Abstract
The intracellular parasite Toxoplasma gondii infects a large proportion of humans worldwide and can cause adverse complications in the settings of immune-compromise and pregnancy. T. gondii thrives within many different cell types due in part to its residence within a specialized and heavily modified compartment in which the parasite divides, termed the parasitophorous vacuole. Within this vacuole, numerous proteins optimize intracellular survival following their secretion by the parasite. We investigated the contribution of one of these proteins, TgPPM3C, predicted to contain a PP2C-class serine/threonine phosphatase domain and previously shown to interact with the protein MYR1, an essential component of a putative vacuolar translocon that mediates effector export into the host cell. Parasites lacking the TgPPM3C gene exhibit a minor growth defect in vitro, are avirulent during acute infection in mice, and form fewer cysts in mouse brain during chronic infection. Phosphoproteomic assessment of TgPPM3C deleted parasite cultures demonstrated alterations in the phosphorylation status of many secreted vacuolar proteins including two exported effector proteins, GRA16 and GRA28, as well as MYR1. Parasites lacking TgPPM3C are defective in GRA16 and GRA28 export, but not in the export of other MYR1-dependant effectors. Phosphomimetic mutation of two GRA16 serine residues results in export defects, suggesting that de-phosphorylation is a critical step in the process of GRA16 export. These findings provide another example of the emerging role of phosphatases in regulating the complex environment of the T. gondii parasitophorous vacuole and influencing the export of specific effector proteins from the vacuolar lumen into the host cell.
Highlights
A large proportion of the human population, varying by region, is predicted to be infected with the parasite Toxoplasma gondii based on seropositivity, posing a life-threatening risk in immune-compromised individuals [1]
Mutations mimicking the phosphorylation of two GRA16 serine residues results in GRA16 export defects in wild type parasites, though importantly not in ΔTgPPM3C parasites. These results demonstrate that TgPPM3C influences the phosphorylation status of many vacuolar proteins and reveal de-phosphorylation as a potentially critical process that facilitates the export of GRA16 from the parasitophorous vacuole
TgPPM3C is a putative protein phosphatase secreted into the lumen of the parasitophorous vacuole
Summary
A large proportion of the human population, varying by region, is predicted to be infected with the parasite Toxoplasma gondii based on seropositivity, posing a life-threatening risk in immune-compromised individuals [1]. The rapidly growing tachyzoite life stage disseminates to various host tissues through repeated rounds of invasion, replication, and host cell lysis [3]. Advances have been made in understanding how the parasite thrives within host cells and avoids host cell defenses, much remains to be discovered regarding how the unique compartment in which parasites replicate, the parasitophorous vacuole (containing tachyzoites) or the tissue cyst (containing bradyzoites), is optimized and remodeled throughout the infection process of either life stage
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