Journal of Proteomics 148 (2016) 12–19

Contents lists available at ScienceDirect

Journal of Proteomics
journal homepage: www.elsevier.com/locate/jprot

Global iTRAQ-based proteomic profiling of Toxoplasma gondii oocysts
during sporulation
Chun-Xue Zhou a,b, Xing-Quan Zhu b, Hany M. Elsheikha c, Shuai He b,d, Qian Li e, Dong-Hui Zhou b,⁎, Xun Suo a,⁎
a

National Animal Protozoa Laboratory and College of Veterinary Medicine, China Agricultural University, Beijing 100193, PR China
State Key Laboratory of Veterinary Etiological Biology, Key Laboratory of Veterinary Parasitology of Gansu Province, Lanzhou Veterinary Research Institute, Chinese Academy of Agricultural
Sciences, Lanzhou, Gansu Province 730046, PR China
c
Faculty of Medicine and Health Sciences, School of Veterinary Medicine and Science, University of Nottingham, Sutton Bonington Campus, Loughborough, LE12 5RD, UK
d
College of Animal Science and Technology, Anhui Agricultural University, Hefei, Anhui Province 230036, PR China
e
School of Basic Medicine, Dali University, Dali, Yunnan Province 671000, PR China
b

a r t i c l e

i n f o

Article history:
Received 19 March 2016
Received in revised form 15 June 2016
Accepted 11 July 2016
Available online 12 July 2016
Keywords:
Toxoplasma gondii
Oocyst
Sporulation
iTRAQ
Proteomics

a b s t r a c t
Toxoplasma gondii is a medically and economically important protozoan parasite. However, the molecular mechanisms of its sporulation remain largely unknown. Here, we applied iTRAQ coupled with 2D LC–MS/MS proteomic analysis to investigate the proteomic expression profile of T. gondii oocysts during sporulation. Of the 2095
non-redundant proteins identified, 587 were identified as differentially expressed proteins (DEPs). Based on
Gene Ontology enrichment and KEGG pathway analyses the majority of these DEPs were found related to the metabolism of amino acids, carbon and energy. Protein interaction network analysis generated by STRING identified
ATP-citrate lyase (ACL), GMP synthase, IMP dehydrogenase (IMPDH), poly (ADP-ribose) glycohydrolase (PARG),
and bifunctional dihydrofolate reductase-thymidylate synthase (DHFR-TS) as the top five hubs. We also identified 25 parasite virulence factors that were expressed at relatively high levels in sporulated oocysts compared
to non-sporulated oocysts, which might contribute to the infectivity of mature oocysts. Considering the importance of oocysts in the dissemination of toxoplasmosis these findings may help in the search of protein targets
with a key role in infectiousness and ecological success of oocysts, creating new opportunities for the development of better means for disease prevention.
Biological significance: The development of new preventative interventions against T. gondii infection relies on an
improved understanding of the proteome and chemical pathways of this parasite. To identify proteins required
for the development of environmentally resistant and infective T. gondii oocysts, we compared the proteome
of non-sporulated (immature) oocysts with the proteome of sporulated (mature, infective) oocysts. iTRAQ 2DLC-MS/MS analysis revealed proteomic changes that distinguish non-sporulated from sporulated oocysts.
Many of the differentially expressed proteins were involved in metabolic pathways and 25 virulence factors
were identified upregulated in the sporulated oocysts. This work provides the first quantitative characterization
of the proteomic variations that occur in T. gondii oocyst stage during sporulation.
© 2016 Published by Elsevier B.V.

