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 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.

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 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 A Comparison of Stage Conversion in the Coccidian Apicomplexans Toxoplasma gondii, Hammondia hammondi, and Neospora caninum

2020 ◽  
Vol 10 ◽  
Author(s):  
Sarah L. Sokol-Borrelli ◽  
Rachel S. Coombs ◽  
Jon P. Boyle
Keyword(s):  
Life Cycle ◽  
Toxoplasma Gondii ◽  
Stress Responses ◽  
Neospora Caninum ◽  
Life Forms ◽  
New Host ◽  
Environmental Signals ◽  
Stage Conversion ◽  
Genetic Components ◽  
Species Specific

Stage conversion is a critical life cycle feature for several Apicomplexan parasites as the ability to switch between life forms is critical for replication, dissemination, pathogenesis and ultimately, transmission to a new host. In order for these developmental transitions to occur, the parasite must first sense changes in their environment, such as the presence of stressors or other environmental signals, and then respond to these signals by initiating global alterations in gene expression. As our understanding of the genetic components required for stage conversion continues to broaden, we can better understand the conserved mechanisms for this process and unique components and their contribution to pathogenesis by comparing stage conversion in multiple closely related species. In this review, we will discuss what is currently known about the mechanisms driving stage conversion in Toxoplasma gondii and its closest relatives Hammondia hammondi and Neospora caninum. Work by us and others has shown that these species have some important differences in the way that they (1) progress through their life cycle and (2) respond to stage conversion initiating stressors. To provide a specific example of species-specific complexities associated with stage conversion, we will discuss our recent published and unpublished work comparing stress responses in T. gondii and H. hammondi.

scholarly journals Toxoplasma gondii PPM3C, a secreted protein phosphatase, affects parasitophorous vacuole effector export

2020 ◽  
Author(s):  
Joshua A. Mayoral ◽  
Tadakimi Tomita ◽  
Vincent Tu ◽  
Jennifer T. Aguilan ◽  
Simone Sidoli ◽  
...  
Keyword(s):  
Toxoplasma Gondii ◽  
Parasitophorous Vacuole ◽  
Acute Infection ◽  
Critical Role ◽  
Cell Types ◽  
Effector Proteins ◽  
Secreted Proteins ◽  
Growth Defect ◽  
Label Free

ABSTRACTToxoplasma gondii is a highly successful parasite that infects a significant portion of the human population. As an intracellular parasite, T. gondii thrives within many different cell types due to its residence in the parasitophorous vacuole, a specialized and heavily modified compartment in which parasites divide. Within this vacuole, numerous secreted proteins facilitate functions that optimize intracellular survival. We characterized one such protein, TgPPM3C, which is predicted to contain a domain belonging to the PP2C class of serine/threonine phosphatases and is secreted by both tachyzoites and differentiating bradyzoites into the vacuolar lumen. Genetic deletion of TgPPM3C established that parasites lacking this predicted phosphatase exhibit a minor growth defect in vitro, are avirulent during acute infection in mice, and form fewer cysts in mouse brain during chronic infection. A label-free phosphoproteomic approach was utilized to identify putative TgPPM3C substrates and demonstrated several secreted proteins with altered phosphorylation status in the absence of TgPPM3C. Altered phosphorylation status was seen in MYR1, a protein essential to the process of protein effector export from the parasitophorous vacuole into the host cell, and in GRA16 and GRA28, two exported effector proteins. Defects were seen in the export of GRA16 and GRA28, but not the effector TgIST, in the TgPPM3C knockout strain. Parasites lacking TgPPM3C also exhibited defects in host c-Myc induction, a process influenced by effector export. Phosphomimetic mutations of GRA16 serine residues recapitulated export defects, implicating de-phosphorylation as an important process in facilitating the export of GRA16. These findings provide an example of the emerging critical role that phosphatases play in regulating the complex environment of the T. gondii parasitophorous vacuole.

scholarly journals Toxoplasma gondiisalvages sphingolipids from the host Golgi through the rerouting of selected Rab vesicles to the parasitophorous vacuole

2013 ◽  
Vol 24 (12) ◽  
pp. 1974-1995 ◽  
Author(s):  
Julia D. Romano ◽  
Sabrina Sonda ◽  
Emily Bergbower ◽  
Maria Elisa Smith ◽  
Isabelle Coppens
Keyword(s):  
Toxoplasma Gondii ◽  
Mammalian Cells ◽  
De Novo ◽  
Parasitophorous Vacuole ◽  
Parasite Growth ◽  
Sphingolipid Metabolism ◽  
Obligate Intracellular ◽  
Membrane Bound ◽  
Parasite Replication

