The
                    Toxoplasma gondii
                    Rhoptry Kinome Is Essential for Chronic Infection

ABSTRACT Ingestion of the obligate intracellular protozoan parasite Toxoplasma gondii causes an acute infection that leads to chronic infection of the host. To facilitate the acute phase of the infection, T. gondii manipulates the host response by secreting rhoptry organelle proteins (ROPs) into host cells during its invasion. A few key ROP proteins with signatures of kinases or pseudokinases (ROPKs) act as virulence factors that enhance parasite survival against host gamma interferon-stimulated innate immunity. However, the roles of these and other ROPK proteins in establishing chronic infection have not been tested. Here, we deleted 26 ROPK gene loci encoding 31 unique ROPK proteins of type II T. gondii and show that numerous ROPK proteins influence the development of chronic infection. Cyst burdens were increased in the Δ rop16 knockout strain or moderately reduced in 11 ROPK knockout strains. In contrast, deletion of ROP5 , ROP17 , ROP18 , ROP35 , or ROP38 / 29 / 19 ( ROP38 , ROP29 , and ROP19 ) severely reduced cyst burdens. Δ rop5 and Δ rop18 knockout strains were less resistant to host immunity-related GTPases (IRGs) and exhibited >100-fold-reduced virulence. ROP18 kinase activity and association with the parasitophorous vacuole membrane were necessary for resistance to host IRGs. The Δ rop17 strain exhibited a >12-fold defect in virulence; however, virulence was not affected in the Δ rop35 or Δ rop38 / 29 / 19 strain. Resistance to host IRGs was not affected in the Δ rop17 , Δ rop35 , or Δ rop38 / 29 / 19 strain. Collectively, these findings provide the first definitive evidence that the type II T. gondii ROPK proteome functions as virulence factors and facilitates additional mechanisms of host manipulation that are essential for chronic infection and transmission of T. gondii . IMPORTANCE Reactivation of chronic Toxoplasma gondii infection in individuals with weakened immune systems causes severe toxoplasmosis. Existing treatments for toxoplasmosis are complicated by adverse reactions to chemotherapy. Understanding key parasite molecules required for chronic infection provides new insights into potential mechanisms that can interrupt parasite survival or persistence in the host. This study reveals that key secreted rhoptry molecules are used by the parasite to establish chronic infection of the host. Certain rhoptry proteins were found to be critical virulence factors that resist innate immunity, while other rhoptry proteins were found to influence chronic infection without affecting virulence. This study reveals that rhoptry proteins utilize multiple mechanisms of host manipulation to establish chronic infection of the host. Targeted disruption of parasite rhoptry proteins involved in these biological processes opens new avenues to interfere with chronic infection with the goal to either eliminate chronic infection or to prevent recrudescent infections.

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Nonreplicating, Cyst-Defective Type II Toxoplasma gondii Vaccine Strains Stimulate Protective Immunity against Acute and Chronic Infection

Infection and Immunity ◽

10.1128/iai.02756-14 ◽

2015 ◽

Vol 83 (5) ◽

pp. 2148-2155 ◽

Cited By ~ 20

Author(s):

Barbara A. Fox ◽

David J. Bzik

Keyword(s):