1. Introduction
The apicomplexan protozoan parasite Toxoplasma gondii is the causative agent of one of the most medically important parasitic zoonoses
worldwide [1]. T. gondii can infect all warm-blooded animals and
humans as the intermediate host and felids as the definitive host [2].
This parasite has a considerable public health impact because it can
cause hydrocephaly, retinochoroiditis, mental retardation, and even
death in developing fetus as well as life-threatening encephalitis in immunocompromised individuals [3,4]. Waterborne transmission,
foodborne transmission and congenital (transplacental) infection are
⁎ Corresponding authors.
E-mail addresses: zhoudonghui@caas.cn (D.-H. Zhou), suoxun@cau.edu.cn (X. Suo).

http://dx.doi.org/10.1016/j.jprot.2016.07.010
1874-3919/© 2016 Published by Elsevier B.V.

the common means of T. gondii acquisition [5]. Historically, the consumption of raw or undercooked meat containing T. gondii tissue cysts
has been deemed as the major route for spread of T. gondii infection in
humans [6]. However, the role of the environmental stage of T. gondii
(oocyst) as an important source of human and animal infection cannot
be underestimated [7,8].
T. gondii has a complicated life cycle that encompasses both asexual
(takes place in any warm-blooded vertebrate animal) and sexual (occurs only in felids) reproductive cycles. The sexual cycle of T. gondii occurs exclusively in the enteroepithelial cells of the intestine of its
definitive felid host and culminates in the production of oocyst, which
is well-adapted to its adverse environmental habitats [9]. T. gondii oocysts are shed non-sporulated (non-infective) in cat feces and in a few
days they undergo an aerobic sporulation process in the environment

C.-X. Zhou et al. / Journal of Proteomics 148 (2016) 12–19

with appropriate climatic conditions and become infective sporulated
oocysts. Oocyst-related infections in warm-blooded animals and
humans have been increasingly reported worldwide [10–13] and can
lead to deleterious ocular and neurological complications and potentially death in immunocompromised individuals [14].
Considering the importance of T. gondii oocysts, some progress has
been made towards the understanding of the basic structures [15], mechanics [16] and biological processes of oocysts [17,18]. However, little
is known about the molecular mechanisms underlying sporulation
and the remarkable ability of T. gondii oocyst to infect a wide range of intermediate vertebrate hosts. Because the infectiousness of oocysts is
largely determined by events occurring during its development, understanding protein regulation during oocyst sporulation is of fundamental
importance for the understanding of the parasite infection biology and
for the identification of new therapeutic targets.
In this study we tested the hypothesis that proteins whose expression changes during oocyst sporulation are essential for the development of this environmental stage of T. gondii life cycle. We employed,
for the first time, isobaric tag for relative and absolute quantitation
(iTRAQ) coupled with two-dimensional liquid chromatography–tandem mass spectrometry to analyze proteomic changes in T. gondii oocysts during sporulation. This method can provide a more accurate
identification and quantification of proteins than low-throughput proteomic methods, such as traditional two-dimensional gel-based approaches [19]. Our goals were to identify proteins that were
differentially expressed in oocysts during sporulation and to elucidate
the affected pathways and processes that operate during the development of T. gondii oocysts. Our results identified 587 differentially
expressed proteins (DEPs), the majority of which are involved in metabolic activities.

13

onto a discontinuous CsCl gradient (specific gravity from 1.05 to
1.125), and centrifuged at 12,000g for 60 min at 4 °C with no brake. Oocysts were collected from the opaque-to-white layer (between 1.05 and
1.11 layers) and washed twice with 30–40 ml 0.85% saline. The final pellet was re-suspended in 1× phosphate buffered saline (PBS). For sporulation, the sample was centrifuged at 350g and pellet was mixed with 2%
H2SO4 and aerated on a shaker for 7 days at ambient temperature. Finally, the sporulated oocysts were washed twice in 0.85% saline and
suspended in 2% H2SO4 at 4 °C for storage until use.
2.3. Protein preparation and iTRAQ labeling
Protein was extracted from an equal number (~107) of sporulated and
non-sporulated oocysts as follows. Oocysts were centrifuged at 350g for
15 min. The pellet was re-suspended in radioimmunoprecipitation assay
(RIPA) lysis buffer (20 mM Tris-HCl pH 7.5, 150 mM sodium chloride,
2 mM EDTA, 1% DOC, 1% Triton X-100) supplemented with protease inhibitors (Roche), briefly sonicated and centrifuged at 10,625g for 20 min
at 4 °C, and the supernatants were analyzed as total lysates. Total protein
concentration was determined using the QuantiPro™ BCA Assay Kit
(Sigma) according to the manufacturer's instructions. Bovine serum albumin (BSA) was used as a standard.
iTRAQ tagging and analysis was performed as described previously
[23]. Briefly, 100 μg proteins from each sample were reduced, alkylated,
digested with trypsin (Promega). The digested peptides were then dried
and reconstituted in 50 μl 0.5 M triethyl ammonium bicarbonate
(TEAB). The dried peptides were labeled in line with the iTRAQ recommended protocol (AB Sciex, Foster City, Calif, USA). Non-sporulated oocysts were labeled with 113 and 114 while sporulated oocysts with 115
and 116 iTRAQ reagents. The labeled samples were finally mixed in a
single vial and dried with a rotary vacuum concentrator.

