scholarly journals Acquisition of Host Cytosolic Protein by Toxoplasma gondii Bradyzoites

mSphere ◽  
2021 ◽  
Vol 6 (1) ◽  
Author(s):  
Geetha Kannan ◽  
Pariyamon Thaprawat ◽  
Tracey L. Schultz ◽  
Vern B. Carruthers
Keyword(s):  
Toxoplasma Gondii ◽  
Cyst Wall ◽  
Parasitophorous Vacuole ◽  
Protozoan Parasite ◽  
Acute Stage ◽  
Host Proteins ◽  
Reporter Protein ◽  
Content Type ◽  
Chronic Stage ◽  
Infected Cells

ABSTRACT Toxoplasma gondii is a protozoan parasite that persists in the central nervous system as intracellular chronic-stage bradyzoites that are encapsulated by a thick cyst wall. While the cyst wall separates bradyzoites from the host cytosol, it has been posited that small solutes can traverse the cyst wall to sustain bradyzoites. Recently, it was found that host cytosolic macromolecules can cross the parasitophorous vacuole and are ingested and digested by actively replicating acute-stage tachyzoites. However, the extent to which bradyzoites have an active ingestion pathway remained unknown. To interrogate this, we modified previously published protocols that look at tachyzoite acquisition and digestion of host proteins by measuring parasite accumulation of a host-expressed reporter protein after impairment of an endolysosomal protease (cathepsin protease L [CPL]). Using two cystogenic parasite strains (ME49 and Pru), we demonstrate that T. gondii bradyzoites can ingest host-derived cytosolic mCherry. Bradyzoites acquire host mCherry within 4 h of invasion and after cyst wall formation. This study provides direct evidence that host macromolecules can be internalized by T. gondii bradyzoites across the cyst wall in infected cells. IMPORTANCE Chronic infection of humans with Toxoplasma gondii is common, but little is known about how this intracellular parasite obtains the resources that it needs to persist indefinitely inside neurons and muscle cells. Here, we provide evidence that the chronic-stage form of T. gondii can internalize proteins from the cytosol of infected cells despite residing within an intracellular cyst that is surrounded by a cyst wall. We also show that accumulation of host-derived protein within the chronic-stage parasites is enhanced by disruption of a parasite protease, suggesting that such protein is normally degraded to generate peptides and amino acids. Taken together, our findings imply that chronic-stage T. gondii can ingest and digest host proteins, potentially to support its persistence.

scholarly journals Acquisition of Host Cytosolic Protein by Toxoplasma gondii Bradyzoites

2020 ◽  
Author(s):  
Geetha Kannan ◽  
Pariyamon Thaprawat ◽  
Tracey L. Schultz ◽  
Vern B. Carruthers
Keyword(s):  
Toxoplasma Gondii ◽  
Cyst Wall ◽  
Parasitophorous Vacuole ◽  
Protozoan Parasite ◽  
Acute Stage ◽  
Host Proteins ◽  
Reporter Protein ◽  
The Central Nervous System ◽  
Infected Cells ◽  
Cathepsin Protease

ABSTRACTToxoplasma gondii is a protozoan parasite that persists in the central nervous system as intracellular chronic stage bradyzoites that are encapsulated by a thick cyst wall. While the cyst wall separates bradyzoites from the host cytosol, it has been posited that small solutes can traverse the cyst wall to sustain bradyzoites. Recently it was found that host cytosolic macromolecules can cross the parasitophorous vacuole and are ingested and digested by actively replicating acute stage tachyzoites. However, the extent to which bradyzoites have an active ingestion pathway remained unknown. To interrogate this, we modified previously published protocols that look at tachyzoite acquisition and digestion of host proteins by measuring parasite accumulation of a host-expressed reporter protein after impairment of an endolysosomal protease (Cathepsin Protease L, CPL). Using two cystogenic parasite strains (ME49 and Pru), we demonstrate that T. gondii bradyzoites can ingest host-derived cytosolic mCherry. Bradyzoites acquire host mCherry within 4 hours of invasion and post-cyst wall formation. This study provides direct evidence that host macromolecules can be internalized by T. gondii bradyzoites across the cyst wall in infected cells.

