Comparative Phosphoproteomic Analysis of Sporulated Oocysts and Tachyzoites of Toxoplasma gondii Reveals Stage-Specific Patterns.

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.

Global phosphoproteome analysis reveals significant differences between sporulated oocysts of virulent and avirulent strains of Toxoplasma 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.

Generation and application of a monoclonal antibody against the 18-kDa oncosphere antigen of Taenia pisiformis.

Taenia pisiformis is a parasite that causes cysticercosis pisiformis, which has acquired economic relevance because of its effects on animal welfare and production. A useful assay for the detection of T. pisiformis is needed for the prevention of cysticercosis pisiformis and control of the parasite. The 18-kDa oncosphere antigen is expressed in the oncosphere of several cysticerci in species of the genus Taenia, including T. pisiformis. This protein plays an important role in tissue invasion and has extensive applications in diagnosis. In this study, the T. pisiformis 18-kDa oncosphere antigen (TPO18) was expressed in soluble form and successfully purified for use in the production of monoclonal antibodies (MAbs) against TPO18. Twenty hybridomas were obtained using ELISA, and the subcloning process identified three positive hybridoma cell lines, which were designated as 4E8, 5G5, and 7E8. MAb 7E8 exhibited the highest titer and had an IgG2b heavy chain and a kappa light chain. Western blot analysis demonstrated that MAb 7E8 reacted with GST-TPO18. Immunohistochemistry showed that TPO18 was widely distributed in the drape and wall of uteri in adults of T. pisiformis adults and in the fibrous layer of the sucker and cyst cavity of T. pisiformis cysticerci. This research will provide a foundation for the development of diagnostic tools and will contribute to a better understanding of the functions of TPO18.

Acetylome analysis of the feline small intestine following Toxoplasma gondii infection.

Toxoplasma gondii is a protozoan parasite capable of infecting a large number of warm-blooded animals and causes serious health complications in immunocompromised patients. T. gondii infection of the feline small intestine is critical for the completion of the life cycle and transmission of T. gondii. Protein acetylation is an important posttranslational modification, which plays roles in the regulation of various cellular processes. Therefore, understanding of how T. gondii reprograms the protein acetylation status of feline definitive host can help to thwart the production and spread of T. gondii. Here, we used affinity enrichment and high-resolution liquid chromatography with tandem mass spectrometry to profile the alterations of the acetylome in cat small intestine 10 days after infection by T. gondii Prugniuad (Pru) strain. Our analysis showed that T. gondii induced significant changes in the acetylation of proteins in the cat intestine. We identified 2606 unique lysine acetylation sites in 1357 acetylated proteins. The levels of 334 acetylated peptides were downregulated, while the levels of 82 acetylated peptides were increased in the infected small intestine. The proteins with differentially acetylated peptides were particularly enriched in the bioenergetics-related processes, such as tricarboxylic acid cycle, oxidative phosphorylation, and oxidation-reduction. These results provide the first baseline of the global acetylome of feline small intestine following T. gondii infection and should facilitate further analysis of the role of acetylated protein in the pathogenesis of T. gondii infection in its definitive host.

Calcium formate assisted catalytic pyrolysis of pine for enhanced production of monocyclic aromatic hydrocarbons over bimetal-modified HZSM-5.

An efficient process was developed to selectively produce monocyclic aromatic hydrocarbons (MAHs) from ex-situ catalytic fast pyrolysis (CFP) of pine assisted with calcium formate (CF) over bimetal-modified HZSM-5. Mo and another metal (Mg, Ga or Zn) were used to modify the HZSM-5, and the as-synthesized bimetal-modified HZSM-5 catalysts were utilized for both pyrolysis-gas chromatography/mass spectrometry (Py-GC/MS) and lab-scale CFP tests with CF as a hydrogen donor to selectively obtain MAHs. The results revealed that the presence of CF and Mg-Mo modified HZSM-5 (0.5Mg1Mo/HZ) exhibited excellent capability for MAHs production with tiny generation of polycyclic aromatic hydrocarbons (PAHs). The maximum MAHs yield attained 12.79 wt% at 650 °C from Py-GC/MS with the CF-to-pine (CF-to-PN) ratio of 3 and catalyst-to-pine (CA-to-PN) ratio of 11, and became 9.67 wt% from lab-scale device with CF-to-PN and CA-to-PN ratios of 0.5 and 4, respectively. In addition, compared with HZSM-5, 0.5Mg1Mo/HZ possessed better anti-deactivation ability.

Label-Free Quantitative Acetylome Analysis Reveals Toxoplasma gondii Genotype-Specific Acetylomic Signatures.

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, xxxxxKAcHxxxx, xxxxxKAcFxxxx, and xxxxGKAcSxxxx, were detected in the acetylome of the three strains. However, xxxxxKAcNxxxx (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.

iTRAQ-Based Global Phosphoproteomics Reveals Novel Molecular Differences Between Toxoplasma gondii Strains of Different Genotypes.

