Secretion by Toxoplasma gondii of an antigen that appears to become associated with the parasitophorous vacuole membrane upon invasion of the host cell

1987 ◽  
Vol 88 (2) ◽  
pp. 231-239
Author(s):  
I. Kimata ◽  
K. Tanabe
Keyword(s):  
Toxoplasma Gondii ◽  
Host Cell ◽  
Anterior Part ◽  
Parasitophorous Vacuole ◽  
Host Cells ◽  
Immunoperoxidase Staining ◽  
Cell Plasma ◽  
Sds Page ◽  
Infected Cells ◽  
Triton X

Monoclonal antibodies against Toxoplasma gondii were prepared to characterize antigens of the parasite. Immunoperoxidase staining of parasites fixed with paraformaldehyde and glutaraldehyde (PFAGA) followed by Triton X-100 treatment showed that the antibody of clone I-63 recognized an antigen located in the anterior part of the parasite. When analysed by SDS-PAGE and immunoblotting, the antigen migrated in a 66 × 10(3) Mr region. The parasite antigen diminished greatly in parasites after invasion of host cells, but reappeared around a time when intracellular T. gondii multiplied. Immunodetection on PFAGA-fixed T. gondii-infected cells, whose membranes were permeabilized by freeze-thawing in the presence of 5% glycerol, demonstrated that, immediately after parasite invasion, the I-63 antibody-reactive antigen appeared to become associated with the parasitophorous vacuole (PV) membrane, that had been formed mainly by invagination of the host-cell plasma membrane so as to surround an invading parasite. The antigen remained associated with the PV membrane for some time, but disappeared later when the PV increased in size after the parasites had multiplied several times. These results were strengthened by immunoelectron microscopic observations: the antigen that had been localized at the anterior part of the parasite before invasion appeared in an area of the host cell cytoplasm around the tips of penetrating parasites and, thereafter, extended throughout the surface of the PV membrane when parasites completed invasion. Thus, it appears that the I-63-reactive antigen is secreted by T. gondii upon invasion of the host cell and becomes associated with the PV membrane shortly after invasion.

scholarly journals Evidence for Host Cells as the Major Contributor of Lipids in the Intravacuolar Network of Toxoplasma-Infected Cells

2011 ◽  
Vol 10 (8) ◽  
pp. 1095-1099 ◽  
Author(s):  
Carolina E. Caffaro ◽  
John C. Boothroyd
Keyword(s):  
Host Cell ◽  
Fluorescence Recovery After Photobleaching ◽  
Parasitophorous Vacuole ◽  
Dense Granule ◽  
Host Cells ◽  
Cell Plasma Membrane ◽  
Content Type ◽  
Cell Plasma ◽  
Continuous Stream ◽  
Infected Cells

ABSTRACT The intracellular parasite Toxoplasma gondii develops inside a parasitophorous vacuole (PV) that derives from the host cell plasma membrane during invasion. Previous electron micrograph images have shown that the membrane of this vacuole undergoes an extraordinary remodeling with an extensive network of thin tubules and vesicles, the intravacuolar network (IVN), which fills the lumen of the PV. While dense granule proteins, secreted during and after invasion, are the main factors for the organization and tubulation of the network, little is known about the source of lipids used for this remodeling. By selectively labeling host cell or parasite membranes, we uncovered evidence that strongly supports the host cell as the primary, if not exclusive, source of lipids for parasite IVN remodeling. Fluorescence recovery after photobleaching (FRAP) microscopy experiments revealed that lipids are surprisingly dynamic within the parasitophorous vacuole and are continuously exchanged or replenished by the host cell. The results presented here suggest a new model for development of the parasitophorous vacuole whereby the host provides a continuous stream of lipids to support the growth and maturation of the PVM and IVN.

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 Coimmunoprecipitation with MYR1 Identifies Three Additional Proteins within the Toxoplasma gondii Parasitophorous Vacuole Required for Translocation of Dense Granule Effectors into Host Cells

mSphere ◽  
2020 ◽  
Vol 5 (1) ◽  
Author(s):  
Alicja M. Cygan ◽  
Terence C. Theisen ◽  
Alma G. Mendoza ◽  
Nicole D. Marino ◽  
Michael W. Panas ◽  
...  
Keyword(s):  
Toxoplasma Gondii ◽  
Protein Translocation ◽  
Affinity Purification ◽  
Parasitophorous Vacuole ◽  
Dense Granule ◽  
Effector Proteins ◽  
Host Cells ◽  
Content Type ◽  
Cyclin E1 ◽  
Infected Cells

