scholarly journals A Noncanonical Autophagy Pathway Restricts Toxoplasma gondii Growth in a Strain-Specific Manner in IFN-γ-Activated Human Cells

mBio ◽  
2015 ◽  
Vol 6 (5) ◽  
Author(s):  
Elizabeth M. Selleck ◽  
Robert C. Orchard ◽  
Kara G. Lassen ◽  
Wandy L. Beatty ◽  
Ramnik J. Xavier ◽  
...  
Keyword(s):  
Toxoplasma Gondii ◽  
Human Cells ◽  
Intracellular Pathogens ◽  
Innate Immune Responses ◽  
Content Type ◽  
Downstream Effectors ◽  
Core Set ◽  
Autophagy Pathway ◽  
Parasite Replication ◽  
Ifn Γ

ABSTRACTA core set of autophagy proteins is required for gamma interferon (IFN-γ)-mediated clearance ofToxoplasma gondiiin the mouse because of their control of several downstream effectors, including immunity-related GTPases (IRGs) and guanylate-binding proteins (GBPs). However, these effectors are absent (i.e., IRGs) from or nonessential (i.e., GBPs) in IFN-γ-activated human cells, raising the question of how these cells control parasite replication. Here, we define a novel role for ubiquitination and recruitment of autophagy adaptors in the strain-specific control ofT. gondiireplication in IFN-γ-activated human cells. Vacuoles containing susceptible strains ofT. gondiibecame ubiquitinated, recruited the adaptors p62 and NDP52, and were decorated with LC3. Parasites within LC3-positive vacuoles became enclosed in multiple layers of host membranes, resulting in stunting of parasite replication. However, LC3-positiveT. gondii-containing vacuoles did not fuse with endosomes and lysosomes, indicating that this process is fundamentally different from xenophagy, a form of autophagy involved in the control of intracellular bacterial pathogens. Genetic knockout of ATG16L or ATG7 reverted the membrane encapsulation and restored parasite replication, indicating that core autophagy proteins involved in LC3 conjugation are important in the control of parasite growth. Despite a role for the core autophagy machinery in this process, upstream activation through Beclin 1 was not sufficient to enhance the ubiquitination ofT. gondii-containing vacuoles, suggesting a lack of reliance on canonical autophagy. These findings demonstrate a new mechanism for IFN-γ-dependent control ofT. gondiiin human cells that depends on ubiquitination and core autophagy proteins that mediate membrane engulfment and restricted growth.IMPORTANCEAutophagy is a process of cellular remodeling that allows the cell to recycle senescent organelles and recapture nutrients. During innate immune responses in the mouse, autophagy is recruited to help target intracellular pathogens and thus eliminate them. However, the antimicrobial mediators that depend on autophagy in the mouse are not conserved in humans, raising the issue of how human cells control intracellular pathogens. Our study defines a new pathway for the control of the ubiquitous intracellular parasiteT. gondiiin human cells activated by IFN-γ. Recruitment of autophagy adaptors resulted in engulfment of the parasite in multiple membranes and growth impairment. Although susceptible type 2 and 3 stains ofT. gondiiwere captured by this autophagy-dependent pathway, type 1 strains were able to avoid entrapment.

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

mBio ◽  
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.

scholarly journals Loss of the Conserved Alveolate Kinase MAPK2 Decouples Toxoplasma Cell Growth from Cell Division

mBio ◽  
2020 ◽  
Vol 11 (6) ◽  
Author(s):  
Xiaoyu Hu ◽  
William J. O’Shaughnessy ◽  
Tsebaot G. Beraki ◽  
Michael L. Reese
Keyword(s):  
Toxoplasma Gondii ◽  
Protein Kinases ◽  
Daughter Cell ◽  
Protozoan Parasite ◽  
Productive Infection ◽  
Centrosome Duplication ◽  
Loss Of Function ◽  
Content Type ◽  
Mitogen Activated Protein ◽  
Parasite Replication

