Distinct genotypic and pathogenic differences exist between Toxoplasma gondii genotypes. For example, genotype I is highly virulent, whereas genotype II and genotype III are less virulent. Moreover, Chinese 1 genotype (ToxoDB#9) is also virulent. Here, we compare the acetylomes of genotype 1 (RH strain) and Chinese 1 genotype (ToxoDB#9, PYS strain) of T. gondii. Using mass spectrometry enriched for acetylated peptides, we found a relationship between the levels of protein acetylation and parasite genotype-specific virulence. Notably, lysine acetylation was the largest (458 acetylated proteins) in RH strain, followed by PYS strain (188 acetylated proteins), whereas only 115 acetylated proteins were detected in PRU strain. Our analysis revealed four, three, and four motifs in RH strain, PRU strain and PYS strain, respectively. Three conserved sequences around acetylation sites, namely, xxxxxK^AcHxxxx, xxxxxK^AcFxxxx, and xxxxGK^AcSxxxx, were detected in the acetylome of the three strains. However, xxxxxK^AcNxxxx (asparagine) was found in RH and PYS strains but was absent in PRU strain. Our analysis also identified 15, 3, and 26 differentially expressed acetylated proteins in RH strain vs. PRU strain, PRU strain vs. PYS strain and PYS strain vs. RH strain, respectively. KEGG pathway analysis showed that a large proportion of the acetylated proteins are involved in metabolic processes. Pathways for the biosynthesis of secondary metabolites, biosynthesis of antibiotics and microbial metabolism in diverse environments were featured in the top five enriched pathways in all three strains. However, acetylated proteins from the virulent strains (RH and PYS) were more enriched in the pyruvate metabolism pathway compared to acetylated proteins from PRU strain. Increased levels of histone-acetyl-transferase and glycyl-tRNA synthase were detected in RH strain compared to PRU strain and PYS strain. Both enzymes play roles in stress tolerance and proliferation, key features in the parasite virulence. These findings reveal novel insight into the acetylomic profiles of major T. gondii genotypes and provide a new important resource for further investigations of the roles of the acetylated parasite proteins in the modulation of the host cell response to the infection of T. gondii.

We characterized the porcine tissue transcriptional landscapes that follow Toxoplasma gondii infection. RNAs were isolated from liver, spleen, cerebral cortex, lung, and mesenteric lymph nodes (MLNs) of T. gondii-infected and uninfected (control) pigs at days 6 and 18 postinfection, and were analyzed using next-generation sequencing (RNA-seq). T. gondii altered the expression of 178, 476, 199, 201, and 362 transcripts at 6 dpi and 217, 223, 347, 119, and 161 at 18 dpi in the infected brain, liver, lung, MLNs and spleen, respectively. The differentially expressed transcripts (DETs) were grouped into five expression patterns and 10 sub-clusters. Gene Ontology enrichment and pathway analysis revealed that immune-related genes dominated the overall transcriptomic signature and that metabolic processes, such as steroid biosynthesis, and metabolism of lipid and carboxylic acid, were downregulated in infected tissues. Co-expression network analysis identified transcriptional modules associated with host immune response to infection. These findings not only show how T. gondii infection alters porcine transcriptome in a tissue-specific manner, but also offer a gateway for testing new hypotheses regarding human response to T. gondii infection.

Cysticercus pisiformis is a kind of tapeworm larvae of Taenia pisiformis, which parasitizes the liver envelope, omentum, mesentery, and rectum of rodents such as rabbits. Cysteine protease inhibitors derived from helminth were immunoregulatory molecules of intermediate hosts and had an immunomodulatory function that regulates the production of inflammatory factors. Thus, in the present research, the recombinant Stefin of C. pisiformis was confirmed to have the potential to fight inflammation in LPS-Mediated RAW264.7 murine macrophages. CCK8 test showed that rCpStefin below 50 μg/mL concentration did not affect cellular viability. Moreover, the NO production level determined by the Griess test was decreased. In addition, the secretion levels of IL-1β, IL-6, and TNF-α as measured by ELISA were decreased. Furthermore, it exerted anti-inflammatory activity by decreasing the production of proinflammatory cytokines and proinflammatory mediators, including IL-1β, IL-6, TNF-α, iNOS, and COX-2 at the gene transcription level, as measured by qRT-PCR. Therefore, Type I cystatin derived from C. pisiformis suppresses the LPS-Mediated inflammatory response of the intermediate host and is a potential candidate for the treatment of inflammatory diseases.

