scholarly journals Lewis rat NLRP1 inflammasome activation is mediated by threeToxoplasma gondiidense granule proteins

2018 ◽  
Author(s):  
Yifan Wang ◽  
Kimberly M. Cirelli ◽  
Patricio D.C. Barros ◽  
Lamba Omar Sangaré ◽  
Vincent Butty ◽  
...  
Keyword(s):  
Cell Death ◽  
Parasitophorous Vacuole ◽  
Dense Granule ◽  
Inflammasome Activation ◽  
Parasitophorous Vacuole Membrane ◽  
Lewis Rat ◽  
Vacuole Membrane ◽  
Nlrp1 Inflammasome ◽  
Granule Proteins

AbstractThe Lewis rat is the only known warm-blooded animal that has sterile immunity toToxoplasma. Upon invasion of Lewis rat macrophagesToxoplasmarapidly activates the nucleotide-binding oligomerization domain, leucine-rich repeat and pyrin domain containing 1 (NLRP1) inflammasome resulting in interleukin (IL)-1β secretion and a form of cell death known as pyroptosis, which preventsToxoplasmareplication. Using a chemical mutagenesis screen we identifiedToxoplasmamutants that no longer induced pyroptosis. Whole genome sequencing led to the identification of threeToxoplasmaparasitophorous vacuole-localized dense granule proteins, GRA35, GRA42 and GRA43 that are individually required for inflammasome activation in Lewis rat macrophages. Macrophage infection with Δgra35, Δgra42, and Δgra43parasites leads to greatly reduced cell death and reduced IL-1β secretion. Lewis rat macrophage infected with parasites containing single, double or triple deletion of these GRAs showed similar levels of cell viability suggesting the three GRAs function in the same pathway that activates the inflammasome. Deletion ofGRA42andGRA43resulted in GRA35, and other GRAs, being retained inside the parasitophorous vacuole instead of being localized to the parasitophorous vacuole membrane.Toxoplasmadeficient in GRA35, GRA42 or GRA43 do not establish chronic infection in Lewis rats, but have reduced cyst number in parasite-susceptible F344 rats, in whichToxoplasmadoes not activate the NLRP1 inflammasome, revealing these GRAs determine parasitein vivofitness independent of their role in inflammasome activation. Overall, our data suggest thatToxoplasmadense granule proteins that localize to the parasitophorous vacuole membrane are novel mediators of host NLRP1 inflammasome activation.ImportanceInflammasomes are a major component of the innate immune system and responsible for detecting various microbial and environmental danger signals. The Lewis rat has sterile immunity toToxoplasmabecause upon invasion of Lewis rat macrophages the parasite rapidly activates the NLRP1 inflammasome resulting in cell death and parasite elimination. The work reported here identified thatToxoplasmaGRA35, GRA42 and GRA43 are required for activation of the Lewis rat NLRP1 inflammasome. GRA42 and GRA43 mediate the correct localization of other GRAs, including GRA35, to the parasitophorous vacuole membrane. In addition to their role in inflammasome activation, these three GRAs are also important for parasitein vivofitness in aToxoplasma-susceptible rat strain. Thus, these results give new insight into NLRP1 inflammasome activation byToxoplasmaeffectors and identified three GRAs that are required for pathogenesis of the parasite.

scholarly journals ThreeToxoplasma gondiiDense Granule Proteins Are Required for Induction of Lewis Rat Macrophage Pyroptosis

mBio ◽  
2019 ◽  
Vol 10 (1) ◽  
Author(s):  
Yifan Wang ◽  
Kimberly M. Cirelli ◽  
Patricio D. C. Barros ◽  
Lamba Omar Sangaré ◽  
Vincent Butty ◽  
...  
Keyword(s):  
Cell Death ◽  
Chronic Infection ◽  
Parasitophorous Vacuole ◽  
Dense Granule ◽  
Parasitophorous Vacuole Membrane ◽  
Lewis Rat ◽  
Vacuole Membrane ◽  
Granule Proteins ◽  
F344 Rats

