scholarly journals Toxoplasma Uses GRA16 To Upregulate Host c-Myc

mSphere ◽  
2020 ◽  
Vol 5 (3) ◽  
Author(s):  
Michael W. Panas ◽  
John C. Boothroyd
Keyword(s):  
Host Cell ◽  
Protein Phosphatase ◽  
Crucial Role ◽  
Neospora Caninum ◽  
Parasitophorous Vacuole ◽  
Regulatory Subunit ◽  
Close Relative ◽  
Effector Protein ◽  
Animal Cells ◽  
Content Type

ABSTRACT Manipulation of the host cell is a crucial part of life for many intracellular organisms. We have recently come to appreciate the extent to which the intracellular pathogen Toxoplasma gondii reprograms its host cell, and this is illustrated by the marked upregulation of the central regulator c-Myc, an oncogene that coordinates myriad cellular functions. In an effort to identify an effector protein capable of regulating c-Myc, our laboratory constructed a screen for mutant parasites unable to accomplish this upregulation. Interestingly, this screen identified numerous components of a complex located in/on the parasitophorous vacuole membrane necessary to translocate Toxoplasma proteins out into the host cytosol, but it never identified a specific effector protein. Thus, how the parasite upregulates c-Myc has largely been a mystery. Previously, the Toxoplasma dense granule protein GRA16 has been described to bind to one isoform of PP2A-B, a regulatory subunit that coordinates the activity of the catalytic protein phosphatase PP2A. As other PP2A subunits have been reported to target PP2A protein phosphatase activity to c-Myc, subsequently leading to c-Myc destabilization, we examined whether GRA16 has an impact on host c-Myc accumulation. Expression of Toxoplasma’s GRA16 protein in Neospora caninum, a close relative of Toxoplasma that does not naturally upregulate host c-Myc, conferred the ability on Neospora to do this now. Further support was obtained by deleting the GRA16 gene from Toxoplasma and observing a severely diminished ability of Toxoplasma tachyzoites to upregulate host c-Myc. Thus, GRA16 is an effector protein central to Toxoplasma’s ability to upregulate host c-Myc. IMPORTANCE The proto-oncogene c-Myc plays a crucial role in the growth and division of many animal cells. Previous studies have identified an active upregulation of c-Myc by Toxoplasma tachyzoites, suggesting the existence of one or more exported “effector” proteins. The identity of such an effector, however, has not previously been known. Here, we show that a previously known secreted protein, GRA16, plays a crucial role in c-Myc upregulation. This finding will enable further dissection of the precise mechanism and role of c-Myc upregulation in Toxoplasma-infected cells.

Characterisation of NcGRA7 and NcSAG4 proteins: Immunolocalisation and their role in the host cell invasion by Neospora caninum tachyzoites

2010 ◽  
Vol 55 (4) ◽  
Author(s):  
Adriana Aguado-Martínez ◽  
Gema Álvarez-García ◽  
Gereon Schares ◽  
Verónica Risco-Castillo ◽  
Aurora Fernández-García ◽  
...  
Keyword(s):  
Monoclonal Antibody ◽  
Host Cell ◽  
Cell Invasion ◽  
Neospora Caninum ◽  
Parasitophorous Vacuole ◽  
Membrane Surface ◽  
Chronic Phase ◽  
Host Cell Invasion ◽  
Invasion Mechanisms ◽  
The Matrix

AbstractNeospora caninum negatively impacts bovine reproductive performance around the world. Addressing this problem requires a greater understanding of the parasite’s molecular biology. In this study, monoclonal antibodies against recombinant proteins were successfully developed and employed to characterise two different proteins of N. caninum: the acute phase-associated NcGRA7 and the chronic phase-associated NcSAG4. Immunofluorescence with the anti-rNcGRA7 monoclonal antibody suggested that NcGRA7 trafficks from tachyzoite dense granules to the matrix of the parasitophorous vacuole and parasite’s surroundings. Furthermore, NcGRA7 is also expressed in the bradyzoite stage and localised on the matrix of bradyzoite-positive vacuoles. NcGRA7 appears to be partially involved in the tachyzoite-invasion mechanisms, as an anti-rNcGRA7 monoclonal antibody partially inhibited in vitro tachyzoite-invasion. A monoclonal antibody specific for NcSAG4 confirmed this protein’s bradyzoitespecific expression both by western blot and immunofluorescence. However, some bradyzoite-positive vacuoles only weakly expressed NcSAG4, if it was expressed at all. The specificity of the anti-rNcSAG4 monoclonal antibody was confirmed by the recognition of the NcSAG4 in the membrane surface of Nc-1SAG4c transgenic tachyzoites, which constitutively expresses NcSAG4. Blocking NcSAG4 of Nc-1SAG4c tachyzoites with the monoclonal antibody did not affect host cell invasion. However, its implication on the host cell adhesion or host immune evasion should not be discarded.

