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 ( My c r egulation 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.

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Association of host cell endoplasmic reticulum and mitochondria with the Toxoplasma gondii parasitophorous vacuole membrane: a high affinity interaction

Journal of Cell Science ◽

10.1242/jcs.110.17.2117 ◽

1997 ◽

Vol 110 (17) ◽

pp. 2117-2128 ◽

Cited By ~ 8

Author(s):

A.P. Sinai ◽

P. Webster ◽

K.A. Joiner

Keyword(s):

Endoplasmic Reticulum ◽

Toxoplasma Gondii ◽

Host Cell ◽

Parasitophorous Vacuole ◽

Protozoan Parasite ◽

Host Cells ◽

Parasitophorous Vacuole Membrane ◽

Vacuole Membrane ◽

Protein Protein Interaction ◽

High Affinity

The parasitophorous vacuole membrane (PVM) of the obligate intracellular protozoan parasite Toxoplasma gondii forms tight associations with host mitochondria and the endoplasmic reticulum (ER). We have used a combination of morphometric and biochemical approaches to characterize this unique phenomenon, which we term PVM-organelle association. The PVM is separated from associated mitochondria and ER by a mean distance of 12 and 18 nm, respectively. The establishment of PVM-organelle association is dependent on active parasite entry, but does not require parasite viability for its maintenance. Association is not a consequence of spatial constraints imposed on the growing vacuole. Morphometric analysis indicates that the extent of mitochondrial association with the PVM stays constant as the vacuole enlarges, whereas the extent of ER association decreases. Disruption of host cell microtubules partially blocks the establishment but not the maintenance of PVM-mitochondrial association, and has no significant effect on PVM-ER association. PVM-organelle association is maintained following disruption of infected host cells, as assessed by electron microscopy and by sub-cellular fractionation showing co-migration of fixed PVM and organelle markers. Taken together, the data suggest that a high affinity, potentially protein-protein interaction between parasite and organelle components is responsible for PVM-organelle association.

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The Toxoplasma gondii protein ROP2 mediates host organelle association with the parasitophorous vacuole membrane

The Journal of Cell Biology ◽

10.1083/jcb.200101073 ◽

2001 ◽

Vol 154 (1) ◽

pp. 95-108 ◽

Cited By ~ 146

Author(s):

Anthony P. Sinai ◽

Keith A. Joiner

Keyword(s):

Toxoplasma Gondii ◽

Fluorescent Protein ◽

Parasitophorous Vacuole ◽

Host Cells ◽

In Vitro Assays ◽

Mitochondrial Outer Membrane ◽

Mitochondrial Targeting ◽

Parasitophorous Vacuole Membrane ◽

Parasite Protein ◽

Vacuole Membrane

Toxoplasma gondii replicates within a specialized vacuole surrounded by the parasitophorous vacuole membrane (PVM). The PVM forms intimate interactions with host mitochondria and endoplasmic reticulum (ER) in a process termed PVM–organelle association. In this study we identify a likely mediator of this process, the parasite protein ROP2. ROP2, which is localized to the PVM, is secreted from anterior organelles termed rhoptries during parasite invasion into host cells. The NH2-terminal domain of ROP2 (ROP2hc) within the PVM is exposed to the host cell cytosol, and has characteristics of a mitochondrial targeting signal. In in vitro assays, ROP2hc is partially translocated into the mitochondrial outer membrane and behaves like an integral membrane protein. Although ROP2hc does not translocate across the ER membrane, it does exhibit carbonate-resistant binding to this organelle. In vivo, ROP2hc expressed as a soluble fragment in the cytosol of uninfected cells associates with both mitochondria and ER. The 30–amino acid (aa) NH2-terminal sequence of ROP2hc, when fused to green fluorescent protein (GFP), is sufficient for mitochondrial targeting. Deletion of the 30-aa NH2-terminal signal from ROP2hc results in robust localization of the truncated protein to the ER. These results demonstrate a new mechanism for tight association of different membrane-bound organelles within the cell cytoplasm.

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The Toxoplasma gondii rhoptry protein ROP 2 is inserted into the parasitophorous vacuole membrane, surrounding the intracellular parasite, and is exposed to the host cell cytoplasm.

