Title: Global Lysine Crotonylation and 2-Hydroxyisobutyrylation in Phenotypically Different Toxoplasma gondii Parasites
Abstract: Toxoplasma gondii is a unicellular protozoan parasite of the phylum Apicomplexa. The parasite repeatedly goes through a cycle of invasion, division and induction of host cell rupture, which is an obligatory process for proliferation inside warm-blooded animals. It is known that the biology of the parasite is controlled by a variety of mechanisms ranging from genomic to epigenetic to transcriptional regulation. In this study, we investigated the global protein posttranslational lysine crotonylation and 2-hydroxyisobutyrylation of two T. gondii strains, RH and ME49, which represent distinct phenotypes for proliferation and pathogenicity in the host. Proteins with differential expression and modification patterns associated with parasite phenotypes were identified. Many proteins in T. gondii were crotonylated and 2-hydroxyisobutyrylated, and they were localized in diverse subcellular compartments involved in a wide variety of cellular functions such as motility, host invasion, metabolism and epigenetic gene regulation. These findings suggest that lysine crotonylation and 2-hydroxyisobutyrylation are ubiquitous throughout the T. gondii proteome, regulating critical functions of the modified proteins. These data provide a basis for identifying important proteins associated with parasite development and pathogenicity. Toxoplasma gondii is a unicellular protozoan parasite of the phylum Apicomplexa. The parasite repeatedly goes through a cycle of invasion, division and induction of host cell rupture, which is an obligatory process for proliferation inside warm-blooded animals. It is known that the biology of the parasite is controlled by a variety of mechanisms ranging from genomic to epigenetic to transcriptional regulation. In this study, we investigated the global protein posttranslational lysine crotonylation and 2-hydroxyisobutyrylation of two T. gondii strains, RH and ME49, which represent distinct phenotypes for proliferation and pathogenicity in the host. Proteins with differential expression and modification patterns associated with parasite phenotypes were identified. Many proteins in T. gondii were crotonylated and 2-hydroxyisobutyrylated, and they were localized in diverse subcellular compartments involved in a wide variety of cellular functions such as motility, host invasion, metabolism and epigenetic gene regulation. These findings suggest that lysine crotonylation and 2-hydroxyisobutyrylation are ubiquitous throughout the T. gondii proteome, regulating critical functions of the modified proteins. These data provide a basis for identifying important proteins associated with parasite development and pathogenicity. Toxoplasma gondii is an intracellular parasite that infects all warm-blooded animals and is disseminated frequently via contaminated meat (1Marino N.D. Panas M.W. Franco M. Theisen T.C. Naor A. Rastogi S. Buchholz K.R. Lorenzi H.A. Boothroyd J.C. Identification of a novel protein complex essential for effector translocation across the parasitophorous vacuole membrane of Toxoplasma gondii.PLoS Pathog. 2018; 14: e1006828Crossref PubMed Scopus (48) Google Scholar, 2Kwong W.K. del Campo J. Mathur V. Vermeij M.J.A. Keeling P.J. A widespread coral-infecting apicomplexan with chlorophyll biosynthesis genes.Nature. 2019; 568: 103-107Crossref PubMed Scopus (62) Google Scholar). Toxoplasmosis is one of the most prevalent zoonoses worldwide. Feline animals, particularly cats, are the definitive hosts of the parasite and where T. gondii undergoes sexual reproduction within the intestine epithelial cells, and millions of oocysts are shed in the faeces of infected cats. Animals and human beings are primarily infected by ingestion of oocyst-contaminated water and feed or cyst-contaminated meat. In infected animals, the parasites invade nucleated cells such as macrophages, dendritic cells (DCs) 1The abbreviations used are:DCdendritic cellPTMpost-translational modificationIPimmunoprecipitationNSInanospray ionizationIRGimmunity-related GTPase. 