The Rhoptry Pseudokinase ROP54 Modulates Toxoplasma gondii Virulence and Host GBP2 Loading

ABSTRACT The interactions between intracellular microbes and their host cells can lead to the discovery of novel drug targets. During Toxoplasma infections, host cells express an array of immunity-related GTPases (IRGs) and guanylate binding proteins (GBPs) that load onto the parasite-containing vacuole to clear the parasite. To counter this mechanism, the parasite secretes effector proteins that traffic to the vacuole to disarm the immunity-related loading proteins and evade the immune response. While the interplay between host IRGs and Toxoplasma effector proteins is well understood, little is known about how Toxoplasma neutralizes the GBP response. We describe here a T. gondii pseudokinase effector, ROP54, that localizes to the vacuole upon invasion and is critical for parasite virulence. Toxoplasma vacuoles lacking ROP54 display an increased loading of the host immune factor GBP2, but not IRGb6, indicating that ROP54 plays a distinct role in immune evasion. Toxoplasma gondii uses unique secretory organelles called rhoptries to inject an array of effector proteins into the host cytoplasm that hijack host cell functions. We have discovered a novel rhoptry pseudokinase effector, ROP54, which is injected into the host cell upon invasion and traffics to the cytoplasmic face of the parasitophorous vacuole membrane (PVM). Disruption of ROP54 in a type II strain of T. gondii does not affect growth in vitro but results in a 100-fold decrease in virulence in vivo, suggesting that ROP54 modulates some aspect of the host immune response. We show that parasites lacking ROP54 are more susceptible to macrophage-dependent clearance, further suggesting that ROP54 is involved in evasion of innate immunity. To determine how ROP54 modulates parasite virulence, we examined the loading of two known innate immune effectors, immunity-related GTPase b6 (IRGb6) and guanylate binding protein 2 (GBP2), in wild-type and ∆rop54II mutant parasites. While no difference in IRGb6 loading was seen, we observed a substantial increase in GBP2 loading on the parasitophorous vacuole (PV) of ROP54-disrupted parasites. These results demonstrate that ROP54 is a novel rhoptry effector protein that promotes Toxoplasma infections by modulating GBP2 loading onto parasite-containing vacuoles. IMPORTANCE The interactions between intracellular microbes and their host cells can lead to the discovery of novel drug targets. During Toxoplasma infections, host cells express an array of immunity-related GTPases (IRGs) and guanylate binding proteins (GBPs) that load onto the parasite-containing vacuole to clear the parasite. To counter this mechanism, the parasite secretes effector proteins that traffic to the vacuole to disarm the immunity-related loading proteins and evade the immune response. While the interplay between host IRGs and Toxoplasma effector proteins is well understood, little is known about how Toxoplasma neutralizes the GBP response. We describe here a T. gondii pseudokinase effector, ROP54, that localizes to the vacuole upon invasion and is critical for parasite virulence. Toxoplasma vacuoles lacking ROP54 display an increased loading of the host immune factor GBP2, but not IRGb6, indicating that ROP54 plays a distinct role in immune evasion.

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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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Alveolar Macrophages and Neutrophils Are the Primary Reservoirs for Legionella pneumophila and Mediate Cytosolic Surveillance of Type IV Secretion

Infection and Immunity ◽

10.1128/iai.01891-14 ◽

2014 ◽

Vol 82 (10) ◽

pp. 4325-4336 ◽

Cited By ~ 31

Author(s):

Alan M. Copenhaver ◽

Cierra N. Casson ◽

Hieu T. Nguyen ◽

Thomas C. Fung ◽

Matthew M. Duda ◽

...

Keyword(s):

Immune Response ◽

Alveolar Macrophages ◽

Legionella Pneumophila ◽

Pulmonary Infection ◽

Severe Pneumonia ◽

Effector Proteins ◽

Type Iv Secretion ◽

Host Cells ◽

Content Type ◽

Type Iv

ABSTRACTLegionella pneumophila, an intracellular pathogen responsible for the severe pneumonia Legionnaires' disease, uses itsdot/icm-encoded type IV secretion system (T4SS) to translocate effector proteins that promote its survival and replication into the host cell cytosol. However, by introducing bacterial products into the host cytosol,L. pneumophilaalso activates cytosolic immunosurveillance pathways, thereby triggering robust proinflammatory responses that mediate the control of infection. Thus, the pulmonary cell types thatL. pneumophilainfects not only may act as an intracellular niche that facilitates its pathogenesis but also may contribute to the immune response againstL. pneumophila. The identity of these host cells remains poorly understood. Here, we developed a strain ofL. pneumophilaproducing a fusion protein consisting of β-lactamase fused to the T4SS-translocated effector RalF, which allowed us to track cells injected by the T4SS. Our data reveal that alveolar macrophages and neutrophils both are the primary recipients of T4SS-translocated effectors and harbor viableL. pneumophiladuring pulmonary infection of mice. Moreover, both alveolar macrophages and neutrophils from infected mice produced tumor necrosis factor and interleukin-1α in response to T4SS-sufficient, but not T4SS-deficient,L. pneumophila. Collectively, our data suggest that alveolar macrophages and neutrophils are both an intracellular reservoir forL. pneumophilaand a source of proinflammatory cytokines that contribute to the host immune response againstL. pneumophiladuring pulmonary infection.