The Journal of Cell Biology ◽

10.1083/jcb.127.4.947 ◽

1994 ◽

Vol 127 (4) ◽

pp. 947-961 ◽

Cited By ~ 151

Author(s):

C J Beckers ◽

J F Dubremetz ◽

O Mercereau-Puijalon ◽

K A Joiner

Keyword(s):

Toxoplasma Gondii ◽

Host Cell ◽

Direct Evidence ◽

Parasitophorous Vacuole ◽

Velocity Sedimentation ◽

Parasitophorous Vacuole Membrane ◽

Cell Cytoplasm ◽

Vacuole Membrane ◽

Specific Antibodies ◽

Host Cell Cytoplasm

The origin of the vacuole membrane surrounding the intracellular protozoan parasite Toxoplasma gondii is not known. Although unique secretory organelles, the rhoptries, discharge during invasion of the host cell and may contribute to the formation of this parasitophorous vacuole membrane (PVM), no direct evidence for this hypothesis exists. Using a novel approach we have determined that parasite-encoded proteins are present in the PVM, exposed to the host cell cytoplasm. In infected cells incubated with streptolysin-O or low concentrations of digitonin, the host cell plasma membrane was selectively permeabilized without significantly affecting the integrity of the PVM. Antisera prepared against whole parasites or a parasite fraction enriched in rhoptries and dense granules reacted with the PVM in these permeabilized cells, indicating that parasite-encoded antigens were exposed on the cytoplasmic side of the PVM. Parasite antigens responsible for this staining of the PVM were identified by fractionating total parasite proteins by SDS-PAGE and velocity sedimentation, and then affinity purifying "fraction-specific" antibodies from the crude antisera. Proteins responsible for the PVM-staining, identified with fraction-specific antibodies, cofractionated with known rhoptry proteins. The gene encoding one of the rhoptry proteins, ROP 2, was cloned and sequenced, predicting and integral membrane protein. Antibodies specific for ROP 2 reacted with the intact PVM. These results provide the first direct evidence that rhoptry contents participate in the formation of the PVM of T. gondii and suggest a possible role of ROP 2 in parasite-host cell interactions.

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Differential Impacts on Host Transcription by ROP and GRA Effectors from the Intracellular Parasite Toxoplasma gondii

10.1101/2020.02.04.934570 ◽

2020 ◽

Author(s):

Suchita Rastogi ◽

Yuan Xue ◽

Stephen R. Quake ◽

John C. Boothroyd

Keyword(s):

Toxoplasma Gondii ◽

Host Cell ◽

Cell Biology ◽

Parasitophorous Vacuole ◽

Dense Granule ◽

Effector Proteins ◽

Transcriptomic Analysis ◽

Host Cells ◽

Intracellular Parasite ◽

I Cells

ABSTRACTThe intracellular parasite Toxoplasma gondii employs a vast array of effector proteins from the rhoptry and dense granule organelles to modulate host cell biology; these effectors are known as ROPs and GRAs, respectively. To examine the individual impacts of ROPs and GRAs on host gene expression, we developed a robust, novel protocol to enrich for ultra-pure populations of a naturally occurring and reproducible population of host cells called uninfected-injected (U-I) cells, which Toxoplasma injects with ROPs but subsequently fails to invade. We then performed single cell transcriptomic analysis at 1-3 hours post-infection on U-I cells (as well as on uninfected and infected controls) arising from infection with either wild type parasites or parasites lacking the MYR1 protein, which is required for soluble GRAs to cross the parasitophorous vacuole membrane (PVM) and reach the host cell cytosol. Based on comparisons of infected and U-I cells, the host’s earliest response to infection appears to be driven primarily by the injected ROPs, which appear to induce immune and cellular stress pathways. These ROP-dependent pro-inflammatory signatures appear to be counteracted by at least some of the MYR1-dependent GRAs and may be enhanced by the MYR-independent GRAs, (which are found embedded within the PVM). Finally, signatures detected in uninfected bystander cells from the infected monolayers suggests that MYR1-dependent paracrine effects also counteract inflammatory ROP-dependent processes.IMPORTANCEThis work performs the first transcriptomic analysis of U-I cells, captures the earliest stage of a host cell’s interaction with Toxoplasma gondii, and dissects the effects of individual classes of parasite effectors on host cell biology.