scholarly journals Phosphorylation of Toxoplasma gondii Secreted Proteins during Acute and Chronic Stages of Infection

mSphere ◽  
2020 ◽  
Vol 5 (5) ◽  
Author(s):  
Joanna C. Young ◽  
Malgorzata Broncel ◽  
Helena Teague ◽  
Matt R. G. Russell ◽  
Olivia L. McGovern ◽  
...  
Keyword(s):  
Toxoplasma Gondii ◽  
Cyst Wall ◽  
Dominant Role ◽  
Parasitophorous Vacuole ◽  
Cyst Formation ◽  
Secreted Proteins ◽  
New Host ◽  
Content Type ◽  
Chronic Stage ◽  
Membrane Bound

ABSTRACT The intracellular parasite Toxoplasma gondii resides within a membrane-bound parasitophorous vacuole (PV) and secretes an array of proteins to establish this replicative niche. It has been shown previously that Toxoplasma secretes kinases and that numerous proteins are phosphorylated after secretion. Here, we assess the role of the phosphorylation of strand-forming protein 1 (SFP1) and the related protein GRA29, two secreted proteins with unknown function. We show that both proteins form stranded structures in the PV that are independent of the previously described intravacuolar network or actin. SFP1 and GRA29 can each form these structures independently of other Toxoplasma secreted proteins, although GRA29 appears to regulate SFP1 strands. We show that an unstructured region at the C termini of SFP1 and GRA29 is required for the formation of strands and that mimicking the phosphorylation of this domain of SFP1 negatively regulates strand development. When tachyzoites convert to chronic-stage bradyzoites, both proteins show a dispersed localization throughout the cyst matrix. Many secreted proteins are reported to dynamically redistribute as the cyst forms, and secreted kinases are known to play a role in cyst formation. Using quantitative phosphoproteome and proteome analyses comparing tachyzoite and early bradyzoite stages, we reveal widespread differential phosphorylation of secreted proteins. While we found no direct evidence for phosphorylation playing a dominant role for SFP1/GRA29 redistribution in the cyst, these data support a model in which secreted kinases and phosphatases contribute to the regulation of secreted proteins during stage conversion. IMPORTANCE Toxoplasma gondii is a common parasite that infects up to one-third of the human population. Initially, the parasite grows rapidly, infecting and destroying cells of the host, but subsequently switches to a slow-growing form and establishes chronic infection. In both stages, the parasite lives within a membrane-bound vacuole within the host cell, but in the chronic stage, a durable cyst wall is synthesized, which provides protection to the parasite during transmission to a new host. Toxoplasma secretes proteins into the vacuole to build its replicative niche, and previous studies identified many of these proteins as phosphorylated. We investigate two secreted proteins and show that a phosphorylated region plays an important role in their regulation in acute stages. We also observed widespread phosphorylation of secreted proteins when parasites convert from acute to chronic stages, providing new insight into how the cyst wall may be dynamically regulated.

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.

Toxoplasma gondii reorganizes the host cell architecture during spontaneous cyst formation in vitro

Parasitology ◽  
2017 ◽  
Vol 145 (8) ◽  
pp. 1027-1038 ◽  
Author(s):  
T. C. Paredes-Santos ◽  
E. S. Martins-Duarte ◽  
W. de Souza ◽  
M. Attias ◽  
R. C. Vommaro
Keyword(s):  
Toxoplasma Gondii ◽  
Host Cell ◽  
Cyst Wall ◽  
Cyst Formation ◽  
Acute Stage ◽  
Host Cells ◽  
Cell Cytoplasm ◽  
Chronic Stage ◽  
Host Cell Cytoplasm