To gain insights into differences in the virulence among T. gondii strains at the post-translational level, we conducted a quantitative analysis of the phosphoproteome profile of T. gondii strains belonging to three different genotypes. Phosphopeptides from three strains, type I (RH strain), type II (PRU strain) and ToxoDB#9 (PYS strain), were enriched by titanium dioxide (TiO2) affinity chromatography and quantified using iTRAQ technology. A total of 1,441 phosphopeptides, 1,250 phosphorylation sites and 759 phosphoproteins were detected. In addition, 392, 298, and 436 differentially expressed phosphoproteins (DEPs) were identified in RH strain when comparing RH/PRU strains, in PRU strain when comparing PRU/PYS strains, and in PYS strain when comparing PYS/RH strains, respectively. Functional characterization of the DEPs using GO, KEGG, and STRING analyses revealed marked differences between the three strains. In silico kinase substrate motif analysis of the DEPs revealed three (RxxS, SxxE, and SxxxE), three (RxxS, SxxE, and SP), and five (SxxE, SP, SxE, LxRxxS, and RxxS) motifs in RH strain when comparing RH/PRU strains, in PRU strain when comparing PRU/PYS, and in PYS strain when comparing PYS/RH strains, respectively. This suggests that multiple overrepresented protein kinases including PKA, PKG, CKII, IKK, and MAPK could be involved in such a difference between T. gondii strains. Kinase associated network analysis showed that ROP5, ROP16, and cell-cycle-associated protein kinase CDK were the most connected kinase peptides. Our data reveal significant changes in the abundance of phosphoproteins between T. gondii genotypes, which explain some of the mechanisms that contribute to the virulence heterogeneity of this parasite.

Global Transcriptome Profiling of Multiple Porcine Organs Reveals Toxoplasma gondii-Induced Transcriptional Landscapes.

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.

Comparative proteomic analysis of virulent and avirulent strains of Toxoplasma gondii reveals strain-specific patterns.

Research exploring the proteome of Toxoplasma gondii oocysts has gained momentum over the past few years. However, little is known about the oocyst's protein repertoires that contribute to differential virulence among T. gondii strains. Here, we used isobaric tag for relative and absolute quantitation-based proteomic analysis of oocysts of two T. gondii strains exhibiting the virulent PYS (ToxoDB#9) phenotype versus the less virulent PRU (Type II, ToxoDB#1) phenotype. Our aim was to determine protein expression patterns that contribute to the virulence of a particular phenotype. A total of 2,551 proteins were identified, of which 374 were differentially expressed proteins (DEPs) (|log2 fold change| ≥ 0.58 and P < 0.05). DEPs included 192 increased and 182 decreased proteins. Gene Ontology and KEGG pathway analyses revealed a large number of DEPs enriched in various metabolic processes. Protein interaction network analysis using STRING identified inosine monophosphate dehydrogenase (IMPDH), Bifunctional GMP synthase/glutamine amidotransferase protein, Glucose-6-phosphate 1-dehydrogenase, and Citrate synthase as the top four hubs. Of the 22 virulence proteins commonly expressed in the oocysts of the two strains, 13 and 2 proteins were increased in PYS strain and PRU strain, respectively. Also, 10 and 3 of the 22 identified oocyst wall proteins showed higher expression in oocysts of PRU strain and PYS strain, respectively. These findings revealed new proteomic differences in the oocysts of T. gondii strains of different genotypic backgrounds.

Proteomic Differences between Developmental Stages of Toxoplasma gondii Revealed by iTRAQ-Based Quantitative Proteomics.

Toxoplasma gondii has a complex two-host life-cycle between intermediate host and definitive host. Understanding proteomic variations across the life-cycle stages of T. gondii may improve the understanding of molecular adaption mechanism of T. gondii across life-cycle stages, and should have implications for the development of new treatment and prevention interventions against T. gondii infection. Here, we utilized LC-MS/MS coupled with iTRAQ labeling technology to identify differentially expressed proteins (DEPs) specific to tachyzoite (T), bradyzoites-containing cyst (C) and sporulated oocyst (O) stages of the cyst-forming T. gondii Prugniuad (Pru) strain. A total of 6285 proteins were identified in the three developmental stages of T. gondii. Our analysis also revealed 875, 656, and 538 DEPs in O vs. T, T vs. C, and C vs. O, respectively. The up- and down-regulated proteins were analyzed by Gene Ontology enrichment, KEGG pathway and STRING analyses. Some virulence-related factors and ribosomal proteins exhibited distinct expression patterns across the life-cycle stages. The virulence factors expressed in sporulated oocysts and the number of up-regulated virulence factors in the cyst stage were about twice as many as in tachyzoites. Of the 79 ribosomal proteins identified in T. gondii, the number of up-regulated ribosomal proteins was 33 and 46 in sporulated oocysts and cysts, respectively, compared with tachyzoites. These results support the hypothesis that oocyst and cystic stages are able to adapt to adverse environmental conditions and selection pressures induced by the host's immune response, respectively. These findings have important implications for understanding of the developmental biology of T. gondii, which may facilitate the discovery of novel therapeutic targets to better control toxoplasmosis.