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 A Family of Toxoplasma gondii Genes Related to GRA12 Regulate Cyst Burdens and Cyst Reactivation

mSphere ◽  
2021 ◽  
Vol 6 (2) ◽  
Author(s):  
Rebekah B. Guevara ◽  
Barbara A. Fox ◽  
David J. Bzik
Keyword(s):  
Toxoplasma Gondii ◽  
Cyst Wall ◽  
Chronic Infection ◽  
Acute Infection ◽  
Dense Granule ◽  
Toxoplasmic Encephalitis ◽  
Content Type ◽  
The Central Nervous System ◽  
Related Proteins ◽  
Cyst Development

ABSTRACT Toxoplasma gondii causes a chronic infection that renders the immunocompromised human host susceptible to toxoplasmic encephalitis triggered by cyst reactivation in the central nervous system. The dense granule protein GRA12 is a major parasite virulence factor required for parasite survival during acute infection. Here, we characterized the role of four GRA12-related genes in acute and chronic stages of infection. While GRA12A, GRA12B, and GRA12D were highly expressed in asexual stage tachyzoites and bradyzoites, expression of GRA12C appeared to be restricted to the sexual stages. In contrast to deletion of GRA12 (Δgra12), no major defects in acute virulence were observed in Δgra12A, Δgra12B, or Δgra12D parasites, though Δgra12B parasites exhibited an increased tachyzoite replication rate. Bradyzoites secreted GRA12A, GRA12B, and GRA12D and incorporated these molecules into the developing cyst wall, as well as the cyst matrix in distinct patterns. Similar to GRA12, GRA12A, GRA12B, and GRA12D colocalized with the dense granules in extracellular tachyzoites, with GRA2 and the intravacuolar network in the tachyzoite stage parasitophorous vacuole and with GRA2 in the cyst matrix and cyst wall. Chronic stage cyst burdens were decreased in mice infected with Δgra12A parasites and were increased in mice infected with Δgra12B parasites. However, Δgra12B cysts were not efficiently maintained in vivo. Δgra12A, Δgra12B, and Δgra12D in vitro cysts displayed a reduced reactivation efficiency, and reactivation of Δgra12A cysts was delayed. Collectively, our results suggest that a family of genes related to GRA12 play significant roles in the formation, maintenance, and reactivation of chronic stage cysts. IMPORTANCE If host immunity weakens, Toxoplasma gondii cysts recrudesce in the central nervous system and cause a severe toxoplasmic encephalitis. Current therapies target acute stage infection but do not eliminate chronic cysts. Parasite molecules that mediate the development and persistence of chronic infection are poorly characterized. Dense granule (GRA) proteins such as GRA12 are key virulence factors during acute infection. Here, we investigated four GRA12-related genes. GRA12-related genes were not major virulence factors during acute infection. Instead, GRA12-related proteins localized at the cyst wall and cyst matrix and played significant roles in cyst development, persistence, and reactivation during chronic infection. Similar to GRA12, the GRA12-related proteins selectively associated with the intravacuolar network of membranes inside the vacuole. Collectively, our results support the hypothesis that GRA12 proteins associated with the intravacuolar membrane system support parasite virulence during acute infection and cyst development, persistence, and reactivation during chronic infection.

scholarly journals Toxoplasma gondii Intravacuolar-Network-Associated Dense Granule Proteins Regulate Maturation of the Cyst Matrix and Cyst Wall

mSphere ◽  
2019 ◽  
Vol 4 (5) ◽  
Author(s):  
Rebekah B. Guevara ◽  
Barbara A. Fox ◽  
Alejandra Falla ◽  
David J. Bzik
Keyword(s):  
Toxoplasma Gondii ◽  
Cyst Wall ◽  
Protein Localization ◽  
Dense Granule ◽  
Wall Material ◽  
Content Type ◽  
Wall Structures ◽  
Cyst Development ◽  
Rate Of Accumulation

