Secretion of anEimeria tenella sporozoite antigen during host-cell invasion: Visualization of the parasitophorous vacuole membrane and parasitophorous duct-like structures

1993 ◽  
Vol 79 (1) ◽  
pp. 77-79 ◽  
Author(s):  
G. Zgrzebski ◽  
W. Raether ◽  
J. Hofmann ◽  
R. Entzeroth
Keyword(s):  
Host Cell ◽  
Cell Invasion ◽  
Parasitophorous Vacuole ◽  
Host Cell Invasion ◽  
Parasitophorous Vacuole Membrane ◽  
Vacuole Membrane

Characterisation of NcGRA7 and NcSAG4 proteins: Immunolocalisation and their role in the host cell invasion by Neospora caninum tachyzoites

2010 ◽  
Vol 55 (4) ◽  
Author(s):  
Adriana Aguado-Martínez ◽  
Gema Álvarez-García ◽  
Gereon Schares ◽  
Verónica Risco-Castillo ◽  
Aurora Fernández-García ◽  
...  
Keyword(s):  
Monoclonal Antibody ◽  
Host Cell ◽  
Cell Invasion ◽  
Neospora Caninum ◽  
Parasitophorous Vacuole ◽  
Membrane Surface ◽  
Chronic Phase ◽  
Host Cell Invasion ◽  
Invasion Mechanisms ◽  
The Matrix

AbstractNeospora caninum negatively impacts bovine reproductive performance around the world. Addressing this problem requires a greater understanding of the parasite’s molecular biology. In this study, monoclonal antibodies against recombinant proteins were successfully developed and employed to characterise two different proteins of N. caninum: the acute phase-associated NcGRA7 and the chronic phase-associated NcSAG4. Immunofluorescence with the anti-rNcGRA7 monoclonal antibody suggested that NcGRA7 trafficks from tachyzoite dense granules to the matrix of the parasitophorous vacuole and parasite’s surroundings. Furthermore, NcGRA7 is also expressed in the bradyzoite stage and localised on the matrix of bradyzoite-positive vacuoles. NcGRA7 appears to be partially involved in the tachyzoite-invasion mechanisms, as an anti-rNcGRA7 monoclonal antibody partially inhibited in vitro tachyzoite-invasion. A monoclonal antibody specific for NcSAG4 confirmed this protein’s bradyzoitespecific expression both by western blot and immunofluorescence. However, some bradyzoite-positive vacuoles only weakly expressed NcSAG4, if it was expressed at all. The specificity of the anti-rNcSAG4 monoclonal antibody was confirmed by the recognition of the NcSAG4 in the membrane surface of Nc-1SAG4c transgenic tachyzoites, which constitutively expresses NcSAG4. Blocking NcSAG4 of Nc-1SAG4c tachyzoites with the monoclonal antibody did not affect host cell invasion. However, its implication on the host cell adhesion or host immune evasion should not be discarded.

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 revised mechanism for how Plasmodium falciparum recruits and exports proteins into its erythrocytic host cell.

2021 ◽  
Author(s):  
Mikha Gabriela ◽  
Kathryn Matthews ◽  
Cas Boshoven ◽  
Betty Kouskousis ◽  
David Steer ◽  
...  
Keyword(s):  
Plasmodium Falciparum ◽  
Host Cell ◽  
Parasitophorous Vacuole ◽  
Specific Interactions ◽  
Parasitophorous Vacuole Membrane ◽  
Conducting Channel ◽  
Trophozoite Stage ◽  
Vacuole Membrane ◽  
N Terminus ◽  
Exported Protein