AbstractToxoplasma gondii is an intracellular protozoan parasite that causes toxoplasmosis, a prevalent infection related to abortion, ocular diseases and encephalitis in immuno-compromised individuals. In the untreatable (and life-long) chronic stage of toxoplasmosis, parasitophorous vacuoles (PVs, containing T. gondii tachyzoites) transform into tissue cysts, containing slow-dividing bradyzoite forms. While acute-stage infection with tachyzoites involves global rearrangement of the host cell cytoplasm, focused on favouring tachyzoite replication, the cytoplasmic architecture of cells infected with cysts had not been described. Here, we characterized (by fluorescence and electron microscopy) the redistribution of host cell structures around T. gondii cysts, using a T. gondii strain (EGS) with high rates of spontaneous cystogenesis in vitro. Microtubules and intermediate filaments (but not actin microfilaments) formed a ‘cage’ around the cyst, and treatment with taxol (to inhibit microtubule dynamics) favoured cystogenesis. Mitochondria, which appeared adhered to the PV membrane, were less closely associated with the cyst wall. Endoplasmic reticulum (ER) profiles were intimately associated with folds in the cyst wall membrane. However, the Golgi complex was not preferentially localized relative to the cyst, and treatment with tunicamycin or brefeldin A (to disrupt Golgi or ER function, respectively) had no significant effect on cystogenesis. Lysosomes accumulated around cysts, while early and late endosomes were more evenly distributed in the cytoplasm. The endocytosis tracer HRP (but not BSA or transferrin) reached bradyzoites after uptake by infected host cells. These results suggest that T. gondii cysts reorganize the host cell cytoplasm, which may fulfil specific requirements of the chronic stage of infection.

scholarly journals Differential protein phosphorylation affects the localisation of two secreted Toxoplasma proteins and is widespread during stage conversion

2020 ◽  
Author(s):  
Joanna Young ◽  
Malgorzata Broncel ◽  
Helena Teague ◽  
Matt Russell ◽  
Olivia McGovern ◽  
...  
Keyword(s):  
Toxoplasma Gondii ◽  
Cyst Wall ◽  
Parasitophorous Vacuole ◽  
Proteome Analysis ◽  
Cyst Formation ◽  
Secreted Proteins ◽  
New Host ◽  
Chronic Stage ◽  
Stage Conversion ◽  
Membrane Bound

ABSTRACTThe intracellular parasite Toxoplasma gondii resides within a membrane bound parasitophorous vacuole (PV) and secretes an array of proteins to establish this replicative niche. It has been shown previously that Toxoplasma both secretes kinases and that numerous proteins are phosphorylated after secretion. Here we assess the role of phosphorylation of SFP1 and the related GRA29, two secreted proteins with unknown function. We show that both proteins form stranded structures in the PV that are independent of the previously described intravacuolar network or actin. GRA29 likely acts as a seed for SFP1 strand formation, and these structures can form independently of other Toxoplasma secreted proteins. We show that an unstructured region at the C-terminus of SFP1 and GRA29 is required for the formation of strands and that mimicking phosphorylation of this domain negatively regulates strand development. When tachyzoites convert to chronic stage bradyzoites, both proteins show a dispersed localisation throughout the cyst matrix. Many secreted proteins are reported to dynamically redistribute as the cyst forms and secreted kinases are known to play a role in cyst formation. Using quantitative phosphoproteome and proteome analysis comparing tachyzoite and early bradyzoite stages, we reveal widespread differential phosphorylation of secreted proteins. These data support a model in which secreted kinases and phosphatases are important to dynamically regulate parasite secreted proteins during stage conversion.IMPORTANCEToxoplasma gondii is a common parasite that infects up to one third of the human population. Initially the parasite grows rapidly, infecting and destroying cells of the host, but subsequently switches to a slow-growing form and establishes chronic infection. In both stages the parasite lives within a membrane bound vacuole within the host cell, but in the chronic stage a durable cyst wall is synthesized that provides protection to the parasite during transmission to a new host. Toxoplasma secretes proteins into the vacuole to build its replicative niche and previous studies identified many of these proteins as phosphorylated. We investigate two secreted proteins and show that phosphorylation plays an important role in their regulation. We also observed widespread phosphorylation of secreted proteins when parasites convert from acute to chronic stages, providing new insight into how the cyst wall may be dynamically regulated.