ABSTRACT Little is known regarding how the chronic Toxoplasma gondii cyst develops. Here, we investigated intravacuolar-network-associated dense granule (GRA) proteins GRA1, GRA2, GRA4, GRA6, GRA9, and GRA12 during cyst development in vitro after differentiation of the tachyzoite-stage parasitophorous vacuole. By day 1 postdifferentiation, GRA1, GRA4, GRA6, GRA9, and GRA12 colocalized with Dolichos biflorus agglutinin stain at the cyst periphery. In contrast, GRA2 remained in the cyst matrix. By day 2 postdifferentiation, coinciding with localization of GRA2 to the cyst periphery, GRA1, GRA4, GRA6, and GRA9 established a continuous matrix pattern in the cyst. In contrast, GRA2 and GRA12 were colocalized in prominent cyst matrix puncta throughout cyst development. While GRA2, GRA6, and GRA12 localized in outer and inner layers of the cyst wall, GRA1, GRA4, and GRA9 localized predominantly in the inner layers of the cyst wall. GRA2 and GRA12 were colocalized in the cyst wall by day 7 postdifferentiation. However, by day 10 postdifferentiation, GRA12 was relocalized from the cyst wall to puncta in the cyst matrix. Differentiation of Δgra2 parasites revealed a defect in the ability to establish a normal cyst matrix. In addition, the deletion of any intravacuolar-network-associated GRA protein, except GRA1, reduced the rate of accumulation of cyst wall proteins at the cyst periphery relative to the cyst interior. Our findings reveal dynamic patterns of GRA protein localization during cyst development and suggest that intravacuolar-network-associated GRA proteins regulate the formation and maturation of the cyst matrix and cyst wall structures. IMPORTANCE Toxoplasma gondii establishes chronic infection in humans by forming thick-walled cysts that persist in the brain. If host immunity wanes, cysts reactivate to cause severe, and often lethal, toxoplasmic encephalitis. There is no available therapy to eliminate cysts or to prevent their reactivation. Moreover, how the vital and characteristic cyst matrix and cyst wall structures develop is poorly understood. Here, we visualized and tracked the localization of Toxoplasma intravacuolar-network-associated dense granule (GRA) proteins during cyst development in vitro. Intravacuolar-network GRAs were present within the cyst matrix and at the cyst wall in developing cysts, and genetic deletion of intravacuolar-network-associated GRAs reduced the rate of accumulation of cyst wall material at the cyst periphery. Our results show that intravacuolar-network-associated GRAs, particularly GRA2 and GRA12, play dynamic and essential roles in the development and maturation of the cyst matrix and the cyst wall structures.

scholarly journals Toxoplasma gondii Parasitophorous Vacuole Membrane-Associated Dense Granule Proteins Regulate Maturation of the Cyst Wall

mSphere ◽  
2020 ◽  
Vol 5 (1) ◽  
Author(s):  
Rebekah B. Guevara ◽  
Barbara A. Fox ◽  
David J. Bzik
Keyword(s):  
Toxoplasma Gondii ◽  
Cyst Wall ◽  
Parasitophorous Vacuole ◽  
Dense Granule ◽  
Wall Region ◽  
Parasitophorous Vacuole Membrane ◽  
Vacuole Membrane ◽  
Cyst Membrane ◽  
Cyst Development

ABSTRACT After differentiation is triggered, the tachyzoite-stage Toxoplasma gondii parasitophorous vacuole membrane (PVM) has been hypothesized to transition into the cyst membrane that surrounds the cyst wall and encloses bradyzoites. Here, we tracked the localization of two PVM dense granule (GRA) proteins (GRA5 and GRA7) after in vitro differentiation of the tachyzoite stage parasitophorous vacuole into the mature cyst. GRA5 and GRA7 were visible at the cyst periphery at 6 h and at all later times after differentiation, suggesting that the PVM remained intact as it transitioned into the cyst membrane. By day 3 postdifferentiation, GRA5 and GRA7 were visible in a continuous pattern at the cyst periphery. In mature 7- and 10-day-old cysts permeabilized with a saponin pulse, GRA5 and GRA7 were localized to the cyst membrane and the cyst wall regions. Cysts at different stages of cyst development exhibited differential susceptibility to saponin permeabilization, and, correspondingly, saponin selectively removed GRA5 from the cyst membrane and cyst wall region in 10-day-old cysts. GRA5 and GRA7 were localized at the cyst membrane and cyst wall region at all times after differentiation of the parasitophorous vacuole, which supports a previous model proposing that the PVM develops into the cyst membrane. In addition, evaluation of Δgra3, Δgra5, Δgra7, Δgra8, and Δgra14 mutants revealed that PVM-localized GRAs were crucial to support the normal rate of accumulation of cyst wall proteins at the cyst periphery. IMPORTANCE Toxoplasma gondii establishes chronic infection in humans by forming thick-walled cysts that persist in the brain. Once host immunity wanes, cysts reactivate to cause severe, and often lethal, toxoplasmic encephalitis. There is no available therapy to eliminate cysts or to prevent their reactivation. Furthermore, how the cyst membrane and cyst wall structures develop is poorly understood. Here, we visualized and tracked the localization of Toxoplasma parasitophorous vacuole membrane (PVM) dense granules (GRA) proteins during cyst development in vitro. PVM-localized GRA5 and GRA7 were found at the cyst membrane and cyst wall region throughout cyst development, suggesting that the PVM remains intact and develops into the cyst membrane. In addition, our results show that genetic deletion of PVM GRAs reduced the rate of accumulation of cyst wall cargo at the cyst periphery and suggest that PVM-localized GRAs mediate the development and maturation of the cyst wall and cyst membrane.