ABSTRACT Toxoplasma gondii is a ubiquitous, intracellular protozoan that extensively modifies infected host cells through secreted effector proteins. Many such effectors must be translocated across the parasitophorous vacuole (PV), in which the parasites replicate, ultimately ending up in the host cytosol or nucleus. This translocation has previously been shown to be dependent on five parasite proteins: MYR1, MYR2, MYR3, ROP17, and ASP5. We report here the identification of several MYR1-interacting and novel PV-localized proteins via affinity purification of MYR1, including TGGT1_211460 (dubbed MYR4), TGGT1_204340 (dubbed GRA54), and TGGT1_270320 (PPM3C). Further, we show that three of the MYR1-interacting proteins, GRA44, GRA45, and MYR4, are essential for the translocation of the Toxoplasma effector protein GRA16 and for the upregulation of human c-Myc and cyclin E1 in infected cells. GRA44 and GRA45 contain ASP5 processing motifs, but like MYR1, processing at these sites appears to be nonessential for their role in protein translocation. These results expand our understanding of the mechanism of effector translocation in Toxoplasma and indicate that the process is highly complex and dependent on at least eight discrete proteins. IMPORTANCE Toxoplasma is an extremely successful intracellular parasite and important human pathogen. Upon infection of a new cell, Toxoplasma establishes a replicative vacuole and translocates parasite effectors across this vacuole to function from the host cytosol and nucleus. These effectors play a key role in parasite virulence. The work reported here newly identifies three parasite proteins that are necessary for protein translocation into the host cell. These results significantly increase our knowledge of the molecular players involved in protein translocation in Toxoplasma-infected cells and provide additional potential drug targets.

Besnoitia besnoiti and Toxoplasma gondii: two apicomplexan strategies to manipulate the host cell centrosome and Golgi apparatus

Parasitology ◽  
2014 ◽  
Vol 141 (11) ◽  
pp. 1436-1454 ◽  
Author(s):  
RITA CARDOSO ◽  
SOFIA NOLASCO ◽  
JOÃO GONÇALVES ◽  
HELDER C. CORTES ◽  
ALEXANDRE LEITÃO ◽  
...  
Keyword(s):  
Toxoplasma Gondii ◽  
Golgi Apparatus ◽  
Host Cell ◽  
Parasitophorous Vacuole ◽  
Host Cells ◽  
Besnoitia Besnoiti ◽  
Microtubule Cytoskeleton ◽  
Cytoskeleton Organization ◽  
Evolutionary Paths ◽  
The Impact

SUMMARYBesnoitia besnoiti and Toxoplasma gondii are two closely related parasites that interact with the host cell microtubule cytoskeleton during host cell invasion. Here we studied the relationship between the ability of these parasites to invade and to recruit the host cell centrosome and the Golgi apparatus. We observed that T. gondii recruits the host cell centrosome towards the parasitophorous vacuole (PV), whereas B. besnoiti does not. Notably, both parasites recruit the host Golgi apparatus to the PV but its organization is affected in different ways. We also investigated the impact of depleting and over-expressing the host centrosomal protein TBCCD1, involved in centrosome positioning and Golgi apparatus integrity, on the ability of these parasites to invade and replicate. Toxoplasma gondii replication rate decreases in cells over-expressing TBCCD1 but not in TBCCD1-depleted cells; while for B. besnoiti no differences were found. However, B. besnoiti promotes a reorganization of the Golgi ribbon previously fragmented by TBCCD1 depletion. These results suggest that successful establishment of PVs in the host cell requires modulation of the Golgi apparatus which probably involves modifications in microtubule cytoskeleton organization and dynamics. These differences in how T. gondii and B. besnoiti interact with their host cells may indicate different evolutionary paths.

scholarly journals Host ER–parasitophorous vacuole interaction provides a route of entry for antigen cross-presentation in Toxoplasma gondii–infected dendritic cells

2009 ◽  
Vol 206 (2) ◽  
pp. 399-410 ◽  
Author(s):  
Romina S. Goldszmid ◽  
Isabelle Coppens ◽  
Avital Lev ◽  
Pat Caspar ◽  
Ira Mellman ◽  
...  
Keyword(s):  
T Cells ◽  
Dendritic Cells ◽  
Toxoplasma Gondii ◽  
Mhc Class I ◽  
Cd8 T Cells ◽  
Parasitophorous Vacuole ◽  
Host Cells ◽  
Class I ◽  
Cross Presentation ◽  
Infected Cells

