Phosphorylation Profiles of T. gondii Oocysts
In an effort to reveal genotype-specific differences in the phosphoproteomes of sporulated oocysts of T. gondii, we performed a comparative phosphoproteomic profiling of sporulated oocysts between virulent PYS strain and avriluent PRU strain. In total, we identified 10,645 unique phosphopeptides, 8,181 nonredundant phosphorylation sites and 2,792 phosphoproteins from sporulated oocysts of T. gondii virulent PYS strain and avriluent PRU strain with a FDR (false-discovery rate) < 0.01 for phosphopeptide and phosphoRS probability > 0.75 for phosphorylation site. The 8,181 phosphorylation sites consisted of 7,051 phosphoserine (86.27%), 1,092 phosphothreonine (9.28%), and 38 phosphotyrosine (0.42%) (Fig. 1a). Out of the 10,645 phosphopeptides, 10,206 (95.88%) phosphopeptides had one phosphorylation site, 426 phosphopeptides (4.00%) had double phosphorylation sites, 12 phosphopeptide (0.11%) possessed 3 triple phosphorylation sites and 1 phosphopeptide (0.01%) possessed quadruple phosphorylation sites (Fig. 1b). The number of phosphopeptides matched with 1 spectrum, 2 spectra, 3 spectra, 4 spectra, 5 spectra, and ≥ 6 spectra were 6, 201, 2, 124, 897, 462, 304, and 657, respectively (Fig. 1c). Additionally, the number of phosphoproteins harboring 1, 2, 3, 4, 5 and ≥ 6 phosphorylation sites were 1,031, 550, 323, 208, 124 and 367, respectively (Fig. 1d). Among the total phosphoproteins, 99% phosphoproteins had CV (Coefficient of variation) value < 50% among three replicates (Fig. 2).
Phosphopeptides Quantification and Clustering Analysis
The quantitative analysis identified 4,129 differentially expressed phosphopeptides (DEPs) between sporulated oocysts of PYS strain and PRU strain (|log1.5 fold change| > 1 and p < 0.05), including 2,485 upregulated and 1,644 downregulated phosphopeptides (Fig. 3a, Additional file 1: Table S1 and Additional file 2: Table S2). Among these DEPs, 1,541, 665, 205, 61 and 13 phosphoproteins increased by > 1, 2, 3, 4, and 5 log1.5 fold changes. In contrast, 1,273, 269, 82, 16 and 4 phosphoproteins decreased by > 1, 2, 3, 4 and 5 log1.5 fold changes (Fig. 3b). The result of hierarchical clustering analysis of DEPs between sporulated oocysts of PYS strain and PRU strain is shown in Fig. 4.
Motifs Analysis of Phosphorylation Sites of PYS Strain Versus PRU Strain
Phosphosite motifs are crucial amino acid sequences, which are involved in the recognition of substrate by the corresponding kinase [16–17]. To investigate the potential motifs of upregulated or downregulated phosphorylated peptides of sporulated oocysts between PYS strain and PRU strain, substantial preference of amino-acid residue in sequence from − 7 to + 7 surrounding the phosphorylation sites was analyzed by the motif-X software tool. Motif analysis identified 24 motifs from the upregulated phosphorylated peptides in PYS strain when comparing PYS/PRU, including 22 serine motifs (VSP, SPR, SPxG, SPT, PxSP, LxxxRxxS, RxxSP, SDxE, SP, LxRxxS, RxxSxG, RxxSD, SExE, RxxS, SDxD, SGxE, GSE, DSD, GS, KxxS, SxxS and SxE) and 2 threonine motifs (TPE and TP) (Fig. 5). In regard to motif analysis of downregulated phosphorylated peptides in PYS strain when comparing PYS/PRU, 12 serine motifs (SPxG, AxSP, LSP, ExxxxxSP, DxSP, SPG, SPS, SPV, SP, RxxSL, RxxS and SxxxxxK) and 3 threonine motifs (TP, RxxT and KxxT) were overrepresented (Fig. 6). Because each motif corresponding to one or several category kinases, kinases consistent with motifs of upregulated or downregulated phosphorylated peptides are listed in Table 1 and Table 2, respectively. These results reflect the difference in the substrate-recognition abilities and kinase secretory preference of sporulated oocysts between virulent PYS strain and avirulent PRU strain.