Toxoplasma gondii transmits through various routes, rapidly proliferates during acute infection and causes toxoplasmosis, which is an important zoonotic disease in human and veterinary medicine. T. gondii can produce nitric oxide and derivatives, and S-nitrosylation contributes to their signaling transduction and post-translation regulation. To date, the S-nitrosylation proteome of T. gondii remains mystery. In this study, we reported the first S-nitrosylated proteome of T. gondii using mass spectrometry in combination with resin-assisted enrichment. We found that 637 proteins were S-nitrosylated, more than half of which were localized in the nucleus or cytoplasm. Motif analysis identified seven motifs. Of these motifs, five and two contained lysine and isoleucine, respectively. Gene Ontology enrichment revealed that S-nitrosylated proteins were primarily located in the inner membrane of mitochondria and other organelles. These S-nitrosylated proteins participated in diverse biological and metabolic processes, including organic acid binding, carboxylic acid binding ribose and phosphate biosynthesis. T. gondii S-nitrosylated proteins significantly contributed to glycolysis/gluconeogenesis and aminoacyl-tRNA biosynthesis. Moreover, 27 ribosomal proteins and 11 microneme proteins were identified as S-nitrosylated proteins, suggesting that proteins in the ribosome and microneme were predominantly S-nitrosylated. Protein–protein interaction analysis identified three subnetworks with high-relevancy ribosome, RNA transport and chaperonin complex components. These results imply that S-nitrosylated proteins of T. gondii are associated with protein translation in the ribosome, gene transcription, invasion and proliferation of T. gondii. Our research is the first to identify the S-nitrosylated proteomic profile of T. gondii and will provide direction to the ongoing investigation of the functions of S-nitrosylated proteins in T. gondii.

Toxoplasma gondii is an obligate intracellular protozoan of severe threat to humans and livestock, whose life history harbors both gamic and apogamic stages. Chinese 1 (ToxoDB#9) was a preponderant genotype epidemic in food-derived animals and humans in China, with a different pathogenesis from the strains from the other nations of the world. Posttranslational modifications (PTMs) of proteins were critical mediators of the biology, developmental transforms, and pathogenesis of protozoan parasites. The phosphoprotein profiling and the difference between the developmental phases of T. gondii, contributing to development and infectivity, remain unknown. A quantitative phosphoproteomic approach using IBT integrated with TiO₂ affinity chromatography was applied to identify and analyze the difference in the phosphoproteomes between the sporulated oocysts and the tachyzoites of the virulent ToxoDB#9 (PYS) strain of T. gondii. A total of 4058 differential phosphopeptides, consisting of 2597 upregulated and 1461 downregulated phosphopeptides, were characterized between sporulated the oocysts and tachyzoites. Twenty-one motifs extracted from the upregulated phosphopeptides contained 19 serine motifs and 2 threonine motifs (GxxTP and TP), whereas 16 motifs identified from downregulated phosphopeptides included 13 serine motifs and 3 threonine motifs (KxxT, RxxT, and TP). Beyond the traditional kinases, some infrequent classes of kinases, including Ab1, EGFR, INSR, Jak, Src and Syk, were found to be corresponding to motifs from the upregulated and downregulated phosphopeptides. Remarkable functional properties of the differentially expressed phosphoproteins were discovered by GO analysis, KEGG pathway analysis, and STRING analysis. S8GFS8 (DNMT1-RFD domain-containing protein) and S8F5G5 (Histone kinase SNF1) were the two most connected peptides in the kinase-associated network. Out of these, phosphorylated modifications in histone kinase SNF1 have functioned in mitosis and interphase of T. gondii, as well as in the regulation of gene expression relevant to differentiation. Our study discovered a remarkable difference in the abundance of phosphopeptides between the sporulated oocysts and tachyzoites of the virulent ToxoDB#9 (PYS) strain of T. gondii, which may provide a new resource for understanding stage-specific differences in PTMs and may enhance the illustration of the regulatory mechanisms contributing to the development and infectivity of T. gondii.

In this study, the differences in the phosphoproteomic landscape of sporulated oocysts between virulent and avirulent strains of Toxoplasma gondii were examined using a global phosphoproteomics approach. Phosphopeptides from sporulated oocysts of the virulent PYS strain (Chinese ToxoDB#9) and the avirulent PRU strain (type II) were enriched by titanium dioxide (TiO₂) affinity chromatography and quantified using IBT approach. A total of 10,645 unique phosphopeptides, 8181 nonredundant phosphorylation sites and 2792 phosphoproteins were identified. We also detected 4129 differentially expressed phosphopeptides (DEPs) between sporulated oocysts of PYS strain and PRU strain (|log1.5 fold change| > 1 and p < 0.05), including 2485 upregulated and 1644 downregulated phosphopeptides. Motif analysis identified 24 motifs from the upregulated phosphorylated peptides including 22 serine motifs and two threonine motifs (TPE and TP), and 15 motifs from the downregulated phosphorylated peptides including 12 serine motifs and three threonine motifs (TP, RxxT and KxxT) in PYS strain when comparing PYS strain to PRU strain. Several kinases were consistent with motifs of overrepresented phosphopeptides, such as PKA, PKG, CKII, IKK, MAPK, EGFR, INSR, Jak, Syk, Src, Ab1. GO enrichment, KEGG pathway analysis and STRING analysis revealed DEPs significantly enriched in many biological processes and pathways. Kinase related network analysis showed that AGC kinase was the most connected kinase peptide. Our findings reveal significant difference in phosphopeptide profiles of sporulated oocysts between virulent and avirulent T. gondii strains, providing new resources for further elucidation of the mechanisms underpinning the virulence of T. gondii.