scholarly journals Translocation of Dense Granule Effectors across the Parasitophorous Vacuole Membrane in Toxoplasma-Infected Cells Requires the Activity of ROP17, a Rhoptry Protein Kinase

mSphere ◽  
2019 ◽  
Vol 4 (4) ◽  
Author(s):  
Michael W. Panas ◽  
Abel Ferrel ◽  
Adit Naor ◽  
Elizabeth Tenborg ◽  
Hernan A. Lorenzi ◽  
...  
Keyword(s):  
Protein Kinase ◽  
Host Cell ◽  
Parasitophorous Vacuole ◽  
Dense Granule ◽  
Effector Proteins ◽  
Large Set ◽  
Gene Encoding ◽  
Cell Functions ◽  
Infected Cells ◽  
Rhoptry Protein

ABSTRACT Toxoplasma gondii tachyzoites co-opt host cell functions through introduction of a large set of rhoptry- and dense granule-derived effector proteins. These effectors reach the host cytosol through different means: direct injection for rhoptry effectors and translocation across the parasitophorous vacuolar membrane (PVM) for dense granule (GRA) effectors. The machinery that translocates these GRA effectors has recently been partially elucidated, revealing three components, MYR1, MYR2, and MYR3. To determine whether other proteins might be involved, we returned to a library of mutants defective in GRA translocation and selected one with a partial defect, suggesting it might be in a gene encoding a new component of the machinery. Surprisingly, whole-genome sequencing revealed a missense mutation in a gene encoding a known rhoptry protein, a serine/threonine protein kinase known as ROP17. ROP17 resides on the host cytosol side of the PVM in infected cells and has previously been known for its activity in phosphorylating and thereby inactivating host immunity-related GTPases. Here, we show that null or catalytically dead mutants of ROP17 are defective in GRA translocation across the PVM but that translocation can be rescued “in trans” by ROP17 delivered by other tachyzoites infecting the same host cell. This strongly argues that ROP17’s role in regulating GRA translocation is carried out on the host cytosolic side of the PVM, not within the parasites or lumen of the parasitophorous vacuole. This represents an entirely new way in which the different secretory compartments of Toxoplasma tachyzoites collaborate to modulate the host-parasite interaction. IMPORTANCE When Toxoplasma infects a cell, it establishes a protective parasitophorous vacuole surrounding it. While this vacuole provides protection, it also serves as a barrier to the export of parasite effector proteins that impact and take control of the host cell. Our discovery here that the parasite rhoptry protein ROP17 is necessary for export of these effector proteins provides a distinct, novel function for ROP17 apart from its known role in protecting the vacuole. This will enable future research into ways in which we can prevent the export of effector proteins, thereby preventing Toxoplasma from productively infecting its animal and human hosts.

scholarly journals Translocation of dense granule effectors across the parasitophorous vacuole membrane inToxoplasma-infected cells requires the activity of ROP17, a rhoptry protein kinase

2019 ◽  
Author(s):  
Michael W. Panas ◽  
Abel Ferrel ◽  
Adit Naor ◽  
Elizabeth Tenborg ◽  
Hernan A. Lorenzi ◽  
...  
Keyword(s):  
Protein Kinase ◽  
Host Cell ◽  
Parasitophorous Vacuole ◽  
Dense Granule ◽  
Effector Proteins ◽  
Large Set ◽  
Gene Encoding ◽  
Cell Functions ◽  
Infected Cells ◽  
Rhoptry Protein

AbstractToxoplasma gondiitachyzoites co-opt host cell functions through introduction of a large set of rhoptry- and dense granule-derived effector proteins. These effectors reach the host cytosol through different means: direct injection for rhoptry effectors and translocation across the parasitophorous vacuolar membrane (PVM) for dense granule (GRA) effectors. The machinery that translocates these GRA effectors has recently been partially elucidated, revealing 3 components, MYR1, MYR2 and MYR3. To determine if other proteins might be involved, we returned to a library of mutants defective in GRA translocation and selected one with a partial defect, suggesting it might be in a gene encoding a new component of the machinery. Surprisingly, whole-genome sequencing revealed a missense mutation in a gene encoding a known rhoptry protein, a serine/threonine protein kinase known as ROP17. ROP17 resides on the host-cytosol side of the PVM in infected cells and has previously been known for its activity in phosphorylating and, thereby, inactivating host immunity-related GTPases. Here, we show that null or catalytically dead mutants of ROP17 are defective in GRA translocation across the PVM, but that translocation can be rescued “intrans"by ROP17 delivered by other tachyzoites infecting the same host cell. This strongly argues that ROP17’s role in regulating GRA translocation is carried out on the host-cytosolic side of the PVM, not within the parasites or lumen of the parasitophorous vacuole. This represents an entirely new way in which the different secretory compartments ofToxoplasmatachyzoites collaborate to modulate the host-parasite interaction.ImportanceWhenToxoplasmainfects a cell it establishes a protective parasitophorous vacuole surrounding it. While this vacuole provides protection, it also serves as a barrier to the export of parasite effector proteins that impact and take control of the host cell. Our discovery here that the parasite rhoptry protein, ROP17, is necessary for export of these effector proteins provides a distinct, novel function for ROP17 apart from its known role in protecting the vacuole. This will enable future research into ways in which we can prevent the export of effector proteins thereby preventingToxoplasmafrom productively infecting its animal and human hosts.