scholarly journals Fierce Competition between Toxoplasma and Chlamydia for Host Cell Structures in Dually Infected Cells

2012 ◽  
Vol 12 (2) ◽  
pp. 265-277 ◽  
Author(s):  
Julia D. Romano ◽  
Catherine de Beaumont ◽  
Jose A. Carrasco ◽  
Karen Ehrenman ◽  
Patrik M. Bavoil ◽  
...  
Keyword(s):  
Host Cell ◽  
Parasitophorous Vacuole ◽  
Productive Infection ◽  
Microtubule Organizing Center ◽  
Nutrient Pools ◽  
Content Type ◽  
Cell Structures ◽  
Organizing Center ◽  
A Cell ◽  
Fierce Competition

ABSTRACTThe prokaryoteChlamydia trachomatisand the protozoanToxoplasma gondii, two obligate intracellular pathogens of humans, have evolved a similarmodus operandito colonize their host cell and salvage nutrients from organelles. In order to gain fundamental knowledge on the pathogenicity of these microorganisms, we have established a cell culture model whereby single fibroblasts are coinfected byC. trachomatisandT. gondii. We previously reported that the two pathogens compete for the same nutrient pools in coinfected cells and thatToxoplasmaholds a significant competitive advantage overChlamydia. Here we have expanded our coinfection studies by examining the respective abilities ofChlamydiaandToxoplasmato co-opt the host cytoskeleton and recruit organelles. We demonstrate that the two pathogen-containing vacuoles migrate independently to the host perinuclear region and rearrange the host microtubular network around each vacuole. However,ToxoplasmaoutcompetesChlamydiato the host microtubule-organizing center to the detriment of the bacterium, which then shifts to a stress-induced persistent state. Solely in cells preinfected withChlamydia, the centrosomes become associated with the chlamydial inclusion, while theToxoplasmaparasitophorous vacuole displays growth defects. Both pathogens fragment the host Golgi apparatus and recruit Golgi elements to retrieve sphingolipids. This study demonstrates that the productive infection by bothChlamydiaandToxoplasmadepends on the capability of each pathogen to successfully adhere to a finely tuned developmental program that aims to remodel the host cell for the pathogen's benefit. In particular, this investigation emphasizes the essentiality of host organelle interception by intravacuolar pathogens to facilitate access to nutrients.

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 Toxoplasmacontrols host cyclin E expression through the use of a novel MYR1-dependent effector protein, HCE1

2019 ◽  
Author(s):  
Michael W. Panas ◽  
Adit Naor ◽  
Alicja M. Cygan ◽  
John C. Boothroyd
Keyword(s):  
Transcription Factor ◽  
Target Genes ◽  
Neospora Caninum ◽  
Parasitophorous Vacuole ◽  
Cyclin E ◽  
Host Cells ◽  
Growth Defect ◽  
Effector Protein ◽  
Dependent Manner ◽  
E2f Transcription Factor

AbstractToxoplasma gondiiis an obligate intracellular parasite that establishes a favorable environment in the host cells in which it replicates. We have previously reported that it uses MYR-dependent translocation of dense granule proteins to elicit a key set of host responses related to the cell cycle, specifically E2F transcription factor targets including cyclin E. We report here the identification of a novelToxoplasmaeffector protein that is exported from the parasitophorous vacuole in a MYR1-dependent manner and localizes to the host’s nucleus. Parasites lacking this inducer ofHostCyclinE(HCE1) are unable to modulate E2F transcription factor target genes and exhibit a substantial growth defect. Immunoprecipitation of HCE1 from infected host cells shows that HCE1 efficiently binds elements of the cyclin E regulatory complex, DP1 and its partners E2F3 and E2F4. Expression of HCE1 inNeospora caninum, or in uninfected HFFs, shows localization of the expressed protein to the host nuclei and strong cyclin E up-regulation. Thus, HCE1 is a novel effector protein that is necessary and sufficient to impact the E2F-axis of transcription resulting in co-opting of host functions toToxoplasma’sadvantage.