Table 1
Types of kinases represented by the motifs of up-regulated phosphor-peptides as determined by KinasePho.
Motif | Kinase Classes |
VSP | PKA, PKB, PKG, CKI, IKK, CaM-II, ATM, MAPK, CDK, CDC2 |
SPR | PKA, PKB, PKG, CKI, CKII, IKK, ATM, MAPK, CDK, CDC2, Syk, Jak, INSR |
SP.G | PKA, PKB, PKG, CKI, CKII, IKK, CaM-II, ATM, MAPK, CDK, CDC2, INSR |
SPT | PKA, PKB, PKG, CKI, IKK, ATM, MAPK, CDK, CDC2 |
P.SP | PKA, PKB, PKG, CKI, IKK, ATM, MAPK, CDK, CDC2 |
L…R..S | PKA, PKB, PKC, PKG, IKK, MAPK, CKI, CKII, CaM-II, ATM, CDC2 |
R..SP | PKA, PKB, PKC, PKG, CDK, CDC2, MAPK, ATM, IKK, CKI, CKII, CaM-II, INSR |
SD..E | PKA, PKB, PKC, PKG, CKI, CKII, ATM, MAPK, IKK, CaM-II, EGFR, Src, CDC2 |
SP | PKA, PKB, PKC, PKG, CKI, CKII, ATM, MAPK, IKK, CaM-II, CDK, CDC2, Jak, INSR, Syk, Src, Ab1 |
L.R..S | PKA, PKB, PKC, PKG, CKI, CKII, IKK, CaM-II, CDK, CDC2, ATM |
R..S.G | PKA, PKB, PKC, PKG, CKI, CKII, IKK, CaM-II, CDK, CDC2, ATM, MAPK, INSR, Src |
R..SD | PKA, PKB, PKC, PKG, CKI, CKII, IKK, CaM-II, CDC2, ATM, MAPK, INSR, EGFR, Syk |
SE.E | PKA, PKB, PKC, PKG, CKI, CKII, IKK, CaM-II, CDK, CDC2, ATM, MAPK, EGFR, INSR, Syk |
R..S | PKA, PKB, PKC, PKG, CKI, CKII, IKK, CaM-II, CDK, CDC2, ATM, MAPK, INSR, Syk |
SD.D | PKA, PKG, CKI, CKII, IKK, CDC2, ATM, MAPK, INSR |
SG.E | PKA, PKB, PKC, PKG, CKI, CKII, IKK, CaM-II, CDK, CDC2, ATM, MAPK, INSR, Syk |
GSE | PKA, PKG, CKI, CKII, IKK, CaM-II, CDC2, ATM, MAPK, Syk |
DSD | PKA, PKC, PKG, CKI, CKII, IKK, ATM, MAPK |
GS | PKA, PKB, PKC, PKG, CKI, CKII, IKK, CaM-II, CDK, CDC2, ATM, MAPK, INSR, Syk, Src, Ab1 |
K..S | PKA, PKB, PKC, PKG, CKI, CKII, IKK, CaM-II, CDK, CDC2, ATM, MAPK, EGFR, INSR |
S..S | PKA, PKB, PKC, PKG, CKI, CKII, IKK, CaM-II, CDK, CDC2, ATM, MAPK, INSR, Syk, Src, Ab1 |
S.E | PKA, PKC, PKG, CKI, CKII, IKK, CDK, CDC2, ATM, MAPK, INSR, Syk, Other_MDD |
TPE | PKA, PKB, PKC, PKG, CKI, CKII, IKK, CaM-II, CDK, CDC2, ATM, MAPK, INSR, Syk, Src, Ab1 |
TP | PKA, PKB, PKC, PKG, CKII, IKK, CaM-II, CDK, CDC2, ATM, MAPK, INSR |
Abbreviations: |
PKA: cAMP-dependent Protein Kinase; PKB: Protein Kinase B; PKC: Protein Kinase C; PKG: cGMP-dependent Protein Kinase; CKI: Casein Kinase I; CKII: Casein Kinase II; IKK: IkappaB Kinase; CaM-II: Calmodulin-dependent Protein Kinase II; ATM: Ataxia Telangiectasia Mutated Kinase; MAPK: Mitogen-Activated Protein Kinase; CDC2: Cell division Cycle Protein Kinase p34; CDK: Cyclin-Dependent Kinase; INSR: Insulin Receptor; EGFR: Epithelial Growth Factor Receptor; Syk: Cytoplasmic Tyrosine Kinases; Jak: Janus kinase; Src: Sarcoma gene kinase; Ab1: Abelson kinase . |
Table 2
Types of kinases represented by the motifs of down-regulated phosphor-peptides as determined by KinasePho.
