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.

Importance: Toxoplasma gondii is an important human pathogen and a model for the study of intracellular parasitism. Infection of the host cell with Toxoplasma tachyzoites involves the introduction of protein effectors, including many that are initially secreted into the parasitophorous vacuole but must ultimately translocate to the host cell cytosol to function. The work reported here identified a novel protein that is required for this translocation. These results give new insight into a very unusual cell biology process as well as providing a potential handle on a pathway that is necessary for virulence and, therefore, a new potential target for chemotherapy.

FIG 1

Genetic screen to isolate Toxoplasma mutants that fail to induce c-Myc. (A) Flow cytometry histogram of BMMs expressing c-Myc–GFP infected with Toxoplasma wild-type tachyzoites (RH) or Neospora tachyzoites or mock-infected. The gate was set on highly infected cells. The y axis shows relative cell count for each population (normalized to mode), and the x axis shows green fluorescence intensity. (B) Selection of mutants that fail to induce c-Myc. BMM c-Myc–GFP reporter cells were infected with a mutagenized population of RH tachyzoites and sorted for low GFP fluorescence. This was repeated until a homogeneous population was obtained that induced low GFP fluorescence. The GFP fluorescence profiles for the mutagenized population after 3, 5, and 7 rounds of selection are shown. (Histograms for first two sorts were similar to the one for sort 3 and were omitted.) The profiles for cells infected with wild-type RH and Neospora tachyzoites are shown for comparison. The x and y axes are as described for panel A.

FIG 2

Characterization of Toxoplasma mutants that fail to up-regulate c-Myc in host cells. (A) Human foreskin fibroblasts (HFFs) were infected with wild-type parasites (RH) or mutant strains MFM1.1, MFM2.1, and MFM2.2. Neospora- and mock-infected fibroblasts were used as negative controls for c-Myc induction. Lysates from infected cells were prepared at 20 hpi, and host c-Myc levels were analyzed by Western blotting using anti-human c-Myc antibodies. Anti-hGAPDH staining was used as a loading control for host (human) protein, and anti-SAG1 staining was used to show comparable levels of Toxoplasma infection. Each row is derived from the same exposure of the same blot; the white vertical separator shows where irrelevant data were removed from the image. (B) Schematic diagram of wild-type and mutant TGGT1_254470 (MYR1) protein. Numbers indicate amino acid positions relative to the predicted N terminus of the unprocessed protein. The signal peptide (SP) is as predicted by the SignalP 4.1 tool (http://www.cbs.dtu.dk/services/SignalP/) and ToxoDB (http://www.toxodb.org) and phosphorylated serine (S*) residues were determined by phosphoproteomic analysis (24). The region expressed as a recombinant version of MYR1 representing amino acids 155 to 328 to generate an N-terminus-specific antibody is indicated by the solid line [rMYR1(155–328)]. Transmembrane domains are as predicted by the TMHMM algorithm and ToxoDB (http://www.cbs.dtu.dk/services/TMHMM-2.0/), and the position of the first residue of each is indicated. A likely ASP5 cleavage site (RRLSE) is as described in the text. Mutant versions of MYR1, as predicted by sequencing strains MFM1.1, MFM2.1, and MFM2.2, are also shown. The letters and numbers to the right of each schematic show the original coding function and position of the nonsense mutation in each.

FIG 3

MYR1 is a secreted protein that is necessary for c-Myc induction. (A) HFFs were infected at an MOI of 1 with the indicated strain and lysed at 20 hpi, and lysates were analyzed by Western blotting using anti-MYR1 antibodies and anti-HA antibodies to detect endogenous and recombinant MYR1 levels. Rabbit anti-c-Myc antibodies were used to assess the levels of c-Myc in each sample. Anti-GAPDH and anti-SAG1 antibodies served as loading controls for cells and parasites, respectively. (B) As described for panel A, except the monolayers were scrape-syringed and tachyzoites were purified away from host cell material prior to the lysate being prepared. An additional band at ~105 kDa is apparent in these lysates. (C) Anti-MYR1 and anti-HA blots of cell lysate prepared from HFFs infected with complemented (RHΔmyr1::MYR1) parasites. These blots were overexposed so the slow-running band (~105 kDa) corresponding to full-length MYR1 and detected by both antibodies can be seen.

FIG 4

MYR1 N-terminal and C-terminal domains localize to the parasitophorous vacuole membrane and PV space. (A) C-terminal HA-tagging of the endogenous MYR1 locus does not affect its expression levels. Extracellular tachyzoites of the RH and C-terminally tagged RH:MYR1-HA strains were lysed in radioimmunoprecipitation assay (RIPA) buffer and analyzed by Western blotting using anti-MYR1(155–328) and anti-HA antibodies. GRA7 antibodies were used to assess equal loading. (B) The N-terminal and C-terminal domains of MYR1 localize to the PV and PVM. HFFs were infected with the RH:MYR1-HA strain and fixed with methanol at 12 hpi. The fixed monolayers were stained with DAPI and antibodies to HA or MYR1 N-terminus (green) and GRA7 (red).