scholarly journals Neighbors Working Together: a Toxoplasma Rhoptry Protein That Facilitates Dense Granule Protein Translocation into the Host Cell

mSphere ◽  
2019 ◽  
Vol 4 (4) ◽  
Author(s):  
Gustavo Arrizabalaga
Keyword(s):  
Host Cell ◽  
Protein Translocation ◽  
Parasitophorous Vacuole ◽  
Opportunistic Pathogen ◽  
Dense Granule ◽  
Effector Proteins ◽  
Content Type ◽  
Dense Granules ◽  
Cell Functions ◽  
Host Signaling

ABSTRACT The opportunistic pathogen Toxoplasma gondii is highly adept at manipulating host cell functions. While inside a host cell, Toxoplasma divides within a parasitophorous vacuole from which it secretes numerous effector proteins from its dense granules. Many of these so-called GRA proteins are translocated from the parsitophorous vacuole into the host cell where they directly disrupt host signaling pathways. The machinery that drives the translocation of GRA proteins across the parasitophorous vacuole membrane is being elucidated through both genetic and biochemical approaches. A new mSphere research article (M. W. Panas, A. Ferrel, A. Naor, E. Tenborg, et al., mSphere 4:e00276-19, 2019, https://doi.org/10.1128/mSphere.00276-19) describes how the kinase ROP17, which is secreted from the parasite’s rhoptries into the host cell during invasion, regulates the translocation of GRA effectors.

scholarly journals Molecular cloning and characterization ofNcROP2Fam-1, a member of the ROP2 family of rhoptry proteins inNeospora caninumthat is targeted by antibodies neutralizing host cell invasionin vitro

Parasitology ◽  
2013 ◽  
Vol 140 (8) ◽  
pp. 1033-1050 ◽  
Author(s):  
FERIAL ALAEDDINE ◽  
ANDREW HEMPHILL ◽  
KARIM DEBACHE ◽  
CHRISTOPHE GUIONAUD
Keyword(s):  
Host Cell ◽  
Family Member ◽  
Vaccine Candidate ◽  
Neospora Caninum ◽  
Parasitophorous Vacuole ◽  
Host Cells ◽  
General Belief ◽  
Intracellular Parasites ◽  
Promising Vaccine Candidate

SUMMARYRecent publications demonstrated that a fragment of aNeospora caninumROP2 family member antigen represents a promising vaccine candidate. We here report on the cloning of the cDNA encoding this protein,N. caninumROP2 family member 1 (NcROP2Fam-1), its molecular characterization and localization. The protein possesses the hallmarks of ROP2 family members and is apparently devoid of catalytic activity. NcROP2Fam-1 is synthesized as a pre-pro-protein that is matured to 2 proteins of 49 and 55 kDa that localize to rhoptry bulbs. Upon invasion the protein is associated with the nascent parasitophorous vacuole membrane (PVM), evacuoles surrounding the host cell nucleus and, in some instances, the surface of intracellular parasites. Staining was also observed within the cyst wall of ‘cysts’ producedin vitro. Interestingly, NcROP2Fam-1 was also detected on the surface of extracellular parasites entering the host cells and antibodies directed against NcROP2Fam-1-specific peptides partially neutralized invasionin vitro. We conclude that, in spite of the general belief that ROP2 family proteins are intracellular antigens, NcROP2Fam-1 can also be considered as an extracellular antigen, a property that should be taken into account in further experiments employing ROP2 family proteins as vaccines.

scholarly journals The Secreted Acid Phosphatase Domain-Containing GRA44 from Toxoplasma gondii Is Required for c-Myc Induction in Infected Cells

mSphere ◽  
2020 ◽  
Vol 5 (1) ◽  
Author(s):  
William J. Blakely ◽  
Michael J. Holmes ◽  
Gustavo Arrizabalaga
Keyword(s):  
Acid Phosphatase ◽  
Toxoplasma Gondii ◽  
Host Cell ◽  
Secretory Pathway ◽  
Parasitophorous Vacuole ◽  
Severe Disease ◽  
Immune Recognition ◽  
Phosphatase Domain ◽  
Content Type ◽  
Cell Defense

