Faculty Opinions recommendation of The Toxoplasma gondii cyst wall protein CST1 is critical for cyst wall integrity and promotes bradyzoite persistence.

A positive Feulgen reaction has been seen in all stages of Toxoplasma gondii. The intense basophilia seen in the intracellular and the extracellular stages is removable by treatment with ribonuclease. Granules which reacted positively with the periodic acid-Schiff technique were found in abundance in the cytoplasm of the cysts, whereas the same granules were not universally present in the intracellular and the extracellular stages at all days of infection. The cyst wall stained with this technique with varying degree of intensity.It is thought that the variations seen in the individual parasites may be attributed to various causes, for example, strain differences, differences in the reaction of the host to the parasite, differences in the period of infection or to other unknown causes.

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The Toxoplasma gondii Cyst Wall Interactome

mBio ◽

10.1128/mbio.02699-19 ◽

2020 ◽

Vol 11 (1) ◽

Cited By ~ 5

Author(s):

Vincent Tu ◽

Tadakimi Tomita ◽

Tatsuki Sugi ◽

Joshua Mayoral ◽

Bing Han ◽

...

Keyword(s):

Toxoplasma Gondii ◽

Protein Interactions ◽

Cyst Wall ◽

Affinity Purification ◽

Dense Granule ◽

Hypothetical Proteins ◽

Content Type

ABSTRACT A characteristic of the latent cyst stage of Toxoplasma gondii is a thick cyst wall that forms underneath the membrane of the bradyzoite vacuole. Previously, our laboratory group published a proteomic analysis of purified in vitro cyst wall fragments that identified an inventory of cyst wall components. To further refine our understanding of the composition of the cyst wall, several cyst wall proteins were tagged with a promiscuous biotin ligase (BirA*), and their interacting partners were screened by streptavidin affinity purification. Within the cyst wall pulldowns, previously described cyst wall proteins, dense granule proteins, and uncharacterized hypothetical proteins were identified. Several of the newly identified hypothetical proteins were validated to be novel components of the cyst wall and tagged with BirA* to expand the model of the cyst wall interactome. Community detection of the cyst wall interactome model revealed three distinct clusters: a dense granule, a cyst matrix, and a cyst wall cluster. Characterization of several of the identified cyst wall proteins using genetic strategies revealed that MCP3 affects in vivo cyst sizes. This study provides a model of the potential protein interactions within the cyst wall and the groundwork to understand cyst wall formation. IMPORTANCE A model of the cyst wall interactome was constructed using proteins identified through BioID. The proteins within this cyst wall interactome model encompass several proteins identified in a prior characterization of the cyst wall proteome. This model provides a more comprehensive understanding of the composition of the cyst wall and may lead to insights on how the cyst wall is formed.

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Toxoplasma gondii induces robust humoral immune response against cyst wall antigens in chronically infected animals and humans

Microbial Pathogenesis ◽

10.1016/j.micpath.2020.104643 ◽

2020 ◽

pp. 104643

Author(s):

Abhijit S. Deshmukh ◽

Rajkumar Gurupwar ◽

Pallabi Mitra ◽

Kalyani Aswale ◽

Shilpshri Shinde ◽

...

Keyword(s):

Immune Response ◽

Toxoplasma Gondii ◽

Cyst Wall ◽

Humoral Immune Response ◽

Humoral Immune

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Making Home Sweet and Sturdy: Toxoplasma gondii ppGalNAc-Ts Glycosylate in Hierarchical Order and Confer Cyst Wall Rigidity

mBio ◽

10.1128/mbio.02048-16 ◽

2017 ◽

Vol 8 (1) ◽

Cited By ~ 30

Author(s):

Tadakimi Tomita ◽

Tatsuki Sugi ◽

Rama Yakubu ◽

Vincent Tu ◽

Yanfen Ma ◽

...

