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.

Keywords: Parasite; Toxoplasma gondii; pathway analysis; phenotype; posttranslational modification; protein-protein interactions; proteogenomics; proteome; regulation; virulence.

Graphical abstract

Fig. 1.

Quantitative analysis differences in proteomes between the T. gondii RH strain and the ME49 strain. A, Schematic illustration of the proteomic analytical steps for protein purification, trypsinization, antibody-based enrichment, and bioinformatic analysis after LC-MS/MS. B, Summary of qualitative data identified in the phenotypically different T. gondii strains (supplemental Data S1, S2). C, Volcano plot of differentially expressed proteins in the two T. gondii strains. Magenta dots represent upregulated proteins in the T. gondii RH strain (RH/ME49 ratio > 1.2; t test, p < 0.05). Green dots represent upregulated proteins in the T. gondii ME49 strain (RH/ME49 ratio < 1/1.2; t test, p < 0.05). Detailed data are listed in supplemental Data S3. D, Pie charts showing upregulated proteins classified by localization in the T. gondii RH strain, excluding unknown proteins. Detailed data are listed in supplemental Data S4. E, Pie charts showing upregulated proteins classified by localization in the T. gondii ME49 strain, excluding unknown proteins. Detailed data are listed in supplemental Data S4.

Fig. 2.

Western blotting and immunofluorescence analysis of lysine crotonylation and 2-hydroxyisobutyrylation in T. gondii strains. A, SDS-PAGE analysis of tachyzoite lysates of the T. gondii RH and ME49 strains. B, and C, Western blot analysis of 20 μg of tachyzoite lysate probed with anti-crotonyllysine and anti-2-hydroxybutyryllysine antibodies. D, Immunofluorescence staining of paraformaldehyde-fixed tachyzoites of T. gondii RH and T. gondii ME49 with an anti-crotonyllysine antibody (red). Immunofluorescence staining of pre-chilled paraformaldehyde-fixed tachyzoites of T. gondii RH and T. gondii ME49 with an anti-2-hydroxybutyryllysine antibody (green). Nuclei were stained with DAPI (blue).

Fig. 3.

Qualitative analysis of lysine crotonylation and 2-hydroxyisobutyrylation in T. gondii. A, Venn diagram showing overlap between lysine crotonylated and 2-hydroxyisobutyrylated proteins in T. gondii strains. Ellipses in green and pink represent the numbers of crotonylated proteins and 2-hydroxyisobutyrylated proteins identified in RH strain T. gondii, respectively. Overlap regions represent the number of proteins with both modifications in the two parasite strains. Ellipses in blue and yellow represent the numbers of crotonylated proteins and 2-hydroxyisobutyrylated proteins identified in ME49 strain T. gondii, respectively. B, Visual presentation of lysine crotonylation and 2-hydroxyisobutyrylation based on subcellular location in the two T. gondii strains. The outermost circle represents the number of proteins identified. The proteins associated with subcellular location are displayed by heat maps (black frame). The circle (navy blue) represents the number of modification sites. The relative abundance of each protein within a given organelle is represented by the red histograms. Circles in light blue and orange represent the number of 2-hydroxyisobutyrylation sites and crotonylation sites, respectively. A deeper color represents higher enrichment of the modification. Note that the height of each bar reflects the subcellular organelle abundance of each protein or PTM site, i.e. longer bars represent a greater degree of abundance. C, KEGG pathway-based enrichment analysis of the crotonylated and 2-hydroxyisobutyrylated proteins in the T. gondii strains (p < 0.05). Detailed data are listed in supplemental Data S8.

Fig. 4.

Quantitative analysis of crotonylation and 2-hydroxyisobutyrylation in the phenotypically different T. gondii parasites. A, The differentially crotonylated and 2-hydroxyisobutyrylated proteins were classified based on subcellular location in T. gondii. Detailed data are listed in supplemental Data S13. B, The differentially crotonylated and 2-hydroxyisobutyrylated proteins in the phenotypically different T. gondii strains were further analyzed by the KOG (Eukaryotic Orthologous Groups) database. The abscissa is the content of each category of KOG, and the ordinate is the number of proteins (Fisher's exact test, p < 0.05). Detailed data are listed in supplemental Data S14. C, KEGG pathway-based enrichment analysis at the differential modification level of crotonylated and 2-hydroxyisobutyrylated proteins in T. gondii (Fisher's exact test, p < 0.05). Detailed data are listed in supplemental Data S16 and S17.

Fig. 5.

Overview of lysine crotonylation and 2-hydroxyisobutyrylation sites identified on histones of the two T. gondii strains. Green ellipses represent the lysine crotonylation sites. Purple ellipses represent the lysine 2-hydroxyisobutyrylation sites. Orange ellipses represent the lysine acetylation sites. Numbers in the middle of the sequences indicate the amino acid position on histon.

Fig. 6.

Lysine crotonylation and 2-hydroxyisobutyrylation of invasion-related proteins. Differential modification of key sites between the T. gondii RH and ME49 strains during invasion of host cells. 0 < Log2 of the RH/ME49 ratio < 2 represents upregulated modification of sites in the T. gondii RH strain. Log2 = 2 of the RH/ME49 ratio represents specific modification of sites in the T. gondii RH strain. −2 < Log2 of the RH/ME49 ratio < 0 represents upregulated modification of sites in the T. gondii ME49 strain. Log2 = −2 of the RH/ME49 ratio represents specific modification of sites in the T. gondii ME49 strain. Detailed data are listed in supplemental Data S19.

Fig. 7.

Overview of lysine crotonylation and 2-hydroxyisobutyrylation sites identified on micronemes of the two T. gondii strains. Green ellipses represent lysine crotonylation sites. Purple ellipses represent lysine 2-hydroxyisobutyrylation sites. C-terminal cleavage sites for AMA1, M2AP, MIC2, MIC6 and MIC8 are marked with arrows.