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

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.

Author summary

The flexible life cycle of the intracellular parasite Toxoplasma gondii allows it to infect many different types of warm-blooded hosts, as well as diverse cell types once inside the host organism. This formidable achievement is partly mediated by the establishment of a unique compartment following host cell invasion, termed the parasitophorous vacuole. While advancements have been made in cataloguing Toxoplasma secreted proteins that reside within this vacuole, the specific functions and contributions of many of these secreted parasite “tools” remain elusive. Here, we assessed the contribution of a parasite vacuolar protein called TgPPM3C, predicted to function as an enzyme that dephosphorylates other proteins. We found that deleting the TgPPM3C gene in the parasite results in a profound virulence defect during infection in mice, likely due to the dysregulated phosphorylation status of many vacuolar proteins detected by phosphoproteomic analysis of TgPPM3C-deleted parasites. We found that the phosphorylation status of one such protein, GRA16, influences its ability to cross the parasitophorous vacuole membrane and enter the host cell, where it is known to induce host transcriptional changes that benefit parasite growth. These findings illustrate the emerging role of Toxoplasma vacuolar phosphatases in regulating host-parasite interactions during infection.