scholarly journals 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.

1994 ◽  
Vol 127 (4) ◽  
pp. 947-961 ◽  
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.

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

1997 ◽  
Vol 110 (17) ◽  
pp. 2117-2128 ◽  
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.

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

mBio ◽  
2016 ◽  
Vol 7 (1) ◽  
Author(s):  
Magdalena Franco ◽  
Michael W. Panas ◽  
Nicole D. Marino ◽  
Mei-Chong Wendy Lee ◽  
Kerry R. Buchholz ◽  
...  
Keyword(s):  
Toxoplasma Gondii ◽  
Host Cell ◽  
Cell Biology ◽  
Parasitophorous Vacuole ◽  
Critical Role ◽  
Host Cells ◽  
Secreted Protein ◽  
Parasitophorous Vacuole Membrane ◽  
Vacuole Membrane ◽  
Novel Protein

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.

Subcellular localization of Pfs16, a Plasmodium falciparum gametocyte antigen

Parasitology ◽  
1994 ◽  
Vol 108 (2) ◽  
pp. 129-137 ◽  
Author(s):  
D. A. Baker ◽  
O. Daramola ◽  
M. V. McCrossan ◽  
J. Harmer ◽  
G. A. T. Targett
Keyword(s):  
Plasmodium Falciparum ◽  
Parasitophorous Vacuole ◽  
Subcellular Location ◽  
Cellular Structures ◽  
Parasitophorous Vacuole Membrane ◽  
Cell Cytoplasm ◽  
Vacuole Membrane ◽  
Host Cell Cytoplasm ◽  
Food Vacuoles ◽  
Membrane Region

SUMMARYWe have used immunoelectron microscopy to investigate the subcellular location of Pfs16 in Plasmodium falciparum. It was detected in the outer membrane region of gametocytes and more specifically on the parasitophorous vacuole membrane (pvm), since, during gametogenesis when the pvm disintegrates, the majority of the antigen was detected on the remains of this membrane in multilaminated whorls and not on the gamete plasma membrane. The antigen was also present on other gametocyte cellular structures, including those which we believe to be Garnham bodies, present in the host cell cytoplasm of some gametocytes. The antigen was present too on the membrane surrounding cytostomes and the resulting food vacuoles in the parasite cytoplasm.

Eimeria canadensis (Protozoa, Sporozoea): ultrastructure of the host–parasite interface during development of first-generation schizonts in vitro

1980 ◽  
Vol 58 (11) ◽  
pp. 2018-2025 ◽  
Author(s):  
Bodo E. G. Mueller
Keyword(s):  
Host Cell ◽  
Parasitophorous Vacuole ◽  
Eimeria Species ◽  
Outer Zone ◽  
Ultrastructural Observation ◽  
Cell Cytoplasm ◽  
Cell Organelles ◽  
Host Cell Cytoplasm ◽  
Host Parasite

Eimeria canadensis sporozoites were inoculated into monolayer cultures of Madin–Darby bovine kidney and primary bovine embryonic kidney cells. Sporozoites retained their shape for at least 9 days. At that time, the nucleus was enlarged and contained a prominent nucleolus, and amylopectin granules were no longer apparent. The width of the parasitophorous vacuole (pv) between host cell cytoplasm and parasite pellicle widened during transformation of sporozoites into multinucleate schizonts. Areas of altered host cell cytoplasm immediately adjacent to the pv membrane increased in size and became confluent, resulting in the formation of two distinct layers of cytoplasm. The outer zone contained the host cell nucleus, mitochondria, Golgi stacks, and ER, whereas the inner layer appeared granular and was void of all cell organelles except structures resembling ribosomes. Microfilaments were abundant at the border between inner and outer zone. In the most advanced stages observed, host cell organelles persisted only in the perinuclear region. The remaining, attenuated cytoplasm resembled the former inner zone.The novel ultrastructural observation of a bilayered cytoplasm of cells harbouring E. canadensis schizonts is compared with light microscope reports of similar effects caused by other Eimeria species of ruminants and with electron microscope findings of altered intestinal and abomasal cells of sheep harbouring "globidial" schizonts.

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

2001 ◽  
Vol 154 (1) ◽  
pp. 95-108 ◽  
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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