scholarly journals Targeted Disruption of Toxoplasma gondii Serine Protease Inhibitor 1 Increases Bradyzoite Cyst FormationIn Vitroand Parasite Tissue Burden in Mice

2011 ◽  
Vol 80 (3) ◽  
pp. 1156-1165 ◽  
Author(s):  
Viviana Pszenny ◽  
Paul H. Davis ◽  
Xing W. Zhou ◽  
Christopher A. Hunter ◽  
Vern B. Carruthers ◽  
...  
Keyword(s):  
Toxoplasma Gondii ◽  
Protease Inhibitor ◽  
Host Cell ◽  
Serine Protease ◽  
Parasitophorous Vacuole ◽  
Parasite Burden ◽  
Serine Protease Inhibitor ◽  
Genomic Locus ◽  
Content Type

As an intracellular protozoan parasite,Toxoplasma gondiiis likely to exploit proteases for host cell invasion, acquisition of nutrients, avoidance of host protective responses, escape from the parasitophorous vacuole, differentiation, and other activities.T. gondiiserine protease inhibitor 1 (TgPI1) is the most abundantly expressed protease inhibitor in parasite tachyzoites. We show here that alternative splicing produces twoTgPI1 isoforms, both of which are secreted via dense granules into the parasitophorous vacuole shortly after invasion, become progressively more abundant over the course of the infectious cycle, and can be detected in the infected host cell cytoplasm. To investigateTgPI1 function, the endogenous genomic locus was disrupted in the RH strain background. ΔTgPI1 parasites replicate normally as tachyzoites but exhibit increased bradyzoite gene transcription and labeling of vacuoles withDolichos bifloruslectin under conditions promotingin vitrodifferentiation. The differentiation phenotype can be partially complemented by eitherTgPI1 isoform. Mice infected with the ΔTgPI1 mutant display ∼3-fold-increased parasite burden in the spleen and liver, and thisin vivophenotype is also complemented by eitherTgPI1 isoform. These results demonstrate thatTgPI1 influences both parasite virulence and bradyzoite differentiation, presumably by inhibiting parasite and/or host serine proteases.

scholarly journals Migratory Activation of Primary Cortical Microglia upon Infection with Toxoplasma gondii

2011 ◽  
Vol 79 (8) ◽  
pp. 3046-3052 ◽  
Author(s):  
Isabel Dellacasa-Lindberg ◽  
Jonas M. Fuks ◽  
Romanico B. G. Arrighi ◽  
Henrik Lambert ◽  
Robert P. A. Wallin ◽  
...  
Keyword(s):  
T Cells ◽  
Toxoplasma Gondii ◽  
Mhc Class Ii ◽  
Glial Cells ◽  
Costimulatory Molecule ◽  
Toxoplasmic Encephalitis ◽  
Immune Functions ◽  
Content Type