Motif | Kinase Classes |
SP.G | PKA、PKB、PKC、PKG、CKI、CKII、IKK、CDK、CDC2、ATM、MAPK、Ab1 |
A.SP | PKA、PKB、PKC、PKG、CKII、IKK、CaM-II、CDK、CDC2、ATM、MAPK、INSR、Syk、Src、Ab1 |
LSP | PKA、PKB、PKC、PKG、CKI、CKII、IKK、CaM-II、CDK、CDC2、ATM、MAPK |
E…..SP | PKA、PKB、PKG、CKI、CKII、IKK、CaM-II、CDK、CDC2、ATM、MAPK |
D.SP | PKA、PKG、IKK、CaM-II、CDC2、ATM、MAPK、Jak |
SPG | PKB、PKC、PKG、CKI、IKK、CaM-II、CDK、CDC2、ATM、MAPK |
SPS | PKA、PKB、PKC、PKG、CKI、CKII、IKK、CaM-II、CDK、CDC2、ATM、MAPK、INSR、Syk |
SPV | PKA、PKB、PKC、PKG、CKI、CKII、IKK、CaM-II、CDK、CDC2、ATM、MAPK、INSR、Ab1 |
SP | PKA、PKB、PKC、PKG、CKI、CKII、IKK、CaM-II、CDK、CDC2、ATM、MAPK、EGFR、INSR、Syk、Src |
R..SL | PKA、PKB、PKC、PKG、CKI、IKK、CaM-II、CDK、CDC2、ATM、MAPK |
R..S | PKA、PKB、PKC、PKG、CKI、CKII、IKK、CaM-II、CDK、CDC2、ATM、MAPK、EGFR、INSR、Jak、Syk、Src |
S…..K | PKA、PKB、PKC、PKG、CKI、CKII、IKK、CaM-II、CDK、CDC2、ATM、MAPK、INSR、Jak、Syk |
TP | PKA、PKB、PKC、PKG、CKI、CKII、IKK、CaM-II、CDK、CDC2、ATM、MAPK、Jak |
R..T | PKA、PKB、PKC、PKG、IKK、CaM-II、CDC2、MAPK、EGFR、INSR、Src、Ab1 |
K..T | PKA、PKC、PKG、CKII、IKK、CDK、MAPK |
Abbreviations: |
PKA: cAMP-dependent Protein Kinase; PKB: Protein Kinase B; PKC: Protein Kinase C; PKG: cGMP-dependent Protein Kinase; CKI: Casein Kinase I; CKII: Casein Kinase II; IKK: IkappaB Kinase; CaM-II: Calmodulin-dependent Protein Kinase II; ATM: Ataxia Telangiectasia Mutated Kinase; MAPK: Mitogen-Activated Protein Kinase; CDC2: Cell division Cycle Protein Kinase p34; CDK: Cyclin-Dependent Kinase; INSR: Insulin Receptor; EGFR: Epithelial Growth Factor Receptor; Syk: Cytoplasmic Tyrosine Kinases; Jak: Janus Kinase; Src: Sarcoma Gene Kinase; Ab1: Abelson Kinase. |
Functional Enrichment Analysis
To uncover the differences in the functions of the differentially expressed phosphoproteins (DEPs) of sporulated oocysts between PYS strain and PRU strain, GO enrichment analysis was performed. The enriched GO terms stratified into three GO categories (BP, CC and MF) are listed for upregulated and downregulated phosphoproteins (Fig. 7a-7b). The top five significantly enriched GO terms under BP for upregulated DEPs included catalytic activity, binding, transporter activity, structural molecule activity and transcription. In contrast, catalytic activity, binding, transporter activity, structural molecule activity and signal transducer activity were the top five enriched GO terms under BP for downregulated phosphoproteins.