Toxoplasma Gondii ◽

Chronic Infection ◽

Acute Infection ◽

Cyst Formation ◽

Type I ◽

Type Ii ◽

Live Attenuated Vaccine ◽

Content Type ◽

Monophosphate Decarboxylase ◽

Uracil Auxotroph

Live attenuated vaccine strains, such as type I nonreplicating uracil auxotroph mutants, are highly effective in eliciting lifelong immunity to virulent acute infection byToxoplasma gondii. However, it is currently unknown whether vaccine-elicited immunity can provide protection against acute infection and also prevent chronic infection. To address this problem, we developed nonreverting, nonreplicating, live attenuated uracil auxotroph vaccine strains in the type II Δku80genetic background by targeting the deletion of the orotidine 5′-monophosphate decarboxylase (OMPDC) and uridine phosphorylase (UP) genes. Deletion ofOMPDCinduced a severe uracil auxotrophy with loss of replication, loss of virulence in mice, and loss of the ability to develop cysts and chronic infection. Vaccination of mice using type II Δku80Δompdcmutants stimulated a fully protective CD8+T cell-dependent immunity that prevented acute infection by type I and type II strains ofT. gondii, and this vaccination also severely reduced or prevented cyst formation after type II challenge infection. Nonreverting, nonreplicating, and non-cyst-forming Δompdcmutants provide new tools to examine protective immune responses elicited by vaccination with a live attenuated type II vaccine.

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Transcriptional repression by ApiAP2 factors is central to chronic toxoplasmosis

10.1101/100628 ◽

2017 ◽

Cited By ~ 5

Author(s):

Joshua B. Radke ◽

Danielle Worth ◽

Dong-Pyo Hong ◽

Sherri Huang ◽

William J. Sullivan ◽

...

Keyword(s):

Gene Expression ◽

Transcriptional Repression ◽

Chronic Infection ◽

Parasitophorous Vacuole ◽

Acute Infection ◽

Cyst Formation ◽

Tissue Cyst ◽

Genetic Lineages ◽

Inflammatory Monocytes ◽

Bradyzoite Differentiation

AbstractBradyzoite differentiation is marked by major changes in gene expression resulting in a parasite that expresses a new repertoire of surface antigens hidden inside a modified parasitophorous vacuole called the tissue cyst. The factors that control this important life cycle transition are not well understood. Here we describe an importantToxoplasmatranscriptional repressor mechanism controlling bradyzoite differentiation that operates exclusively in the tachyzoite stage. The ApiAP2 factor, AP2IV-4, is a nuclear factor dynamically expressed in late S phase through mitosis/cytokinesis of the tachyzoite cell cycle. Remarkably, deletion of the AP2IV-4 locus resulted in the increased expression of bradyzoite mRNAs in replicating tachyzoites, and in two different genetic lineages we confirmed the misexpression of tissue cyst wall components (e.g. BPK1, MCP4, CST1) and the bradyzoite surface antigen SRS9 in the tachyzoite stage. In the murine animal model, the loss of AP2IV-4 had profound biological consequences. Type II prugniaud strain parasites lacking AP2IV-4 were unable to form tissue cysts in brain tissue and the absence of this factor also recruited a potent immune response characterized by increases inflammatory monocytes, IFN-γ and higher numbers of both CD8+ and CD4+ T-cells. Altogether, these results indicate that suppression of bradyzoite antigens by AP2IV-4 during acute infection is required forToxoplasmato establish a chronic infection in the immune-competent host.Author SummaryTheToxoplasmabiology that underlies the establishment of a chronic infection is developmental conversion of the acute tachyzoite stage into the latent bradyzoite-tissue cyst stage. Despite the important clinical consequences of this developmental pathway, the molecular basis of the switch mechanisms that control formation of the tissue cyst is still poorly understood. A fundamental feature of tissue cyst formation is the expression of bradyzoite-specific genes. Here we show the transcription factor AP2IV-4 directly silences bradyzoite mRNA and protein expression in the acute tachyzoite stage demonstrating that developmental control of tissue cyst formation is as much about when not to express bradyzoite genes as it is about when to activate them. Loosing the suppression of bradyzoite gene expression in the acute tachyzoite stage caused by deleting AP2IV-4 blocked the establishment of chronic disease in healthy animals through the pre-arming of the immune system suggesting a possible strategy for preventing chronicToxoplasmainfections.