ABSTRACT During host cell invasion, the eukaryotic pathogen Toxoplasma gondii forms a parasitophorous vacuole to safely reside within the cell, while it is partitioned from host cell defense mechanisms. From within this safe niche, parasites sabotage multiple host cell systems, including gene expression, apoptosis, and intracellular immune recognition, by secreting a large arsenal of effector proteins. Many parasite proteins studied for active host cell manipulative interactions have been kinases. The translocation of effectors from the parasitophorous vacuole into the host cell is mediated by a putative translocon complex, which includes the proteins MYR1, MYR2, and MYR3. Whether other proteins are involved in the structure or regulation of this putative translocon is not known. We have discovered that the secreted protein GRA44, which contains a putative acid phosphatase domain, interacts with members of this complex and is required for host cell effects downstream of effector secretion. We have determined that GRA44 is processed in a region with homology to sequences targeted by protozoan proteases of the secretory pathway and that both major cleavage fragments are secreted into the parasitophorous vacuole. Immunoprecipitation experiments showed that GRA44 interacts with a large number of secreted proteins, including MYR1. Importantly, conditional knockdown of GRA44 resulted in a lack of host cell c-Myc upregulation, which mimics the phenotype seen when members of the translocon complex are genetically disrupted. Thus, the putative acid phosphatase GRA44 is crucial for host cell alterations during Toxoplasma infection and is associated with the translocon complex which Toxoplasma relies upon for success as an intracellular pathogen. IMPORTANCE Approximately one-third of humans are infected with the parasite Toxoplasma gondii. Toxoplasma infections can lead to severe disease in those with a compromised or suppressed immune system. Additionally, infections during pregnancy present a significant health risk to the developing fetus. Drugs that target this parasite are limited, have significant side effects, and do not target all disease stages. Thus, a thorough understanding of how the parasite propagates within a host is critical in the discovery of novel therapeutic targets. Toxoplasma replication requires that it enter the cells of the infected organism. In order to survive the environment inside a cell, Toxoplasma secretes a large repertoire of proteins, which hijack a number of important cellular functions. How these Toxoplasma proteins move from the parasite into the host cell is not well understood. Our work shows that the putative phosphatase GRA44 is part of a protein complex responsible for this process.

scholarly journals Evidence for Host Cells as the Major Contributor of Lipids in the Intravacuolar Network of Toxoplasma-Infected Cells

2011 ◽  
Vol 10 (8) ◽  
pp. 1095-1099 ◽  
Author(s):  
Carolina E. Caffaro ◽  
John C. Boothroyd
Keyword(s):  
Host Cell ◽  
Fluorescence Recovery After Photobleaching ◽  
Parasitophorous Vacuole ◽  
Dense Granule ◽  
Host Cells ◽  
Cell Plasma Membrane ◽  
Content Type ◽  
Cell Plasma ◽  
Continuous Stream ◽  
Infected Cells

ABSTRACT The intracellular parasite Toxoplasma gondii develops inside a parasitophorous vacuole (PV) that derives from the host cell plasma membrane during invasion. Previous electron micrograph images have shown that the membrane of this vacuole undergoes an extraordinary remodeling with an extensive network of thin tubules and vesicles, the intravacuolar network (IVN), which fills the lumen of the PV. While dense granule proteins, secreted during and after invasion, are the main factors for the organization and tubulation of the network, little is known about the source of lipids used for this remodeling. By selectively labeling host cell or parasite membranes, we uncovered evidence that strongly supports the host cell as the primary, if not exclusive, source of lipids for parasite IVN remodeling. Fluorescence recovery after photobleaching (FRAP) microscopy experiments revealed that lipids are surprisingly dynamic within the parasitophorous vacuole and are continuously exchanged or replenished by the host cell. The results presented here suggest a new model for development of the parasitophorous vacuole whereby the host provides a continuous stream of lipids to support the growth and maturation of the PVM and IVN.

scholarly journals First Characterization of theNeospora caninumDense Granule Protein GRA9

2017 ◽  
Vol 2017 ◽  
pp. 1-15 ◽  
Author(s):  
Margret Leineweber ◽  
Katrin Spekker-Bosker ◽  
Vanessa Ince ◽  
Gereon Schares ◽  
Andrew Hemphill ◽  
...  
Keyword(s):  
Host Cell ◽  
Cell Invasion ◽  
Neospora Caninum ◽  
Parasitophorous Vacuole ◽  
Dense Granule ◽  
Immunogold Electron Microscopy ◽  
Host Cell Invasion ◽  
Dense Granules ◽  
Granule Protein

