iTRAQ-based phosphoproteomic analysis reveals host cell's specific responses to Toxoplasma gondii at the phases of invasion and prior to egress

https://doi.org/10.1016/j.bbapap.2018.12.004Get rights and content

Highlights

  • A phosphoproteomic analysis based on iTRAQ was performed on host cells infected with T. gondii for 30 min and 28 h.

  • Significantly regulated phosphoproteins were identified on comparing between-groups of uninfected, 30 min, and 28 h infection.

  • Cytoskeleton and membrane changes were emphasized at 30 m infection and the cell cycle regulation was enriched at 28 h infection.

  • Host cell biological processes were extensively regulated, and dominated differently at various phases of infection.

Abstract

Protein phosphorylation plays a key role in host cell-T. gondii interaction. However, the phosphoproteome data of host cell at various phases of T. gondii infection has not been thoroughly described. In this study, we assessed the host phosphoproteome data with isobaric tags for relative and absolute quantification (iTRAQ) method during the phases of T. gondii invasion (30 min post infection, PI) and prior to egress (28 h PI). Our iTRAQ analysis revealed a total of 665 phosphoproteins, among which the significantly regulated phosphoproteins in different between-group comparisons were further analyzed. Functional analysis of these significantly regulated phosphoproteins suggested that T. gondii modulated host cell processes through phosphorylation including cell cycle regulation, inducing apoptosis, blocking the synthesis of some inflammatory factors, mediating metabolism to support its proliferation at the infection phase prior to egress, and utilizing membrane and energy from host cell, reorganizing cytoskeleton to favor its invasion and PV formation at the phase of invasion. The phosphorylation level of Smad2, CTNNA1, and HSPB1 identified with western blot revealed a consistent trend of change with iTRAQ result. These newly identified and significantly regulated phosphoproteins from our phosphoproteome data may provide new clues to unravel the host cell's complex reaction against T. gondii infection and the interaction between the host cell and T. gondii.

Introduction

Toxoplasma gondii is an intracellular apicomplexan parasite that can infect almost all types of nucleated cells of warm-blooded vertebrates [1]. About one-third of the world's population is chronically infected with this parasite [2], and toxoplasmosis has been regarded as a major neglected parasitic infection world-wide [3]. As an obligate intracellular parasite, the three basic steps of invasion, replication, and egress are involved in the T. gondii infective cycle, and these steps are critical for its survival, dissemination, and transmission [4,5].

Protein phosphorylation is a reversible process that involves phosphate being esterified to amino acids by protein kinases, and it is estimated to affect approximately one-third of all proteins in eukaryotic cells at any given time [[6], [7], [8]]. Phosphorylation is considered to be a key regulatory post-translational protein modification, which often leads to structural changes in the protein that can directly switch the protein activity on or off, thereby inducing changes in its interacting molecules or subcellular localization [9]. For example, T. gondii infection alone can induce sustained host cell STAT1 phosphorylation and mediate its translocation from the cytosol to the nucleus [10,11]. In addition, ROP18 of virulent T. gondii strains can bind to and directly phosphorylate host cell Irga6 and Irgb6, as a result to prevent their accumulation on the parasitophorous vacuole (PV) membrane, thereby blocking the clearance of parasites [12,13]. Protein phosphorylation and dephosphorylation are also important processes of complex cellular signal sensing and transduction networks. Complex host cell signal activation/deactivation is regulated by the phosphorylation of phosphoproteins, which may be exploited by T. gondii for successful infection. For example, T. gondii infection leads to Bad phosphorylation via the PI3K-PKB/Akt pathway to prevent host cell apoptosis [14].

Qualitative and quantitative proteomic analysis has been applied to identify T. gondii proteins [[15], [16], [17], [18], [19], [20]] and are extensively used for host protein profiling [[21], [22], [23]] to characterize the key parasitic and host factors functioning in host-pathogen interaction. Although proteomics has been used extensively to identify and characterize both T. gondii and host cell proteins, not much is known about the phosphorylation modification of the host proteins involved in cell signaling occurring in various phases of infection, when T. gondii are cultured in vitro.

This current study focused on the different host cell responses to T. gondii infection in the process of invasion (i.e. 30 min PI) and prior to egress (i.e. 28 h PI). At 30 min PI, most of the T. gondii tachyzoites just invade the host cell and the parasitophorous vacuoles (PVs) are just formed, representing the early phase of infection; while at 28 h PI, the tachyzoites have been thoroughly proliferated and are ready to egress from the host cells, representing the late phase of infection [24]. This current study will be helpful to elucidate the host cell reactions at different phases of T. gondii infection, and may favor to advance our understanding to unravel the complicated molecular mechanism of host-parasite interaction in T. gondii infection.

Section snippets

Culture of HFF cells and T. gondii tachyzoites

HFF cells were purchased from the American Type Culture Collection (ATCC) and cultured in complete Dulbecco's modified Eagle's medium (DMEM, Invitrogen) supplemented with 10% fetal calf serum (FBS; Gibco) and 100 μg/mL gentamicin. T. gondii RH strain tachyzoites were cultured in HFF cells supplemented with 1% FBS and 100 μg/mL gentamicin.

HFF cell infection with T. gondii and preparation of samples

The HFF cells were infected with T. gondii RH tachyzoites with a multiplicity of infection of 3 (MOI = 3) for 30 min (i.e., early-stage infection [EI]) and

Phosphoproteomic identification and characterization of uninfected HFF cells and those infected with T. gondii tachyzoites

All eight samples were labeled with iTRAQ reagents. The phosphorylated peptides from these labeled samples were enriched using TiO2 and identified with LC-MS/MS. The workflow of this study is presented in Fig. 1. As a result, a total of 1207 phosphopeptides matching to 665 phosphoproteins were identified, and 1031 phosphorylation sites were detected (Fig. 2A). Among the phosphorylation sites, phospho-Serine (pS) was the most abundant and accounted for 85.35% of all the phosphorylated amino

Discussion

The cell lytic cycle of T. gondii tachyzoites consists of invasion, replication and egress [4]. However, the phosphoproteome data of host cell during the infection phases of T. gondii invasion and prior to egress have not been identified yet.

Tachyzoites of the T. gondii RH strain reside in the PVs, which are formed during the invasion and grows to full vacuoles for about 28 h after invading the host cell [24]. Therefore, the infection time points of 30 min and 28 h represent two significantly

Conclusions

In summary, the phosphoproteomic data of HFF cell at the infective phase of T. gondii just invading and prior to egress is reported here, which supplemented the phosphoproteome data of host cells infected by T. gondii at different infective phases. During prior to egress phase, T. gondii may regulate the host cellular processes to favor its proliferation, including regulating the cell cycle through TGF-β/smad pathway, inducing the host apoptosis by phosphorylating lamin, and mediating host cell

Conflict of interest

This submission is not under review at any other publication, in whole or in part, and all the authors listed have approved the enclosed manuscript. All authors declare no financial conflict of interest and have fulfilled the criteria of authorship for the manuscript.

Acknowledgement

This research is supported by the funding of National Key R&D program of China (2017YFD0500400), National Natural Science Foundation of China (81772217, 20180907, 81572012), Guangdong Provincial Natural Science Foundation Project (2016A030311025, 2017A030313694), Science and Technology Planning Project of Guangdong Province (2018A050506038) and Guangzhou health and medical collaborative innovation major special project (201604020011) to HJP.

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