ABSTRACTDisseminated toxoplasmosis in the central nervous system (CNS) is often accompanied by a lethal outcome. Studies with murine models of infection have focused on the role of systemic immunity in control of toxoplasmic encephalitis, while knowledge remains limited on the contributions of resident cells with immune functions in the CNS. In this study, the role of glial cells was addressed in the setting of recrudescentToxoplasmainfection in mice. Activated astrocytes and microglia were observed in the close vicinity of foci with replicating parasitesin situin the brain parenchyma.Toxoplasma gondiitachyzoites were allowed to infect primary microglia and astrocytesin vitro. Microglia were permissive to parasite replication, and infected microglia readily transmigrated across transwell membranes and cell monolayers. Thus, infected microglia, but not astrocytes, exhibited a hypermotility phenotype reminiscent of that recently described for infected dendritic cells. In contrast to gamma interferon-activated microglia,Toxoplasma-infected microglia did not upregulate major histocompatibility complex (MHC) class II molecules and the costimulatory molecule CD86. YetToxoplasma-infected microglia and astrocytes exhibited increased sensitivity to T cell-mediated killing, leading to rapid parasite transfer to effector T cellsin vitro. We hypothesize that glial cells and T cells, besides their role in triggering antiparasite immunity, may also act as “Trojan horses,” paradoxically facilitating dissemination ofToxoplasmawithin the CNS. To our knowledge, this constitutes the first report of migratory activation of a resident CNS cell by an intracellular parasite.

scholarly journals The Toxoplasma gondii Cyst Wall Interactome

mBio ◽  
2020 ◽  
Vol 11 (1) ◽  
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.

scholarly journals Significant Reduction of Brain Cysts Caused by Toxoplasma gondii after Treatment with Spiramycin Coadministered with Metronidazole in a Mouse Model of Chronic Toxoplasmosis

2012 ◽  
Vol 56 (4) ◽  
pp. 1762-1768 ◽  
Author(s):  
Wai Kit Chew ◽  
Ignacio Segarra ◽  
Stephen Ambu ◽  
Joon Wah Mak
Keyword(s):  
Toxoplasma Gondii ◽  
Vero Cells ◽  
Toxoplasmic Encephalitis ◽  
Combination Group ◽  
Control Group ◽  
Time Curve ◽  
Content Type ◽  
Brain Cysts ◽  
The Brain

ABSTRACTToxoplasma gondiiis a parasite that generates latent cysts in the brain; reactivation of these cysts may lead to fatal toxoplasmic encephalitis, for which treatment remains unsuccessful. We assessed spiramycin pharmacokinetics coadministered with metronidazole, the eradication of brain cysts and thein vitroreactivation. Male BALB/c mice were fed 1,000 tachyzoites orally to develop chronic toxoplasmosis. Four weeks later, infected mice underwent different treatments: (i) infected untreated mice (n= 9), which received vehicle only; (ii) a spiramycin-only group (n= 9), 400 mg/kg daily for 7 days; (iii) a metronidazole-only group (n= 9), 500 mg/kg daily for 7 days; and (iv) a combination group (n= 9), which received both spiramycin (400 mg/kg) and metronidazole (500 mg/kg) daily for 7 days. An uninfected control group (n= 10) was administered vehicle only. After treatment, the brain cysts were counted, brain homogenates were cultured in confluent Vero cells, and cysts and tachyzoites were counted after 1 week. Separately, pharmacokinetic profiles (plasma and brain) were assessed after a single dose of spiramycin (400 mg/kg), metronidazole (500 mg/kg), or both. Metronidazole treatment increased the brain spiramycin area under the concentration-time curve from 0 h to ∞ (AUC0–∞) by 67% without affecting its plasma disposition. Metronidazole plasma and brain AUC0–∞values were reduced 9 and 62%, respectively, after spiramycin coadministration. Enhanced spiramycin brain exposure after coadministration reduced brain cysts 15-fold (79 ± 23 for the combination treatment versus 1,198 ± 153 for the untreated control group [P< 0.05]) and 10-fold versus the spiramycin-only group (768 ± 125). Metronidazole alone showed no effect (1,028 ± 149). Tachyzoites were absent in the brain. Spiramycin reducedin vitroreactivation. Metronidazole increased spiramycin brain penetration, causing a significant reduction ofT. gondiibrain cysts, with potential clinical translatability for chronic toxoplasmosis treatment.

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

2011 ◽  
Vol 10 (12) ◽  
pp. 1637-1647 ◽  
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.

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 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.

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