2. Materials and methods
2.4. Two-dimensional liquid chromatography and MS/MS analysis
2.1. Ethics statement
All animal procedures were approved by Animal Ethics Committee
of Lanzhou Veterinary Research Institute, Chinese Academy of Agricultural Sciences (Permit No. LVRIAEC2014-002). All experiments were
performed in accordance with the requirements of the Animal Ethics
Procedures and Guidelines of the People's Republic of China. All efforts
have been made to alleviate suffering and minimize the numbers of animals used in this study.
2.2. Oocyst production and purification
Mouse infection was performed according to previously described
method [20]. Briefly, T. gondii type II Prugniuad (Pru) strain was maintained via oral inoculation of T. gondii cysts in BALB/c mice. Following
general anesthesia, mice brains were removed and homogenized in a
sterile tissue homogenizer. After verification of the cyst status, via histological examination, a 10-week-old specific-pathogen-free kitten was
fed with freshly prepared cysts. The modified agglutination (MAT) test
was utilized to confirm the seronegtive status of the kitten prior to infection [21].
Feces were collected daily and examined with a light microscope
(Olympus, Japan). All procedures were conducted following strict safety
measures. Once oocysts are detected they were purified using sucrose
suspension followed by a caesium chloride (CsCl) centrifugation [22].
Briefly, fecal samples were mixed with water and shaken on a shaker
until they were completely broken. The mixture was filtered through
a 250 μm mesh and centrifuged at 60g for 15 min, and then the supernatant was discarded. This step was repeated three times and the final supernatant was decanted, and the sediment was mixed with 5 volumes
of sucrose solution (specific gravity 1.15) and centrifuged for 10 min
at 350 g. Oocysts were collected from the supernatant and suspended
in TE buffer. Approximately 10 ml of oocyst suspension was overlaid

2D liquid chromatography was carried out according to previously
reported procedure with slight modifications [24]. Briefly, strong cation
exchange (SCX) fractionation was performed with a high performance
liquid chromatography (HPLC) system (Phenomenex columns; Gemini-NX 3u C18 110 A; 150 × 2.00 mm). Peptides were separated by a linear gradient formed by mobile phase A (20 mM HCOONH4, pH = 10)
and mobile phase B (20 mM HCOONH4,80% acetonitrile (ACN),pH =
10). The UV detector was set at 214/280 nm, and fractions were collected every 1 min. In total, 24 fractions were collected, acidified with 50%
trifluoroacetic acid (TFA) and dried by vacuum centrifuge. Peptides
were than eluted using 65 min gradient of buffer A (0.1% formic acid)
to buffer B (80% ACN containing 0.1% formic acid) at 300 nl/min. The
MS/MS analysis was performed on Q Exactive system (Thermo Scientific) in information dependent acquisition mode. MS spectra were acquired across the scan range of 350–1800 m/z in a resolution of
70,000 using maximum injection time (40 ms) per spectrum. Twenty
most intense precursors per MS cycle were selected for fragmentation
detected with 60 ms maximum injection time. Tandem mass spectra
were recorded in a resolution of 17,500 with rolling collision energy
on and iTRAQ reagent collision energy adjustment on. For accurate
mass measurements, the lock mass option was enabled.
2.5. Protein identification and quantitation
Protein identification and quantification were performed with
ProteinPilot™ Software 4.5 (AB SCIEX) using the Paragon™ algorithm
(4.5.0.0, 1654) for the peptide identification, which was further processed by Pro GroupTM algorithm where isoform-specific quantification
was adopted to trace the differences between expressions of various
isoforms. Some search parameters were listed as follows: (1) Detected
Protein Threshold: 0.05; (2) Competitor Error Margin: 2.00; (3) Revision Number: 1656; (4) Instrument: Orbi MS (1-3 ppm), Orbi MS/MS;