ABSTRACT Mitogen-activated protein kinases (MAPKs) are a conserved family of protein kinases that regulate signal transduction, proliferation, and development throughout eukaryotes. The apicomplexan parasite Toxoplasma gondii expresses three MAPKs. Two of these, extracellular signal-regulated kinase 7 (ERK7) and MAPKL1, have been implicated in the regulation of conoid biogenesis and centrosome duplication, respectively. The third kinase, MAPK2, is specific to and conserved throughout the Alveolata, although its function is unknown. We used the auxin-inducible degron system to determine phenotypes associated with MAPK2 loss of function in Toxoplasma. We observed that parasites lacking MAPK2 failed to duplicate their centrosomes and therefore did not initiate daughter cell budding, which ultimately led to parasite death. MAPK2-deficient parasites initiated but did not complete DNA replication and arrested prior to mitosis. Surprisingly, the parasites continued to grow and replicate their Golgi apparatus, mitochondria, and apicoplasts. We found that the failure in centrosome duplication is distinct from the phenotype caused by the depletion of MAPKL1. As we did not observe MAPK2 localization at the centrosome at any point in the cell cycle, our data suggest that MAPK2 regulates a process at a distal site that is required for the completion of centrosome duplication and the initiation of parasite mitosis. IMPORTANCE Toxoplasma gondii is a ubiquitous intracellular protozoan parasite that can cause severe and fatal disease in immunocompromised patients and the developing fetus. Rapid parasite replication is critical for establishing a productive infection. Here, we demonstrate that a Toxoplasma protein kinase called MAPK2 is conserved throughout the Alveolata and essential for parasite replication. We found that parasites lacking MAPK2 protein were defective in the initiation of daughter cell budding and were rendered inviable. Specifically, T. gondii MAPK2 (TgMAPK2) appears to be required for centrosome replication at the basal end of the nucleus, and its loss causes arrest early in parasite division. MAPK2 is unique to the Alveolata and not found in metazoa and likely is a critical component of an essential parasite-specific signaling network.

scholarly journals Infection-Induced Intestinal Dysbiosis Is Mediated by Macrophage Activation and Nitrate Production

mBio ◽  
2019 ◽  
Vol 10 (3) ◽  
Author(s):  
Shuai Wang ◽  
Ayah El-Fahmawi ◽  
David A. Christian ◽  
Qun Fang ◽  
Enrico Radaelli ◽  
...  
Keyword(s):  
Toxoplasma Gondii ◽  
Oral Infection ◽  
Nitrate Respiration ◽  
Characteristic Change ◽  
Intestinal Immunity ◽  
Parasite Control ◽  
Shotgun Metagenomics ◽  
Content Type ◽  
Distal Small Intestine ◽  
Ifn Γ

ABSTRACT Oral infection of C57BL/6J mice with Toxoplasma gondii results in a marked bacterial dysbiosis and the development of severe pathology in the distal small intestine that is dependent on CD4+ T cells and interferon gamma (IFN-γ). This dysbiosis and bacterial translocation contribute to the development of ileal pathology, but the factors that support the bloom of bacterial pathobionts are unclear. The use of microbial community profiling and shotgun metagenomics revealed that Toxoplasma infection induces a dysbiosis dominated by Enterobacteriaceae and an increased potential for nitrate respiration. In vivo experiments using bacterial metabolic mutants revealed that during this infection, host-derived nitrate supports the expansion of Enterobacteriaceae in the ileum via nitrate respiration. Additional experiments with infected mice indicate that the IFN-γ/STAT1/iNOS axis, while essential for parasite control, also supplies a pool of nitrate that serves as a source for anaerobic respiration and supports overgrowth of Enterobacteriaceae. Together, these data reveal a trade-off in intestinal immunity after oral infection of C57BL/6J mice with T. gondii, in which inducible nitric oxide synthase (iNOS) is required for parasite control, while this host enzyme is responsible for specific modification of the composition of the microbiome that contributes to pathology. IMPORTANCE Toxoplasma gondii is a protozoan parasite and a leading cause of foodborne illness. Infection is initiated when the parasite invades the intestinal epithelium, and in many host species, this leads to intense inflammation and a dramatic disruption of the normal microbial ecosystem that resides in the healthy gut (the so-called microbiome). One characteristic change in the microbiome during infection with Toxoplasma—as well as numerous other pathogens—is the overgrowth of Escherichia coli or similar bacteria and a breakdown of commensal containment leading to seeding of peripheral organs with gut bacteria and subsequent sepsis. Our findings provide one clear explanation for how this process is regulated, thereby improving our understanding of the relationship between parasite infection, inflammation, and disease. Furthermore, our results could serve as the basis for the development of novel therapeutics to reduce the potential for harmful bacteria to bloom in the gut during infection.

scholarly journals Immediate Interferon Gamma Induction Determines Murine Host Compatibility Differences between Toxoplasma gondii and Neospora caninum

