Global iTRAQ-based proteomic profiling of Toxoplasma gondii oocysts
during sporulation
Chun-Xue Zhou, Xing-Quan Zhu, Hany M. Elsheikha, Shuai He, Qian
Li, Dong-Hui Zhou, Xun Suo
PII:
DOI:
Reference:

S1874-3919(16)30306-2
doi: 10.1016/j.jprot.2016.07.010
JPROT 2623

To appear in:

Journal of Proteomics

Received date:
Revised date:
Accepted date:

19 March 2016
15 June 2016
11 July 2016

Please cite this article as: Zhou Chun-Xue, Zhu Xing-Quan, Elsheikha Hany M., He
Shuai, Li Qian, Zhou Dong-Hui, Suo Xun, Global iTRAQ-based proteomic profiling
of Toxoplasma gondii oocysts during sporulation, Journal of Proteomics (2016), doi:
10.1016/j.jprot.2016.07.010

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Global iTRAQ-based proteomic profiling of Toxoplasma gondii

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oocysts during sporulation

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Chun-Xue Zhou1, 2, Xing-Quan Zhu2, Hany M. Elsheikha3, Shuai He2, 4, Qian Li5,

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Dong-Hui Zhou 2*& Xun Suo1*

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National Animal Protozoa Laboratory and College of Veterinary Medicine, China

Agricultural University, Beijing 100193, PR China
2

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State Key Laboratory of Veterinary Etiological Biology, Key Laboratory of Veterinary

Parasitology of Gansu Province, Lanzhou Veterinary Research Institute, Chinese Academy of

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Agricultural Sciences, Lanzhou, Gansu Province 730046, PR China
3

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Faculty of Medicine and Health Sciences, School of Veterinary Medicine and Science,

University of Nottingham, Sutton Bonington Campus, Loughborough, LE12 5RD, UK
4

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College of Animal Science and Technology, Anhui Agricultural University, Hefei,

Anhui Province 230036, PR China
5

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School of Basic Medicine, Dali University, Dali, Yunnan Province 671000, PR China

*Corresponding authors: Xun Suo, Tel: +86-10-62734325, Email: suoxun@cau.edu.cn;
Dong-Hui Zhou, Tel: +86-931-8342837, Email: zhoudonghui@caas.cn;
Corresponding authors at: 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.

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Abstract
Toxoplasma gondii is a medically and economically important protozoan parasite.

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However, the molecular mechanisms of its sporulation remain largely unknown. Here,

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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

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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,

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carbon and energy. Protein interaction network analysis generated by STRING
identified ATP-citrate lyase (ACL), GMP synthase, IMP dehydrogenase (IMPDH),
glycohydrolase

(PARG),

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poly (ADP-ribose)

and

bifunctional

dihydrofolate

reductase-thymidylate synthase (DHFR-TS) as the top five hubs. We also identified

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25 parasite virulence factors that were expressed at relatively high levels in sporulated

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oocysts compared to non-sporulated oocysts, which might contribute to the infectivity
of mature oocysts. Considering the importance of oocysts in the dissemination of

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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

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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 2D-LC-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
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stage during sporulation.

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Keyword: Toxoplasma gondii; oocyst; sporulation; iTRAQ; proteomics

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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

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infect all warm-blooded animals and humans as the intermediate host and felids as the

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definitive host [2]. This parasite has a considerable public health impact because it

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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 the common means of T. gondii

acquisition [5].

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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

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[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

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any warm-blooded vertebrate animal) and sexual (occurs only in felids) reproductive

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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,

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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 with appropriate climatic conditions

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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.
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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

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quantitation (iTRAQ) coupled with two-dimensional liquid chromatography–tandem

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mass spectrometry to analyze proteomic changes in T. gondii oocysts during
sporulation. This method can provide a more accurate identification and

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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

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affected pathways and processes that operate during the development of T. gondii
oocysts. Our results identified 587 differentially expressed proteins (DEPs), the

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2. Materials and Methods

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majority of which are involved in metabolic activities.

2.1. Ethics statement

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All animal procedures were approved by Animal Ethics Committee of Lanzhou
Veterinary Research Institute, Chinese Academy of Agricultural Sciences (Permit No.

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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

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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
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(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 15min, and then the supernatant was

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discarded. This step was repeated three times and the final supernatant was decanted,

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and the sediment was mixed with 5 volumes of sucrose solution (specific gravity 1.15)
and centrifuged for 10 min at 350g. Oocysts were collected from the supernatant and

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suspended in TE buffer. Approximately 10 ml of oocyst suspension was overlaid onto
a discontinuous CsCl gradient (specific gravity from 1.05 to 1.125), and centrifuged at
12000g for 60 min at 4 °C with no brake. Oocysts were collected from the

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opaque-to-white layer (between 1.05 and 1.11 layers) and washed twice with 30-40ml
0.85% saline. The final pellet was re-suspended in 1X phosphate buffered saline

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(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

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4 °C for storage until use.

