Abstract
The invasion and egress are two key steps in lytic cycle vital to the propagation of Toxoplasma gondii infection, and phosphorylation is believed to play important roles in these processes. However, the phosphoproteome of T. gondii at these two stages has not been characterized. In this study, we profiled the phosphoproteome of tachyzoites at the stages of “just invading” (JI) and “prior to egress” (PE) based on iTRAQ quantitative analysis, in which a total of 46 phosphopeptides, 42 phosphorylation sites, and 38 phosphoproteins were detected. In the comparison of PE vs. JI, 10 phosphoproteins were detected with their phosphorylation level significantly changed, and four of them were demonstrated to be significantly down-regulated at the transcriptional level. Bioinformatic analysis of these identified phosphoproteins suggested that phosphorylation-mediated modulation of protein function was employed to regulate the pathway of toxoplasmosis and metabolism and cellular processes correlated with tachyzoite’s binding, location, and metabolism, and thus play vital roles in the parasite lytic cycle. Moreover, cytoskeletal network (CN)-associated Inner Membrane Complex (IMC1, IMC4, IMC6 and IMC12), Intravascular Network (IVN)-related GRAs (GRA2, GRA3, GRA7 and GRA12), and Parasitophorous Vacuole Membrane (PVM)-localized ROP5 were shown to be enriched at the central nodes in the protein interaction network generated by bioinformatic analysis, in which the phosphorylation level of IMC4, GRA2, GRA3, and GRA12 were found to be significantly regulated. This study revealed the main cellular processes and key phosphoproteins crucial for the invasion and egress of T. gondii, which will provide new insights into the developmental biology of T. gondii in vitro and contribute to the understanding of pathogen-host interaction from the parasite perspective.
Highlights
Toxoplasma gondii is an obligate intracellular apicomplexan parasite that chronically infects approximately one-third of the world’s human population, and the toxoplasmosis caused by its infection has been regarded as one of the major neglected parasitic infections (Hotez, 2014; Wei et al, 2016)
All six samples were labeled with iTRAQ reagents, and the phosphopeptides were enriched by TiO2, which were analyzed with LC-MS/MS
A total of 46 phosphopeptides matching to 38 phosphoproteins were identified with a falsediscovery rate (FDR) ≤0.01 in phosphopeptide level, and 42 phosphorylation sites were detected with phosphoRS probability ≥0.75 in phosphorylation site level (Figure 2A)
Summary
Toxoplasma gondii is an obligate intracellular apicomplexan parasite that chronically infects approximately one-third of the world’s human population, and the toxoplasmosis caused by its infection has been regarded as one of the major neglected parasitic infections (Hotez, 2014; Wei et al, 2016). Most infections in healthy people do not show obvious clinical symptoms. Severe complications, such as encephalitis and eye disease, even death can be caused by the infection of T. gondii in immunocompromised patients (Weiss and Dubey, 2009). As an obligate intracellular parasite, the successful invasion and egress of T. gondii from its host cell are critical for survival, dissemination and transmission, and are believed to be essential for the propagation of parasite infection (Lavine and Arrizabalaga, 2007; Hortua Triana et al, 2018). Only a few phosphorylated proteins of T. gondii have been identified, and the quantitative phosphoproteomic analysis of T. gondii at the different lytic cycle stages is very few
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