1The abbreviations used are:DCdendritic cellPTMpost-translational modificationIPimmunoprecipitationNSInanospray ionizationIRGimmunity-related GTPase. and muscle cells. T. gondii poses a great threat to individuals whose immune systems are compromised, such as patients with HIV infections or those receiving immune suppression treatment (3Osunkalu V.O. Akanmu S.A. Ofomah N.J. Onyiaorah I.V. Adediran A.A. Akinde R.O. Onwuezobe I.A. Seroprevalence of Toxoplasma gondii IgG antibody in HIV-infected patients at the Lagos University Teaching Hospital.HIV AIDS. 2011; 3: 101-105Google Scholar). The direct damage caused by the parasites is massive cell lysis and tissue dysfunction. The parasite can traverse the placenta and proliferate in the fetus, making it one of the pathogens threatening the health of pregnant women and newborns (4Pappas G. Roussos N. Falagas M.E. Toxoplasmosis snapshots: global status of Toxoplasma gondii seroprevalence and implications for pregnancy and congenital toxoplasmosis.Int. J. Parasitol. 2009; 39: 1385-1394Crossref PubMed Scopus (702) Google Scholar). dendritic cell post-translational modification immunoprecipitation nanospray ionization immunity-related GTPase. dendritic cell post-translational modification immunoprecipitation nanospray ionization immunity-related GTPase. After invasion into a host cell, T. gondii replicates into several daughter cells named tachyzoites, which rapidly invade new host cells after egress. In immune-competent hosts, the parasites convert into a semidormant state with slow development, named bradyzoites, in tissue cysts, which can be activated immediately if the host immune capacity is weakened. Currently, effective drugs of choice against T. gondii are limited to a few metabolic inhibitors such as pyrimethamine, sulfonamides, spiramycin, and clindamycin (5Valentini P. Buonsenso D. Barone G. Serranti D. Calzedda R. Ceccarelli M. Speziale D. Ricci R. Masini L. Spiramycin/cotrimoxazole versus pyrimethamine/sulfonamide and spiramycin alone for the treatment of toxoplasmosis in pregnancy.J. Perinatol. 2015; 35: 90-94Crossref PubMed Scopus (30) Google Scholar). They are effective in only replicative tachyzoites, with little or no effect on the semidormant bradyzoites, and some are quite cytotoxic to the hosts. The discovery of new drug targets is essential but relies on a deep understanding of parasite biology. The genome of T. gondii is ∼70 Mb, encoding more than 7000 proteins, which may be expressed at distinct developmental stages in various quantities (6Weiss L.M. Fiser A. Angeletti R.H. Kim K. Toxoplasma gondii proteomics.Expert Rev. Proteomics. 2009; 6: 303-313Crossref PubMed Scopus (27) Google Scholar) (www.ToxoDB.org). As in other organisms, a majority of T. gondii proteins are localized in functionally specialized organelles, such as food vacuoles, rhoptries, dense granules and micronemes. Further, T. gondii proteins are also post-translationally modified to achieve their functionality. Post-translational modification (PTM) is a process in which a chemical moiety is covalently added to certain amino acid groups after a protein is translated from its mRNA template. These modifications include acetylation, glycosylation, ubiquitination, nitrosylation, methylation, phosphorylation, and lipidation, which influence almost all aspects of cell biology and pathogenesis. Therefore, identification of and understanding the mechanisms of PTMs is critical in the study of cell function and disease treatment and prevention (7Witze E.S. Old W.M. Resing K.A. Ahn N.G. Mapping protein post-translational modifications with mass spectrometry.Nat. Method. 2007; 4: 798-806Crossref PubMed Scopus (602) Google Scholar, 8Yakubu R.R. Weiss L.M. Silmon de Monerri N.C. Post-translational modifications as key regulators of apicomplexan biology: insights from proteome-wide studies.Mol. Microbiol. 2018; 107: 1-23Crossref PubMed Scopus (32) Google Scholar). In recent years, PTM studies in parasitology have been on mainly Plasmodium falciparum, the causative agent of malaria, and T. gondii (8Yakubu R.R. Weiss L.M. Silmon de Monerri N.C. Post-translational modifications as key regulators of apicomplexan biology: insights from proteome-wide studies.Mol. Microbiol. 2018; 107: 1-23Crossref PubMed Scopus (32) Google Scholar, 9Croken M.M. Nardelli S.C. Kim K. Chromatin modifications, epigenetics, and how protozoan parasites regulate their lives.Trends Parasitol. 