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C.-X. Zhou et al. / Journal of Proteomics 148 (2016) 12–19

(5) Sample Type: iTRAQ 8 plex (Peptide Labeled); (6) Cysteine Alkylation: MMTS; (7) Digestion: Trypsin; (8) Special Factors: None; (9) ID
Focus: Biological modification; (10) Search Effort: Thorough ID; (11)
FDR Analysis: Yes; (12) User Modified Parameter Files: No. The required
MS/MS spectra were searched against ToxoDB13_T. gondii ME49.fasta
(http://www.toxodb.org/common/downloads/release-10.0/T. gondii
ME49 /fasta/data/) and the total number of protein sequences recorded
in this database was 8322. Unique protein with at least one unique peptide was identified and FDR was set to b0.01 for the identification of
both peptides and proteins. Peptide confidence level of 95% or unused
confidence score N 1.3 was used as the qualification criteria. Relative
quantification of proteins was calculated based on the ratio of peak
areas from MS/MS spectra. To designate significant changes in protein
expression, log2 fold change ≥ 1was deemed as up-regulated and log2
fold change ≤ −1 classified as downregulated.

3. Results

2.6. Gene ontology and KEGG pathway enrichment analysis

3.1. Protein identification and quantification

To better understand the biological functions of significantly altered
proteins, these proteins are analyzed using web-based GO software
(http://www.geneontology.org) for gene ontology (GO) annotation
and enrichment analysis [25]. The GO project includes three main modules: biological process, cellular component and molecular function.
Pathway analysis was done using the web-based Kyoto Encyclopedia
of Genes and Genomes (KEGG, http://www.genome.p/kegg) [26]. Hierarchical clustering was performed using Cluster 3.0, and the results
were presented using java Tree view [27]. Protein-protein interaction
networks were built based the publicly available program, the Search
Tool for the Retrieval of Interacting Genes/Proteins (STRING) database
[28]. Each interaction has a combined score (edge score), which represents the reliability of the interaction between proteins. The PPI interactions with a combined score (0: lowest confidence; 1: highest
confidence) larger than 0.7 were used for further network analysis. To
visualize the interaction networks, Cytoscape tool was utilized [29].

To understand how the proteome changes with T. gondii oocyst development, we utilized an iTRAQ-based quantitative proteomic approach. Oocyst morphological changes during sporulation process are
shown in Fig. S1. Global proteomic analysis detected a total of 2095 proteins, each has at least one peptide identified with a minimum unused
score of ≥ 1.3, which indicates N95% confidence in correct sequence
identification (Table S2). About 89.3% of these proteins had a coefficient
of variation (CV) of b50% among replicates (Fig. 1A). The following analysis is based on proteins with a CV value b50%. Fig. 1B shows a hierarchical clustering analysis of all experiments (including 2 biological
replicates, each containing 2 technical replicates) and includes 1823
proteins with a CV value b50%. These proteins were then assigned to
functional cluster of orthologous group (COG) categories. As shown in
Fig. 2, about 69% of the proteins were covered and divided into 25 categories. “Translation, ribosomal structure and biogenesis”, “General function prediction only”, “Posttranslational modification, protein turnover,
chaperones”, and “Signal transduction mechanisms” are the four categories with the most identified proteins. In total, 587 proteins were
identified as differentially expressed proteins (DEPs) (| log2 fold
change | N 1 and P b 0.05). Among these DEPs, 208, 120, 119, and 28
proteins reproducibly produced N1, 2, 3, and 4 log2 fold changes,