The obligate intracellular apicomplexan parasiteNeospora caninum (N. caninum)is closely related toToxoplasma gondii (T. gondii). The dense granules, which are present in all apicomplexan parasites, are important secretory organelles. Dense granule (GRA) proteins are released into the parasitophorous vacuole (PV) following host cell invasion and are known to play important roles in the maintenance of the host-parasite relationship and in the acquisition of nutrients. Here, we provide a detailed characterization of theN. caninumdense granule protein NcGRA9. The in silico genomic organization and key protein characteristics are described. Immunofluorescence-based localization studies revealed that NcGRA9 is located in the dense granules and is released into the interior of the PV following host cell invasion. Immunogold-electron microscopy confirmed the dense granule localization and showed that NcGRA9 is associated with the intravacuolar network. In addition, NcGRA9 is found in the “excreted secreted antigen” (ESA) fraction ofN. caninum. Furthermore, by analysing the distribution of truncated versions of NcGRA9, we provide evidence that the C-terminal region of this protein is essential for the targeting of NcGRA9 into the dense granules ofN. caninum, and the truncated proteins show reduced secretion.

scholarly journals Microsporidia Interact with Host Cell Mitochondria via Voltage-Dependent Anion Channels Using Sporoplasm Surface Protein 1

mBio ◽  
2019 ◽  
Vol 10 (4) ◽  
Author(s):  
Bing Han ◽  
Yanfen Ma ◽  
Vincent Tu ◽  
Tadakimi Tomita ◽  
Joshua Mayoral ◽  
...  
Keyword(s):  
Host Cell ◽  
Parasitophorous Vacuole ◽  
Surface Protein ◽  
Host Cells ◽  
Anion Channels ◽  
Polar Tube ◽  
Content Type ◽  
Proteomic Approach ◽  
Voltage Dependent ◽  
Microsporidian Infection

ABSTRACT Microsporidia are opportunistic intracellular pathogens that can infect a wide variety of hosts ranging from invertebrates to vertebrates. During invasion, the microsporidian polar tube pushes into the host cell, creating a protective microenvironment, the invasion synapse, into which the sporoplasm extrudes. Within the synapse, the sporoplasm then invades the host cell, forming a parasitophorous vacuole (PV). Using a proteomic approach, we identified Encephalitozoon hellem sporoplasm surface protein 1 (EhSSP1), which localized to the surface of extruded sporoplasms. EhSSP1 was also found to interact with polar tube protein 4 (PTP4). Recombinant EhSSP1 (rEhSSP1) bound to human foreskin fibroblasts, and both anti-EhSSP1 and rEhSSP1 caused decreased levels of host cell invasion, suggesting that interaction of SSP1 with the host cell was involved in invasion. Coimmunoprecipitation (Co-IP) followed by proteomic analysis identified host cell voltage-dependent anion channels (VDACs) as EhSSP1 interacting proteins. Yeast two-hybrid assays demonstrated that EhSSP1 was able to interact with VDAC1, VDAC2, and VDAC3. rEhSSP1 colocalized with the host mitochondria which were associated with microsporidian PVs in infected cells. Transmission electron microscopy revealed that the outer mitochondrial membrane interacted with meronts and the PV membrane, mitochondria clustered around meronts, and the VDACs were concentrated at the interface of mitochondria and parasite. Knockdown of VDAC1, VDAC2, and VDAC3 in host cells resulted in significant decreases in the number and size of the PVs and a decrease in mitochondrial PV association. The interaction of EhSSP1 with VDAC probably plays an important part in energy acquisition by microsporidia via its role in the association of mitochondria with the PV. IMPORTANCE Microsporidia are important opportunistic human pathogens in immune-suppressed individuals, such as those with HIV/AIDS and recipients of organ transplants. The sporoplasm is critical for establishing microsporidian infection. Despite the biological importance of this structure for transmission, there is limited information about its structure and composition that could be targeted for therapeutic intervention. Here, we identified a novel E. hellem sporoplasm surface protein, EhSSP1, and demonstrated that it can bind to host cell mitochondria via host VDAC. Our data strongly suggest that the interaction between SSP1 and VDAC is important for the association of mitochondria with the parasitophorous vacuole during microsporidian infection. In addition, binding of SSP1 to the host cell is associated with the final steps of invasion in the invasion synapse.

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