Plasmodium falciparum exports ~10% of its proteome into its host erythrocyte to modify the host cell’s physiology. The Plasmodium export element (PEXEL) motif contained within the N-terminus of most exported proteins directs the trafficking of those proteins into the erythrocyte. To reach the host cell, the PEXEL motif of exported proteins are processed by the endoplasmic reticulum (ER) resident aspartyl protease plasmepsin V. Then, following secretion into the parasite-encasing parasitophorous vacuole, the mature exported protein must be unfolded and translocated across the parasitophorous vacuole membrane by the Plasmodium translocon of exported proteins (PTEX). PTEX is a protein-conducting channel consisting of the pore-forming protein EXP2, the protein unfoldase HSP101, and structural component PTEX150. The mechanism of how exported proteins are specifically trafficked from the parasite’s ER following PEXEL cleavage to PTEX complexes on the parasitophorous vacuole membrane is currently not understood. Here, we present evidence that EXP2 and PTEX150 form a stable subcomplex that facilitates HSP101 docking. We also demonstrate that HSP101 localises both within the parasitophorous vacuole and within the parasite’s ER throughout the ring and trophozoite stage of the parasite, coinciding with the timeframe of protein export. Interestingly, we found that HSP101 can form specific interactions with model PEXEL proteins in the parasite ER, irrespective of their PEXEL processing status. Collectively, our data suggest that HSP101 recognises and chaperones PEXEL proteins from the ER to the parasitophorous vacuole and given HSP101’s specificity for the EXP2-PTEX150 subcomplex, this provides a mechanism for how exported proteins are specifically targeted to PTEX for translocation into the erythrocyte.

scholarly journals Origins of the parasitophorous vacuole membrane of the malaria parasite, Plasmodium falciparum, in human red blood cells

1992 ◽  
Vol 102 (3) ◽  
pp. 527-532 ◽  
Author(s):  
A.R. Dluzewski ◽  
G.H. Mitchell ◽  
P.R. Fryer ◽  
S. Griffiths ◽  
R.J. Wilson ◽  
...  
Keyword(s):  
Plasmodium Falciparum ◽  
Cell Membrane ◽  
Host Cell ◽  
Malaria Parasite ◽  
Parasitophorous Vacuole ◽  
Parasitophorous Vacuole Membrane ◽  
Host Cell Membrane ◽  
Vacuole Membrane ◽  
Parasite Plasmodium ◽  
Parasite Plasmodium Falciparum

We have attempted to determine whether the parasitophorous vacuole membrane, in which the malaria parasite (merozoite) encapsulates itself when it enters a red blood cell, is derived from the host cell plasma membrane, as the appearance of the invasion process in the electron microscope has been taken to suggest, or from lipid material stored in the merozoite. We have incorporated into the red cell membrane a haptenic phospholipid, phosphatidylethanolamine, containing an NBD (N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)) group, substituted in the acyl chain, and allowed it to translocate into the inner bilayer leaflet. After invasion of these labelled cells by the parasite, Plasmodium falciparum, immuno-gold electron microscopy was used to follow the distribution of the labelled lipid; this was found to be overwhelmingly in favour of the host cell membrane relative to the parasitophorous vacuole. Merozoites of P. knowlesi were allowed to attach irreversibly to red cells without invasion, using the method of pretreatment with cytochalasin. The region of contact between the merozoite and the host cell membrane was in all cases devoid of the labelled phosphatidylethanolamine. These results lead us to infer that the parasitophorous vacuole membrane is derived wholly or partly from lipid preexisting in the merozoite.

The origin of parasitophorous vacuole membrane lipids in malaria-infected erythrocytes

1993 ◽  
Vol 106 (1) ◽  
pp. 237-248 ◽  
Author(s):  
G.E. Ward ◽  
L.H. Miller ◽  
J.A. Dvorak
Keyword(s):  
Host Cell ◽  
Erythrocyte Membrane ◽  
Membrane Lipids ◽  
Parasitophorous Vacuole ◽  
Light Level ◽  
Surface Proteins ◽  
Parasitophorous Vacuole Membrane ◽  
Host Cell Membrane ◽  
Vacuole Membrane ◽  
Erythrocyte Surface