2.7. qPCR analysis
Quantitative PCR (qPCR) was employed to compare gene expression
in non-sporulated and sporulated T. gondii oocysts. Total RNA was

extracted from non-sporulated and sporulated samples using TRIzol
method according to the manufacturer's recommendations (Invitrogen).
DNase-treated RNA was reverse transcribed using a reverse transcription
kit GoScriptTM Reverse Transcription System (Promega). The reactions
were performed on QIAGEN's real-time PCR cycler, the Rotor-Gene Q
using SYBR Green GoTaq® qPCR Master Mix (Promega). The amplification reactions were performed with the following conditions: 2 min at
95 °C, 40 cycles of 95 °C for 15 s, 55 °C for 30 s, 72 °C for 30 s. The T. gondii
glyceraldehyde-3-phosphatedehydrogenase gene (GAPDH) was used as
an endogenous control to normalize all qPCR data [30]. Primers were designed and synthesized by Takara Bio company and sequences of primer
sets used for qPCR are included in Table S1. The relative expression was
calculated using the 2−ΔΔCT method [31].

Fig. 1. iTRAQ coupled with LC-MS/MS analysis of sporulated and non-sporulated oocysts. (A) Determination of experimental variation using all detected proteins common in all biological
replicates. The cumulative percentage (red line) is defined as the cumulative number of proteins falling within the defined variation range against the total number of protein. The x-axis
represents % coefficient of variation (CV) of the same protein from different biological replicate samples. The left vertical axis represents the number of proteins. The right vertical axis
represents the cumulative % of the counted proteins (red line). (B) Hierarchical clustering of proteins falling in b50% variation range. TR1 and TR2 indicate technical replicates 1 and 2,
respectively. Each technical replicate includes two biological replicates. (C) Number of proteins detected up and downregulated between sporulated and non-sporulated oocysts samples.

C.-X. Zhou et al. / Journal of Proteomics 148 (2016) 12–19

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Fig. 2. Cluster of Orthologous Groups (COG) analysis of the identified proteins in T. gondii oocysts. The horizontal axis represents the number of proteins. The vertical axis represents
different COG classes. A, RNA processing and modification; B, chromatin structure and dynamics; C, energy production and conversion, D, cell cycle control, cell division, chromosome
partitioning; E, amino acid transport and metabolism; F, nucleotide transport and metabolism; G, carbohydrate transport and metabolism; H, coenzyme transport and metabolism; I,
lipid transport and metabolism; J, translation, ribosomal structure and biogenesis; K, transcription; L, replication, recombination and repair; M, cell wall/membrane/envelope
biogenesis; N, cell motility; O, posttranslational modification, protein turnover, chaperones; P, inorganic ion transport and metabolism; Q, secondary metabolites biosynthesis,
transport and catabolism; R, general function prediction only; S, function unknown; T, signal transduction mechanisms; U, intracellular trafficking, secretion, and vesicular transport;
V, defense mechanisms; W, extracellular structures; X, mobilome: prophages, transposons; Y, nuclear structure; Z, cytoskeleton.

respectively. In contrast, the expression levels of 94, 17 and 1 protein
decreased by N1, 2 and 3 log2 fold changes, respectively (Fig. 1C).
3.2. GO annotation and KEGG analysis of DEPs
To obtain a comprehensive view of DEPs, we carried out the GO analysis using GOseq R package [32] and obtained a total of 81 GO terms, including 34 biological process terms, 16 cellular component terms and
31 molecular function terms. The top 10 enriched GO terms within
each major functional category are shown in Fig. 3. The top four most
enriched GO terms under “biological process” included “biosynthetic
process”, “cellular protein modification process”, “cellular nitrogen
compound metabolic process”, and “small molecule metabolic process”
(Fig. 3A). We noted that half of the top ten enriched GO terms were related to metabolic activity, including “cellular nitrogen compound metabolic process”, “small molecule metabolic process”, “carbohydrate

metabolic process”, “cellular amino acid metabolic process”, and “cofactor metabolic process”. An analysis of identified DEPs under “cellular
component” allowed the differentiation of 16 different sub-categories
and top 10 categories are shown in Fig. 3B. “Protein complex” had the
most DEPs, followed by “intracellular” and “cytoplasm”. In the molecular function category, “ion binding”, “oxidoreductase activity” and “kinase activity” were the most prominent categories (Fig. 3C).
Interestingly, the “ion binding” term included 110 DEPs, which makes
it the most enriched GO term among other sub-categories under the
three major functional categories.
DEGs were then mapped to the reference pathway in the KEGG database in order to identify the parasite biological pathway operating
during the sporulation process. Among these 587 identified DEPs, only
51 DEPs (19 down-regulated and 32 up-regulated) had a KEGG
Orthology (KO) ID and were involved in 50 pathways. The top 12
enriched pathways shown in Fig. 3D are almost all closely related to

