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

Cygan, Alicja M.; Theisen, Terence C.; Mendoza, Alma G.; Marino, Nicole D.; Panas, Michael W.; Boothroyd, John C.

doi:10.1128/msphere.00858-19

Cited by 61 publications

(60 citation statements)

References 52 publications

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“…One crucial linchpin of gene expression that the parasite targets is c-Myc, a central regulator capable of controlling more than 15% of the genes in the mammalian genome (6). Previous genetic screens showed that upregulation of c-Myc is dependent on a number of Toxoplasma genes, including four dubbed "Myc regulatory" genes (MYRs) (7). In all cases, however, these MYR genes were shown to be necessary for the translocation of many GRA effectors across the PVM, rather than encoding the protein directly controlling c-Myc upregulation.…”

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confidence: 99%

Toxoplasma Uses GRA16 To Upregulate Host c-Myc

Panas

Boothroyd

2020

mSphere

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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.

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Toxoplasma Uses GRA16 To Upregulate Host c-Myc

Panas

Boothroyd

2020

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“…6). Interestingly, as we were studying the interactome of GRA44, we learned of ongoing work in John C. Boothroyd's lab showing a physical and functional interaction between MYR1 and GRA44 (34). MYR1 was initially identified through a forward genetic screen for Toxoplasma mutants unable to activate host c-Myc and translocate effectors, such as GRA16 and GRA24 (22).…”

Section: Discussionmentioning

confidence: 99%

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

Blakely

Holmes

Arrizabalaga

2020

mSphere

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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.

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“…A fourth protein, the secreted parasitic kinase ROP17, is also required for translocation [ 108 ]; ROP17 is likely to phosphorylate one or more of the components of the MYR translocon and thus potentially energize translocation. Recently, three additional proteins, MYR4, GRA44, and, GRA45, were also identified as essential for the export of GRA effectors in Toxoplasma [ 109 ]. GRA44 is a putative phosphatase, while GRA45, predicted to contain a small heat shock protein domain and a transthyretin-like domain, is critical in preventing other GRA effectors from aggregating and thus functions as a chaperone (Wang Y. et al, 2019. bioRxiv doi:10.1101/867705) ( Figure 10 , Figure S7 ).…”

Section: Secretion Across the Parasitophorous Vacuole In Other Apimentioning

confidence: 99%

“…All MYR translocon proteins (MYR1-4, ROP17, GRA44, and GRA45) identified in Toxoplasma gondii have orthologues in other apicomplexan parasites, such as Neospora caninum and Hammondia hammondi , although the levels of sequence divergence suggest rapid evolutionary pressure [ 10 , 106 , 109 ]. However, no homologues of MYR proteins have been identified in Sarcocystis (also an Eucoccidioridia) or the more distantly related Plasmodium; thus, MYR proteins appear somehow restricted to a subset of Coccidia, perhaps as an adaptation to the rapid evolution of their specific effectors and life cycle (host cell).…”

Section: Parasitic Vacuolar Secretion Pathways As Drug Targetsmentioning

confidence: 99%

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Crossing the Vacuolar Rubicon: Structural Insights into Effector Protein Trafficking in Apicomplexan Parasites

Egea

2020

Microorganisms

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Apicomplexans form a large phylum of parasitic protozoa, including the genera Plasmodium, Toxoplasma, and Cryptosporidium, the causative agents of malaria, toxoplasmosis, and cryptosporidiosis, respectively. They cause diseases not only in humans but also in animals, with dramatic consequences in agriculture. Most apicomplexans are vacuole-dwelling and obligate intracellular parasites; as they invade the host cell, they become encased in a parasitophorous vacuole (PV) derived from the host cellular membrane. This creates a parasite–host interface that acts as a protective barrier but also constitutes an obstacle through which the pathogen must import nutrients, eliminate wastes, and eventually break free upon egress. Completion of the parasitic life cycle requires intense remodeling of the infected host cell. Host cell subversion is mediated by a subset of essential effector parasitic proteins and virulence factors actively trafficked across the PV membrane. In the malaria parasite Plasmodium, a unique and highly specialized ATP-driven vacuolar secretion system, the Plasmodium translocon of exported proteins (PTEX), transports effector proteins across the vacuolar membrane. Its core is composed of the three essential proteins EXP2, PTEX150, and HSP101, and is supplemented by the two auxiliary proteins TRX2 and PTEX88. Many but not all secreted malarial effector proteins contain a vacuolar trafficking signal or Plasmodium export element (PEXEL) that requires processing by an endoplasmic reticulum protease, plasmepsin V, for proper export. Because vacuolar parasitic protein export is essential to parasite survival and virulence, this pathway is a promising target for the development of novel antimalarial therapeutics. This review summarizes the current state of structural and mechanistic knowledge on the Plasmodium parasitic vacuolar secretion and effector trafficking pathway, describing its most salient features and discussing the existing differences and commonalities with the vacuolar effector translocation MYR machinery recently described in Toxoplasma and other apicomplexans of significance to medical and veterinary sciences.

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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

Cited by 61 publications

References 52 publications

Toxoplasma Uses GRA16 To Upregulate Host c-Myc

Toxoplasma Uses GRA16 To Upregulate Host c-Myc

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

Crossing the Vacuolar Rubicon: Structural Insights into Effector Protein Trafficking in Apicomplexan Parasites

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