In regard to the CC category, the five enriched GO terms of upregulated phosphoproteins were cell part, cell, organelle, membrane and macromolecular complex, whereas cell part, cell, organelle, macromolecular complex and membrane were the top five significantly enriched GO terms for the downregulated phosphoproteins. Regarding the MF of upregulated phosphoproteins, metabolic process, cellular process, localization, biological regulation and regulation of biological process were the top five enriched GO terms. In contrast, the five mostly enriched GO terms for downregulated phosphoproteins included metabolic process, cellular process, biological regulation, localization and regulation of biological process.
The co-expression networks of GO terms belonging to the three functional categories were constructed using the R Igraph package in order to investigate the level of association among GO terms. The significantly enriched GO terms in the network of upregulated phosphoproteins in PYS when comparing PYS/PRU are shown in Additional file 3: Figure S1. In the MF category, six GO terms, including pseudouridine synthase activity, 2-alkenal reductase [NAD(P)] activity, metal ion transmembrane transporter activity, calcium ion transmembrane transporter activity, calcium-transporting ATPase activity, and motor activity, were significantly enriched. Also, in the CC category, six GO terms, including extracellular region, protein complex, membrane part, membrane protein complex, coated membrane, and membrane coat, were significantly enriched.
In the BP category, 40 GO terms were significantly enriched, including biological regulation, regulation of biological process, cell cycle process, response to stress, transmembrane transport, G-protein coupled receptor signaling pathway, glycerolipid metabolic process, glycerophospholipid metabolic process, phosphatidylinositol metabolic process, lipid phosphorylation, phosphatidylinositol phosphorylation, organophosphate metabolic process, aromatic compound catabolic process, cellular nitrogen compound catabolic process, heterocycle catabolic process, nucleobase-containing compound catabolic process, organic cyclic compound catabolic process, negative regulation of biological process, negative regulation of cellular process, regulation of cellular component organization, regulation of organelle organization, RNA modification, pseudouridine synthesis, regulation of metabolic process, regulation of cellular metabolic process, regulation of primary metabolic process, regulation of biosynthetic process, regulation of nitrogen compound metabolic process, regulation of cellular biosynthetic process, regulation of cellular amide metabolic process, regulation of macromolecule metabolic process, regulation of RNA metabolic process, regulation of macromolecule biosynthetic process, regulation of RNA biosynthetic process, regulation of cellular macromolecule biosynthetic process, regulation of nucleic acid-templated transcription, regulation of translation, regulation of gene expression, regulation of transcription, and posttranscriptional regulation of gene expression.
The significantly enriched GO terms in the network of downregulated phosphoproteins in PYS when comparing PYS/PRU are shown in Additional file 4: Figure S2. In the MF category, 17 GO terms, including binding, small molecular binding, nucleoside phosphate binding, nucleotide binding, pattern binding, polysaccharide binding, starch binding ligase activity, transferase activity, oxidoredutase, kinase regulatory activity, protein kinase regulatory activity, antiporter activity, cation antiporter activity, proton antiporter activity, translation initiation factor activity and translation factor activity, were significantly enriched. Only three GO terms (cell, cell part and intracellular) in the CC category were enriched. However, in the BP category there were 21 significantly enriched GO terms, including locomotion, developmental process, cellular developmental process, anatomical structure morphogenesis, anatomical structure development, tissue development, nervous system development, response to external stimulus, regulation of phosphorus metabolic process, regulation of phosphate metabolic process, regulation of phosphorylation, regulation of protein phosphorylation, regulation of protein kinase activity, regulation of kinase activity, macromolecule metabolic process, pyruvate metabolic process, cellular carbohydrate biosynthetic process, cellular amide metabolic process, peptide metabolic process, peptide biosynthetic process, and translation metabolic process establishment of localization and transport.