scholarly journals Succinylated Wheat Germ Agglutinin Colocalizes with the Toxoplasma gondii Cyst Wall Glycoprotein CST1

mSphere ◽  
2020 ◽  
Vol 5 (2) ◽  
Author(s):  
Rebekah B. Guevara ◽  
Barbara A. Fox ◽  
David J. Bzik
Keyword(s):  
Toxoplasma Gondii ◽  
Cyst Wall ◽  
Wheat Germ Agglutinin ◽  
Wheat Germ ◽  
Parasitophorous Vacuole ◽  
Biological Information ◽  
Toxoplasmic Encephalitis ◽  
Content Type ◽  
Cyst Development

ABSTRACT The glycosylated mucin domain of the Toxoplasma gondii cyst wall glycoprotein CST1 is heavily stained by Dolichos biflorus agglutinin, a lectin that binds to N-acetylgalactosamine. The cyst wall is also heavily stained by the chitin binding lectin succinylated wheat germ agglutinin (s-WGA), which selectively binds to N-acetylglucosamine-decorated structures. Here, we tracked the localization of N-acetylglucosamine-decorated structures that bind to s-WGA in immature and mature in vitro cysts. s-WGA localization was observed at the cyst periphery 6 h after the differentiation of the tachyzoite-stage parasitophorous vacuole. By day 1 and at all later times after differentiation, s-WGA was localized in a continuous staining pattern at the cyst wall. Coinciding with the maturation of the cyst matrix by day 3 of cyst development, s-WGA also localized in a continuous matrix pattern inside the cyst. s-WGA localized in both the outer and inner layer regions of the cyst wall and in a continuous matrix pattern inside mature 7- and 10-day-old cysts. In addition, s-WGA colocalized in the cyst wall with CST1, suggesting that N-acetylglucosamine- and N-acetylgalactosamine-decorated molecules colocalized in the cyst wall. In contrast to CST1, GRA4, and GRA6, the relative accumulation of the molecules that bind s-WGA in the cyst wall was not dependent on the expression of GRA2. Our results suggest that GRA2-dependent and GRA2-independent mechanisms regulate the trafficking and accumulation of glycosylated molecules that colocalize in the cyst wall. IMPORTANCE Chronic Toxoplasma gondii infection is maintained in the central nervous system by thick-walled cysts. If host immunity wanes, cysts recrudesce and cause severe and often lethal toxoplasmic encephalitis. Currently, there are no therapies to eliminate cysts, and little biological information is available regarding cyst structure(s). Here, we investigated cyst wall molecules recognized by succinylated wheat germ agglutinin (s-WGA), a lectin that specifically binds to N-acetylglucosamine-decorated structures. N-Acetylglucosamine regulates cell signaling and plays structural roles at the cell surface in many organisms. The cyst wall and cyst matrix were heavily stained by s-WGA in mature cysts and were differentially stained during cyst development. The relative accumulation of molecules that bind to s-WGA in the cyst wall was not dependent on the expression of GRA2. Our findings suggest that glycosylated cyst wall molecules gain access to the cyst wall via GRA2-dependent and GRA2-independent mechanisms and colocalize in the cyst wall.

