A Novel Secreted Protein, MYR1, Is Central to Toxoplasma ’s Manipulation of Host Cells

Abstract

The intracellular protozoan Toxoplasma gondii dramatically reprograms the transcriptome of host cells it infects, including substantially up-regulating the host oncogene c-myc. By applying a flow cytometry-based selection to infected mouse cells expressing green fluorescent protein fused to c-Myc (c-Myc–GFP), we isolated mutant tachyzoites defective in this host c-Myc up-regulation. Whole-genome sequencing of three such mutants led to the identification of MYR1 (Myc regulation 1; TGGT1_254470) as essential for c-Myc induction. MYR1 is a secreted protein that requires TgASP5 to be cleaved into two stable portions, both of which are ultimately found within the parasitophorous vacuole and at the parasitophorous vacuole membrane. Deletion of MYR1 revealed that in addition to its requirement for c-Myc up-regulation, the MYR1 protein is needed for the ability of Toxoplasma tachyzoites to modulate several other important host pathways, including those mediated by the dense granule effectors GRA16 and GRA24. This result, combined with its location at the parasitophorous vacuole membrane, suggested that MYR1 might be a component of the machinery that translocates Toxoplasma effectors from the parasitophorous vacuole into the host cytosol. Support for this possibility was obtained by showing that transit of GRA24 to the host nucleus is indeed MYR1-dependent. As predicted by this pleiotropic phenotype, parasites deficient in MYR1 were found to be severely attenuated in a mouse model of infection. We conclude, therefore, that MYR1 is a novel protein that plays a critical role in how Toxoplasma delivers effector proteins to the infected host cell and that this is crucial to virulence.

An in vitro model of intestinal infection reveals a developmentally regulated transcriptome of Toxoplasma sporozoites and a NF-κB-like signature in infected host cells

“…GRA15 , whose gene product is known to modulate NF-κB signaling in tachyzoites [62], has a significantly higher expression (2.2-fold, q -value = 0.77%) in infecting sporozoites compared to tachyzoites. Sporozoites also had higher levels of MYR1 , which encodes a recently described protein necessary for translocation of dense granule proteins beyond the parasitophorous vacuole membrane (PVM) [63]. There were only 8 genes encoding dense granule proteins with higher expression in tachyzoites compared to infecting sporozoites (Table 10), with one isoform of GRA11 [64] and GRA36 [33] having the highest fold changes (~9- and 7-fold higher in the TZ, relative to SPZ samples, respectively).…”

Toxoplasmacontrols host cyclin E expression through the use of a novel MYR1-dependent effector protein, HCE1

“…Several publications have described the active interference of Toxoplasma tachyzoites with host cell cycle machinery (1, 2, 27, 28), including in MYR1-dependent ways (12). The results presented here for HCE1 likely provide at least a partial explanation for these effects since among the E2F-regulated genes whose expression is HCE1-dependent, we see several genes related to the pre-replication complex such as the minichromosome maintenance genes (MCM), DNA polymerase E and Origin Replication complex subunit 1 (ORC1).…”

“…The following strains were used in this study: Toxoplasma RHΔ hxgprt (2), RHΔ myr1 (2), RHΔ asp5 (30), RHΔ gra16 (6), RHΔ gra16::GRA16HA (6) and Neospora caninum NC-1 (31). Toxoplasma and Neospora tachyzoites were propagated in human foreskin fibroblasts (HFFs) cultured in complete Dulbecco’s Modified Eagle Medium (cDMEM) supplemented with 10% heat-inactivated fetal bovine serum (FBS; HyClone, Logan, UT), 2 mM L-glutamine, 100 U ml −1 penicillin and 100 µg ml −1 streptomycin at 37 °C with 5% CO 2 .…”

“…Indeed, this ubiquitous member of the phylum Apicomplexa has previously been described to cause host cells to stall in states ranging between S phase and the G2/M transition (1). In some host cells, this manifests in the host cell endocycling, duplicating its DNA without subsequent cytokinesis, and previous evidence has suggested that this is mediated by an active, but unknown, parasite-derived activity (2).…”

“…Toxoplasma tachyzoites use secreted effectors derived from the dense granules to manipulate host cell functions while replicating in the parasitophorous vacuole (PV)(3, 4). A select set of these dense granule proteins can cross the parasitophorous vacuole membrane (PVM) and enter the host cytoplasm (5) in a process that is dependent on at least four parasite proteins; three of these are located at the PVM and have been termed MYR1, MYR2, and MYR3 (2, 6) while a fourth, aspartyl protease 5 (ASP5), is found within the Golgi and catalyzes proteolysis at a conserved Arg-Arg-Leu (RRL) sequence (7, 8). To date, GRA16 (6, 9), GRA24 (2) and GRA18 (10) have been shown to employ this machinery and the loss of the TgIST-induced phenotype in Myr − mutants is consistent with it being a fourth such protein (11).…”