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

mSphere ◽  
2020 ◽  
Vol 5 (1) ◽  
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.

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 Co-immunoprecipitation with MYR1 identifies three additional proteins within the Toxoplasma parasitophorous vacuole required for translocation of dense granule effectors into host cells

2019 ◽  
Author(s):  
Alicja M. Cygan ◽  
Terence C. Theisen ◽  
Alma G. Mendoza ◽  
Nicole D. Marino ◽  
Michael W. Panas ◽  
...  
Keyword(s):  
Drug Targets ◽  
Protein Translocation ◽  
Affinity Purification ◽  
Parasitophorous Vacuole ◽  
Dense Granule ◽  
Effector Proteins ◽  
Host Cells ◽  
Cyclin E1 ◽  
Infected Cells ◽  
Potential Drug Targets

AbstractToxoplasma 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.ImportanceToxoplasma 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.

scholarly journals Differential Impacts on Host Transcription by ROP and GRA Effectors from the Intracellular Parasite Toxoplasma gondii

2020 ◽  
Author(s):  
Suchita Rastogi ◽  
Yuan Xue ◽  
Stephen R. Quake ◽  
John C. Boothroyd
Keyword(s):  
Toxoplasma Gondii ◽  
Host Cell ◽  
Cell Biology ◽  
Parasitophorous Vacuole ◽  
Dense Granule ◽  
Effector Proteins ◽  
Transcriptomic Analysis ◽  
Host Cells ◽  
Intracellular Parasite ◽  
I Cells

ABSTRACTThe intracellular parasite Toxoplasma gondii employs a vast array of effector proteins from the rhoptry and dense granule organelles to modulate host cell biology; these effectors are known as ROPs and GRAs, respectively. To examine the individual impacts of ROPs and GRAs on host gene expression, we developed a robust, novel protocol to enrich for ultra-pure populations of a naturally occurring and reproducible population of host cells called uninfected-injected (U-I) cells, which Toxoplasma injects with ROPs but subsequently fails to invade. We then performed single cell transcriptomic analysis at 1-3 hours post-infection on U-I cells (as well as on uninfected and infected controls) arising from infection with either wild type parasites or parasites lacking the MYR1 protein, which is required for soluble GRAs to cross the parasitophorous vacuole membrane (PVM) and reach the host cell cytosol. Based on comparisons of infected and U-I cells, the host’s earliest response to infection appears to be driven primarily by the injected ROPs, which appear to induce immune and cellular stress pathways. These ROP-dependent pro-inflammatory signatures appear to be counteracted by at least some of the MYR1-dependent GRAs and may be enhanced by the MYR-independent GRAs, (which are found embedded within the PVM). Finally, signatures detected in uninfected bystander cells from the infected monolayers suggests that MYR1-dependent paracrine effects also counteract inflammatory ROP-dependent processes.IMPORTANCEThis work performs the first transcriptomic analysis of U-I cells, captures the earliest stage of a host cell’s interaction with Toxoplasma gondii, and dissects the effects of individual classes of parasite effectors on host cell biology.

scholarly journals Functional Analysis of the Salmonella Pathogenicity Island 2-Mediated Inhibition of Antigen Presentation in Dendritic Cells

2008 ◽  
Vol 76 (11) ◽  
pp. 4924-4933 ◽  
Author(s):  
Serkan Halici ◽  
Sebastian F. Zenk ◽  
Jonathan Jantsch ◽  
Michael Hensel
Keyword(s):  
Dendritic Cells ◽  
Antigen Presentation ◽  
Host Cell ◽  
Pathogenicity Island ◽  
Effector Proteins ◽  
Dependent Manner ◽  
Murine Bone Marrow ◽  
Mhc Ii ◽  
Cell Functions ◽  
Infected Cells

ABSTRACTSalmonella entericais a facultative intracellular pathogen that is able to modify host cell functions by means of effector proteins translocated by the type III secretion system (T3SS) encoded bySalmonellaPathogenicity Island 2 (SPI2). The SPI2-T3SS is also active inSalmonellaafter uptake by murine bone marrow-derived dendritic cells (BM-DC). We have previously shown that intracellularSalmonellainterfere with the ability of BM-DC to stimulate antigen-dependent T-cell proliferation in an SPI2-T3SS-dependent manner. We observed thatSalmonella-mediated inhibition of antigen presentation could be restored by external addition of peptides on major histocompatibility complex class II (MHC-II). The processing of antigens inSalmonella-infected cells was not altered; however, the intracellular loading of peptides on MHC-II was reduced as a function of the SPI2-T3SS. We set out to identify the effector proteins of the SPI2-T3SS involved in inhibition of antigen presentation and demonstrated that effector proteins SifA, SspH2, SlrP, PipB2, and SopD2 were equally important for the interference with antigen presentation, whereas SseF and SseG contributed to a lesser extent to this phenotype. These observations indicate the presence of a host cell-specific virulence function of a novel subset of SPI2-effector proteins.

