ROP16-Mediated Activation of STAT6 Suppresses Host Cell Reactive Oxygen Species Production, Facilitating Type III Toxoplasma gondii Growth and Survival

ABSTRACT Polymorphic effector proteins determine the susceptibility of Toxoplasma gondii strains to IFN-γ-mediated clearance mechanisms deployed by murine host cells. However, less is known about the influence of these polymorphic effector proteins on IFN-γ-independent clearance mechanisms. Here, we show that deletion of one such polymorphic effector protein, ROP16, from a type III background leads to a defect in parasite growth and survival in unstimulated human fibroblasts and murine macrophages. Rescue of these defects requires a ROP16 with a functional kinase domain and the ability to activate a specific family of host cell transcription factors (STAT3, 5a, and 6). The growth and survival defects correlate with an accumulation of host cell reactive oxygen species (ROS) and are prevented by treatment with an ROS inhibitor. Exogenous activation of STAT3 and 6 suppresses host cell ROS production during infection with ROP16-deficient parasites and depletion of STAT6, but not STAT3 or 5a, causes an accumulation of ROS in cells infected with wild-type parasites. Pharmacological inhibition of NOX2 and mitochondrially derived ROS also rescues growth and survival of ROP16-deficient parasites. Collectively, these findings reveal an IFN-γ-independent mechanism of parasite restriction in human cells that is subverted by injection of ROP16 by type III parasites. IMPORTANCE Toxoplasma gondii is an obligate intracellular parasite that infects up to one-third of the world’s population. Control of the parasite is largely accomplished by IFN-γ-dependent mechanisms that stimulate innate and adaptive immune responses. Parasite suppression of IFN-γ-stimulated responses has been linked to proteins that the parasite secretes into its host cell. These secreted proteins vary by T. gondii strain and determine strain-specific lethality in mice. How these strain-specific polymorphic effector proteins affect IFN-γ-independent parasite control mechanisms in human and murine cells is not well known. This study shows that one such secreted protein, ROP16, enables efficient parasite growth and survival by suppressing IFN-γ-independent production of ROS by human and mouse cells.

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AToxoplasmaProlyl Hydroxylase Mediates Oxygen Stress Responses by Regulating Translation Elongation

mBio ◽

10.1128/mbio.00234-19 ◽

2019 ◽

Vol 10 (2) ◽

Cited By ~ 3

Author(s):

Celia Florimond ◽

Charlotte Cordonnier ◽

Rahil Taujale ◽

Hanke van der Wel ◽

Natarajan Kannan ◽

...

Keyword(s):

Reactive Oxygen Species ◽

Protein Synthesis ◽

Toxoplasma Gondii ◽

Oxygen Sensing ◽

Reactive Oxygen ◽

Protozoan Parasite ◽

Cellular Responses ◽

Parasite Growth ◽

Oxygen Species ◽

Prolyl Hydroxylases

ABSTRACTAs the protozoan parasiteToxoplasma gondiidisseminates through its host, it responds to environmental changes by altering its gene expression, metabolism, and other processes. Oxygen is one variable environmental factor, and properly adapting to changes in oxygen levels is critical to prevent the accumulation of reactive oxygen species and other cytotoxic factors. Thus, oxygen-sensing proteins are important, and among these, 2-oxoglutarate-dependent prolyl hydroxylases are highly conserved throughout evolution.Toxoplasmaexpresses two such enzymes, TgPHYa, which regulates the SCF-ubiquitin ligase complex, and TgPHYb. To characterize TgPHYb, we created aToxoplasmastrain that conditionally expresses TgPHYb and report that TgPHYb is required for optimal parasite growth under normal growth conditions. However, exposing TgPHYb-depleted parasites to extracellular stress leads to severe decreases in parasite invasion, which is likely due to decreased abundance of parasite adhesins. Adhesin protein abundance is reduced in TgPHYb-depleted parasites as a result of inactivation of the protein synthesis elongation factor eEF2 that is accompanied by decreased rates of translational elongation. In contrast to most other oxygen-sensing proteins that mediate cellular responses to low O2, TgPHYb is specifically required for parasite growth and protein synthesis at high, but not low, O2tensions as well as resistance to reactive oxygen species.In vivo, reduced TgPHYb expression leads to lower parasite burdens in oxygen-rich tissues. Taken together, these data identify TgPHYb as a sensor of high O2levels, in contrast to TgPHYa, which supports the parasite at low O2.IMPORTANCEBecause oxygen plays a key role in the growth of many organisms, cells must know how much oxygen is available. O2-sensing proteins are therefore critical cellular factors, and prolyl hydroxylases are the best-studied type of O2-sensing proteins. In general, prolyl hydroxylases trigger cellular responses to decreased oxygen availability. But, how does a cell react to high levels of oxygen? Using the protozoan parasiteToxoplasma gondii, we discovered a prolyl hydroxylase that allows the parasite to grow at elevated oxygen levels and does so by regulating protein synthesis. Loss of this enzyme also reduces parasite burden in oxygen-rich tissues, indicating that sensing both high and low levels of oxygen impacts the growth and physiology ofToxoplasma.