Fig. 3. GO annotation and KEGG pathway analysis of the DEPs. (A) Top 10 enriched GO terms under “biological process”. (B) Top 10 enriched GO terms under “cellular components”. (C)
Top 10 enriched GO terms “molecular function”. (D) Top 12 enriched KEGG pathways. Up, number of upregulated proteins; down, number of downregulated proteins.

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Fig. 4. Differentially expressed virulence factors identified between sporulated and non-sporulated oocysts. TR1 and TR2 indicate technical replicates 1 and 2, respectively.

metabolic activities except “phagosome”, “protein processing in endoplasmic reticulum” and “toxoplasmosis”. TGME49_301120 (acetyil
CoA acetyltransferase/thiolase) is involved in the most KEGG pathways,
followed by TGME49_227090 (aspartate kinase), TGME49_221320
(acetyl-CoA carboxylase ACC1), TGME49_264030 (aminotransferase),
TGME49_318310 (transketolase), and TGME49_318230 (phosphoglycerate kinase PGKI).
T. gondii virulence is dependent on a number of factors that affect the
severity of the disease [33,34]. By literature retrieval and analysis, virulence factors that are differentially expressed in this study are shown in
Fig. 4. All these virulence factors were found upregulated in the sporulated oocysts in our study, which might contribute to the infectivity of the
mature oocyst. Sporozoite proteins, thrombospondin repeats (SPATR),
rhoptry protein 18 (ROP18) and microneme protein 1 (MIC1), were the
top three most differentially expressed virulence factors.
3.3. Protein–protein interaction analysis of DEPs
The protein-protein interaction (PPI) network of the DEPs identified
in our study was analyzed using STRING 10 [35]. By removing

unconnected proteins and self-loops, the resulting PPI network
contained 156 protein nodes and 410 edges (Fig. 5). Proteins with
higher connectivity than others in the network are referred to as
“hubs”. These hubs may play crucial role in the regulation of network.
PPI analysis revealed the following molecular hubs: ATP-citrate lyase
(ACL), GMP synthase, IMP dehydrogenase (IMPDH), poly (ADP-ribose)
glycohydrolase (PARG), and bifunctional dihydrofolate reductasethymidylate synthase (DHFR-TS). The expressions of genes encoding
these five hub proteins were verified by qPCR and were found consistent with the proteome data (Fig. 6).
Next, we presented the predicted modes of actions between hubs
and their associated DEPs (Fig. 7). Edge actions, including activation,
binding, catalysis, expression, inhibition and post-translational modification (ptmod), were predicted based on the computational integration
of relevant datasets in T. gondii or from other organisms, which provided
probabilistic-based directionality for each protein-protein relationship.
In Fig. 7A, DHFR-TS interacts with proliferating cell nuclear antigen
(PCNA2) and ribonucleoside-diphosphate reductase small subunit
(RnrS) in the reaction mode with action score of 0.767 and 0.720, respectively. Acetyl-CoA carboxylase, containing two main isoforms

Fig. 5. The protein-protein interaction network generated with STRING and visualized with Cytoscape for DEPs. The PPI network with a high combined score N 0.7 was prepared using the
STRING 10 database program. DEPs are represented as round nodes. The red node indicates upregulation or green node indicates downregulation of the DEPs. The node size indicates high
interaction degree (large) or low degree (small). Proteins that are associated to each other are linked by an edge. The color of the edge indicates the combined interaction score (edge
score).