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Association of host cell endoplasmic reticulum and mitochondria with the Toxoplasma gondii parasitophorous vacuole membrane: a high affinity interaction

Journal of Cell Science ◽

10.1242/jcs.110.17.2117 ◽

1997 ◽

Vol 110 (17) ◽

pp. 2117-2128 ◽

Cited By ~ 8

Author(s):

A.P. Sinai ◽

P. Webster ◽

K.A. Joiner

Keyword(s):

Endoplasmic Reticulum ◽

Toxoplasma Gondii ◽

Host Cell ◽

Parasitophorous Vacuole ◽

Protozoan Parasite ◽

Host Cells ◽

Parasitophorous Vacuole Membrane ◽

Vacuole Membrane ◽

Protein Protein Interaction ◽

High Affinity

The parasitophorous vacuole membrane (PVM) of the obligate intracellular protozoan parasite Toxoplasma gondii forms tight associations with host mitochondria and the endoplasmic reticulum (ER). We have used a combination of morphometric and biochemical approaches to characterize this unique phenomenon, which we term PVM-organelle association. The PVM is separated from associated mitochondria and ER by a mean distance of 12 and 18 nm, respectively. The establishment of PVM-organelle association is dependent on active parasite entry, but does not require parasite viability for its maintenance. Association is not a consequence of spatial constraints imposed on the growing vacuole. Morphometric analysis indicates that the extent of mitochondrial association with the PVM stays constant as the vacuole enlarges, whereas the extent of ER association decreases. Disruption of host cell microtubules partially blocks the establishment but not the maintenance of PVM-mitochondrial association, and has no significant effect on PVM-ER association. PVM-organelle association is maintained following disruption of infected host cells, as assessed by electron microscopy and by sub-cellular fractionation showing co-migration of fixed PVM and organelle markers. Taken together, the data suggest that a high affinity, potentially protein-protein interaction between parasite and organelle components is responsible for PVM-organelle association.

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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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Besnoitia besnoiti and Toxoplasma gondii: two apicomplexan strategies to manipulate the host cell centrosome and Golgi apparatus

Parasitology ◽

10.1017/s0031182014000493 ◽

2014 ◽

Vol 141 (11) ◽

pp. 1436-1454 ◽

Cited By ~ 5

Author(s):

RITA CARDOSO ◽

SOFIA NOLASCO ◽

JOÃO GONÇALVES ◽

HELDER C. CORTES ◽

ALEXANDRE LEITÃO ◽

...

Keyword(s):

Toxoplasma Gondii ◽

Golgi Apparatus ◽

Host Cell ◽

Parasitophorous Vacuole ◽

Host Cells ◽

Besnoitia Besnoiti ◽

Microtubule Cytoskeleton ◽

Cytoskeleton Organization ◽

Evolutionary Paths ◽

The Impact

SUMMARYBesnoitia besnoiti and Toxoplasma gondii are two closely related parasites that interact with the host cell microtubule cytoskeleton during host cell invasion. Here we studied the relationship between the ability of these parasites to invade and to recruit the host cell centrosome and the Golgi apparatus. We observed that T. gondii recruits the host cell centrosome towards the parasitophorous vacuole (PV), whereas B. besnoiti does not. Notably, both parasites recruit the host Golgi apparatus to the PV but its organization is affected in different ways. We also investigated the impact of depleting and over-expressing the host centrosomal protein TBCCD1, involved in centrosome positioning and Golgi apparatus integrity, on the ability of these parasites to invade and replicate. Toxoplasma gondii replication rate decreases in cells over-expressing TBCCD1 but not in TBCCD1-depleted cells; while for B. besnoiti no differences were found. However, B. besnoiti promotes a reorganization of the Golgi ribbon previously fragmented by TBCCD1 depletion. These results suggest that successful establishment of PVs in the host cell requires modulation of the Golgi apparatus which probably involves modifications in microtubule cytoskeleton organization and dynamics. These differences in how T. gondii and B. besnoiti interact with their host cells may indicate different evolutionary paths.

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Host ER–parasitophorous vacuole interaction provides a route of entry for antigen cross-presentation in Toxoplasma gondii–infected dendritic cells

Journal of Experimental Medicine ◽

10.1084/jem.20082108 ◽

2009 ◽

Vol 206 (2) ◽

pp. 399-410 ◽

Cited By ~ 115

Author(s):

Romina S. Goldszmid ◽

Isabelle Coppens ◽

Avital Lev ◽

Pat Caspar ◽

Ira Mellman ◽

...