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NADPH Oxidase and Guanylate Binding Protein 5 Restrict Survival of Avirulent Type III Strains of Toxoplasma gondii in Naive Macrophages

mBio ◽

10.1128/mbio.01393-18 ◽

2018 ◽

Vol 9 (4) ◽

Cited By ~ 10

Author(s):

Sumit K. Matta ◽

Kelley Patten ◽

Quiling Wang ◽

Bae-Hoon Kim ◽

John D. MacMicking ◽

...

Keyword(s):

Toxoplasma Gondii ◽

Nadph Oxidase ◽

Binding Protein ◽

Parasitic Infections ◽

Type I ◽

Content Type ◽

Innate Defense ◽

Type Iii ◽

Ifn Γ ◽

Guanylate Binding Protein

ABSTRACT Phagocytic cells are the first line of innate defense against intracellular pathogens, and yet Toxoplasma gondii is renowned for its ability to survive in macrophages, although this paradigm is based on virulent type I parasites. Surprisingly, we find that avirulent type III parasites are preferentially cleared in naive macrophages, independent of gamma interferon (IFN-γ) activation. The ability of naive macrophages to clear type III parasites was dependent on enhanced activity of NADPH oxidase (Nox)-generated reactive oxygen species (ROS) and induction of guanylate binding protein 5 (Gbp5). Macrophages infected with type III parasites (CTG strain) showed a time-dependent increase in intracellular ROS generation that was higher than that induced by type I parasites (GT1 strain). The absence of Nox1 or Nox2, gp91 subunit isoforms of the Nox complex, reversed ROS-mediated clearance of CTG parasites. Consistent with this finding, both Nox1−/− and Nox2−/− mice showed higher susceptibility to CTG infection than wild-type mice. Additionally, Gbp5 expression was induced upon infection and the enhanced clearance of CTG strain parasites was reversed in Gbp5−/− macrophages. Expression of a type I ROP18 allele in CTG prevented clearance in naive macrophages, suggesting that it plays a role counteracting Gbp5. Although ROS and Gbp5 have been linked to activation of the NLRP3 inflammasome, clearance of CTG parasites did not rely on induction of pyroptosis. Collectively, these findings reveal that not all strains of T. gondii are adept at avoiding clearance in macrophages and define new roles for ROS and Gbps in controlling this important intracellular pathogen. IMPORTANCE Toxoplasma infections in humans and other mammals are largely controlled by IFN-γ produced by the activated adaptive immune system. However, we still do not completely understand the role of cell-intrinsic functions in controlling Toxoplasma or other apicomplexan infections. The present work identifies intrinsic activities in naive macrophages in counteracting T. gondii infection. Using an avirulent strain of T. gondii, we highlight the importance of Nox complexes in conferring protection against parasite infection both in vitro and in vivo. We also identify Gbp5 as a novel macrophage factor involved in limiting intracellular infection by avirulent strains of T. gondii. The rarity of human infections caused by type III strains suggests that these mechanisms may also be important in controlling human toxoplasmosis. These findings further extend our understanding of host responses and defense mechanisms that act to control parasitic infections at the cellular level.

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Interaction of Interferon Gamma-Induced Reactive Oxygen Species with Ceftazidime Leads to Synergistic Killing of Intracellular Burkholderia pseudomallei

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.02781-14 ◽

2014 ◽

Vol 58 (10) ◽

pp. 5954-5963 ◽

Cited By ~ 9

Author(s):

Kara Mosovsky ◽

Ediane Silva ◽

Ryan Troyer ◽

Katie Propst-Graham ◽

Steven Dow

Keyword(s):