C.-X. Zhou et al. / Journal of Proteomics 148 (2016) 12–19

17

Fig. 6. Relative abundance of the top 5 hub DEPs determined by qPCR and iTRAQ analysis, respectively. X-axis indicates different DEPs and Y-axis indicates log2 fold change values.

ACC1 and ACC2, is a biotin-dependent enzyme that functions in the production of malonyl-CoA. As shown in Fig. 7B, IMPDH interacts with ACC
two isoforms in multiple ways, including activation, inhibition, catalysis
and ptmod. ACL is a key lipogenic enzyme that converts citrate in the cytoplasm to acetyl-CoA, the precursor to yield malonyl-CoA for fatty acid
biosynthesis. As shown in Fig. 7C, it engages with cell-cycle-associated
protein kinase GSK in expression mode and interacts with ATP synthase
beta subunit (ATP-B) and ATPase synthase subunit alpha in reaction
mode. Besides the three hubs, GMP synthase was found to interact
with UV excision repair protein RAD23 in the binding mode, which
was previously validated by an affinity capture-MS [36].

4. Discussion
Food and water contaminated with T. gondii oocysts from infected
cat feces has been considered as the main source of infection dissemination [8,9]. Hence, understanding differential expression of proteins during oocyst sporulation is of considerable importance for the elucidation
of the remarkable capability of T. gondii to adapt to and establish infection in a broad range of intermediate hosts. Despite the importance of
this significant biological phenomenon literature on proteomic expression profiling in T. gondii oocysts during sporulation is limited. Previous
research utilized one-dimensional gel electrophoresis coupled with LC-

Fig. 7. Modes of action predicted between hub DEPs and their directly interacted DEPs. Modes of action include “binding”, “reaction”, “post-translational modification (ptmod)”, “catalysis”,
“inhibition”, “activation” and “expression”, and each of these actions is shown in different shapes. Node color is green if the protein expression is decreased and red if increased. The color of
the edge indicates the combined interaction score (edge score).

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MS/MS identified 1247 non-redundant proteins in sporulated oocyst,
and some of these proteins were related to environmental resistance
[37]. Using the same method, Possenti and colleagues identified 1304
non-redundant proteins, of which 154 proteins were unique to oocyst/sporozoite compared to the proteome of T. gondii tachyzoite stage
[38].
In the present study, we used an iTRAQ-based proteomic approach
to quantitatively profile T. gondii oocyst proteins during sporulation in
order to reveal the central metabolic proteins involved in oocyst maturation. The iTRAQ coupled to 2D-LC–MS/MS analysis allowed 2095 proteins to be identified, of which 587 (28%) were differentially expressed
(DEPs). To understand how the expression of these proteins changed in
response to sporulation, they were divided into two categories: metabolism-related proteins and virulence-related proteins.

The majority of the top 10 enriched GO terms under “biological process” (Fig. 3A) and the majority of the top 12 enriched KEGG pathways
(Fig. 3D) were found related to metabolic activities, indicating the important role of metabolism in the development of oocysts. Interestingly,
some of the DEPs involved in metabolic pathways had been also shown
to be potential drug targets against T. gondii [39]. For instance, acetylCoA carboxylase (ACC1), which is known to be involved in fatty acid
metabolism and is found, in our study, to be down-regulated (log2
fold change = − 1.683) during sporulation. Inhibition of ACC using
aryloxyphenoxypropionates (an inhibitor of ACC) has caused significant
reduction in the growth of parasite [40].