2.3. Protein preparation and iTRAQ labeling

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Protein was extracted from an equal number (~107) of sporulated and
non-sporulated oocysts as follows. Oocysts were centrifuged at 350g for 15min. The
pellet was re-suspended in radioimmunoprecipitation assay (RIPA) lysis buffer (20

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mM Tris-HCl pH7.5, 150 mM sodium chloride, 2 mM EDTA, 1% DOC, 1% Triton
X-100) supplemented with protease inhibitors (Roche), briefly sonicated and
centrifuged at 10625g for 20min at 4 °C, and the supernatants were analyzed as total
lysates. Total protein concentration was determined using the QuantiProTM 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).
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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

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and dried with a rotary vacuum concentrator.

2.4. Two-dimensional liquid chromatography and MS/MS analysis

procedure with

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2D liquid chromatography was carried out according to previously reported
slight modifications [24]. Briefly, strong cation exchange (SCX)

fractionation was performed with a high performance liquid chromatography (HPLC)

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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 (20mM HCOONH4,

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pH=10) and mobile phase B (20mM HCOONH4,80% acetonitrile (ACN),pH=10).
The UV detector was set at 214/280 nm, and fractions were collected every 1 min. In

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total, 24 fractions were collected, acidified with 50% trifluoroacetic acid (TFA) and

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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

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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

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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)
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Detected Protein Threshold: 0.05; (2) Competitor Error Margin: 2.00; (3) Revision
Number: 1656; (4) Instrument: Orbi MS (1-3ppm), Orbi MS/MS; (5) Sample Type:
iTRAQ 8 plex (Peptide Labeled); (6) Cysteine Alkylation: MMTS; (7) Digestion:

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Trypsin; (8) Special Factors: None; (9) ID Focus: Biological modification; (10)
Search Effort: Thorough ID; (11) FDR Analysis: Yes; (12) User Modified Parameter

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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

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8322. Unique protein with at least one unique peptide was identified and FDR was set
to <0.01 for the identification of both peptides and proteins. Peptide confidence level

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of 95% or unused confidence score >1.3 was used as the qualification criteria.
Relative quantification of proteins was calculated based on the ratio of peak areas

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from MS/MS spectra. To designate significant changes in protein expression, log2

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downregulated.

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fold change≥1was deemed as up-regulated and log2 fold change≤ -1 classified as

2.6. Gene Ontology and KEGG pathway enrichment analysis
To better understand the biological functions of significantly altered proteins, these

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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
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networks, Cytoscape tool was utilized [29].

2.7. qPCR analysis

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

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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

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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

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amplification reactions were performed with the following conditions: 2 min at 95 °C,
40 cycles of 95 °C for 15 s, 55°C for 30s, 72°C for 30s. The T. gondii
gene

(GAPDH)

was

used

as

an

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glyceraldehyde-3-phosphatedehydrogenase

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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

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included in Table S1. The relative expression was calculated using the 2-△△CT method

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[31].

3. Results

3.1. Protein identification and quantification
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 >95% confidence in correct
sequence identification (Table S2). About 89.3% of these proteins had a coefficient of
variation (CV) of less than 50% among replicates (Fig. 1A). The following analysis is
based on proteins with a CV value <50%. Fig. 1B shows a hierarchical clustering
analysis of all experiments (including 2 biological replicates, each containing 2
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technical replicates) and includes 1823 proteins with a CV value <50%. 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

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into 25 categories. “Translation, ribosomal structure and biogenesis”, “General

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function prediction only”, “Posttranslational modification, protein turnover,

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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|>1 and P<0.05). Among these DEPs,

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208, 120, 119, and 28 proteins reproducibly produced more than 1, 2, 3, and 4 log2
fold changes, respectively. In contrast, the expression levels of 94, 17 and 1 protein

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decreased by more than 1, 2 and 3 log2 fold changes, respectively (Fig. 1C).

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3.2. GO annotation and KEGG analysis of DEPs

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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

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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

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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
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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

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to identify the parasite biological pathway operating during the sporulation process.
Among these 587 identified DEPs, only 51 DEPs (19 down-regulated and 32

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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

metabolic activities except “phagosome”, “protein processing in endoplasmic
and

“toxoplasmosis”.

TGME49_301120

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reticulum”

(acetyil

CoA

acetyltransferase/thiolase) is involved in the most KEGG pathways, followed by

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TGME49_227090 (aspartate kinase), TGME49_221320 (acetyl-CoA carboxylase
ACC1), TGME49_264030 (aminotransferase), TGME49_318310 (transketolase), and

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TGME49_318230 (phosphoglycerate kinase PGKI).

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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

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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

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(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)
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glycohydrolase

(PARG),

and

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bifunctional

dihydrofolate

reductase-thymidylate

synthase

(DHFR-TS).

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expressions of genes encoding these five hub proteins were verified by qPCR and
were found consistent with the proteome data (Fig. 6).

