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

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

10.1101/2020.04.09.035279 ◽

2020 ◽

Cited By ~ 2

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.

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Acquisition of Host Cytosolic Protein by Toxoplasma gondii Bradyzoites

mSphere ◽

10.1128/msphere.00934-20 ◽

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.

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

mSphere ◽

10.1128/msphere.00031-20 ◽

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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Toxoplasma gondii MAF1b Binds the Host Cell MIB Complex To Mediate Mitochondrial Association

mSphere ◽

10.1128/msphere.00183-17 ◽

2017 ◽

Vol 2 (3) ◽

Cited By ~ 4

Author(s):

Felice D. Kelly ◽

Brian M. Wei ◽

Alicja M. Cygan ◽

Michelle L. Parker ◽

Martin J. Boulanger ◽

...

Keyword(s):

Toxoplasma Gondii ◽

Host Cell ◽

Close Association ◽

Parasitophorous Vacuole ◽

Secreted Proteins ◽

Intracellular Pathogens ◽

Mitochondrial Complex ◽

Content Type ◽

The Common ◽

Insight Into

ABSTRACT Parasites interact intimately with their hosts, and the interactions shape both parties. The common human parasite Toxoplasma gondii replicates exclusively in a vacuole in a host cell and alters its host cell’s environment through secreted proteins. One of these secreted proteins, MAF1b, acts to concentrate mitochondria around the parasite’s vacuole, and this relocalization alters the host immune response. Many other intracellular pathogens also recruit host mitochondria, but the identities of the partners that mediate this interaction have not previously been described in any infection. Here, we show that Toxoplasma MAF1b binds to the multifunctional MIB protein complex on the host mitochondria. Reducing the levels of the proteins in this mitochondrial complex reduces the close association of host cell mitochondria and the parasite’s vacuole. This work provides new insight into a key host-pathogen interaction and identifies possible targets for future therapeutic intervention as well as a more molecular understanding of important biology. Many diverse intracellular pathogens, such as Legionella pneumophila, Chlamydia psittaci, Encephalitozoon sp., and Toxoplasma gondii, manipulate and relocate host cell organelles, including mitochondria. Toxoplasma tachyzoites use a secreted protein, mitochondrial association factor 1b (MAF1b), to drive the association between the host mitochondria and the membrane of the parasitophorous vacuole, in which the parasites grow. The identity of the host partner in this interaction, however, has not previously been identified. By exogenously expressing tagged MAF1b in mouse embryonic fibroblasts, we were able to isolate host cell proteins that specifically interact with MAF1b. We then verified these interactions in the MAF1b-expressing fibroblasts, as well as in the context of parasite infection in human fibroblasts and HeLa cells. The results show that a host cell mitochondrial complex, the mitochondrial intermembrane space bridging (MIB) complex, specifically interacts with MAF1b. We further demonstrate that a version of MAF1b that is deficient in host-mitochondrial association does not efficiently coprecipitate the MIB complex. Validation of the importance of the MAF1b-MIB interaction came from showing that knockdown of two MIB complex components, MIC60 and SAM50, substantially reduces mitochondrial association with the parasitophorous vacuole membrane. This interaction between a secreted membrane-integral parasite protein and a membrane-bound complex of a host organelle represents the first instance of organelle relocalization in which both the host and pathogen molecules are known and provides the foundation for more detailed biochemical studies. IMPORTANCE Parasites interact intimately with their hosts, and the interactions shape both parties. The common human parasite Toxoplasma gondii replicates exclusively in a vacuole in a host cell and alters its host cell’s environment through secreted proteins. One of these secreted proteins, MAF1b, acts to concentrate mitochondria around the parasite’s vacuole, and this relocalization alters the host immune response. Many other intracellular pathogens also recruit host mitochondria, but the identities of the partners that mediate this interaction have not previously been described in any infection. Here, we show that Toxoplasma MAF1b binds to the multifunctional MIB protein complex on the host mitochondria. Reducing the levels of the proteins in this mitochondrial complex reduces the close association of host cell mitochondria and the parasite’s vacuole. This work provides new insight into a key host-pathogen interaction and identifies possible targets for future therapeutic intervention as well as a more molecular understanding of important biology.

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Toxoplasma gondii Matrix Antigen 1 Is a Secreted Immunomodulatory Effector

mBio ◽

10.1128/mbio.00603-21 ◽

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.

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Coimmunoprecipitation with MYR1 Identifies Three Additional Proteins within the Toxoplasma gondii Parasitophorous Vacuole Required for Translocation of Dense Granule Effectors into Host Cells

mSphere ◽

10.1128/msphere.00858-19 ◽

2020 ◽

Vol 5 (1) ◽

Cited By ~ 9

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.