scholarly journals Toxoplasma gondii Matrix Antigen 1 Is a Secreted Immunomodulatory Effector

mBio ◽  
2021 ◽  
Vol 12 (3) ◽  
Author(s):  
Tadakimi Tomita ◽  
Debanjan Mukhopadhyay ◽  
Bing Han ◽  
Rama Yakubu ◽  
Vincent Tu ◽  
...  
Keyword(s):  
Toxoplasma Gondii ◽  
Cyst Wall ◽  
Matrix Protein ◽  
Immune Reaction ◽  
Parasitophorous Vacuole ◽  
Host Cells ◽  
Inflammasome Activation ◽  
Host Immune System ◽  
Structural Protein ◽  
Content Type

ABSTRACT Our studies on novel cyst wall proteins serendipitously led us to the discovery that cyst wall and vacuolar matrix protein MAG1, first identified a quarter of a century ago, functions as a secreted immunomodulatory effector. MAG1 is a dense granular protein that is found in the parasitophorous vacuolar matrix in tachyzoite vacuoles and the cyst wall and matrix in bradyzoite vacuoles. In the current study, we demonstrated that MAG1 is secreted beyond the parasitophorous vacuole into the host cytosol in both tachyzoites and bradyzoites. Secretion of MAG1 gradually decreases as the parasitophorous vacuole matures, but prominent MAG1 puncta are present inside host cells even at 4 and 6 days following infection. During acute murine infection, Δmag1 parasites displayed significantly reduced virulence and dissemination. In the chronic stage of infection, Δmag1 parasites generated almost no brain cysts. To identify the mechanism behind the attenuated pathology seen with Δmag1 parasites, various immune responses were screened in vitro using bone marrow-derived macrophages (BMDM). Infection of BMDM with Δmag1 parasites induced a significant increase in interleukin 1β (IL-1β) secretion, which is a hallmark of inflammasome activation. Heterologous complementation of MAG1 in BMDM cells prevented this Δmag1 parasite-induced IL-1β release, indicating that secreted MAG1 in host cytosol dampens inflammasome activation. Furthermore, knocking out GRA15 (an inducer of IL-1β release) in Δmag1 parasites completely inhibited all IL-1β release by host cells following infection. These data suggest that MAG1 has a role as an immunomodulatory molecule and that by suppressing inflammasome activation, it would favor survival of the parasite and the establishment of latent infection. IMPORTANCE Toxoplasma gondii is an Apicomplexan that infects a third of humans, causing encephalitis in AIDS patients and intellectual disabilities in congenitally infected patients. We determined that one of the cyst matrix proteins, MAG1, which had been thought to be an innate structural protein, can be secreted into the host cell and suppress the host immune reaction. This particular immune reaction is initiated by another parasite-secreted protein, GRA15. The intricate balance of inflammasome activation by GRA15 and suppression by MAG1 protects mice from acute death while enabling parasites to disseminate and establish chronic cysts. Our finding contributes to our understanding of how parasites persist in the host and how T. gondii modulates the host immune system.

scholarly journals Loss of the Conserved Alveolate Kinase MAPK2 Decouples Toxoplasma Cell Growth from Cell Division

mBio ◽  
2020 ◽  
Vol 11 (6) ◽  
Author(s):  
Xiaoyu Hu ◽  
William J. O’Shaughnessy ◽  
Tsebaot G. Beraki ◽  
Michael L. Reese
Keyword(s):  
Toxoplasma Gondii ◽  
Protein Kinases ◽  
Daughter Cell ◽  
Protozoan Parasite ◽  
Productive Infection ◽  
Centrosome Duplication ◽  
Loss Of Function ◽  
Content Type ◽  
Mitogen Activated Protein ◽  
Parasite Replication