2012; 28: 202-213Abstract Full Text Full Text PDF PubMed Scopus (52) Google Scholar). In T. gondii, earlier studies suggested that methylation, acetylation, ubiquitylation, succinylation, SUMOylation and glycosylation were widely distributed in the proteome of the parasite (8Yakubu R.R. Weiss L.M. Silmon de Monerri N.C. Post-translational modifications as key regulators of apicomplexan biology: insights from proteome-wide studies.Mol. Microbiol. 2018; 107: 1-23Crossref PubMed Scopus (32) Google Scholar). Several key proteins, such as TgGAP40, TgGAP45, TgGAP50, and TgMLC1, involved in tachyzoite gliding motility, moving junction (MJ) formation, and other functions that are essential for host-cell invasion are modified by ubiquitination, phosphorylation and N-glycosylation (8Yakubu R.R. Weiss L.M. Silmon de Monerri N.C. Post-translational modifications as key regulators of apicomplexan biology: insights from proteome-wide studies.Mol. Microbiol. 2018; 107: 1-23Crossref PubMed Scopus (32) Google Scholar, 10Fauquenoy S.M.W. Hovasse A. Bednarczyk A. Slomianny C. Schaeffer C. Van Dorsselaer A. Tomavo S. Proteomics and glycomics analyses of N-glycosylated structures involved in Toxoplasma gondii–host cell interactions.Mol. Cell Proteomes. 2008; 7: 891-910Abstract Full Text Full Text PDF PubMed Scopus (69) Google Scholar, 11Silmon de Monerri N.C. Yakubu R.R. Chen A.L. Bradley P.J. Nieves E. Weiss L.M. Kim K. The ubiquitin proteome of Toxoplasma gondii reveals roles for protein ubiquitination in cell-cycle transitions.Cell Host Microbe. 2015; 18: 621-633Abstract Full Text Full Text PDF PubMed Scopus (44) Google Scholar). These modifications obviously regulate parasite movement by affecting protein distribution or polymerization. With FLAG-affinity chromatography techniques, 120 proteins in the categories of chromatin and transcriptional machinery, ribosomal biogenesis, translation-related proteins (ROPs), stress-related proteins and bradyzoite parasitophorous vacuole membrane proteins were found to be SUMOylated (12Braun L. Cannella D. Pinheiro A.M. Kieffer S. Belrhali H. Garin J. Hakimi M.A. The small ubiquitin-like modifier (SUMO)-conjugating system of Toxoplasma gondii.Int. J. Parasitol. 2009; 39: 81-90Crossref PubMed Scopus (40) Google Scholar). Recently, with the application of high-throughput mass spectrometry, histone modifications, “the histone code” and core markers of epigenetic regulation, of several protozoan parasites have been gradually dissected (9Croken M.M. Nardelli S.C. Kim K. Chromatin modifications, epigenetics, and how protozoan parasites regulate their lives.Trends Parasitol. 2012; 28: 202-213Abstract Full Text Full Text PDF PubMed Scopus (52) Google Scholar). To date, chromatin modifications are the most well-studied PTMs in most organisms. Histone modifications that affect gene activation and suppression include methylation, acetylation, ubiquitination, glycosylation, phosphorylation, ADP-ribosylation and SUMOylation, which frequently occur at the N-terminal tails of histone units (9Croken M.M. Nardelli S.C. Kim K. Chromatin modifications, epigenetics, and how protozoan parasites regulate their lives.Trends Parasitol. 2012; 28: 202-213Abstract Full Text Full Text PDF PubMed Scopus (52) Google Scholar). The functions of many histone PTMs appear to be evolutionarily conserved in the Apicomplexan parasite. In general, histone acetylation is associated with gene activation, whereas methylation can be associated with either gene repression or activation depending upon the residues modified. In T. gondii, the most conserved modification is trimethylation of histone 3 lysine 4 (H3K4me3), which is a marker of transcriptionally active promoters. Further, it is likely that the classic gene activation marks H3K4me3, H4ac, and H3K9ac are co-localized and mark the promoters of actively transcribed genes in euchromatins of T. gondii (13Gissot M. Kelly K.A. Ajioka J.W. Greally J.M. Kim K. Epigenomic modifications predict active promoters and gene structure in Toxoplasma gondii.PLoS Pathog. 