FIG 5

The cell cycle is dysregulated in cells infected by wild-type parasites but not in cells infected with Δmyr1 mutants. Propidium iodide assessment of cell DNA content was performed in RAW264.7 cells at 19 hpi with RH and RHΔmyr1 parasites expressing mCherry. Cells were fixed with methanol, stained with propidium iodide, and then assessed on an LSRII flow cytometer. The three peaks represent cells in G₀/G₁ (2n), cells in G₂/M (4n), and aneuploid cells (~8n).

FIG 6

MYR1 is not necessary for the ability of Toxoplasma tachyzoites to recruit host mitochondria or induce NF-κB. (A) Confirmation of c-Myc phenotype. HFFs grown in low-serum medium were infected with the indicated parasites expressing mCherry for 14 h before being fixed with formaldehyde and stained with DAPI and antibodies to c-Myc. UI, uninfected. Bar, 5 µm. (B) Effect of MYR1 on host mitochondrial association. The images are as described for panel A except that HFFs were infected with the indicated Toxoplasma strains for 13 h. Host cell mitochondria were detected using an anti-TOM20 antibody (green), and parasites were detected using the constitutively expressed mCherry marker in each strain (red). (C) Effect of MYR1 on nuclear NF-κB accumulation. The images are as described for panel A except that ME49, ME49Δmyr1, and ME49Δmyr1::MYR1 parasites were used to infect HFFs for 30 h, at which time cells were fixed and stained with anti-SAG1 antibody (red) and anti-NFκB antibody (green). (D, E, and F) c-Myc induction, HMA, and nuclear NF-κB accumulation were quantified. Data are means and standard errors of the means (SEM) from three independent assessments. **, P < 0.01.

FIG 7

MYR1 is necessary for the function of several effectors, including GRA16 and GRA24. (A) Effect of MYR1 on GRA24-mediated p38 MAPK activation. An immunofluorescence assay was performed on confluent HFFs that were uninfected or infected with the indicated strains. At 20 hpi, cells were fixed and stained using anti-phospho-p38 MAPK antibody. Bar, 5 µm. (B) Effect of MYR1 on GRA16-mediated PP2A nuclear translocation. The images are as described for panel A except that cells were stained using antibody to the PP2A-B subunit. (C) Role for MYR1 in the block of IFN-γ-induced IRF1 activation. The images are as described for panel A except that HFFs were infected for 14 h with the indicated parasites expressing mCherry, stimulated with 200 units/ml of IFN-γ for 6 h, and fixed with formaldehyde. Cells were stained using anti-IRF1 antibody. White arrowheads highlight the host nuclei in infected cells. UI, uninfected. (D, E, and F) p38 phosphorylation and nuclear accumulation, PP2A nuclear translocation, and IRF1 nuclear accumulation were quantified. Data are means and SEM from three independent assessments. **, P < 0.01.

FIG 8

MYR1 plays a role in translocation of GRA24 to the host nucleus. HFFs were infected with RH-WT, RHΔmyr1, or RHΔmyr1::MYR1 parasites expressing a Myc-tagged GRA24, and at 20 hpi, the cultures were fixed and stained with antibodies to the Myc tag. Bar, 5 µm.

FIG 9

MYR1 mutants exhibit wild-type growth in vitro but decreased virulence in mice. (A) Growth comparison of ME49 strains in vitro. HFF were infected with the indicated strains of parasites for 22 h, and the number of parasites in each vacuole was then assessed. The percentage of vacuoles demonstrating 2, 4, 8, or >8 parasites per vacuole is displayed. Data are averages for two independent replicates. (B) Kaplan-Meier survival curve for mice infected with ME49 parasites with or without MYR1. Five mice in each group were injected intraperitoneally with PBS or with 100 tachyzoites of Toxoplasma ME49, ME49Δmyr1, or ME49Δmyr1::MYR1. The results are representative of two experiments with similar outcomes. (C) As for panel A, growth of RH strains was assessed in HFFs at 16 h. The percentage of vacuoles demonstrating 2, 4, 8, or >8 parasites per vacuole is displayed. Data are averages for two independent replicates. (D) As for panel B, survival was assessed in mice injected intraperitoneally with PBS or with 100 tachyzoites of Toxoplasma RH (5 mice per group), RHΔmyr1 (6 mice per group), or RHΔmyr1::MYR1 (5 mice per group). The results are representative of two experiments with similar outcomes. *, P < 0.05; **, P < 0.01.