Reactive Oxygen Species ◽

Interferon Gamma ◽

Burkholderia Pseudomallei ◽

Actin Polymerization ◽

Reactive Oxygen ◽

Buthionine Sulfoximine ◽

Oxygen Species ◽

Intracellular Killing ◽

Content Type ◽

Ifn Γ

ABSTRACTBurkholderia pseudomallei, a facultative intracellular pathogen, causes severe infections and is inherently refractory to many antibiotics. Previous studies from our group have shown that interferon gamma (IFN-γ) interacts synergistically with the antibiotic ceftazidime to kill bacteria in infected macrophages. The present study aimed to identify the underlying mechanism of that interaction. We first showed that blocking reactive oxygen species (ROS) pathways reversed IFN-γ- and ceftazidime-mediated killing, which led to our hypothesis that IFN-γ-induced ROS interacted with ceftazidime to synergistically killBurkholderiabacteria. Consistent with this hypothesis, we also observed that buthionine sulfoximine (BSO), another inducer of ROS, could substitute for IFN-γ to similarly potentiate the effect of ceftazidime on intracellular killing. Next, we observed that IFN-γ induced ROS-mediated killing of intracellular but not extracellular bacteria. On the other hand, ceftazidime effectively reduced extracellular bacteria but was not capable of intracellular killing when applied at 10 μg/ml. We investigated the exact role of IFN-γ-induced ROS responses on intracellular bacteria and notably observed a lack of actin polymerization associated withBurkholderiabacteria in IFN-γ-treated macrophages, which led to our finding that IFN-γ-induced ROS blocks vacuolar escape. Based on these results, we propose a model in which synergistically reduced bacterial burden is achieved primarily through separate and compartmentalized killing: intracellular killing by IFN-γ-induced ROS responses and extracellular killing by ceftazidime. Our findings suggest a means of enhancing antibiotic activity againstBurkholderiabacteria through combination with drugs that induce ROS pathways or otherwise target intracellular spread and/or replication of bacteria.

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Reactive Oxygen Species-Triggered Trophoblast Apoptosis Is Initiated by Endoplasmic Reticulum Stress via Activation of Caspase-12, CHOP, and the JNK Pathway in Toxoplasma gondii Infection in Mice

Infection and Immunity ◽

10.1128/iai.06295-11 ◽

2012 ◽

Vol 80 (6) ◽

pp. 2121-2132 ◽

Cited By ~ 46

Author(s):

Xiucai Xu ◽

Tingting Liu ◽

Aimei Zhang ◽

Xingxing Huo ◽

Qingli Luo ◽

...

Keyword(s):

Oxidative Stress ◽

Reactive Oxygen Species ◽

Endoplasmic Reticulum ◽

Toxoplasma Gondii ◽

Endoplasmic Reticulum Stress ◽

Reactive Oxygen ◽

Oxygen Species ◽

Jnk Pathway ◽

Content Type ◽

Caspase 12

ABSTRACTToxoplasma gondiiinfection in pregnant women may result in abortion or in fetal teratogenesis; however, the underlying mechanisms are still unclear. In this paper, based on a murine model, we showed that maternal infection with RH strainT. gondiitachyzoites induced elevated production of reactive oxygen species (ROS), local oxidative stress, and subsequent apoptosis of placental trophoblasts. PCR array analysis of 84 oxidative stress-related genes demonstrated that 27 genes were upregulated at least 2-fold and that 9 genes were downregulated at least 2-fold in theT. gondiiinfection group compared with levels in the control group. The expression of NADPH oxidase 1 (Nox1) and glutathione peroxidase 6 (Gpx6) increased significantly, about 25-fold. The levels of malondialdehyde (MDA) and 8-hydroxydeoxyguanosine (8-OHdG) increased significantly withT. gondiiinfection, and levels of glutathione (GSH) decreased rapidly.T. gondiiinfection increased the early expression of endoplasmic reticulum stress (ERS) markers, followed by cleavage of caspase-12, activation of ASK1/JNK, and increased apoptosis of trophoblasts, bothin vivoandin vitro. The apoptosis of trophoblasts, the activation of caspase-12 and the ASK1/JNK pathway, and the production of peroxides were dramatically inhibited by pretreatment withN-acetylcysteine (NAC). The upregulation of Nox1 was contact dependent and preceded the increase in levels of ERS markers and the activation of the proapoptosis cascade. Thus, we concluded that apoptosis in placental trophoblasts was initiated predominantly by ROS-mediated ERS via activation of caspase-12, CHOP, and the JNK pathway in acuteT. gondiiinfection. Elevated ROS production is the central event inT. gondii-induced apoptosis of placental trophoblasts.