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Next, we presented the predicted modes of actions between hubs and their
associated DEPs (Fig. 7). Edge actions, including activation, binding, catalysis,

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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

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relationship. In Fig. 7A, DHFR-TS interacts with proliferating cell nuclear antigen
(PCNA2) and ribonucleoside-diphosphate reductase small subunit (RnrS) in the

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reaction mode with action score of 0.767 and 0.720, respectively. Acetyl-CoA
carboxylase, containing two main isoforms ACC1 and ACC2, is a biotin-dependent

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enzyme that functions in the production of malonyl-CoA. As shown in Fig. 7B,

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IMPDH interacts with ACC two isoforms in multiple ways, including activation,
inhibition, catalysis and ptmod. ACL is a key lipogenic enzyme that converts citrate

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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)

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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
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research utilized

one-dimensional gel electrophoresis coupled with LC-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,
and colleagues identified 1304 non-redundant proteins, of which 154

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proteins were unique to oocyst/sporozoite compared to the proteome of T. gondii
tachyzoite stage [38].

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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

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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

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proteins changed in response to sporulation, they were divided into two categories:
metabolism-related proteins and virulence-related proteins.

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4.1. Metabolism-related proteins

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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

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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, acetyl-CoA carboxylase (ACC1), which is known to be involved in fatty

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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].

4.2. Virulence-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 antigen1(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
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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

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and formation of a specialized niche within the host cell. [43-45]. MIC6, MIC4 and

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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

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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

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morphology and diminishes parasite virulence [48, 49].

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4.3. Pathways involved in oocyst sporulation

Central nodes in directed and undirected PPI networks showed overlaps, indicating
that the same protein might be involved in many biological activities, such as

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regulation of signaling pathways, cellular processes and metabolism. Most important

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nodes of DEP-derived interaction networks were determined using degree and
combined score between two nodes. ACL, GMP synthase, IMPDH, PARG and

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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 indicates that ACL enzyme

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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
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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
DHFR-TS, exhibited strong associations with other DEPs. These results suggest that

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DEPs interact at the molecular level with one another in order to sustain the

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developmental program and adaptation of oocysts to their environment during
sporulation. Knowledge generated in this study provided new insights into the

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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

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also for developing new targets for anti-Toxoplasma therapeutic interventions.

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Competing interests

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Authors’ contributions

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The authors declare that they have no competing interests.

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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

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manuscript. All authors read and approved the final version of the manuscript.

Acknowledgments
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).

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.
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Figure Legends:
Fig.1. iTRAQ coupled with LC-MS/MS analysis of sporulated and non-sporulated

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oocysts. (A) Determination of experimental variation using all detected proteins

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common in all biological replicates. The cumulative percentage (red line) is defined

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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

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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 <

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50% variation range. TR1 and TR2 indicate technical replicate 1 and 2, respectively.
Each technical replicate includes two biological replicates. (C) Number of proteins

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detected up and downregulated between sporulated and non-sporulated oocysts

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samples.

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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

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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

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transport; V, defense mechanisms; W, extracellular structures; X, mobilome:

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prophages, transposons; Y, nuclear structure; Z, cytoskeleton.

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Fig. 3. GO annotation and KEGG pathway analysis of the DEPs. (A) Top 10 enriched

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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

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downregulated proteins.

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

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respectively.

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Fig.5. The protein-protein interaction network generated with STRING and visualized
with Cytoscape for DEPs. The PPI network with a high combined score > 0.7 was
prepared using the STRING 10 database program. DEPs are represented as round

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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).

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.

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
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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

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interaction score (edge score).

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Supplementary material

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Fig. S1. Representative images of Toxoplasma gondii oocysts used in the proteomic

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analysis. (A) non-sporulated oocysts are spheroid (10×12 μm in size); (B) sporulated

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oocysts are ovoid (11×13 μm) and have two sporocysts (6×8 μm), each containing
four infective sporozoites (2×6-8 μm).

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Table S2 List of 2095 identified proteins.

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Table S1 Oligonucleotide primers used in quantitative PCR analysis.

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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.

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To identify proteins required for the development of environmentally resistant and
infective T. gondii oocysts, we compared the proteome of non-sporulated (immature)

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oocysts with the proteome of sporulated (mature, infective) oocysts. iTRAQ
2D-LC-MS/MS analysis revealed proteomic changes that distinguish non-sporulated
from sporulated oocysts. Many of the differentially expressed proteins were involved

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in metabolic pathways and 25 virulence factors were identified upregulated in the
sporulated oocysts. This work provides the first quantitative characterization of the

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proteomic variations that occur in T. gondii oocyst stage during sporulation.

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Conflict of interest

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The authors declare that they have no competing interests.

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Graphical abstract

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Highlights

Toxoplasma gondii oocysts adapt to diverse environmental conditions.

We characterized the proteomic changes that occur in the oocysts during

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sporulation.

iTRAQ-based proteomics revealed 2095 proteins, of which 587 were

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differentially expressed.

25 virulence factors were found upregulated in the sporulated ooysts, which
might account for the infectivity of the mature oocysts.

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PPI network analysis identified ACL, GMP synthase, IMPDH, PARG, and

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DHFR-TS as the top hub proteins.

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