2007; 3: e77Crossref PubMed Scopus (95) Google Scholar). Apart from histone modifications, PTMs also occur in proteins of other cellular structures. Xiao et al. found that acetylation, polyglutamylation, and methylation were present in the C-terminal alpha- and beta-tubulin of T. gondii (14Xiao H. El Bissati K. Verdier-Pinard P. Burd B. Zhang H. Kim K. Fiser A. Angeletti R.H. Weiss L.M. Post-translational modifications to Toxoplasma gondii alpha- and beta-tubulins include novel C-terminal methylation.J. Proteome Res. 2010; 9: 359-372Crossref PubMed Scopus (46) Google Scholar). Although strains of the type I (such as the RH strain) are virulent and uniformly lethal to mice, causing severe clinical manifestations of toxoplasmosis, strains of the types II (such as the ME49 strain) are relatively low virulence to murine hosts, which are able to control acute phase of the disease and are able to establish chronic infections (15Grigg M.E. Bonnefoy S. Hehl A.B. Suzuki Y. Boothroyd J.C. Success and virulence in Toxoplasma as the result of sexual recombination between two distinct ancestries.Science. 2001; 294: 161-165Crossref PubMed Scopus (257) Google Scholar, 16Angeloni M.B. Guirelli P.M. Franco P.S. Barbosa B.F. Gomes A.O. Castro A.S. Silva N.M. Martins-Filho O.A. Mineo T.W. Silva D.A. Mineo J.R. Ferro E.A. Differential apoptosis in BeWo cells after infection with highly (RH) or moderately (ME49) virulent strains of Toxoplasma gondii is related to the cytokine profile secreted, the death receptor Fas expression and phosphorylated ERK1/2 expression.Placenta. 2013; 34: 973-982Crossref PubMed Scopus (27) Google Scholar). Thus, the two strains provide ideal models for study genetic and epigenetic regulations in parasite biology. In this study, the proteomes of the extracellular stage of the two phenotypically different T. gondii strains, RH strain and ME49 strain, were systematically analyzed using an LC-MS/MS approach, and the global lysine crotonylation and 2-hydroxyisobutyrylation of the two parasite strains were deeply investigated. Global proteome, crotonylation and 2-hydroxyisobutyrylation of soluble proteins derived from phenotypically different T. gondii parasites (RH and ME49 strains) which were analyzed using label-free mass spectrometry. Three biological replicates of the two T. gondii strains were analyzed in each experiment in order to validate the biological reliability of measurements. Qualitative analysis and quantitative analysis were respectively carried out in different virulent strains. We applied a standard t test to determine if there is a statistically significant difference between the two T. gondii strains. Statistical Testing and generation of graphs was performed with R. Tachyzoites of the T. gondii RH and ME49 strains were purified from peritoneal fluid by passing through 5.0 μm Nucleopore filters and Percoll gradient centrifugation (GE Healthcare, Uppsala, Sweden). The parasite cells were washed using cold, sterile phosphate-buffered saline (PBS-1×). After addition of lysis buffer (1% protease inhibitor mixture, 8 m urea) to purified T. gondii cells, the cells were then sonicated three times on ice with a high-intensity sonicator (Scientz, Ningbo, China). To separate insoluble fragments, the lysate was centrifuged at 12,000 × g at 4 °C for 10 mins. Eventually, the supernatant in the centrifuge tube was collected, and the protein concentration was assayed using a BCA kit (Beyotime, Shanghai, China). Before trypsinization, dithiothreitol was added to the protein solution to a final concentration of 5 mm, and the solution was reduced at 56 °C for 30 mins; then, 11 mm iodoacetamide (Sigma, Saint Louis, MO) was used to alkylate the proteins for 15 mins at 37 °C in a darkroom. The urea concentration of the sample was diluted to less than 2 m with a 100 mm NH4HCO3 solution. Trypsin was added at a mass ratio of 1:50 (pancreatin/protein) for the first time and digested overnight. Then, trypsin was added again at a mass ratio of 1:100 (pancreatin/protein) and digested for 4 h. The tryptic peptides were separated by high-pH reversed-phase fractionation (RPF) with a Thermo Betasil C18 column (5 μm particles, 10 mm ID, 250 mm length). In the final step, peptides were divided into 60 fractions within 60 mins with a concentration gradient of 8% to 32% acetonitrile (pH 9.0) and then further divided into 4 fractions. Afterward, the peptides were subjected to vacuum freeze drying for in-depth analysis. The peptides were dissolved in an immunoprecipitation (IP) buffer solution (100 mm NaCl, 1 mm EDTA, 50 mm Tris-HCl, 0.5% NP-40, pH 8.0), and the supernatant was transferred to a pre-washed dihydroxyisobutyrylated and crotonylated resin (PTM-804 and PTM-502, Hangzhou, China). Then, the solution was placed on a rotary shaker at 4 °C, gently shaken and incubated overnight. After incubation, the resin was washed 4 times with the IP buffer solution and twice with deionized water. Finally, the resin-bound peptides were eluted three times with 0.1% trifluoroacetic acid eluate, and the eluate was collected and vacuum dried. After draining, a desalting operation was carried out according to the C18 ZipTips instructions, and the peptides were vacuum dried and prepared for LC-MS/MS analysis. The peptides were dissolved in liquid phase A (0.1% (v/v) aqueous formic acid) and separated using an EASY-nLC 1000 ultra-performance liquid chromatography (UPLC) system. Solvent B (0.1% formic acid in 98% acetonitrile) was run on the EASY-nLC 1000 UPLC system, maintaining a flow rate of 400 nL/min, and the gradient was increased from 6% to 23% in 26 mins, increased from 23% to 35% in 8 mins and increased to 80% in 3 mins, then maintained at 80% for the last 3 mins. Using UPLC to separate the peptides, they were ionized by a nanospray ionization (NSI) ion source (voltage was 2.0 kV) and finally analyzed using Q ExactiveTM HF-X mass spectrometry. The primary mass spectrometer scan range was 350–1,600 m/z (scan resolution set to 60,000), and the secondary scan resolution was up to 15,000. The top 20 peptides with the highest signal intensity were selected by the data-dependent scan (DDA) program to enter the higher-energy collisional dissociation (HCD) cells and then fragmented using 28% fragmentation energy after the first scan. Then, secondary mass spectrometry was performed in sequence. Automatic gain control (AGC) was set to 5E4. Secondary mass spectral data (repeated in three experiments) were retrieved using MaxQuant (v1.5.2.8). Tandem mass spectra were searched against the UniProtKB Toxoplasma gondii (strain ATCC 50611/Me49) database (ToxoDB 36, 8,315 sequences) concatenated with a reverse decoy database, and an anti-library was added to calculate the false positive rate (FDR) caused by random matching. The enzyme digestion mode was set to Trypsin/P, which allowed up to 4 missing cleavages. The mass error tolerances of the main search and first search were set to 5 ppm and 20 ppm, respectively. The mass error tolerance of the secondary fragment ions was 0.02 Da. For identification of crotonylation, carbamidomethyl on cysteine (Cys) was specified as fixed modification and lysine (Lys) crotonylation modification, oxidation on methionine (Met) and acetylation on protein N-terminal were specified as variable modifications. For identification of 2-hydroxyisobutylation, carbamidomethyl on Cys was specified as fixed modification and Lys 2-hydroxyisobutyrylation modification, oxidation on Met and acetylation on protein N-terminal were specified as variable modifications. The label-free quantification method was LFQ, FDR was set to < 1%, and the lowest score of modified peptides was adjusted to > 40. Protein lysate (20 μg) generated from the tachyzoites of both the RH and ME49 T. gondii strains was separated using 12% Bis-Tris polyacrylamide gels and then transferred to nitrocellulose filter membranes (Bio-Rad, Hercules, CA). Next, 5% skim milk using Tris-buffered saline solution with Tween (TBST) was used to block the membrane for 1 hour at 37 °C, and then the membrane was incubated with antibodies to crotonyllysine (Cat#: PTM-502) (1: 2000; PTM BIO) and 2-hydroxybutyryllysine (Cat#: PTM-801) (1: 1000; PTM BIO) at 4 °C overnight. The membrane was washed three times using TBST buffer before incubation with a secondary antibody at 37 °C for 1 hour (1:10,000; Thermo Scientific™ Pierce, 31430, 31460, MA). To detect modified proteins in the parasites, the tachyzoites of the T. gondii RH and T. gondii ME49 strains were fixed on slides using pre-chilled paraformaldehyde for 20 mins. The parasites were permeabilized by 0.1% Triton X-100 for 20 mins at room temperature. First, the slides were blocked with 5% skim milk for 1 hour at 37 °C and then incubated with primary antibodies recognizing crotonyllysine (Cat#: PTM-502) and 2-hydroxybutyryllysine (Cat#: PTM-801) (PTM BIO) for 1 hour at 37 °C. Then, the cells were incubated for 30 min at 37 °C using a fluorescent secondary antibody (Thermo, MA), and the nuclei were stained as previously described (17Wang W. Huang P. Jiang N. Lu H. Zhang D. Wang D. Zhang K. Wahlgren M. Chen Q. A thioredoxin homologous protein of Plasmodium falciparum participates in erythrocyte invasion.Infect Immun. 