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An Inside Job: Hacking into Janus Kinase/Signal Transducer and Activator of Transcription Signaling Cascades by the Intracellular Protozoan Toxoplasma gondii

Infection and Immunity ◽

10.1128/iai.05974-11 ◽

2011 ◽

Vol 80 (2) ◽

pp. 476-482 ◽

Cited By ~ 40

Author(s):

Eric Y. Denkers ◽

David J. Bzik ◽

Barbara A. Fox ◽

Barbara A. Butcher

Keyword(s):

Toxoplasma Gondii ◽

Host Cell ◽

Secretory Protein ◽

Effector Proteins ◽

Janus Kinase ◽

Host Cells ◽

Cytokine Signaling ◽

Signal Transducer ◽

Content Type ◽

Transcription 3

ABSTRACTThe intracellular protozoanToxoplasma gondiiis well known for its skill at invading and living within host cells. New discoveries are now also revealing the astounding ability of the parasite to inject effector proteins into the cytoplasm to seize control of the host cell. This review summarizes recent advances in our understanding of one such secretory protein called ROP16. This molecule is released from rhoptries into the host cell during invasion. The ROP16 molecule acts as a kinase, directly activating both signal transducer and activator of transcription 3 (STAT3) and STAT6 signaling pathways. In macrophages, an important and preferential target cell of parasite infection, the injection of ROP16 has multiple consequences, including downregulation of proinflammatory cytokine signaling and macrophage deviation to an alternatively activated phenotype.

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Toxoplasma gondii Inhibits Gamma Interferon (IFN-γ)- and IFN-β-Induced Host Cell STAT1 Transcriptional Activity by Increasing the Association of STAT1 with DNA

Infection and Immunity ◽

10.1128/iai.01291-13 ◽

2013 ◽

Vol 82 (2) ◽

pp. 706-719 ◽

Cited By ~ 37

Author(s):

Emily E. Rosowski ◽

Quynh P. Nguyen ◽

Ana Camejo ◽

Eric Spooner ◽

Jeroen P. J. Saeij

Keyword(s):

Toxoplasma Gondii ◽

Host Cell ◽

Transcriptional Activation ◽

Histone Deacetylases ◽

Gamma Interferon ◽

Host Cells ◽

Secondary Response ◽

Type I ◽

Content Type ◽

Ifn Γ

ABSTRACTThe gamma interferon (IFN-γ) response, mediated by the STAT1 transcription factor, is crucial for host defense against the intracellular pathogenToxoplasma gondii, but prior infection withToxoplasmacan inhibit this response. Recently, it was reported that theToxoplasmatype II NTE strain prevents the recruitment of chromatin remodeling complexes containing Brahma-related gene 1 (BRG-1) to promoters of IFN-γ-induced secondary response genes such asCiitaand major histocompatibility complex class II genes in murine macrophages, thereby inhibiting their expression. We report here that a type I strain ofToxoplasmainhibits the expression of primary IFN-γ response genes such asIRF1through a distinct mechanism not dependent on the activity of histone deacetylases. Instead, infection with a type I, II, or III strain ofToxoplasmainhibits the dissociation of STAT1 from DNA, preventing its recycling and further rounds of STAT1-mediated transcriptional activation. This leads to increased IFN-γ-induced binding of STAT1 at theIRF1promoter in host cells and increased global IFN-γ-induced association of STAT1 with chromatin.Toxoplasmatype I infection also inhibits IFN-β-induced interferon-stimulated gene factor 3-mediated gene expression, and this inhibition is also linked to increased association of STAT1 with chromatin. The secretion of proteins into the host cell by a type I strain ofToxoplasmawithout complete parasite invasion is not sufficient to block STAT1-mediated expression, suggesting that the effector protein responsible for this inhibition is not derived from the rhoptries.

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In Vitro Characterization of Protein Effector Export in the Bradyzoite Stage of Toxoplasma gondii

mBio ◽

10.1128/mbio.00046-20 ◽

2020 ◽

Vol 11 (2) ◽

Cited By ~ 5

Author(s):

Joshua Mayoral ◽

Peter Shamamian ◽

Louis M. Weiss

Keyword(s):

