mM)
caused a 2.6-fold increase in CAT activity in Candida albicans supporting
a hypothesis that Cd toxicity is associated with ROS induction within
Table 2
Intracellular levels of reduced (GSH), oxidized (GSSG) and total glutathione (GSH +
GSSG) in R. mucilaginosa grown in MSM supplemented with (100 mg/L) of metals. Data
are expressed as means of triplicate determinations ± S.D.
Growth conditions GSH (mM/g) GSSG (mM/g) GSH + GSSG (mM/g)
Control 7.67 ± 0.95 1.59 ± 0.36 9.26 ± 1.31
CdCl2 18.43 ± 3.44 5.94 ± 1.13 24.22 ± 4.57
NaAsO2 14.76 ± 2.14 5.94 ± 1.13 18.64 ± 2.25
Pb(NO3)2 15.74 ± 5.3 5.23 ± 0.58 20.97 ± 5.88
CuSO4 14.73 ± 2.49 2.28 ± 0.53 17.01 ± 3.02
 S. Ilyas et al. / Journal of Proteomics 141 (2016) 47–56 51

Fig. 2. Effect of heavy metals on glutathione and NPSH in R. mucilaginosa: (A) GSH/GSSG ratio; (B) specific activity of cysteine and NPSH. Cells were exposed to (100 mg/L) metals in MSM
and values were expressed as mean of ±S.D. All experiments were performed in triplicate (*p b 0.05; **p b 0.1 when compared with controls).

yeast cells [42]. Both metals also increased SOD activity (Fig. 3D).
R. mucilaginosa showed increased rates of SOD and CAT on exposure
of elevated Cu (II) concentrations [33]. It is unclear why Cd should
show significantly stronger induction of antioxidant enzymes than As
which led us to hypothesize that As may be detoxified by an alternative
route to Cd such as enzymatic oxidation [27].
The appearance of brownish precipitates on culture plates treated
with AgNO3 (Fig. 4A) is consistent with the presence of arsenate, an in-
dication of arsenite oxidation [43]. KMnO4 assay revealed a pink colour
also suggesting arsenite oxidation. As cannot be destroyed once it has
entered the environment [44]. Arsenite toxicity strongly disrupts pro-
tein -SH groups and interferes with enzyme function whereas As, a
phosphate analogue, can interfere with phosphate uptake and transport
systems. Analysis of relative arsenite and As concentrations in metal-
contaminated cultures revealed that the concentration of arsenite de-
creased while that of As increased pro rata across a 3.5 h time-course
of metal exposure (Fig. 4B). This suggests strongly that arsenite disap-
pearance is due to enzymatic oxidation by arsenite oxidase (ARO).

Fig. 3. Effects of CdCl2 and NaAsO2 on antioxidant enzymes of R. mucilaginosa: (A) GST; (B) GR; (C) CAT; (D) SOD. At least three independent assays were analyzed by Student's t-test and
values are expressed as means ± S.D. (*p b 0.05; **p b 0.001 compared to controls).
52 S. Ilyas et al. / Journal of Proteomics 141 (2016) 47–56

Microorganisms have evolved mechanisms for coping with As toxic-

ity either through ARO activity or by arsenite peroxidation with mem-
brane lipids. On As exposure, ARO activity was increased by 50%
(membrane fraction) and 25% (soluble fraction) over control yeast cul-
tures suggesting that ARO is found predominantly in the membrane
fraction of the yeast cell, whilst modest activity remained in the soluble
fraction (Fig. 4C). It would seem that ARO increases R. mucilaginosa sur-
vival when exposed to As.

3.5. Metal uptake potential by R. mucilaginosa

Cd and As uptakes were determined over a 12-day exposure.

R. mucilaginosa cells continuously accumulated 66 mg/g Cd whereas in-
tracellular As uptake was 65 mg/g at day 12 (Fig. 5A, B). As interaction
with cell surface (26.2 mg/g) was observed in live R. mucilaginosa cells
as compared to Cd (22.08 mg/g). These results clearly indicate that
red yeast has a high affinity for metals and that the uptake mechanisms
may involve metabolism-dependent processes. The minimum and max-
imum removal potential for Cd was 33% and 88% after 2 and 12 days of
incubation, respectively (Fig. 6A). Maximum removal was 91% from the
medium after 12 days (Fig. 6A) under As challenge indicating that the
yeast strain was also capable of As accumulation. Maximum Cd absorp-
tion previously reported for Rhodotorula sp. Y11 cells was 19.38 mg/g
[4]. High Cd and As adsorption by R. mucilaginosa indicates the possible
presence of significant metal binding sites on the cell wall as well as a
potential use for this yeast as a bio-sorbent to remove heavy metal
ions from industrial wastewaters (Figs. 5, 6). Accumulation of metals
by cell wall components usually involves two distinct phases; a
metabolism-independent process, involving metal adsorption around
the cell envelope and metabolism-dependent processes [6]. Yeasts can
accumulate higher concentrations of heavy metal ions by bioaccumula-
tion processes rather than by biosorption. Their enzymes can metabo-
lize toxic heavy metal ions through their metabolic pathways [45].
Schizophyllum commune, Pycnoporus sanguineus, Penicillium sp.,
Pleurotus pulmonarius, Pichia stipitis, Rhizopus arrhizus, Trichoderma
viride and R. mucilaginosa showed Cu adsorption capacities of 1.52,
2.76, 6.2, 15.08, 15.85, 19.0, 19.6 and 26.2 mg/g respectively [46–48].
Fig. 4. Arsenic uptake by R. mucilaginosa: (A) Appearance of brownish precipitates on
culture plates after the application of 0.1 M AgNO3 solution; (B) estimation of arsenite
(○) and arsenate (▲) concentrations in R. mucilaginosa across treatment time course;
3.6. Proteomic analysis by one and two dimensional gel electrophoresis
(C) estimation of arsenite oxidase (Aro) activity. (*p b 0.05).
Fluorescence intensity and band density of proteins prior to and
after staining of 1-DE gels were calculated using PD Quest imageQuant
software (Bio-Rad). A strong reduction in total protein thiol content