ABSTRACTUpon invasion of Lewis rat macrophages,Toxoplasmarapidly induces programmed cell death (pyroptosis), which preventsToxoplasmareplication, possibly explaining the resistance of the Lewis rat toToxoplasma. Using a chemical mutagenesis screen, we identifiedToxoplasmamutants that no longer induced pyroptosis. Whole-genome sequencing led to the identification of threeToxoplasmaparasitophorous vacuole-localized dense granule proteins, GRA35, GRA42, and GRA43, that are individually required for induction of Lewis rat macrophage pyroptosis. Macrophage infection with Δgra35, Δgra42, and Δgra43parasites led to greatly reduced cell death rates and enhanced parasite replication. Lewis rat macrophages infected with parasites containing a single, double, or triple deletion of these GRAs showed similar levels of cell viability, suggesting that the three GRAs function in the same pathway. Deletion ofGRA42orGRA43resulted in GRA35 (and other GRAs) being retained inside the parasitophorous vacuole instead of being localized to the parasitophorous vacuole membrane. Despite having greatly enhanced replication in Lewis rat macrophagesin vitro, Δgra35, Δgra42, and Δgra43parasites did not establish a chronic infection in Lewis rats.Toxoplasmadid not induce F344 rat macrophage pyroptosis, but F344 rats infected with Δgra35, Δgra42, and Δgra43parasites had reduced cyst numbers. Thus, these GRAs determined parasitein vivofitness in F344 rats. Overall, our data suggest that these threeToxoplasmadense granule proteins play a critical role in establishing a chronic infectionin vivo, independently of their role in mediating macrophage pyroptosis, likely due to their importance in regulating protein localization to the parasitophorous vacuole membrane.IMPORTANCEInflammasomes are major components of the innate immune system and are responsible for detecting various microbial and environmental danger signals. Upon invasion of Lewis rat macrophages, the parasite rapidly activates the NLRP1 inflammasome, resulting in pyroptosis and elimination of the parasite’s replication niche. The work reported here revealed thatToxoplasmaGRA35, GRA42, and GRA43 are required for induction of Lewis rat macrophage pyroptosis. GRA42 and GRA43 mediate the correct localization of other GRAs, including GRA35, to the parasitophorous vacuole membrane. These three GRAs were also found to be important for parasitein vivofitness in aToxoplasma-susceptible rat strain, independently of their role in NLRP1 inflammasome activation, suggesting that they perform other important functions. Thus, this study identified three GRAs that mediate the induction of Lewis rat macrophage pyroptosis and are required for pathogenesis of the parasite.

Differential targeting of dense granule proteins in the parasitophorous vacuole ofToxoplasma gondii

Parasitology ◽  
1991 ◽  
Vol 103 (3) ◽  
pp. 321-329 ◽  
Author(s):  
A. Achbarou ◽  
O. Mercereau-Puijalon ◽  
A. Sadak ◽  
B. Fortier ◽  
M. A. Leriche ◽  
...  
Keyword(s):  
Molecular Weight ◽  
Parasitophorous Vacuole ◽  
Dense Granule ◽  
Parasitophorous Vacuole Membrane ◽  
Vacuole Membrane ◽  
Molecular Weights ◽  
Dense Granules ◽  
Metabolic Labelling ◽  
Infected Cells ◽  
Granule Proteins

The biosynthesis and fate of 4 different dense granule proteins ofToxoplasma gondiiwere studied with 3 monoclonal antibodies raised against tachyzoites and 1 polyclonal antibody raised against a recombinant protein. These proteins have the following molecular weights: 27 kDa (GRA 1), 28 kDa (GRA 2), 30 kDa (GRA 3) and 40 kDa (GRA 4). All four proteins were found in dense granules by immunoelectron microscopy; inT. gondii-infected cells, they were found in the vacuolar network but, in addition, GRA 3 was also detected on the parasitophorous vacuole membrane. Therefore, dense granule contents undergo differential targeting when exocytosed in the parasitophorous vacuole. Metabolic labelling and immunoprecipitation showed that GRA 2 and GRA 3 were processed from lower molecular weight precursors, and that GRA 2 and GRA 4 incorporated [3H] glucosamine and are thus likely to be glycosylated.

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

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

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

Molecular structure of a Toxoplasma gondii dense granule antigen (GRA 5) associated with the parasitophorous vacuole membrane

1993 ◽  
Vol 59 (1) ◽  
pp. 143-153 ◽  
Author(s):  
Laurence Lecordier ◽  
Corinne Mercier ◽  
Gérard Torpier ◽  
Béatrice Tourvieille ◽  
Francoise Darcy ◽  
...  
Keyword(s):  
Molecular Structure ◽  
Toxoplasma Gondii ◽  
Parasitophorous Vacuole ◽  
Dense Granule ◽  
Parasitophorous Vacuole Membrane ◽  
Vacuole Membrane

scholarly journals Toxoplasma gondiiParasitophorous Vacuole Membrane-Associated Dense Granule Proteins Orchestrate Chronic Infection and GRA12 Underpins Resistance to Host Gamma Interferon

mBio ◽  
2019 ◽  
Vol 10 (4) ◽  
Author(s):  
Barbara A. Fox ◽  
Rebekah B. Guevara ◽  
Leah M. Rommereim ◽  
Alejandra Falla ◽  
Valeria Bellini ◽  
...  
Keyword(s):  
Chronic Infection ◽  
Parasitophorous Vacuole ◽  
Dense Granule ◽  
Gamma Interferon ◽  
Type I ◽  
Type Ii ◽  
Content Type ◽  
Vacuole Membrane ◽  
Ifn Γ

