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 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 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.

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.

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 From Initiators to Effectors: Roadmap Through the Intestine During Encounter of Toxoplasma gondii With the Mucosal Immune System

2021 ◽  
Vol 10 ◽  
Author(s):  
Lindsay M. Snyder ◽  
Eric Y. Denkers
Keyword(s):  
Toxoplasma Gondii ◽  
Intestinal Mucosa ◽  
Acute Infection ◽  
Protozoan Parasite ◽  
Intraepithelial Lymphocytes ◽  
Complex Interactions ◽  
Adaptive Immune Cells ◽  
Immune Mediated ◽  
The Central Nervous System ◽  
Portal Of Entry

The gastrointestinal tract is a major portal of entry for many pathogens, including the protozoan parasite Toxoplasma gondii. Billions of people worldwide have acquired T. gondii at some point in their life, and for the vast majority this has led to latent infection in the central nervous system. The first line of host defense against Toxoplasma is located within the intestinal mucosa. Appropriate coordination of responses by the intestinal epithelium, intraepithelial lymphocytes, and lamina propria cells results in an inflammatory response that controls acute infection. Under some conditions, infection elicits bacterial dysbiosis and immune-mediated tissue damage in the intestine. Here, we discuss the complex interactions between the microbiota, the epithelium, as well as innate and adaptive immune cells in the intestinal mucosa that induce protective immunity, and that sometimes switch to inflammatory pathology as T. gondii encounters tissues of the gut.

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 Mechanism of Entry Determines the Ability of Toxoplasma gondii To Inhibit Macrophage Proinflammatory Cytokine Production

2002 ◽  
Vol 70 (9) ◽  
pp. 5216-5224 ◽  
Author(s):  
Barbara A. Butcher ◽  
Eric Y. Denkers
Keyword(s):  
Toxoplasma Gondii ◽  
Cytokine Production ◽  
Parasitophorous Vacuole ◽  
Interleukin 12 ◽  
Proinflammatory Cytokine Production ◽  
Necrosis Factor Alpha ◽  
Tnf Α ◽  
Infected Cells ◽  
Factor Alpha ◽  
Necrosis Factor

ABSTRACT Macrophages (Mφ) infected with tachyzoites of the opportunistic protozoan Toxoplasma gondii are blocked in production of the proinflammatory cytokines tumor necrosis factor alpha (TNF-α) and interleukin-12 (IL-12) in response to lipopolysaccharide (LPS) triggering, and this is associated with parasite-induced inhibition of NFκB translocation. Here, we demonstrate a requirement for active invasion in the ability of the parasite to mediate suppression. Neither soluble tachyzoite antigen nor secreted products were suppressive, and heat-inactivated, antibody-coated tachyzoites, which efficiently entered the cell through receptor-mediated uptake, failed to inhibit LPS responses. Cytochalasin D, a drug blocking tachyzoite invasion of, but not adherence to, Mφ, severely curtailed Toxoplasma-induced suppression. In addition, parasite-induced nonresponsiveness, as measured by TNF-α production, was reversed by treating infected cells with the toxoplasmastatic drugs pyrimethamine and 6-thioxanthine prior to LPS stimulation. A divergence in IL-12 and TNF-α responses was found during extended incubation of tachyzoites and Mφ in that 24 h of incubation of infected Mφ resulted in IL-12, but not TNF-α, secretion, and production of the latter cytokine remained suppressed when these cells were subjected to LPS triggering. Our results demonstrate that active invasion and survival of the parasite within the parasitophorous vacuole are required to induce and maintain Mφ cytokine-specific nonresponsiveness to LPS. They also show that the effects of Toxoplasma on IL-12 and TNF-α production are nonidentical, with the parasite exerting a longer-lasting suppression of the latter.

scholarly journals Cathepsin Cs Are Key for the Intracellular Survival of the Protozoan Parasite, Toxoplasma gondii

2006 ◽  
Vol 282 (7) ◽  
pp. 4994-5003 ◽  
Author(s):  
Xuchu Que ◽  
Juan C. Engel ◽  
David Ferguson ◽  
Annette Wunderlich ◽  
Stanislas Tomavo ◽  
...  
Keyword(s):  
Toxoplasma Gondii ◽  
Cathepsin B ◽  
Parasitophorous Vacuole ◽  
Cathepsin L ◽  
Protozoan Parasite ◽  
Cysteine Proteases ◽  
Intracellular Survival ◽  
Chemotherapeutic Agents ◽  
Cathepsin C

Cysteine proteases play key roles in apicomplexan invasion, organellar biogenesis, and intracellular survival. We have now characterized five genes encoding papain family cathepsins from Toxoplasma gondii, including three cathepsin Cs, one cathepsin B, and one cathepsin L. Unlike endopeptidases cathepsin B and L, T. gondii cathepsin Cs are exopeptidases and remove dipeptides from unblocked N-terminal substrates of proteins or peptides. TgCPC1 was the most highly expressed cathepsin mRNA in tachyzoites (by real-time PCR), but three cathepsins, TgCPC1, TgCPC2, and TgCPB, were undetectable in in vivo bradyzoites. The specific cathepsin C inhibitor, Gly-Phe-dimethylketone, selectively inhibited the TgCPCs activity, reducing parasite intracellular growth and proliferation. The targeted disruption of TgCPC1 does not affect the invasion and growth of tachyzoites as TgCPC2 is then up-regulated and may substitute for TgCPC1. TgCPC1 and TgCPC2 localize to constitutive secretory vesicles of tachyzoites, the dense granules. T. gondii cathepsin Cs are required for peptide degradation in the parasitophorous vacuole as the degradation of the marker protein, Escherichia coli β-lactamase, secreted into the parasitophorous vacuole of transgenic tachyzoites was completely inhibited by the cathepsin C inhibitor. Cathepsin C inhibitors also limited the in vivo infection of T. gondii in the chick embryo model of toxoplasmosis. Thus, cathepsin Cs are critical to T. gondii growth and differentiation, and their unique specificities could be exploited to develop novel chemotherapeutic agents.

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.

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