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.

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.

Toxoplasma Mechanisms for Delivery of Proteins and Uptake of Nutrients Across the Host-Pathogen Interface

2020 ◽  
Vol 74 (1) ◽  
pp. 567-586 ◽  
Author(s):  
Yifan Wang ◽  
Lamba Omar Sangaré ◽  
Tatiana C. Paredes-Santos ◽  
Jeroen P. J. Saeij
Keyword(s):  
Pattern Recognition ◽  
Toxoplasma Gondii ◽  
Parasitophorous Vacuole ◽  
Protozoan Parasite ◽  
Intracellular Pathogens ◽  
Parasitophorous Vacuole Membrane ◽  
Vacuole Membrane ◽  
Pathogen Effectors ◽  
Host Pathogen ◽  
Uptake Of Nutrients

Many intracellular pathogens, including the protozoan parasite Toxoplasma gondii, live inside a vacuole that resides in the host cytosol. Vacuolar residence provides these pathogens with a defined niche for replication and protection from detection by host cytosolic pattern recognition receptors. However, the limiting membrane of the vacuole, which constitutes the host-pathogen interface, is also a barrier for pathogen effectors to reach the host cytosol and for the acquisition of host-derived nutrients. This review provides an update on the specialized secretion and trafficking systems used by Toxoplasma to overcome the barrier of the parasitophorous vacuole membrane and thereby allow the delivery of proteins into the host cell and the acquisition of host-derived nutrients.

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.

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

2004 ◽  
Vol 3 (5) ◽  
pp. 1320-1330 ◽  
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.

scholarly journals Implication of Potential Differential Roles of the Two Phosphoglucomutase Isoforms in the Protozoan Parasite Cryptosporidium parvum

Pathogens ◽  
2021 ◽  
Vol 11 (1) ◽  
pp. 21
Author(s):  
Jiawen Nie ◽  
Jigang Yin ◽  
Dongqiang Wang ◽  
Chenchen Wang ◽  
Guan Zhu
Keyword(s):  
Enzyme Activity ◽  
Recombinant Proteins ◽  
Cryptosporidium Parvum ◽  
Plasma Membranes ◽  
Parasitophorous Vacuole ◽  
Protozoan Parasite ◽  
Immunofluorescence Assay ◽  
Parasitophorous Vacuole Membrane ◽  
Vacuole Membrane ◽  
Intracellular Parasites

Phosphoglucomutase 1 (PGM1) catalyzes the conversion between glucose-1-phosphate and glucose-6-phosphate in the glycolysis/glucogenesis pathway. PGM1s are typically cytosolic enzymes in organisms lacking chloroplasts. However, the protozoan Cryptosporidium parasites possess two tandemly duplicated PGM1 genes evolved by a gene duplication after their split from other apicomplexans. Moreover, the downstream PGM1 isoform contains an N-terminal signal peptide, predicting a non-cytosolic location. Here we expressed recombinant proteins of the two PGM1 isoforms from the zoonotic Cryptosporidium parvum, namely CpPGM1A and CpPGM1B, and confirmed their enzyme activity. Both isoforms followed Michaelis–Menten kinetics towards glucose-1-phosphate (Km = 0.17 and 0.13 mM, Vmax = 7.30 and 2.76 μmol/min/mg, respectively). CpPGM1A and CpPGM1B genes were expressed in oocysts, sporozoites and intracellular parasites at a similar pattern of expression, however CpPGM1A was expressed at much higher levels than CpPGM1B. Immunofluorescence assay showed that CpPGM1A was present in the cytosol of sporozoites, however this was enriched towards the plasma membranes in the intracellular parasites; whereas CpPGM1B was mainly present under sporozoite pellicle, although relocated to the parasitophorous vacuole membrane in the intracellular development. These observations indicated that CpPGM1A played a house-keeping function, while CpPGM1B played a different biological role that remains to be defined by future investigations.

scholarly journals Characterization of Stages of Hepatozoon americanum and of Parasitized Canine Host Cells

2005 ◽  
Vol 42 (6) ◽  
pp. 788-796 ◽  
Author(s):  
C. A. Cummings ◽  
R. J. Panciera ◽  
K. M. Kocan ◽  
J. S. Mathew ◽  
S. A. Ewing
Keyword(s):  
Cell Membrane ◽  
Host Cell ◽  
Striated Muscle ◽  
Parasitophorous Vacuole ◽  
Protozoan Parasite ◽  
Host Cells ◽  
Blood Leukocytes ◽  
Host Cell Membrane ◽  
Asexual Multiplication ◽  
Hepatozoon Americanum

American canine hepatozoonosis is caused by Hepatozoon americanum, a protozoan parasite, the definitive host of which is the tick, Amblyomma maculatum. Infection of the dog follows ingestion of ticks that harbor sporulated H. americanum oocysts. Following penetration of the intestinal mucosa, sporozoites are disseminated systemically and give rise to extensive asexual multiplication in cells located predominantly in striated muscle. The parasitized canine cells in “onion skin” cysts and in granulomas situated within skeletal muscle, as well as those in peripheral blood leukocytes (PBL), were identified as macrophages by use of fine structure morphology and/or immunohistochemical reactivity with macrophage markers. Additionally, two basic morphologic forms of the parasite were observed in macrophages of granulomas and PBLs. The forms were presumptively identified as merozoites and gamonts. The presence of a “tail” in some gamonts in PBLs indicated differentiation toward microgametes. Recognition of merozoites in PBLs supports the contention that hematogenously redistributed merozoites initiate repeated asexual cycles and could explain persistence of infection for long periods in the vertebrate host. Failure to clearly demonstrate a host cell membrane defining a parasitophorous vacuole may indicate that the parasite actively penetrates the host cell membrane rather than being engulfed by the host cell, as is characteristic of some protozoans.

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