ABSTRACTToxoplasma gondiievades host immunity to establish a chronic infection. Here, we assessed the role of parasitophorous vacuole (PV) membrane (PVM)- and intravacuolar network (IVN) membrane-localized dense granule (GRA) proteins in the development of acute and chronicToxoplasmainfection. Deletion of PVM-associated GRA3, GRA7, GRA8, and GRA14 or IVN membrane-associated GRA2, GRA9, and GRA12 in the low-virulence type II Prugniaud (Pru) strain induced severe defects in the development of chronic-stage cystsin vivowithout affecting the parasite growth rate or the ability to differentiate into cystsin vitro. Acute virulence of the PruΔgra2, PruΔgra3, and PruΔgra4mutants was reduced but not abolished. In contrast, the PruΔgra12mutant was avirulent in mice and PruΔgra12parasites failed to establish a chronic infection. High-virulence type I strain RHΔgra12parasites also exhibited a major defect in acute virulence. In gamma interferon (IFN-γ)-activated macrophages, type I RHΔgra12and type II PruΔgra12parasites resisted the coating of the PVM with host immunity-related GTPases as effectively as the parental type I RHΔku80and type II PruΔku80strains, respectively. Despite this resistance, Δgra12PVs ultimately succumbed to IFN-γ-activated host cell innate immunity. Our findings uncover a key role for GRA12 in mediating resistance to host IFN-γ and reveal that many other IVN membrane-associated GRA proteins, as well as PVM-localized GRA proteins, play important roles in establishing chronic infection.IMPORTANCEToxoplasma gondiicysts reactivate during immune deficiency and cause fatal encephalitis. Parasite molecules that coordinate the development of acute and chronic infection are poorly characterized. Here, we show that many intravacuolar network membrane and parasitophorous vacuole membrane-associated dense granule (GRA) proteins orchestrate the development of chronic cystsin vivo. A subset of these GRA proteins also modulate acute virulence, and one protein that associates with the intravacuolar network membranes, namely GRA12, was identified as a major virulence factor required for parasite resistance to host gamma interferon (IFN-γ). Our results revealed that many parasitophorous vacuole membrane and intravacuolar network membrane-associated GRA proteins are essential for successful chronic infection.

scholarly journals In Vivo Biotinylation of the Toxoplasma Parasitophorous Vacuole Reveals Novel Dense Granule Proteins Important for Parasite Growth and Pathogenesis

mBio ◽  
2016 ◽  
Vol 7 (4) ◽  
Author(s):  
Santhosh M. Nadipuram ◽  
Elliot W. Kim ◽  
Ajay A. Vashisht ◽  
Andrew H. Lin ◽  
Hannah N. Bell ◽  
...  
Keyword(s):  
Host Cell ◽  
Gene Knockout ◽  
Affinity Purification ◽  
Parasitophorous Vacuole ◽  
Dense Granule ◽  
Parasite Growth ◽  
Host Cells ◽  
Biotin Ligase ◽  
Granule Proteins

ABSTRACT Toxoplasma gondii is an obligate intracellular parasite that invades host cells and replicates within a unique parasitophorous vacuole. To maintain this intracellular niche, the parasite secretes an array of dense granule proteins (GRAs) into the nascent parasitophorous vacuole. These GRAs are believed to play key roles in vacuolar remodeling, nutrient uptake, and immune evasion while the parasite is replicating within the host cell. Despite the central role of GRAs in the Toxoplasma life cycle, only a subset of these proteins have been identified, and many of their roles have not been fully elucidated. In this report, we utilize the promiscuous biotin ligase BirA* to biotinylate GRA proteins secreted into the vacuole and then identify those proteins by affinity purification and mass spectrometry. Using GRA-BirA* fusion proteins as bait, we have identified a large number of known and candidate GRAs and verified localization of 13 novel GRA proteins by endogenous gene tagging. We proceeded to functionally characterize three related GRAs from this group (GRA38, GRA39, and GRA40) by gene knockout. While Δ gra38 and Δ gra40 parasites showed no altered phenotype, disruption of GRA39 results in slow-growing parasites that contain striking lipid deposits in the parasitophorous vacuole, suggesting a role in lipid regulation that is important for parasite growth. In addition, parasites lacking GRA39 showed dramatically reduced virulence and a lower tissue cyst burden in vivo . Together, the findings from this work reveal a partial vacuolar proteome of T. gondii and identify a novel GRA that plays a key role in parasite replication and pathogenesis. IMPORTANCE Most intracellular pathogens reside inside a membrane-bound vacuole within their host cell that is extensively modified by the pathogen to optimize intracellular growth and avoid host defenses. In Toxoplasma , this vacuole is modified by a host of secretory GRA proteins, many of which remain unidentified. Here we demonstrate that in vivo biotinylation of proximal and interacting proteins using the promiscuous biotin ligase BirA* is a powerful approach to rapidly identify vacuolar GRA proteins. We further demonstrate that one factor identified by this approach, GRA39, plays an important role in the ability of the parasite to replicate within its host cell and cause disease.

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.

TgGRA23, a novel Toxoplasma gondii dense granule protein associated with the parasitophorous vacuole membrane and intravacuolar network

2013 ◽  
Vol 62 (4) ◽  
pp. 372-379 ◽  
Author(s):  
Tatsunori Masatani ◽  
Tomohide Matsuo ◽  
Tetsuya Tanaka ◽  
Mohamad Alaa Terkawi ◽  
Eung-Goo Lee ◽  
...  
Keyword(s):  
Toxoplasma Gondii ◽  
Parasitophorous Vacuole ◽  
Dense Granule ◽  
Parasitophorous Vacuole Membrane ◽  
Vacuole Membrane ◽  
Granule Protein
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