Toxoplasma gondii tachyzoites infect host cells by an active invasion process leading to the formation of a specialized compartment, the parasitophorous vacuole (PV). PVs resist fusion with host cell endosomes and lysosomes and are thus distinct from phagosomes. Because the parasite remains sequestered within the PV, it is unclear how T. gondii–derived antigens (Ag’s) access the major histocompatibility complex (MHC) class I pathway for presentation to CD8+ T cells. We demonstrate that recruitment of host endoplasmic reticulum (hER) to the PV in T. gondii–infected dendritic cells (DCs) directly correlates with cross-priming of CD8+ T cells. Furthermore, we document by immunoelectron microscopy the transfer of hER components into the PV, a process indicative of direct fusion between the two compartments. In strong contrast, no association between hER and phagosomes or Ag presentation activity was observed in DCs containing phagocytosed live or dead parasites. Importantly, cross-presentation of parasite-derived Ag in actively infected cells was blocked when hER retrotranslocation was inhibited, indicating that the hER serves as a conduit for the transport of Ag between the PV and host cytosol. Collectively, these findings demonstrate that pathogen-driven hER–PV interaction can serve as an important mechanism for Ag entry into the MHC class I pathway and CD8+ T cell cross-priming.

scholarly journals Invasion by Toxoplasma gondii Establishes a Moving Junction That Selectively Excludes Host Cell Plasma Membrane Proteins on the Basis of Their Membrane Anchoring

1999 ◽  
Vol 190 (12) ◽  
pp. 1783-1792 ◽  
Author(s):  
Dana G. Mordue ◽  
Naishadh Desai ◽  
Michael Dustin ◽  
L. David Sibley
Keyword(s):  
Plasma Membrane ◽  
Toxoplasma Gondii ◽  
Host Cell ◽  
Transmembrane Domain ◽  
Membrane Lipids ◽  
Transmembrane Proteins ◽  
Host Cells ◽  
Cell Plasma Membrane ◽  
Cell Plasma ◽  
Membrane Anchoring

The protozoan parasite Toxoplasma gondii actively penetrates its host cell by squeezing through a moving junction that forms between the host cell plasma membrane and the parasite. During invasion, this junction selectively controls internalization of host cell plasma membrane components into the parasite-containing vacuole. Membrane lipids flowed past the junction, as shown by the presence of the glycosphingolipid GM1 and the cationic lipid label 1.1′-dihexadecyl-3-3′-3-3′-tetramethylindocarbocyanine (DiIC16). Glycosylphosphatidylinositol (GPI)-anchored surface proteins, such as Sca-1 and CD55, were also readily incorporated into the parasitophorous vacuole (PV). In contrast, host cell transmembrane proteins, including CD44, Na+/K+ ATPase, and β1-integrin, were excluded from the vacuole. To eliminate potential differences in sorting due to the extracellular domains, parasite invasion was examined in host cells transfected with recombinant forms of intercellular adhesion molecule 1 (ICAM-1, CD54) that differed in their mechanism of membrane anchoring. Wild-type ICAM-1, which contains a transmembrane domain, was excluded from the PV, whereas both GPI-anchored ICAM-1 and a mutant of ICAM-1 missing the cytoplasmic tail (ICAM-1–Cyt−) were readily incorporated into the PV membrane. Our results demonstrate that during host cell invasion, Toxoplasma selectively excludes host cell transmembrane proteins at the moving junction by a mechanism that depends on their anchoring in the membrane, thereby creating a nonfusigenic compartment.

scholarly journals Paclitaxel Arrests Growth of IntracellularToxoplasma gondii

1998 ◽  
Vol 42 (8) ◽  
pp. 2036-2040 ◽  
Author(s):  
Randee Estes ◽  
Nicolas Vogel ◽  
Douglas Mack ◽  
Rima McLeod
Keyword(s):  
Toxoplasma Gondii ◽  
Host Cell ◽  
Fresh Medium ◽  
Host Cells ◽  
Normal Morphology ◽  
Intracellular Form ◽  
Human Foreskin Fibroblasts ◽  
The Future ◽  
Infected Cells ◽  
Parasite Multiplication

ABSTRACT Addition of paclitaxel (Taxol) at a concentration of 1 μM toToxoplasma gondii-infected human foreskin fibroblasts arrested parasite multiplication. Division of theT. gondii tachyzoite nucleus was inhibited, leading to syncytium-like parasite structures within the fibroblasts by 24 h after infection and treatment of the cultures. By 4 days after infection and treatment of the cultures with paclitaxel, this inhibition was irreversible, since the arrested intracellular form was incapable of leaving the host cell, infecting new cells, and initiating the growth of tachyzoites with normal morphology. Specifically, when paclitaxel was added to infected cells for 4 days and then removed by washing and the infected, paclitaxel-treated cells were cultured for 4 more days, there were no remaining T. gondii organisms with normal morphology. Syncytium-like structures in the cultures that were infected and treated with paclitaxel for 8 days were similar in appearance to those in preparations of infected paclitaxel-treated fibroblasts that had been cultured for 24 to 48 h. Pretreatment of the tachyzoites for 1 h with paclitaxel followed by the removal of the paclitaxel by repeatedly centrifuging and resuspending the parasites in fresh medium without paclitaxel and then adding fresh medium prior to culture of the parasites with fibroblasts did not prevent their invasion of fibroblasts but did affect their subsequent ability to replicate within fibroblasts. Pretreatment of the fibroblasts with paclitaxel also diminished subsequent replication ofT. gondii in such host cells after 8 days. Thus, paclitaxel alters the ability of T. gondii to replicate in host cells. Inhibition of parasite microtubules by such compounds at concentrations which do not interfere with the function of host cell microtubules may be useful for development of novel medicines to treat T. gondii infections in the future.

