Abstract
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.
Highlights
Toxoplasma gondii is a strictly intracellular protozoan parasite, which can infect almost any warm-blooded animal and over two billion people, worldwide
Total proteins obtained from T. gondii tachyzoites of RH, PRU, and PYS strains were digested and phosphopeptides were enriched by TiO2
A total of 1,441 unique phosphopeptides, 1,250 non-redundant phosphorylation sites and 759 phosphoproteins were detected from T. gondii tachyzoites of all three strains (RH, PRU, and PYS) with FDR ≤ 0.01 in phosphopeptide level and phosphoRS probability ≥ 0.75 in phosphorylation site level
Summary
Toxoplasma gondii is a strictly intracellular protozoan parasite, which can infect almost any warm-blooded animal and over two billion people, worldwide. The three clonal lineages of T. gondii commonly found in Eurasia and North America (Type I, II, and II) vary greatly in terms of virulence and phenotypic plasticity (Howe and Sibley, 1995; Sibley and Ajioka, 2008; Sibley et al, 2009a; Khan et al, 2011; Pittman et al, 2014; Shwab et al, 2014). The type I strains are highly virulent for mice, but with limited ability to transform to bradyzoites (Sibley and Boothroyd, 1992; Saeij et al, 2005). Type III strains can transform to bradyzoites and induce chronic infection, but they rarely cause clinical illness in humans.
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