The obligate intracellular protozoan Toxoplasma gondii actively invades mammalian cells and, upon entry, forms its own membrane-bound compartment, named the parasitophorous vacuole (PV). Within the PV, the parasite replicates and scavenges nutrients, including lipids, from host organelles. Although T. gondii can synthesize sphingolipids de novo, it also scavenges these lipids from the host Golgi. How the parasite obtains sphingolipids from the Golgi remains unclear, as the PV avoids fusion with host organelles. In this study, we explore the host Golgi–PV interaction and evaluate the importance of host-derived sphingolipids for parasite growth. We demonstrate that the PV preferentially localizes near the host Golgi early during infection and remains closely associated with this organelle throughout infection. The parasite subverts the structure of the host Golgi, resulting in its fragmentation into numerous ministacks, which surround the PV, and hijacks host Golgi–derived vesicles within the PV. These vesicles, marked with Rab14, Rab30, or Rab43, colocalize with host-derived sphingolipids in the vacuolar space. Scavenged sphingolipids contribute to parasite replication since alterations in host sphingolipid metabolism are detrimental for the parasite's growth. Thus our results reveal that T. gondii relies on host-derived sphingolipids for its development and scavenges these lipids via Golgi-derived vesicles.

scholarly journals Toxoplasma does not secrete the GRA16 and GRA24 effectors beyond the parasitophorous vacuole membrane of tissue cysts

2018 ◽  
Author(s):  
Shruthi Krishnamurthy ◽  
Jeroen PJ Saeij
Keyword(s):  
Cyst Wall ◽  
Parasitophorous Vacuole ◽  
Dense Granule ◽  
Parasitophorous Vacuole Membrane ◽  
Vacuole Membrane ◽  
Stage Conversion ◽  
Granule Proteins ◽  
Tetracycline Inducible System

AbstractAfter invasion, Toxoplasma resides in a parasitophorous vacuole (PV) that is surrounded by the PV membrane (PVM). Once inside the PV, tachyzoites secrete dense granule proteins (GRAs) of which some, such as GRA16 and GRA24, are transported beyond the PVM likely via a putative translocon. However, once tachyzoites convert into bradyzoites within cysts, it is not known if secreted GRAs can traffic beyond the cyst wall membrane. We used the tetracycline inducible system to drive expression of HA epitope tagged GRA16 and GRA24 after inducing stage conversion and show that these proteins are not secreted beyond the cyst wall membrane.

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 Initial phospholipid-dependent Irgb6 targeting to Toxoplasma gondii vacuoles mediates host defense

2019 ◽  
Vol 3 (1) ◽  
pp. e201900549 ◽  
Author(s):  
Youngae Lee ◽  
Hiroshi Yamada ◽  
Ariel Pradipta ◽  
Ji Su Ma ◽  
Masaaki Okamoto ◽  
...  
Keyword(s):  
Toxoplasma Gondii ◽  
Parasitophorous Vacuole ◽  
Protozoan Parasite ◽  
Host Cells ◽  
Interferon Response ◽  
Obligate Intracellular ◽  
Membrane Bound ◽  
Ifn Γ ◽  
Intracellular Protozoan Parasite

Toxoplasma gondii is an obligate intracellular protozoan parasite capable of infecting warm-blooded animals by ingestion. The organism enters host cells and resides in the cytoplasm in a membrane-bound parasitophorous vacuole (PV). Inducing an interferon response enables IFN-γ–inducible immunity-related GTPase (IRG protein) to accumulate on the PV and to restrict parasite growth. However, little is known about the mechanisms by which IRG proteins recognize and destroy T. gondii PV. We characterized the role of IRG protein Irgb6 in the cell-autonomous response against T. gondii, which involves vacuole ubiquitination and breakdown. We show that Irgb6 is capable of binding a specific phospholipid on the PV membrane. Furthermore, the absence of Irgb6 causes reduced targeting of other effector IRG proteins to the PV. This suggests that Irgb6 has a role as a pioneer in the process by which multiple IRG proteins access the PV. Irgb6-deficient mice are highly susceptible to infection by a strain of T. gondii avirulent in wild-type mice.

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.

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