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The Toxoplasma gondii Rhoptry Protein ROP4 Is Secreted into the Parasitophorous Vacuole and Becomes Phosphorylated in Infected Cells

Eukaryotic Cell ◽

10.1128/ec.3.5.1320-1330.2004 ◽

2004 ◽

Vol 3 (5) ◽

pp. 1320-1330 ◽

Cited By ~ 69

Author(s):

Kimberly L. Carey ◽

Artemio M. Jongco ◽

Kami Kim ◽

Gary E. Ward

Keyword(s):

Toxoplasma Gondii ◽

Infected Cell ◽

Host Cell ◽

Transmembrane Protein ◽

Parasitophorous Vacuole ◽

Host Cells ◽

Cell Protein ◽

Type I ◽

Membrane Function ◽

Vacuole Membrane

ABSTRACT Many intracellular pathogens are separated from the cytosol of their host cells by a vacuole membrane. This membrane serves as a critical interface between the pathogen and the host cell, across which nutrients are imported, wastes are excreted, and communication between the two cells takes place. Very little is known about the vacuole membrane proteins mediating these processes in any host-pathogen interaction. During a screen for monoclonal antibodies against novel surface or secreted proteins of Toxoplasma gondii, we identified ROP4, a previously uncharacterized member of the ROP2 family of proteins. We report here on the sequence, posttranslational processing, and subcellular localization of ROP4, a type I transmembrane protein. Mature, processed ROP4 is localized to the rhoptries, secretory organelles at the apical end of the parasite, and is secreted from the parasite during host cell invasion. Released ROP4 associates with the vacuole membrane and becomes phosphorylated in the infected cell. Similar results are seen with ROP2. Further analysis of ROP4 showed it to be phosphorylated on multiple sites, a subset of which result from the action of either host cell protein kinase(s) or parasite kinase(s) activated by host cell factors. The localization and posttranslational modification of ROP4 and other members of the ROP2 family of proteins within the infected cell make them well situated to play important roles in vacuole membrane function.

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Differential Impacts on Host Transcription by ROP and GRA Effectors from the Intracellular Parasite Toxoplasma gondii

mBio ◽

10.1128/mbio.00182-20 ◽

2020 ◽

Vol 11 (3) ◽

Cited By ~ 2

Author(s):

Suchita Rastogi ◽

Yuan Xue ◽

Stephen R. Quake ◽

John C. Boothroyd

Keyword(s):

Toxoplasma Gondii ◽

Host Cell ◽

Cell Biology ◽

Parasitophorous Vacuole ◽

Effector Proteins ◽

Transcriptomic Analysis ◽

Host Cells ◽

Intracellular Parasite ◽

Content Type ◽

I Cells

ABSTRACT The intracellular parasite Toxoplasma gondii employs a vast array of effector proteins from the rhoptry and dense granule organelles to modulate host cell biology; these effectors are known as ROPs and GRAs, respectively. To examine the individual impacts of ROPs and GRAs on host gene expression, we developed a robust, novel protocol to enrich for ultrapure populations of a naturally occurring and reproducible population of host cells called uninfected-injected (U-I) cells, which Toxoplasma injects with ROPs but subsequently fails to invade. We then performed single-cell transcriptomic analysis at 1 to 3 h postinfection on U-I cells (as well as on uninfected and infected controls) arising from infection with either wild-type parasites or parasites lacking the MYR1 protein, which is required for soluble GRAs to cross the parasitophorous vacuole membrane (PVM) and reach the host cell cytosol. Based on comparisons of infected and U-I cells, the host’s earliest response to infection appears to be driven primarily by the injected ROPs, which appear to induce immune and cellular stress pathways. These ROP-dependent proinflammatory signatures appear to be counteracted by at least some of the MYR1-dependent GRAs and may be enhanced by the MYR-independent GRAs (which are found embedded within the PVM). Finally, signatures detected in uninfected bystander cells from the infected monolayers suggest that MYR1-dependent paracrine effects also counteract inflammatory ROP-dependent processes. IMPORTANCE This work performs transcriptomic analysis of U-I cells, captures the earliest stage of a host cell’s interaction with Toxoplasma gondii, and dissects the effects of individual classes of parasite effectors on host cell biology.

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