Keyword(s):

Toxoplasma Gondii ◽

Cyst Wall ◽

Dependent Manner ◽

Intracellular Parasite ◽

Congenital Disease ◽

Brain Cyst ◽

Obligate Intracellular ◽

Obligate Intracellular Parasite ◽

Structural Rigidity ◽

The Brain

ABSTRACT The protozoan intracellular parasite Toxoplasma gondii forms latent cysts in the central nervous system (CNS) and persists for the lifetime of the host. This cyst is cloaked with a glycosylated structure called the cyst wall. Previously, we demonstrated that a mucin-like glycoprotein, CST1, localizes to the cyst wall and confers structural rigidity on brain cysts in a mucin-like domain-dependent manner. The mucin-like domain of CST1 is composed of 20 units of threonine-rich tandem repeats that are O-GalNAc glycosylated. A family of enzymes termed polypeptide N-acetylgalactosaminyltransferases (ppGalNAc-Ts) initiates O-GalNAc glycosylation. To identify which isoforms of ppGalNAc-Ts are responsible for the glycosylation of the CST1 mucin-like domain and to evaluate the function of each ppGalNAc-T in the overall glycosylation of the cyst wall, all five ppGalNAc-T isoforms were deleted individually from the T. gondii genome. The ppGalNAc-T2 and -T3 deletion mutants produced various glycosylation defects on the cyst wall, implying that many cyst wall glycoproteins are glycosylated by T2 and T3. Both T2 and T3 glycosylate the CST1 mucin-like domain, and this glycosylation is necessary for CST1 to confer structural rigidity on the cyst wall. We established that T2 is required for the initial glycosylation of the mucin-like domain and that T3 is responsible for the sequential glycosylation on neighboring acceptor sites, demonstrating hierarchical glycosylation by two distinct initiating and filling-in ppGalNAc-Ts in an intact organism. IMPORTANCE Toxoplasma gondii is an obligate intracellular parasite that infects a third of the world’s population. It can cause severe congenital disease and devastating encephalitis in immunocompromised individuals. We identified two glycosyltransferases, ppGalNAc-T2 and -T3, which are responsible for glycosylating cyst wall proteins in a hierarchical fashion. This glycosylation confers structural rigidity on the brain cyst. Our studies provide new insights into the mechanisms of O-GalNAc glycosylation in T. gondii. IMPORTANCE Toxoplasma gondii is an obligate intracellular parasite that infects a third of the world’s population. It can cause severe congenital disease and devastating encephalitis in immunocompromised individuals. We identified two glycosyltransferases, ppGalNAc-T2 and -T3, which are responsible for glycosylating cyst wall proteins in a hierarchical fashion. This glycosylation confers structural rigidity on the brain cyst. Our studies provide new insights into the mechanisms of O-GalNAc glycosylation in T. gondii.

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

Parasitology ◽

10.1017/s0031182017002050 ◽

2017 ◽

Vol 145 (8) ◽

pp. 1027-1038 ◽

Cited By ~ 5

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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The Toxoplasma gondii Cyst Wall Protein CST1 Is Critical for Cyst Wall Integrity and Promotes Bradyzoite Persistence

PLoS Pathogens ◽

10.1371/journal.ppat.1003823 ◽

2013 ◽

Vol 9 (12) ◽

pp. e1003823 ◽

Cited By ~ 74

Author(s):

Tadakimi Tomita ◽

David J. Bzik ◽

Yan Fen Ma ◽

Barbara A. Fox ◽

Lye Meng Markillie ◽

...