Fig. 5. Uptake of metals from culture media by R. mucilaginosa: Levels of adsorption and uptake over 12 days were estimated for (A) cadmium; (B) arsenic.
 S. Ilyas et al. / Journal of Proteomics 141 (2016) 47–56 53

Fig. 6. Removal of metals from culture media by R. mucilaginosa: Supernatants were sampled at regular intervals at 2–12 days of incubation (controls contained relevant heavy metal ions
but did not contain yeast cells) and percentage removal calculated at each time-point. (A) Cadmium; (B) arsenic.

was revealed on NaAsO2treatment as compared to CdCl2 implying sig-
nificant protein oxidation by the former metal (Fig. 7).
For 2-DE, IAF and colloidal Coomassie-stained gel images were ac-
quired and analyzed by SameSpots software. A total of 1490 spots (ei-
ther on protein staining or by IAF-associated fluorescence) were
evident (Figs. 8, 9). Seventeen spots judged by the software to have sig-
nificantly altered intensities were selected and excised for in-gel tryptic
digestion. Of these, 7 spots gave successful protein identifications by
MALDI-TOF/TOF mass spectrometry (MS) analysis (Table 3). Spot 6
was identified as putative chitin xylose reductase (XR), and matched
well with Rhodotorula sp. In xylose-utilizing fungi, XR reduces xylose
to xylitol using NADH or NADPH as a cofactor, which is then oxidized
to xylulose by xylitol dehydrogenase. Xylose metabolism and ethanol
consumption begin after glucose is exhausted as a carbon-source. The
GRE3 gene which encodes XR is up-regulated under stress conditions,
such as osmotic and oxidative stress, high temperature, and carbon star-
vation [49,50]. XR may have a role in detoxification of methylglyoxal
synthesized in response to stress [50]. The remaining four spots were
identified based on sequence similarity to non-Rhodotorula species.
Spot 1 (see arrow in Fig. 9) matched to Burkholderia xenovorans F0F1
ATP synthase alpha subunit and this proteobacterium is under investi-
gation for its bioremediation potential [51]. The other proteins

Fig. 7. 1-DE analysis of IAF-labeled proteins extracted from R. mucilaginosa: (A) Fluorescence scan prior to staining. Control; lanes 1–4. Cadmium-treated; lanes 5–8. Arsenic-treated, lanes
9–12. (B) IAF-labeled protein fluorescence ratio was calculated from scans of electrophoretic separations quantified by Image-Quant software analysis (*p b 0.05).
54 S. Ilyas et al. / Journal of Proteomics 141 (2016) 47–56

CdCl2 (%change) NaAsO2 (% change)

5.42E+005
6.44E+05

1.17E+05

4.76E+04

5.42E+05

3.52E+04

6.11E+05
(−34)

(−25)

(+52)

(+48)

(−53)

(−15)

(+67)
Average normalized volumes

4.619E−005 3.66E+005 7.34E+005

1.12E+06

1.89E+05

3.82E+04

7.34E+05

3.40E+04

9.65E+05
(+100)

(+100)
(+14)

(+22)

(+22)

(−55)

(+34)
9.80E+05

1.55E+05

3.14E+04

3.66E+05

7.48E+04

7.20E+05
Control

4.62E−05
p-Value

0.004

0.026

0.025

0.037

0.02
Fig. 8. Two dimensional electrophoresis thiol proteome analysis of R. mucilaginosa: Gels
were stained with colloidal Coomassie Brilliant Blue R-250, images scanned with
densitometer GS-800 and analyzed by Progenesis Samespots software. A representative

Number of

identified
gel for Cd exposure is shown. Location of spots 5 and 7 are indicated by arrows.

peptides

5.68 21

29

9.65 10

6.08 16

21
7

7
identified were a 20S proteasome subunit (spot 3) matched to a mould

4.83
6.0

6.0

6.0
pI
Wallemia sebi which has been occasionally reported to cause subcutane-
ous infections in humans [52] and the eukaryotic translation elongation

Sequence
ion confidence interval coverage
factor 2 (spot 2) which is frequently implicated in oxidative stress [53].