During invasion of an erythrocyte by a malaria merozoite, an indentation develops in the erythrocyte surface at the point of contact between the two cells. This indentation deepens as invasion progresses, until the merozoite is completely surrounded by a membrane known as the parasitophorous vacuole membrane (PVM). We incorporated fluorescent lipophilic probes and phospholipid analogs into the erythrocyte membrane, and followed the fate of these probes during PVM formation with low-light-level video fluorescence microscopy. The concentration of probe in the forming PVM was indistinguishable from the concentration of probe in the erythrocyte membrane, suggesting that the lipids of the PVM are continuous with and derived from the host cell membrane during invasion. In contrast, fluorescently labeled erythrocyte surface proteins were largely excluded from the forming PVM. These data are consistent with a model for PVM formation in which the merozoite induces a localized invagination in the erythrocyte lipid bilayer, concomitant with a localized restructuring of the host cell cytoskeleton.

scholarly journals First Characterization of theNeospora caninumDense Granule Protein GRA9

2017 ◽  
Vol 2017 ◽  
pp. 1-15 ◽  
Author(s):  
Margret Leineweber ◽  
Katrin Spekker-Bosker ◽  
Vanessa Ince ◽  
Gereon Schares ◽  
Andrew Hemphill ◽  
...  
Keyword(s):  
Host Cell ◽  
Cell Invasion ◽  
Neospora Caninum ◽  
Parasitophorous Vacuole ◽  
Dense Granule ◽  
Immunogold Electron Microscopy ◽  
Host Cell Invasion ◽  
Dense Granules ◽  
Granule Protein

The obligate intracellular apicomplexan parasiteNeospora caninum (N. caninum)is closely related toToxoplasma gondii (T. gondii). The dense granules, which are present in all apicomplexan parasites, are important secretory organelles. Dense granule (GRA) proteins are released into the parasitophorous vacuole (PV) following host cell invasion and are known to play important roles in the maintenance of the host-parasite relationship and in the acquisition of nutrients. Here, we provide a detailed characterization of theN. caninumdense granule protein NcGRA9. The in silico genomic organization and key protein characteristics are described. Immunofluorescence-based localization studies revealed that NcGRA9 is located in the dense granules and is released into the interior of the PV following host cell invasion. Immunogold-electron microscopy confirmed the dense granule localization and showed that NcGRA9 is associated with the intravacuolar network. In addition, NcGRA9 is found in the “excreted secreted antigen” (ESA) fraction ofN. caninum. Furthermore, by analysing the distribution of truncated versions of NcGRA9, we provide evidence that the C-terminal region of this protein is essential for the targeting of NcGRA9 into the dense granules ofN. caninum, and the truncated proteins show reduced secretion.

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 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.

Host cell invasion by Apicomplexa: an expression of the parasite's contractile system?

Parasitology ◽  
1983 ◽  
Vol 87 (2) ◽  
pp. 199-209 ◽  
Author(s):  
D. G. Russell
Keyword(s):  
Host Cell ◽  
Cell Invasion ◽  
Parasitophorous Vacuole ◽  
Membrane Receptors ◽  
Host Cell Invasion ◽  
Contractile System ◽  
Transmission Electron ◽  
Electron Microscopes ◽  
The One ◽  
Host Parasite

SUMMARYRecent studies on the motility of coccidian sporozoites have demonstrated a membrane-associated contractile system capable of moving certain intramembraneous components down the parasite surface propelling it forwards. The properties of this system resemble recorded observations on host cell invasion. In this study the invasive behaviour ofEimeria tenellaandE. acervulinahas been examined, with reference to the above findings, by light microscope and scanning and transmission electron microscopes. Known inhibitors of motility prevent invasion, though attachment appears unaffected. Invasion itself consists of 3 phases; attachment and orientation, induction of a parasitophorous vacuole and translocation of the parasite into the vacuole. Ultrastructural examination reveals a close membrane/membrane association maintained throughout invasion. From these results it is suggested that the parasite enters the parasitophorous vacuole by ‘capping’ the host/parasite junction down its body, so locomoting into the host cell. Such a model has two main advantages; it requires no additional modifications to either cell, and the specificity of membrane receptors would enable the one membrane-associated contractile system to be responsible for locomotion, antibody capping and host cell invasion.

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