Keyword(s):

T Cells ◽

Dendritic Cells ◽

Toxoplasma Gondii ◽

Mhc Class I ◽

Cd8 T Cells ◽

Parasitophorous Vacuole ◽

Host Cells ◽

Class I ◽

Cross Presentation ◽

Infected Cells

Toxoplasma gondii tachyzoites infect host cells by an active invasion process leading to the formation of a specialized compartment, the parasitophorous vacuole (PV). PVs resist fusion with host cell endosomes and lysosomes and are thus distinct from phagosomes. Because the parasite remains sequestered within the PV, it is unclear how T. gondii–derived antigens (Ag’s) access the major histocompatibility complex (MHC) class I pathway for presentation to CD8+ T cells. We demonstrate that recruitment of host endoplasmic reticulum (hER) to the PV in T. gondii–infected dendritic cells (DCs) directly correlates with cross-priming of CD8+ T cells. Furthermore, we document by immunoelectron microscopy the transfer of hER components into the PV, a process indicative of direct fusion between the two compartments. In strong contrast, no association between hER and phagosomes or Ag presentation activity was observed in DCs containing phagocytosed live or dead parasites. Importantly, cross-presentation of parasite-derived Ag in actively infected cells was blocked when hER retrotranslocation was inhibited, indicating that the hER serves as a conduit for the transport of Ag between the PV and host cytosol. Collectively, these findings demonstrate that pathogen-driven hER–PV interaction can serve as an important mechanism for Ag entry into the MHC class I pathway and CD8+ T cell cross-priming.

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The Toxoplasma gondii protein ROP2 mediates host organelle association with the parasitophorous vacuole membrane

The Journal of Cell Biology ◽

10.1083/jcb.200101073 ◽

2001 ◽

Vol 154 (1) ◽

pp. 95-108 ◽

Cited By ~ 146

Author(s):

Anthony P. Sinai ◽

Keith A. Joiner

Keyword(s):

Toxoplasma Gondii ◽

Fluorescent Protein ◽

Parasitophorous Vacuole ◽

Host Cells ◽

In Vitro Assays ◽

Mitochondrial Outer Membrane ◽

Mitochondrial Targeting ◽

Parasitophorous Vacuole Membrane ◽

Parasite Protein ◽

Vacuole Membrane

Toxoplasma gondii replicates within a specialized vacuole surrounded by the parasitophorous vacuole membrane (PVM). The PVM forms intimate interactions with host mitochondria and endoplasmic reticulum (ER) in a process termed PVM–organelle association. In this study we identify a likely mediator of this process, the parasite protein ROP2. ROP2, which is localized to the PVM, is secreted from anterior organelles termed rhoptries during parasite invasion into host cells. The NH2-terminal domain of ROP2 (ROP2hc) within the PVM is exposed to the host cell cytosol, and has characteristics of a mitochondrial targeting signal. In in vitro assays, ROP2hc is partially translocated into the mitochondrial outer membrane and behaves like an integral membrane protein. Although ROP2hc does not translocate across the ER membrane, it does exhibit carbonate-resistant binding to this organelle. In vivo, ROP2hc expressed as a soluble fragment in the cytosol of uninfected cells associates with both mitochondria and ER. The 30–amino acid (aa) NH2-terminal sequence of ROP2hc, when fused to green fluorescent protein (GFP), is sufficient for mitochondrial targeting. Deletion of the 30-aa NH2-terminal signal from ROP2hc results in robust localization of the truncated protein to the ER. These results demonstrate a new mechanism for tight association of different membrane-bound organelles within the cell cytoplasm.