ABSTRACT Mitogen-activated protein kinases (MAPKs) are a conserved family of protein kinases that regulate signal transduction, proliferation, and development throughout eukaryotes. The apicomplexan parasite Toxoplasma gondii expresses three MAPKs. Two of these, extracellular signal-regulated kinase 7 (ERK7) and MAPKL1, have been implicated in the regulation of conoid biogenesis and centrosome duplication, respectively. The third kinase, MAPK2, is specific to and conserved throughout the Alveolata, although its function is unknown. We used the auxin-inducible degron system to determine phenotypes associated with MAPK2 loss of function in Toxoplasma. We observed that parasites lacking MAPK2 failed to duplicate their centrosomes and therefore did not initiate daughter cell budding, which ultimately led to parasite death. MAPK2-deficient parasites initiated but did not complete DNA replication and arrested prior to mitosis. Surprisingly, the parasites continued to grow and replicate their Golgi apparatus, mitochondria, and apicoplasts. We found that the failure in centrosome duplication is distinct from the phenotype caused by the depletion of MAPKL1. As we did not observe MAPK2 localization at the centrosome at any point in the cell cycle, our data suggest that MAPK2 regulates a process at a distal site that is required for the completion of centrosome duplication and the initiation of parasite mitosis. IMPORTANCE Toxoplasma gondii is a ubiquitous intracellular protozoan parasite that can cause severe and fatal disease in immunocompromised patients and the developing fetus. Rapid parasite replication is critical for establishing a productive infection. Here, we demonstrate that a Toxoplasma protein kinase called MAPK2 is conserved throughout the Alveolata and essential for parasite replication. We found that parasites lacking MAPK2 protein were defective in the initiation of daughter cell budding and were rendered inviable. Specifically, T. gondii MAPK2 (TgMAPK2) appears to be required for centrosome replication at the basal end of the nucleus, and its loss causes arrest early in parasite division. MAPK2 is unique to the Alveolata and not found in metazoa and likely is a critical component of an essential parasite-specific signaling network.

scholarly journals Reduction in the mitochondrial membrane potential of Toxoplasma gondii after invasion of host cells

1984 ◽  
Vol 70 (1) ◽  
pp. 73-81
Author(s):  
K. Tanabe ◽  
K. Murakami
Keyword(s):  
Membrane Potential ◽  
Toxoplasma Gondii ◽  
Mitochondrial Membrane Potential ◽  
Mitochondrial Membrane ◽  
Protozoan Parasite ◽  
Fluorescent Dye ◽  
Host Cells ◽  
Intracellular Parasites ◽  
Neutral Compounds ◽  
Infected Cells

The membrane potential of Toxoplasma gondii, an obligatory intracellular protozoan parasite, was monitored with the cationic permeant fluorescent dye rhodamine 123 (R123). Fluorescence microscopy revealed R123 to be partitioned predominantly in a restricted part of the parasite, which consisted of twisted or branched tubules, or of granular bodies. These structures were frequently connected to each other. The dye retention by these structures was markedly reduced by treating R123-labelled parasites with the proton ionophore, carbonylcyanide m-chlorophenylhydrazone, the potassium ionophore, valinomycin and the inhibitor of electron transport, antimycin A. Thus, these structures are regarded as the parasite mitochondria. Another cationic fluorescent dye, rhodamine 6G, stained the parasite mitochondria, whereas a negatively charged fluorescent dye, fluorescein, and the neutral compounds, rhodamine 110 and rhodamine B, did not. This fact indicates that R123 monitored the parasite mitochondrial membrane potential. T. gondii-infected 3T3 cells were also stained with R123. In contrast to the mitochondria of extracellular parasites, those of intracellular parasites failed to take up the dye. The absence of fluorescence in intracellular parasites persisted until the infected host cells ruptured and liberated daughter parasites 1 day after infection. Parasites, liberated from the host cells, either spontaneously or artificially by passing the infected cells through a 27G needle, regained the ability to take up the dye. After direct microinjection of R123 into the vacuole in which the parasite grows and multiples, the dye appeared in the host-cell mitochondria but not in the parasite's mitochondria. Thus, we conclude that the mitochondrial membrane potential of T. gondii was reduced after invasion of host cells by the parasite.

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.

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