scholarly journals MYR1-Dependent Effectors Are the Major Drivers of a Host Cell’s Early Response to Toxoplasma , Including Counteracting MYR1-Independent Effects

mBio ◽  
2018 ◽  
Vol 9 (2) ◽  
Author(s):  
Adit Naor ◽  
Michael W. Panas ◽  
Nicole Marino ◽  
Michael J. Coffey ◽  
Christopher J. Tonkin ◽  
...  
Keyword(s):  
Toxoplasma Gondii ◽  
Host Cell ◽  
Expression Profiles ◽  
Parasitophorous Vacuole ◽  
Effector Proteins ◽  
Gene Set Enrichment Analysis ◽  
Host Cells ◽  
Content Type ◽  
Cell Functions ◽  
The Impact

ABSTRACT The obligate intracellular parasite Toxoplasma gondii controls its host cell from within the parasitophorous vacuole (PV) by using a number of diverse effector proteins, a subset of which require the aspartyl protease 5 enzyme (ASP5) and/or the recently discovered MYR1 protein to cross the PV membrane. To examine the impact these effectors have in the context of the entirety of the host response to Toxoplasma , we used RNA-Seq to analyze the transcriptome expression profiles of human foreskin fibroblasts infected with wild-type RH (RH-WT), RHΔ myr1 , and RHΔ asp5 tachyzoites. Interestingly, the majority of the differentially regulated genes responding to Toxoplasma infection are MYR1 dependent. A subset of MYR1 responses were ASP5 independent, and MYR1 function did not require ASP5 cleavage, suggesting the export of some effectors requires only MYR1. Gene set enrichment analysis of MYR1-dependent host responses suggests an upregulation of E2F transcription factors and the cell cycle and a downregulation related to interferon signaling, among numerous others. Most surprisingly, “hidden” responses arising in RHΔ myr1 - but not RH-WT-infected host cells indicate counterbalancing actions of MYR1-dependent and -independent activities. The host genes and gene sets revealed here to be MYR1 dependent provide new insight into the parasite’s ability to co-opt host cell functions. IMPORTANCE Toxoplasma gondii is unique in its ability to successfully invade and replicate in a broad range of host species and cells within those hosts. The complex interplay of effector proteins exported by Toxoplasma is key to its success in co-opting the host cell to create a favorable replicative niche. Here we show that a majority of the transcriptomic effects in tachyzoite-infected cells depend on the activity of a novel translocation system involving MYR1 and that the effectors delivered by this system are part of an intricate interplay of activators and suppressors. Removal of all MYR1-dependent effectors reveals previously unknown activities that are masked or hidden by the action of these proteins.

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 Use of a fluorescent probe to assess the activities of candidate agents against intracellular forms of Encephalitozoon microsporidia.

1997 ◽  
Vol 41 (2) ◽  
pp. 337-344 ◽  
Author(s):  
G J Leitch ◽  
M Scanlon ◽  
A Shaw ◽  
G S Visvesvara ◽  
S Wallace
Keyword(s):  
Host Cell ◽  
Fluorescent Probe ◽  
Cultured Cells ◽  
Morphological Changes ◽  
Parasitophorous Vacuole ◽  
Detectable Effect ◽  
Drug Induced ◽  
Encephalitozoon Intestinalis ◽  
Infected Cells

Microsporidia are obligate intracellular protozoan parasites. Three species of the genus Encephalitozoon are among the microsporidia that infect immunodeficient humans. These species, Encephalitozoon cuniculi, Encephalitozoon hellem, and Encephalitozoon intestinalis, all develop in a parasitophorous vacuole within a host cell. The present study describes a method that uses the fluorescent probe calcein and confocal microscopy to detect drug-induced effects in Encephalitozoon-infected green monkey kidney cells. The effects were as follows: (i) changes in parasite organization within the parasitophorous vacuole; (ii) swelling and gross morphological changes of parasite developing stages in situ; (iii) killing of developing parasite stages in situ, detected by their uptake of the fluorescent probe; and (iv) reduction in the viability of the host cell population, assessed by the loss of the probe. Verapamil and itraconazole were used to increase the vital dye loading by both uninfected and infected cells. Agents with known antimicrosporidial activity, albendazole and fumagillin, caused all three types of parasite changes at concentrations that had no detectable effect on host cell viability. The effective doses of albendazole and fumagillin that caused swelling and disorganization of parasite developing stages were 5 x 10(-7) and 10(-6) M respectively. Killing of developing stages was detected at 10-fold-higher concentrations for these agents and at 10(-5) M for metronidazole. This method can be used to screen candidate antimicrosporidial agents in infected cultured cells.

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