2018; 86Crossref Scopus (8) Google Scholar, 18Wang W. Liu F. Jiang N. Lu H. Yang N. Feng Y. Sang X. Cao Y. Chen Q. Plasmodium TatD-like DNase antibodies blocked parasite development in the mosquito gut.Front. Microbiol. 2018; 9: 1023Crossref PubMed Scopus (9) Google Scholar). High-resolution images were captured by a confocal laser scanning microscope (Leica, SP8, Wetzlar, Germany). Intensity of peptides in each sample was performed based on MS peak area, and the intensity of each protein was calculated by MaxQuant LFQ method. For PTM sites, intensity was sum of all the modified peptides containing this PTM site. The relative quantification of each sample was obtained based on the protein (PTM sites) intensity between different samples. Three replicates were performed in this study. The average intensity of three replicates was calculated to represent the overall intensity of the protein (PTM sites) in the sample. Two-sample two-tailed t test method was used to calculate p value of difference abundance in protein and PTM sites level. The spectra were first analyzed by MaxQuant software, and the proteome and modified groups were quantified using label-free quantitation. The quantitative value of RH/ME49 is the average of three replicate quantitative values. The difference significance (p value) was calculated by t test. When RH/ME49 had a fold change > 1.2 and p < 0.05, it was a differential protein. When RH/ME49 had a fold change >1.5 and p < 0.05, it was a differential modification site. When a protein was identified in three replicates of the RH strain and in none of the three replicates of the ME49 strain, it was an RH-specific expressed protein or a specific modification site. In the opposite scenario, it was a specific modification site of the ME49 strain. GO annotations were based on the UniProt-GOA database (www.http://www.ebi.ac.uk/GOA/). The identified protein IDs were matched and converted to the ID of the UniProt database and then mapped to the GO ID using the protein ID. When some of the identified proteins were not annotated with the UniProt-GOA database, the remaining proteins were annotated based on the protein sequence alignment method using InterProScan software. Proteins were classified according to the following three areas of GO annotation: cellular component, molecular function, and biological process. The KEGG database was used to annotate the proteins of relevant pathways. First, the proteins were annotated via the KEGG online service tool KAAS, and they were matched into the corresponding pathways using the KEGG mapper. The KOG annotation proteome was derived from the NCBI-COG database (https://www.ncbi.nlm.nih.gov/COG/). The sequences of differentially modified proteins were matched to the basic local alignment search tool (BLAST) version 2.2.28 KOG database to obtain protein KOG annotation information. Subcellular localization annotation of the submitted proteins was performed using the software wolfpsort (a new version of PSORT/PSORT II), which can be used to predict the subcellular localization of eukaryotic sequences. Motif-x software was used to analyze the motif features of the modified sites. Comparative analysis of a modified 21-mer sequence model consisting of all identified modification sites (ten sites upstream and ten sites downstream) was performed. When the number of peptides in a certain characteristic sequence was > 20 and the statistical test p value was < 0.000001, it was a motif of the modified peptides. For every category, a two-tailed Fisher's exact test was used to test the extent to which differentially modified proteins were enriched for all identified proteins according to the GO function annotation. In addition, a corrected p value < 0.05 was significantly enriched. The enrichment pathways were identified by a two-tailed Fisher exact test using the KEGG