Keyword(s):

Toxoplasma Gondii ◽

Cyst Wall

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In vitro characterization of protein effector export in the bradyzoite stage of Toxoplasma gondii

10.1101/2020.01.08.899773 ◽

2020 ◽

Author(s):

Joshua Mayoral ◽

Peter Shamamian ◽

Louis M. Weiss

Keyword(s):

Toxoplasma Gondii ◽

Host Cell ◽

Cyst Wall ◽

Persistent Infection ◽

Chronic Infection ◽

Life Stage ◽

Effector Proteins ◽

Early Stages ◽

Bradyzoite Differentiation

ABSTRACTThe ubiquitous parasite Toxoplasma gondii exhibits an impressive ability to maintain a chronic infection of its host for prolonged periods. Despite this, little is known regarding if and how T. gondii bradyzoites, a quasi-dormant life-stage residing within intracellular cysts, manipulate the host cell so as to maintain a persistent infection. A previous proteomic study of the cyst wall, an amorphous layer of proteins that forms underneath the cyst membrane, identified MYR1 as a putative cyst wall protein in vitro. As MYR1 is known to be involved in the translocation of parasite derived effector proteins into the host cell, we sought to determine whether parasites transitioning toward the bradyzoite life stage retain the capacity to translocate proteins via this pathway. By epitope tagging the endogenous loci of four known effectors that translocate from the parasitophorous vacuole into the host cell nucleus, we show by immunofluorescence that most effectors accumulate in the host nucleus at early but not late timepoints post-infection during the tachyzoite to bradyzoite transition and when parasites farther along the bradyzoite differentiation continuum invade a new host cell. We demonstrate that the suppression of interferon-gamma (IFN-γ) signaling, previously shown to be mediated by the effector TgIST, also occurs in the context of prolonged infection with bradyzoites, and that TgIST export is a process that occurs beyond the early stages of host cell infection. These findings have important implications as to how this highly successful parasite maintains a persistent infection of its host.IMPORTANCEToxoplasma bradyzoites persist within tissue cysts and are refractory to current treatments, serving as a reservoir for acute complications in settings of compromised immunity. Much remains to be understood regarding how this life-stage successfully establishes and maintains a persistent infection. In this study, we investigated whether the export of parasite effector proteins into the host cell occurs during the development of in vitro tissue cysts. We quantified the presence of four previously described effectors in host cell nuclei at different timepoints post-bradyzoite differentiation and found that they accumulate largely during the early stages of infection. Despite a decline in nuclear accumulation, we found that one of these effectors still mediates its function after prolonged infection with bradyzoites and provide evidence that this effector is exported beyond early infection stages. These findings suggest that effector export from within developing tissue cysts provides one potential mechanism by which this parasite achieves chronic infection.

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Identification of Tissue Cyst Wall Components by Transcriptome Analysis ofIn Vivoand In Vitro Toxoplasma gondii Bradyzoites

Eukaryotic Cell ◽

10.1128/ec.05182-11 ◽

2011 ◽

Vol 10 (12) ◽

pp. 1637-1647 ◽

Cited By ~ 59

Author(s):

Kerry R. Buchholz ◽

Heather M. Fritz ◽

Xiucui Chen ◽

Blythe Durbin-Johnson ◽

David M. Rocke ◽

...

Keyword(s):

Toxoplasma Gondii ◽

Cyst Wall ◽

Tissue Cyst ◽

Content Type ◽

Repeat Domain ◽

Developmental Form ◽

Protein Components ◽

New Protein

ABSTRACTTheToxoplasma gondiibradyzoite is essential to establish persistent infection, yet little is known about what factors this developmental form secretes to establish the cyst or interact with its host cell. To identify candidate bradyzoite-secreted effectors, the transcriptomes ofin vitrotachyzoites 2 days postinfection,in vitrobradyzoites 4 days postinfection, andin vivobradyzoites 21 days postinfection were interrogated by microarray, and the program SignalP was used to identify signal peptides indicating secretion. One hundred two putative bradyzoite-secreted effectors were identified by this approach. Two candidates, bradyzoite pseudokinase 1 and microneme adhesive repeat domain-containing protein 4, were chosen for further investigation and confirmed to be induced and secreted by bradyzoitesin vitroandin vivo. Thus, we report the first analysis of the transcriptomes ofin vitroandin vivobradyzoites and identify two new protein components of theToxoplasmatissue cyst wall.

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