(%)
A hypothetical protein (spot 4) was also identified and its expression

8
30

32

32

31

28
confirmed at the protein level. Conserved domain database (CDD) anal-
ysis suggested that this protein was most likely involved in polysaccha-
Total ion score/total

ride biosynthesis. This family of proteins plays an important role in

xylan biosynthesis in plant cell walls although its role in xylan biosyn-
thesis and its functioning in other organisms remain largely unstudied.
105/100

222/100

125/100

106/100

716/100
72/99

87/96
Lee et al. [54] previously reported an extracellular acetylxylan esterase
produced by R. mucilaginosa that was relatively nonspecific for polysac-
charide or an aliphatic alcohol.
confidence interval (%)

Spots 5 and 7 were found to vary both in protein abundance and in

Protein score/protein

terms of apparent thiol oxidation. They both matched well with acces-
sions from Rhodotorula sp. (Table 3). Spot 5 was identified as a putative
chitin deacetylase which converts chitin to chitosan (the de-acetylated
55,903 148/100

93,362 261/100

26,622 131/100

64,489 108/100

35,669 745/100

56/100
87/96

form of chitin). Chitosan is a key component of this fungus's cell wall

gb|EGU132791 122,129
17,128
(Da)
Mw

gi|395334437

gi|388580573

gi|367015424

gi|342319101

gi|294991932
gi|91785735
Accession
Identification of R. mucilaginosa proteins by MALDI-TOF/TOF MS.

number

Wallemia sebi CBS

xenovorans LB400

LYAD-421 SS1

glutinis ATCC

glutinis ATCC
mucilaginosa
Burkholderia

Rhodotorula

Rhodotorula

Rhodotorula
Torulaspora
Dichomitus

delbrueckii
Organism

squalens

204091

204091
633.66
Eukaryotic translation

Hypothetical protein
elongation factor 2

Valine-tRNA ligase
F0F1 ATP synthase
Identified protein

Xylose reductase
20S proteasome

TDEL_0F01890
Putative chitin
subunit alpha

deacetylase
subunit

Fig. 9. 2-DE analysis of proteins from R. mucilaginosa stained with colloidal Coomassie: A
representative gel for CdCl2 exposure is shown. Spots excised for identification are
Table 3

Spot
no.

indicated by arrows. Circles represent location of spots 5 and 7 which showed variation
1

both in protein expression and in thiol oxidation.

 S. Ilyas et al. / Journal of Proteomics 141 (2016) 47–56
and contributes to its strength and integrity. Chitin has also previously
been reported in Rhodotorula sp. Y11 as the principal cellular compo-
nent responsible for absorbing Cd [55] and acetylation of biomass
from this yeast resulted in a 63% increase in Cd uptake capacity, greater
than that observed for succinylation, modification with ethanolamine or
by esterification [5]. It is therefore likely that down-regulation of chitin
deacetylase would maintain levels of (acetylated) chitin in the
R. mucilaginosa cell wall, thus contributing to Cd uptake. Spot 7 was
identified as valine-tRNA ligase, a class I aminoacyl-tRNA synthetase
[56]. This protein has previously been reported as an oxidation target
in adipocytes [57]. Even though its abundance increased in response
to both CdCl2 (+100%) and NaAsO2 (+67%) (Table 3), a significant de-
crease in IAF fluorescence signal in response to both metals suggested
this protein was selectively oxidized. Aminoacyl-tRNA synthetases are
quite susceptible to oxidation as has previously been reported for Can-
dida albicans [58].
4. Conclusion
This proteomic study explored the biochemical basis of metal-
tolerance in an environmental isolate of R. mucilaginosa. A variety of im-
portant biochemical parameters such as activity of antioxidant en-
zymes, status of GSH, GSSG and protein thiols were found to be
affected by metal exposure. A more detailed analysis of tolerance to ar-
senic and cadmium pointed to a particular role for ASO in arsenic toler-
ance. This is the first time this has been reported in R. mucilaginosa, and
suggests that this isolate may have potential in biosorption of these
metals in the environment. A proteomic analysis revealed that seven
proteins with a variety of roles - ATP synthesis, protein degradation/
synthesis, and metabolism of xylose and chitin - were differentially af-
fected by metal exposure in a manner consistent with oxidative stress.
These may therefore represent a protein expression signature for expo-
sure to cadmium and arsenic.
Conflict of interest
We confirm there is no conflict of interest in this work.
Acknowledgements
SI is grateful to the Higher Education Council of Pakistan (1-/HEC/
HRD/2012/2305) for travel support to DS's laboratory and for support
to perform this research. We confirm there is no conflict of interest in
this work.
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