The Secreted Acid Phosphatase Domain-Containing GRA44 from Toxoplasma gondii Is Required for c-Myc Induction in Infected Cells

Abstract

During host cell invasion, the eukaryotic pathogen Toxoplasma gondii forms a parasitophorous vacuole to safely reside within the cell, while it is partitioned from host cell defense mechanisms. From within this safe niche, parasites sabotage multiple host cell systems, including gene expression, apoptosis, and intracellular immune recognition, by secreting a large arsenal of effector proteins. Many parasite proteins studied for active host cell manipulative interactions have been kinases. The translocation of effectors from the parasitophorous vacuole into the host cell is mediated by a putative translocon complex, which includes the proteins MYR1, MYR2, and MYR3. Whether other proteins are involved in the structure or regulation of this putative translocon is not known. We have discovered that the secreted protein GRA44, which contains a putative acid phosphatase domain, interacts with members of this complex and is required for host cell effects downstream of effector secretion. We have determined that GRA44 is processed in a region with homology to sequences targeted by protozoan proteases of the secretory pathway and that both major cleavage fragments are secreted into the parasitophorous vacuole. Immunoprecipitation experiments showed that GRA44 interacts with a large number of secreted proteins, including MYR1. Importantly, conditional knockdown of GRA44 resulted in a lack of host cell c-Myc upregulation, which mimics the phenotype seen when members of the translocon complex are genetically disrupted. Thus, the putative acid phosphatase GRA44 is crucial for host cell alterations during Toxoplasma infection and is associated with the translocon complex which Toxoplasma relies upon for success as an intracellular pathogen. IMPORTANCE Approximately one-third of humans are infected with the parasite Toxoplasma gondii. Toxoplasma infections can lead to severe disease in those with a compromised or suppressed immune system. Additionally, infections during pregnancy present a significant health risk to the developing fetus. Drugs that target this parasite are limited, have significant side effects, and do not target all disease stages. Thus, a thorough understanding of how the parasite propagates within a host is critical in the discovery of novel therapeutic targets. Toxoplasma replication requires that it enter the cells of the infected organism. In order to survive the environment inside a cell, Toxoplasma secretes a large repertoire of proteins, which hijack a number of important cellular functions. How these Toxoplasma proteins move from the parasite into the host cell is not well understood. Our work shows that the putative phosphatase GRA44 is part of a protein complex responsible for this process.

Coimmunoprecipitation with MYR1 Identifies Three Additional Proteins within the Toxoplasma gondii Parasitophorous Vacuole Required for Translocation of Dense Granule Effectors into Host Cells

“…While this work was in progress, we learned of similar studies by Blakely et al, who also found that GRA44 associates with MYR1 and is necessary for efficient c-Myc upregulation during infection (40). The latter authors used a knockdown approach to study GRA44 and saw a more dramatic impact of GRA44 loss on parasite growth than we and others (30) observed for the GRA44 knockout; this might indicate that compensatory changes were selected for during the prolonged selection necessary to generate and expand our knockout clone, as was reported for AMA1 knockouts, which showed a dramatic upregulation of the paralogue, AMA2 (41).…”

Genome-wide screens identify Toxoplasma gondii determinants of parasite fitness in IFNγ-activated murine macrophages

“…These results demonstrate that GRA45 plays a critical role in the correct trafficking of PVM-integrated or -associated GRAs after exocytosis of dense granules. GRA44, as the known GRA45 interaction partner, has no predicted hydrophobic domain but localizes to the PV 49 , 80 . In contrast to the localization changing from PVM to the PV lumen for above GRAs, GRA44 resided inside the cytosol of Δ gra45 parasites instead of being secreted into the PV (Fig.…”

“…The coverslips were incubated with anti-SAG1 antibodies and anti-HA antibodies or anti-Ty antibodies at 4°C overnight. To observe the localization of GRA44, HFFs infected with wild-type, Δ gra45 or Δ gra45 + GRA45HA strains transiently expressing GRA44-Myc 80 for 20 h were fixed, permeabilized and blocked as described above followed by incubation with anti-Myc antibodies at 4°C overnight. After incubating with Alexa Fluor 488/594 secondary antibodies and DAPI, the coverslips were mounted with Vecta-Shield mounting oil and the microscopy was performed with NIS-Elements software (Nikon) and a digital camera (CoolSNAP EZ; Roper Scientific) connected to an inverted fluorescence microscope (Eclipse Ti-S; Nikon) and either phase contrast or DIC imaging.…”

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

“…Exported effectors such as GRA16 and TgIST have been elegantly characterized and shown to influence host cell signaling in profound ways that benefit the intracellular development of the parasite [23,26,27]. Several effectors exported from the parasitophorous vacuole have been described apart from those explored in this study [9,28,29,46], while the list of proteins necessary for the export of these proteins has also been growing, including an acid phosphatase domain-containing protein in GRA44 [21,[47][48][49]. Cygan et.…”