Toxoplasma Gondii ◽

Host Cell ◽

Persistent Infection ◽

Chronic Infection ◽

Life Stage ◽

Effector Proteins ◽

Content Type ◽

Time Points ◽

Bradyzoite Differentiation

ABSTRACT The ubiquitous parasite Toxoplasma gondii exhibits an impressive ability to maintain chronic infection of its host for prolonged periods. Despite this, little is known regarding whether and how T. gondii bradyzoites, a quasi-dormant life stage residing within intracellular cysts, manipulate the host cell to maintain persistent infection. A previous proteomic study of the cyst wall, an amorphous layer of proteins that forms underneath the cyst membrane, identified MYR1 as a putative cyst wall protein in vitro. Because MYR1 is known to be involved in the translocation of parasite-derived effector proteins into the host cell, we sought to determine whether parasites transitioning toward the bradyzoite life stage retain the capacity to translocate proteins via this pathway. By epitope tagging the endogenous loci of four known effectors that translocate from the parasitophorous vacuole into the host cell nucleus, we show, by immunofluorescence assays, that most effectors accumulate in the host nucleus at early but not late time points after infection, during the tachyzoite-to-bradyzoite transition and when parasites further along the bradyzoite differentiation continuum invade a new host cell. We demonstrate that the suppression of interferon gamma signaling, which was previously shown to be mediated by the effector TgIST, also occurs in the context of prolonged infection with bradyzoites and that TgIST export is a process that occurs beyond the early stages of host cell infection. These findings have important implications regarding how this highly successful parasite maintains persistent infection of its host. IMPORTANCE Toxoplasma bradyzoites persist within tissue cysts and are refractory to current treatments, serving as a reservoir for acute complications in settings of compromised immunity. Much remains to be understood regarding how this life stage successfully establishes and maintains persistent infection. In this study, we investigated whether the export of parasite effector proteins into the host cell occurs during the development of in vitro tissue cysts. We quantified the presence of four previously described effectors in host cell nuclei at different time points after bradyzoite differentiation and found that they accumulated largely during the early stages of infection. Despite a decline in nuclear accumulation, we found that one of these effectors still mediated its function after prolonged infection with bradyzoites, and we provide evidence that this effector is exported beyond early infection stages. These findings suggest that effector export from within developing tissue cysts provides one potential mechanism by which this parasite achieves chronic infection.

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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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ISG15 Connects Autophagy and IFN-γ-Dependent Control of Toxoplasma gondii Infection in Human Cells

mBio ◽

10.1128/mbio.00852-20 ◽

2020 ◽

Vol 11 (5) ◽

Author(s):

Jaya Bhushan ◽

Joshua B. Radke ◽

Yi-Chieh Perng ◽

Michael Mcallaster ◽

Deborah J. Lenschow ◽

...

Keyword(s):

Toxoplasma Gondii ◽

Parasitophorous Vacuole ◽

Interferon Γ ◽

Parasite Growth ◽

Human Cells ◽

Growth Restriction ◽

Intracellular Pathogens ◽

Functional Link ◽

Content Type ◽

Ifn Γ

ABSTRACT The intracellular protozoan parasite Toxoplasma gondii is capable of infecting most nucleated cells, where it survives in a specially modified compartment called the parasitophorous vacuole (PV). Interferon gamma (IFN-γ) is the major cytokine involved in activating cell-autonomous immune responses to inhibit parasite growth within this intracellular niche. In HeLa cells, IFN-γ treatment leads to ubiquitination of susceptible parasite strains, recruitment of the adaptors p62 and NDP52, and engulfment in microtubule-associated protein 1 light chain 3 (LC3)-positive membranes that restrict parasite growth. IFN-γ-mediated growth restriction depends on core members of the autophagy (ATG) pathway but not the initiation or degradative steps in the process. To explore the connection between these different pathways, we used permissive biotin ligation to identify proteins that interact with ATG5 in an IFN-γ-dependent fashion. Network analysis of the ATG5 interactome identified interferon-stimulated gene 15 (ISG15), which is highly upregulated by IFN treatment, as a hub connecting the ATG complex with other IFN-γ-induced genes, suggesting that it forms a functional link between the pathways. Deletion of ISG15 resulted in impaired recruitment of p62, NDP52, and LC3 to the PV and loss of IFN-γ-restricted parasite growth. The function of ISG15 required conjugation, and a number of ISGylated targets overlapped with the IFN-γ-dependent ATG5 interactome, including the adapter p62. Collectively, our findings establish a role for ISG15 in connecting the ATG pathway with IFN-γ-dependent restriction of T. gondii in human cells. IMPORTANCE Interferon(s) provide the primary defense against intracellular pathogens, a property ascribed to their ability to upregulate interferon-stimulated genes. Due to the sequestered niche occupied by Toxoplasma gondii, the host has elaborated intricate ways to target the parasite within its vacuole. One such mechanism is the recognition by a noncanonical autophagy pathway that envelops the parasite-containing vacuole and stunts growth in human cells. Remarkably, autophagy-dependent growth restriction requires interferon-γ, yet none of the classical components of autophagy are induced by interferon. Our studies draw a connection between these pathways by demonstrating that the antiviral protein ISG15, which is normally upregulated by interferons, links the autophagy-mediated control to ubiquitination of the vacuole. These findings suggest a similar link between interferon-γ signaling and autophagy that may underlie defense against other intracellular pathogens.

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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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