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Analysis of Noncanonical Calcium-Dependent Protein Kinases in Toxoplasma gondii by Targeted Gene Deletion Using CRISPR/Cas9

Infection and Immunity ◽

10.1128/iai.01173-15 ◽

2016 ◽

Vol 84 (5) ◽

pp. 1262-1273 ◽

Cited By ~ 35

Author(s):

Shaojun Long ◽

Qiuling Wang ◽

L. David Sibley

Keyword(s):

Toxoplasma Gondii ◽

Protein Kinases ◽

Gene Deletion ◽

Cyst Formation ◽

Content Type ◽

Calcium Dependent ◽

Targeted Gene Deletion ◽

Dependent Protein ◽

Calcium Dependent Protein Kinases

Calcium-dependent protein kinases (CDPKs) are expanded in apicomplexan parasites, especially inToxoplasma gondiiwhere 14 separate genes encoding these enzymes are found. Although previous studies have shown that several CDPKs play a role in controlling invasion, egress, and cell division inT. gondii, the roles of most of these genes are unexplored. Here we developed a more efficient method for gene disruption using CRISPR (clustered regularly interspaced short palindromic repeats)/Cas9 (CRISPR-associated protein 9) that was modified to completely delete large, multiexonic genes from the genome and to allow serial replacement by recycling of the selectable marker using Cre-loxP. Using this system, we generated a total of 24 mutants in type 1 and 2 genetic backgrounds to ascertain the functions of noncanonical CDPKs. Remarkably, although we were able to confirm the essentiality of CDPK1 and CDPK7, the majority of CDPKs had no discernible phenotype for growthin vitroor infection in the mouse model. The exception to this was CDPK6, loss of which leads to reduced plaquing, fitness defect in a competition assay, and reduced tissue cyst formation in chronically infected mice. Our findings highlight the utility of CRISPR/Cas9 for rapid serial gene deletion and also suggest that additional models are needed to reveal the functions of many genes inT. gondii.

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Toxoplasma gondii AP2IX-4 Regulates Gene Expression during Bradyzoite Development

mSphere ◽

10.1128/msphere.00054-17 ◽

2017 ◽

Vol 2 (2) ◽

Cited By ~ 22

Author(s):

Sherri Huang ◽

Michael J. Holmes ◽

Joshua B. Radke ◽

Dong-Pyo Hong ◽

Ting-Kai Liu ◽

...

Keyword(s):

Gene Expression ◽

Toxoplasma Gondii ◽

Cyst Formation ◽

Tissue Cyst ◽

Stress Induction ◽

Content Type ◽

A Cell ◽

Transitional Forms ◽

Mouse Model Of Infection ◽

Cyst Development

ABSTRACT Toxoplasma gondii is a single-celled parasite that persists in its host as a transmissible tissue cyst. How the parasite converts from its replicative form to the bradyzoites housed in tissue cysts is not well understood, but the process clearly involves changes in gene expression. Here we report that parasites lacking a cell cycle-regulated transcription factor called AP2IX-4 display reduced frequencies of tissue cyst formation in culture and in a mouse model of infection. Parasites missing AP2IX-4 lose the ability to regulate bradyzoite genes during tissue cyst development. Expressed in developing bradyzoites still undergoing division, AP2IX-4 may serve as a useful marker in the study of transitional forms of the parasite. Toxoplasma gondii is a protozoan parasite of great importance to human and animal health. In the host, this obligate intracellular parasite persists as a tissue cyst that is imperceptible to the immune response and unaffected by current therapies. The tissue cysts facilitate transmission through predation and give rise to chronic cycles of toxoplasmosis in immunocompromised patients. Transcriptional changes accompany conversion of the rapidly replicating tachyzoites into the encysted bradyzoites, and yet the mechanisms underlying these alterations in gene expression are not well defined. Here we show that AP2IX-4 is a nuclear protein exclusively expressed in tachyzoites and bradyzoites undergoing division. Knockout of AP2IX-4 had no discernible effect on tachyzoite replication but resulted in a reduced frequency of tissue cyst formation following alkaline stress induction—a defect that is reversible by complementation. AP2IX-4 has a complex role in regulating bradyzoite gene expression, as the levels of many bradyzoite mRNAs dramatically increased beyond those seen under conditions of normal stress induction in AP2IX-4 knockout parasites exposed to alkaline media. The loss of AP2IX-4 also resulted in a modest virulence defect and reduced cyst burden in chronically infected mice, which was reversed by complementation. These findings illustrate that the transcriptional mechanisms responsible for tissue cyst development operate across the intermediate life cycle from the dividing tachyzoite to the dormant bradyzoite. IMPORTANCE Toxoplasma gondii is a single-celled parasite that persists in its host as a transmissible tissue cyst. How the parasite converts from its replicative form to the bradyzoites housed in tissue cysts is not well understood, but the process clearly involves changes in gene expression. Here we report that parasites lacking a cell cycle-regulated transcription factor called AP2IX-4 display reduced frequencies of tissue cyst formation in culture and in a mouse model of infection. Parasites missing AP2IX-4 lose the ability to regulate bradyzoite genes during tissue cyst development. Expressed in developing bradyzoites still undergoing division, AP2IX-4 may serve as a useful marker in the study of transitional forms of the parasite.

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

Infection and Immunity ◽

10.1128/iai.06167-11 ◽

2011 ◽

Vol 80 (3) ◽

pp. 1156-1165 ◽

Cited By ~ 22

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.

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