indicates that ACL enzyme catalyzes a critical reaction linking cellular
glucose catabolism and lipid synthesis [50,51]. GMP synthase and
IMPDH, key enzymes known to be involved in de novo purine
nucleotide biosynthesis [52], were both up-regulated in our study.
Dihydrofolate reductase (DHFR) can catalyze the production of tetrahydrofolate from dihydrofolate, whereas thymidylate synthase (TS) plays
a role in transferring a methyl-group from N5, N10-methylene-tetrahydrofolate to dUMP thereby generating dTMP and tetrahydrofolate.
These enzymes are recognized as drug targets due to their involvement
in folate metabolism and pyrimidine synthesis [53].
In conclusion, our results showed that iTRAQ-based quantitative
proteome analysis is a powerful technique for investigating the DEPs involved in T. gondii oocyst maturation. The protein profile between T.
gondii non-sporulated and sporulated oocysts revealed distinct patterns
of DEPs during oocyst sporulation. Several identified DEPs were found
involved in key pathways related to parasite metabolism and virulence.
The top five hub proteins, ACL, GMP synthase, IMPDH, PARG and DHFRTS, exhibited strong associations with other DEPs. These results suggest
that DEPs interact at the molecular level with one another in order to
sustain the developmental program and adaptation of oocysts to their
environment during sporulation. Knowledge generated in this study
provided new insights into the function and interaction of DEPs during
T. gondii oocyst sporulation. Many of the proteomic hits identified in our
study could constitute interesting leads not only for understanding the
molecular mechanism regulating T. gondii oocyst sporulation, but also
for developing new targets for anti-Toxoplasma therapeutic interventions.
Supplementary data to this article can be found online at http://dx.
doi.org/10.1016/j.jprot.2016.07.010.

4.2. Virulence-related proteins

Competing interests

4.1. Metabolism-related proteins

T. gondii needs to invade host cells in order to propagate and establish infection. Virulence factors, which are known to enable parasite invasion and colonization, such as SPATR, POR18, ROP16, ROP5, ROP2,
MIC1, MIC6, MIC3, MIC8, apical membrane antigen 1(AMA1), granule
antigen 7(GRA7), GRA4, GRA1, GRA25, GRA6, rhoptry neck protein
2(RON2), RON5, RON4, protein phosphatase 2C (PP2C), rhomboid
4(ROM4), stripes IMC protein (SIP), photosensitized INA-labeled protein 1 (PhIL1), and v-type H(+)-translocating pyrophosphatase VP1
were found upregulated in our study. Among these proteins, RON2,
RON4, RON5 and AMA1 are secreted from the parasite to mediate the
formation of moving junction [41,42]. T. gondii deploys unique proteins,
ROP2, ROP5, ROP16, ROP18, and PP2C, released from secretory organelles named rhoptries into host cells for internalization of the parasite
and formation of a specialized niche within the host cell. [43–45].
MIC6, MIC4 and MIC1 function as a complex, and deletions of these
genes can result in decreased parasite invasion [46]. In our study, the
virulence factor SPATR, which plays a role in host cell recognition,
showed the highest upregulated expression level. Disruption of this
protein can lead to attenuation of the parasite invasion and enhancement of the mice survival [47]. Likewise, targeted disruption of SIP or
PhIL1 alters parasite morphology and diminishes parasite virulence
[48,49].

Conflict of interest

4.3. Pathways involved in oocyst sporulation

Project support was provided by the National Natural Science Foundation of China (Grant No. 31172316; 31230073) and Yunnan Applied
Basic Research Projects (Grant No. 2015FD042).

Central nodes in directed and undirected PPI networks showed
overlaps, indicating that the same protein might be involved in many
biological activities, such as regulation of signaling pathways, cellular
processes and metabolism. Most important nodes of DEP-derived interaction networks were determined using degree and combined score between two nodes. ACL, GMP synthase, IMPDH, PARG and DHFR-TS were
the top five hub proteins in undirected PPI network. ACL is an important
enzyme that is involved in fatty acid biosynthesis. Seeber and colleagues
showed that cytosolic acetyl-CoA produced from TCA-derived citrate by
ATP-citrate lyase (ACL) is essential for T. gondii proliferation, which

The authors declare that they have no competing interests.
Authors' contributions
XQZ and DHZ conceived and designed the experiments. CXZ and
DHZ performed the experiments. CXZ, DHZ, SH and QL contributed reagents/materials/analysis tools. CXZ analyzed the data and wrote the
paper. XS, HME and XQZ critically revised the manuscript. All authors
read and approved the final version of the manuscript.

The authors declare that they have no competing interests.
Data availability statement
The mass spectrometry proteomics data have been deposited to the
ProteomeXchange Consortium via the PRIDE [54] partner repository
with the data set identifier PXD003765.
Acknowledgments

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