Phosphorylation of Toxoplasma gondii Secreted Proteins during Acute and Chronic Stages of Infection

Abstract

The intracellular parasite Toxoplasma gondii resides within a membrane-bound parasitophorous vacuole (PV) and secretes an array of proteins to establish this replicative niche. It has been shown previously that Toxoplasma secretes kinases and that numerous proteins are phosphorylated after secretion. Here, we assess the role of the phosphorylation of strand-forming protein 1 (SFP1) and the related protein GRA29, two secreted proteins with unknown function. We show that both proteins form stranded structures in the PV that are independent of the previously described intravacuolar network or actin. SFP1 and GRA29 can each form these structures independently of other Toxoplasma secreted proteins, although GRA29 appears to regulate SFP1 strands. We show that an unstructured region at the C termini of SFP1 and GRA29 is required for the formation of strands and that mimicking the phosphorylation of this domain of SFP1 negatively regulates strand development. When tachyzoites convert to chronic-stage bradyzoites, both proteins show a dispersed localization throughout the cyst matrix. Many secreted proteins are reported to dynamically redistribute as the cyst forms, and secreted kinases are known to play a role in cyst formation. Using quantitative phosphoproteome and proteome analyses comparing tachyzoite and early bradyzoite stages, we reveal widespread differential phosphorylation of secreted proteins. While we found no direct evidence for phosphorylation playing a dominant role for SFP1/GRA29 redistribution in the cyst, these data support a model in which secreted kinases and phosphatases contribute to the regulation of secreted proteins during stage conversion. IMPORTANCE Toxoplasma gondii is a common parasite that infects up to one-third of the human population. Initially, the parasite grows rapidly, infecting and destroying cells of the host, but subsequently switches to a slow-growing form and establishes chronic infection. In both stages, the parasite lives within a membrane-bound vacuole within the host cell, but in the chronic stage, a durable cyst wall is synthesized, which provides protection to the parasite during transmission to a new host. Toxoplasma secretes proteins into the vacuole to build its replicative niche, and previous studies identified many of these proteins as phosphorylated. We investigate two secreted proteins and show that a phosphorylated region plays an important role in their regulation in acute stages. We also observed widespread phosphorylation of secreted proteins when parasites convert from acute to chronic stages, providing new insight into how the cyst wall may be dynamically regulated.

Lessons from Toxoplasma: Host responses that mediate parasite control and the microbial effectors that subvert them

“…While long considered to be inert, it is now recognized that bradyzoites undergo episodic bursts of proliferation, internalize host-derived macromolecules, and export parasite effectors (Kannan et al, 2021;Watts et al, 2015). The cyst wall is an important interface between host and parasite that sits just below the PVM (Guevara et al, 2020;Tu et al, 2020;Young et al, 2020), and not every protein that can escape from the PVM in tachyzoites can cross the cyst wall (Krishnamurthy and Saeij, 2018). Nevertheless, the ability of some effectors to cross the cyst wall indicates the presence of a transport mechanism (Mayoral et al, 2020a;Paredes-Santos et al, 2019;Seizova et al, 2019 Preprint;Tomita et al, 2021).…”

Toxoplasma gondii PPM3C, a secreted protein phosphatase, affects parasitophorous vacuole effector export

“…Although we did not find evidence for dysregulated GRA2 activity by inspecting the IVN of vacuoles formed by the ΔTgPPM3C strain ( S2 Fig ), we cannot exclude the possibility that abnormalities in ΔTgPPM3C parasitophorous vacuole architecture may be occurring at a different period during tachyzoite development or may be more evident in tissue cysts. We note that phosphopeptides from two recently characterized vacuolar strand-forming proteins, GRA29 and SFP1 [ 45 ], were detected as significantly more abundant in ΔTgPPM3C cultures, suggesting that these proteins may be substrates of TgPPM3C that are regulated by dephosphorylation events within the vacuolar compartment. Although Young et.…”

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

“…The quantitative phosphoproteomic analysis between the tachyzoite and early bradyzoite identified 7650 phosphosites on 2235 T. gondii proteins, and 144 phosphorylation sites and 193 phosphorylation sites on 170 proteins were deemed to be differentially altered in the tachyzoite and bradyzoite, respectively. Out of these, 51 phosphorylated proteins were predictively secretory proteins [ 46 ]. He et al compared the phosphoproteome of initially penetrating tachyzoites and pre-egressed tachyzoites, employing an iTRAQ-based quantitative approach.…”