Compared to PRU strain, more upregulated phosphoproteins were enriched in GO terms such as transcription regulator activity, macromolecular complex, immune system process, biological adhesion in PYS strain. However, more downregulated phosphoproteins were enriched in GO terms such as signal transducer activity, developmental process, reproductive process, reproduction, locomotion and pigmentation. These results clearly show significant differences in the biological functions of the DEPs of sporulated oocysts between PYS strain and PRU strain.
KEGG Pathway Analysis
Enzymes with phosphorylation sites can regulate key signal pathways in several organisms [11]. To better understand the signal pathways controlled by the phosphorylated proteins and the function of the DEPs, DEPs in sporulated oocysts between PYS strain and PRU strain were annotated and mapped against in the KEGG pathway database. As shown in Figs. 8, 39 and 18 DEPs were significantly enriched in RNA transport and phosphatidylinositol signaling system, respectively.
Protein-Protein Interaction (PPI) Analysis
Using Cytoscape software, the PPI networks (combined score ≥ 0.9) were built in order to identify the mechanisms regulated by phosphorylation and to determine the related functional clusters of the DEPs of sporulated oocysts between the virulent and avirulent T. gondii strains. The PPI network of upregulated and downregulated phosphoproteins between PRU strain and PYS strain included 344 nodes and 670 interactor edges (Fig. 9). Several major hubs were identified, including ribosome biogenesis protein BOP1 (TGME49_301390), nucleolar protein NOP5 (TGME49_205510), eukaryotic initiation factor-2 (TGME49_313230), translation initiation factor 2 beta (TGME49_235540), eukaryotic translation initiation factor 2 gamma subunit (TGME49_235970), translation elongation factor 2 family protein (TGME49_205470), ribosomal protein RPL6 (TGME49_313390), ribosomal protein RPL4 (TGME49_309120), and ATP-dependent RNA helicase (TGME49_312280). Several functional clusters were identified in the PPI network, including DNA replication or RNA transcription, proteasome, metabolism, RNA splicing or translation, ribosomal proteins, molecular chaperones, tRNA related molecular, adapter, coatomer and kinases or enzymes.
Kinase Related Networks
Given that phosphorylation influences the activity of the phosphorylated protein that function as a kinase, potential peptides whose phosphorylation is related to the respective kinase were identified by performing a correlation analysis (Additional file 5: Table S3). After retaining phosphopeptides with > ~ 1.5-fold change abundance, 2 phosphorylated kinases were identified and were associated with 58 phosphopeptides. Positive correlation and negative correlation indicate that kinase phosphorylation positively or negatively impacts its action. These correlations suggest potential differences in kinases and associated substrates of sporulated oocysts between the two T. gondii strains. Among the first cluster of phosphorylated peptides, peptides related to T. gondii AGC kinase A0A125YVN2 (connected with 50 phosphorylated peptides) connected most kinase peptides. The second cluster of phosphorylated peptides encompassed 6 proteins: zinc finger A0A125YGX1, TgCatPRC2 A0A151H897, possible RNA-binding protein Q1JTD1, DNA-directed RNA polymerase subunit S8F5G5, non-specific serine/threonine protein kinase S8F5G5 and the hub protein kinase is histone kinase SNF1 S8F5G5 (Fig. 10). The GO annotation of the most connected kinases peptides (AGC kinase) showed GO terms associated with nucleotide binding, ATP binding, metabolic process, protein serine/threonine kinase activity and phosphorylation, in the MF category.
Potential Phosphorylation-Dependent Interactions
Based on the T. gondii protein interaction network, the putative function of the different phosphorylation sites in our dataset were sought to identify and the effects of mutation or phosphorylation on the interaction between two phosphorylated proteins (enable or disable) were predicted. Using Mechismo and Pearson correlational analysis, a total of 147 interactors of the T. gondii orthologs, enabled or disabled by phosphorylation of the identified sites, were identified (Additional file 6: Table S4). The 9 interacting partners were annotated back to T. gondii proteins and were found to influence interactors in T. gondii (Fig. 10). The T6 and T12 phosphosites of T. gondii A0A151H503 gene were found to influence 7 interactions and phosphosite S10 of T. gondii A0A151GZW7 gene influenced 2 interactions.