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

10.1101/867788 ◽

2019 ◽

Cited By ~ 1

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.

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

mBio ◽

10.1128/mbio.00182-20 ◽

2020 ◽

Vol 11 (3) ◽

Cited By ~ 2

Author(s):

Suchita Rastogi ◽

Yuan Xue ◽

Stephen R. Quake ◽

John C. Boothroyd

Keyword(s):

Toxoplasma Gondii ◽

Host Cell ◽

Cell Biology ◽

Parasitophorous Vacuole ◽

Effector Proteins ◽

Transcriptomic Analysis ◽

Host Cells ◽

Intracellular Parasite ◽

Content Type ◽

I Cells

ABSTRACT The 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 ultrapure 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 to 3 h postinfection 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 proinflammatory 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 suggest that MYR1-dependent paracrine effects also counteract inflammatory ROP-dependent processes. IMPORTANCE This work performs 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.

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Using a Genome-Scale Metabolic Model of Enterococcus faecalis V583 To Assess Amino Acid Uptake and Its Impact on Central Metabolism

Applied and Environmental Microbiology ◽

10.1128/aem.03279-14 ◽

2014 ◽

Vol 81 (5) ◽

pp. 1622-1633 ◽

Cited By ~ 18

Author(s):

Nadine Veith ◽

Margrete Solheim ◽

Koen W. A. van Grinsven ◽

Brett G. Olivier ◽

Jennifer Levering ◽

...

Keyword(s):

Amino Acids ◽

Amino Acid ◽

Metabolic Network ◽

Drug Targets ◽

Metabolic Model ◽

Content Type ◽

A Genome ◽

Genome Scale ◽

Novel Drug ◽

Novel Drug Targets

ABSTRACTIncreasing antibiotic resistance in pathogenic bacteria necessitates the development of new medication strategies. Interfering with the metabolic network of the pathogen can provide novel drug targets but simultaneously requires a deeper and more detailed organism-specific understanding of the metabolism, which is often surprisingly sparse. In light of this, we reconstructed a genome-scale metabolic model of the pathogenEnterococcus faecalisV583. The manually curated metabolic network comprises 642 metabolites and 706 reactions. We experimentally determined metabolic profiles ofE. faecalisgrown in chemically defined medium in an anaerobic chemostat setup at different dilution rates and calculated the net uptake and product fluxes to constrain the model. We computed growth-associated energy and maintenance parameters and studied flux distributions through the metabolic network. Amino acid auxotrophies were identified experimentally for model validation and revealed seven essential amino acids. In addition, the important metabolic hub of glutamine/glutamate was altered by constructing a glutamine synthetase knockout mutant. The metabolic profile showed a slight shift in the fermentation pattern toward ethanol production and increased uptake rates of multiple amino acids, especiallyl-glutamine andl-glutamate. The model was used to understand the altered flux distributions in the mutant and provided an explanation for the experimentally observed redirection of the metabolic flux. We further highlighted the importance of gene-regulatory effects on the redirection of the metabolic fluxes upon perturbation. The genome-scale metabolic model presented here includes gene-protein-reaction associations, allowing a further use for biotechnological applications, for studying essential genes, proteins, or reactions, and the search for novel drug targets.

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Characterization of the erythrocyte GTPase Rac1 in relation to Plasmodium falciparum invasion

Scientific Reports ◽

10.1038/s41598-020-79052-0 ◽

2020 ◽

Vol 10 (1) ◽

Author(s):

Silvio Paone ◽

Sarah D’Alessandro ◽

Silvia Parapini ◽

Francesco Celani ◽

Valentina Tirelli ◽

...

Keyword(s):

Plasmodium Falciparum ◽

Host Cell ◽

Drug Targets ◽

Parasitophorous Vacuole ◽

Erythrocyte Invasion ◽

Inhibitory Compounds ◽

Novel Drug ◽

Novel Drug Targets

AbstractMalaria is still a devastating disease with 228 million cases globally and 405,000 lethal outcomes in 2018, mainly in children under five years of age. The threat of emerging malaria strains resistant to currently available drugs has made the search for novel drug targets compelling. The process by which Plasmodium falciparum parasites invade the host cell has been widely studied, but only a few erythrocyte proteins involved in this process have been identified so far. The erythrocyte protein Rac1 is a GTPase that plays an important role in host cell invasion by many intracellular pathogens. Here we show that Rac1 is recruited in proximity to the site of parasite entry during P. falciparum invasion process and that subsequently localizes to the parasitophorous vacuole membrane. We also suggest that this GTPase may be involved in erythrocyte invasion by P. falciparum, by testing the effect of specific Rac1 inhibitory compounds. Finally, we suggest a secondary role of the erythrocyte GTPase also in parasite intracellular development. We here characterize a new erythrocyte protein potentially involved in P. falciparum invasion of the host cell and propose the human GTPase Rac1 as a novel and promising antimalarial drug target.

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

mBio ◽

10.1128/mbio.02401-17 ◽

2018 ◽

Vol 9 (2) ◽

Cited By ~ 27

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.

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