scholarly journals The Nascent Parasitophorous Vacuole Membrane of Encephalitozoon cuniculi Is Formed by Host Cell Lipids and Contains Pores Which Allow Nutrient Uptake

2008 ◽  
Vol 7 (6) ◽  
pp. 1001-1008 ◽  
Author(s):  
Karin Rönnebäumer ◽  
Uwe Gross ◽  
Wolfgang Bohne
Keyword(s):  
Plasma Membrane ◽  
Host Cell ◽  
Fluorescent Dyes ◽  
Parasitophorous Vacuole ◽  
Time Lapse ◽  
Host Cells ◽  
Encephalitozoon Cuniculi ◽  
Invasion Mechanism ◽  
Cell Plasma ◽  
Metabolite Exchange

ABSTRACT Microsporidia are obligate intracellular pathogens which enter host cells by the discharge of a hollow tube through which the sporoplasma is extruded into the host cell. Since this invasion mechanism is very different from common entry strategies, the formation of the parasitophorous vacuole (PV) in Encephalitozoon species is likely to be distinct from known principles. We investigated the origin of the nascent Encephalitozoon cuniculi PV membrane with the aid of fluorescent lipid probes. When Bodipy 500/510-C12-HPC-labeled spores were used for infection, the emerging PV membrane was unlabeled, suggesting that sporoplasma-derived lipids do not significantly contribute to the formation of the PV membrane. In contrast, when raft and nonraft microdomains of the host cell plasma membrane were selectively labeled with DiIC16 and Speedy DiO, both tracers were detectable in the nascent PV membrane shortly after infection, indicating that the bulk lipids of the PV membrane are host cell derived. Time-lapse fluorescence microscopy revealed that the formation of the PV membrane is a fast event (<1.3 s), which occurred simultaneously with the extrusion of the sporoplasma. The portion of the discharged tube which is in contact with the host cell was found to be coated with labeled host cell lipids, which might be an indication for a plasma membrane invagination at the contact site. To investigate the presence of pores in the E. cuniculi PV membrane, we microinjected fluorescent dyes of different sizes into infected host cells. A 0.5-kDa dextran as well as 0.8- to 1.1-kDa peptides could rapidly enter the PV, while a 10-kDa dextran was stably excluded from the PV lumen, indicating that the PV membrane possesses pores with an exclusion size of <10 kDa, which should allow metabolite exchange.

scholarly journals Class I Major Histocompatibility Complex Presentation of Antigens That Escape from the Parasitophorous Vacuole of Toxoplasma gondii

2005 ◽  
Vol 73 (2) ◽  
pp. 703-711 ◽  
Author(s):  
Marc-Jan Gubbels ◽  
Boris Striepen ◽  
Nilabh Shastri ◽  
Mustafa Turkoz ◽  
Ellen A. Robey
Keyword(s):  
Major Histocompatibility Complex ◽  
Toxoplasma Gondii ◽  
Host Cell ◽  
Parasitophorous Vacuole ◽  
T Cell Response ◽  
Class I ◽  
Peptide Transporter ◽  
Major Histocompatibility ◽  
Histocompatibility Complex ◽  
Infected Cells

ABSTRACT The intracellular parasite Toxoplasma gondii, the causative agent of toxoplasmosis, induces a protective CD8 T-cell response in its host; however, the mechanisms by which T. gondii proteins are presented by the class I major histocompatibility complex remain largely unexplored. T. gondii resides within a specialized compartment, the parasitophorous vacuole, that sequesters the parasite and its secreted proteins from the host cell cytoplasm, suggesting that an alternative cross-priming pathway might be necessary for class I presentation of T. gondii antigens. Here we used a strain of T. gondii expressing yellow fluorescent protein and a secreted version of the model antigen ovalbumin to investigate this question. We found that presentation of ovalbumin secreted by the parasite requires the peptide transporter TAP (transporter associated with antigen processing) and occurs primarily in actively infected cells rather than bystander cells. We also found that dendritic cells are a major target of T. gondii infection in vivo and account for much of the antigen-presenting activity in the spleen. Finally, we obtained evidence that Cre protein secreted by T. gondii can mediate recombination in the nucleus of the host cell. Together, these results indicate that Toxoplasma proteins can escape from the parasitophorous vacuole into the host cytoplasm and be presented by the endogenous class I pathway, leading to direct recognition of infected cells by CD8 T cells.

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.

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