database to test the enrichment of differentially modified proteins for all identified proteins. If the corrected p value of the pathway enrichment was < 0.05, it was considered significant. All functional enriched p values were collected and then screened for functional classifications that were significantly enriched (p value < 0.05) in at least one group. Then, the obtained p value data matrix was first subjected to logarithmic transformation with -log10, which was subjected to Z transform for each function classification. Finally, the data were analyzed by hierarchical clustering (Euclidean distance, average-linkage clustering). The clustering relationship was visualized via the function pheatmap in the R language package. The network of protein-protein interactions was obtained from STRING database (19Szklarczyk D. Franceschini A. Kuhn M. Simonovic M. Roth A. Minguez P. Doerks T. Stark M. Muller J. Bork P. Jensen L.J. von Mering C. The STRING database in 2011: functional interaction networks of proteins, globally integrated and scored.Nucleic Acids Res. 2011; 39: D561-D568Crossref PubMed Scopus (2719) Google Scholar) and visualized using Cytoscape software (20Doerks T. Copley R.R. Schultz J. Ponting C.P. Bork P. Systematic identification of novel protein domain families associated with nuclear functions.Genome Res. 2002; 12: 47-56Crossref PubMed Scopus (462) Google Scholar). The soluble proteins obtained from both the T. gondii RH and ME49 strains were separately analyzed for LC-MS/MS identification with 3 replications (Fig. 1A). Pearson correlation of Log2 LFQ intensity between replications was 1, and 75% coefficient variation of three replications was less than 0.01 (supplemental Fig. S1). In total, 3527 and 3238 proteins were identified in the T. gondii RH and ME49 strains, respectively (Fig. 1B). A total of 2,855 proteins contained quantitative information (supplemental Data S3). Of these, 84 proteins were upregulated in the T. gondii RH strain, and 74 were upregulated in the T. gondii ME49 strain (Fig. 1C). The differentially expressed proteins between the two T. gondii strains were in mainly the nucleus, cytoplasm, plasma membrane, and mitochondria (Fig. 1D and 1E). The 84 upregulated proteins in RH strain T. gondii were significantly enriched in the endoplasmic reticulum, with transferase and oxidoreductase activities. In contrast, in the ME49 strain, the upregulated proteins were more diverse, ranging from ribosomal complex proteins to membrane bound and unbound proteins, which were highly enriched in functions associated with protein dimerization activity and organonitrogen biosynthetic process (supplemental Data S4). Protein modification in tachyzoites was confirmed by Western blotting and immunofluorescence assay (IFA) with anti-crotonyllysine and anti-2-hydroxybutyryllysine antibodies (Fig. 2A–2C). The proteins with 2-hydroxybutyryllysines were widely distributed inside multiple discrete compartments of the two T. gondii strains, whereas proteins with crotonyllysines were abundant in the nucleus (Fig. 2D and 2E). Modified peptides of both parasite strains were enriched with pan-antibodies specifically recognizing lysine residues with either crotonylation or 2-hydroxyisobutyrylation in three replicated experiments. Generally, the modification level of lysine 2-hydroxyisobutyrylation was significantly higher than that of crotonylation in T. gondii, irrespective of phenotype. In total, 1,061 and 984 proteins with 3,735 and 3,396 modified sites in RH and ME49 strain T. gondii, respectively, were crotonylated; 1,950 and 1,720 proteins with 9,502 and 8,092 sites in the two parasite strains were 2-hydroxyisobutyrylated, respectively (Fig. 1B, 3A and supplemental Data S5, S6). The number of proteins with mono-modification, either with crotonylation or 2-hydroxyisobutyrylation, was much less than that of proteins with multiple modifications. Further, 851 proteins (2,481 sites), accounting for 39.8% of all identified proteins, were modified by both crotonylation and 2-hydroxyisobutyrylation in the two parasite strains (supplemental Fig. S2). Gene Ontology (GO) analyses indicated that lysine crotonylation was significantly enriched in the categories of cytoplasm, macromolecular complexes, which were predominantly related