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p47 GTPases Regulate Toxoplasma gondii Survival in Activated Macrophages

Infection and Immunity ◽

10.1128/iai.73.6.3278-3286.2005 ◽

2005 ◽

Vol 73 (6) ◽

pp. 3278-3286 ◽

Cited By ~ 99

Author(s):

Barbara A. Butcher ◽

Robert I. Greene ◽

Stanley C. Henry ◽

Kimberly L. Annecharico ◽

J. Brice Weinberg ◽

...

Keyword(s):

Toxoplasma Gondii ◽

Acute Infection ◽

Host Cells ◽

Intracellular Growth ◽

Latex Beads ◽

Cellular Factors ◽

Ifn Γ ◽

Normal Inhibition

ABSTRACT The cytokine gamma interferon (IFN-γ) is critical for resistance to Toxoplasma gondii. IFN-γ strongly activates macrophages and nonphagocytic host cells to limit intracellular growth of T. gondii; however, the cellular factors that are required for this effect are largely unknown. We have shown previously that IGTP and LRG-47, members of the IFN-γ-regulated family of p47 GTPases, are required for resistance to acute T. gondii infections in vivo. In contrast, IRG-47, another member of this family, is not required. In the present work, we addressed whether these GTPases are required for IFN-γ-induced suppression of T. gondii growth in macrophages in vitro. Bone marrow macrophages that lacked IGTP or LRG-47 displayed greatly attenuated IFN-γ-induced inhibition of T. gondii growth, while macrophages that lacked IRG-47 displayed normal inhibition. Thus, the ability of the p47 GTPases to limit acute infection in vivo correlated with their ability to suppress intracellular growth in macrophages in vitro. Using confocal microscopy and sucrose density fractionation, we demonstrated that IGTP largely colocalizes with endoplasmic reticulum markers, while LRG-47 was mainly restricted to the Golgi. Although both IGTP and LRG-47 localized to vacuoles containing latex beads, neither protein localized to vacuoles containing live T. gondii. These results suggest that IGTP and LRG-47 are able to regulate host resistance to acute T. gondii infections through their ability to inhibit parasite growth within the macrophage.

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Targeted Disruption of the GRA2 Locus inToxoplasma gondii Decreases Acute Virulence in Mice

Infection and Immunity ◽

10.1128/iai.66.9.4176-4182.1998 ◽

1998 ◽

Vol 66 (9) ◽

pp. 4176-4182 ◽

Cited By ~ 61

Author(s):

Corinne Mercier ◽

Daniel K. Howe ◽

Dana Mordue ◽

Maren Lingnau ◽

L. David Sibley

Keyword(s):

Chronic Infection ◽

Parasitophorous Vacuole ◽

Acute Infection ◽

Early Death ◽

Dense Granule ◽

Positive Serology ◽

Targeted Disruption ◽

The Brain

ABSTRACT Following invasion into the host cell, the protozoanToxoplasma gondii secretes a variety of proteins that modify the parasitophorous vacuole. Within the vacuole, the 28-kDa dense granule protein known as GRA2 is specifically targeted to the tubulovesicular network which forms connections with the vacuolar membrane. To investigate the importance of GRA2, we derived from strain RH a mutant T. gondii line in which GRA2 was disrupted by replacement with the marker Ble (selecting for phleomycin resistance). The Δgra2 mutant invaded and grew normally in both fibroblasts and macrophages in vitro; however, it was less virulent during acute infection in mice. The survival rate of mice inoculated with Δgra2 was significantly higher; some infected mice survived the acute infection, whereas all mice infected with the wild-type strain RH succumbed to early death. Chronic infection by Δgra2 was detected by positive serology, immunohistochemical detection of parasites and cysts in the brain, and reisolation of parasites by bioassay at 6 weeks postinfection. Thus, absence of GRA2 partially attenuates the virulence of T. gondii during the acute phase of infection and allows for establishment of chronic infection by the otherwise highly virulent RH strain. These results establish that GRA2 plays an important role during in vivo infection and provide a potential model for examining acute pathogenesis by T. gondii.

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

10.1101/2020.07.06.189308 ◽

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.

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