2020 ◽  
Vol 88 (4) ◽  
Author(s):  
Rachel S. Coombs ◽  
Matthew L. Blank ◽  
Elizabeth D. English ◽  
Yaw Adomako-Ankomah ◽  
Ifeanyi-Chukwu Samuel Urama ◽  
...  
Keyword(s):  
Toxoplasma Gondii ◽  
Immune Responses ◽  
Interferon Gamma ◽  
Neospora Caninum ◽  
Ectopic Expression ◽  
Parasite Burden ◽  
Interleukin 12 ◽  
Content Type ◽  
Ifn Γ ◽  
And Control

ABSTRACT Rodents are critical for the transmission of Toxoplasma gondii to the definitive feline host via predation, and this relationship has been extensively studied as a model for immune responses to parasites. Neospora caninum is a closely related coccidian parasite of ruminants and canines but is not naturally transmitted by rodents. We compared mouse innate immune responses to N. caninum and T. gondii and found marked differences in cytokine levels and parasite growth kinetics during the first 24 h postinfection (hpi). N. caninum-infected mice produced significantly higher levels of interleukin-12 (IL-12) and interferon gamma (IFN-γ) by as early as 4 hpi, but the level of IFN-γ was significantly lower or undetectable in T. gondii-infected mice during the first 24 hpi. “Immediate” IFN-γ and IL-12p40 production was not detected in MyD88−/− mice. However, unlike IL-12p40−/− and IFN-γ−/− mice, MyD88−/− mice survived N. caninum infections at the dose used in this study. Serial measures of parasite burden showed that MyD88−/− mice were more susceptible to N. caninum infections than wild-type (WT) mice, and control of parasite burdens correlated with a pulse of serum IFN-γ at 3 to 4 days postinfection in the absence of detectable IL-12. Immediate IFN-γ was partially dependent on the T. gondii mouse profilin receptor Toll-like receptor 11 (TLR11), but the ectopic expression of N. caninum profilin in T. gondii had no impact on early IFN-γ production or parasite proliferation. Our data indicate that T. gondii is capable of evading host detection during the first hours after infection, while N. caninum is not, and this is likely due to the early MyD88-dependent recognition of ligands other than profilin.

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

mBio ◽  
2018 ◽  
Vol 9 (4) ◽  
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.

scholarly journals GABARAPL2 Is Critical for Growth Restriction of Toxoplasma gondii in HeLa Cells Treated with Gamma Interferon

2020 ◽  
Vol 88 (5) ◽  
Author(s):  
Zhaoxia Zhang ◽  
Haorong Gu ◽  
Qi Li ◽  
Jun Zheng ◽  
Shinuo Cao ◽  
...  
Keyword(s):  
Toxoplasma Gondii ◽  
Hela Cells ◽  
Critical Role ◽  
Gamma Interferon ◽  
Growth Restriction ◽  
Membrane Structures ◽  
Content Type ◽  
Receptor Associated Protein ◽  
Ifn Γ ◽  
And Function

ABSTRACT Gamma interferon (IFN-γ)-induced innate immune responses play important roles in the inhibition of Toxoplasma gondii infection. It has been reported that IFN-γ stimulates non-acidification-dependent growth restriction of T. gondii in HeLa cells, but the mechanism remains unclear. Here, we found that γ-aminobutyric acid (GABA) receptor-associated protein-like 2 (GABARAPL2) plays a critical role in parasite restriction in IFN-γ-treated HeLa cells. GABARAPL2 is recruited to membrane structures surrounding parasitophorous vacuoles (PV). Autophagy adaptors are required for the proper localization and function of GABARAPL2 in the IFN-γ -induced immune response. These findings provide further understanding of a noncanonical autophagy pathway responsible for IFN-γ-dependent inhibition of T. gondii growth in human HeLa cells and demonstrate the critical role of GABARAPL2 in this response.

scholarly journals Toxoplasma gondii Ingests and Digests Host Cytosolic Proteins

mBio ◽  
2014 ◽  
Vol 5 (4) ◽  
Author(s):  
Zhicheng Dou ◽  
Olivia L. McGovern ◽  
Manlio Di Cristina ◽  
Vern B. Carruthers
Keyword(s):  
Toxoplasma Gondii ◽  
Survival Strategies ◽  
Type I ◽  
Intracellular Replication ◽  
Lysosomal Hydrolases ◽  
Cytosolic Proteins ◽  
Content Type ◽  
Genetic Ablation ◽  
Virulence Attenuation ◽  
Parasite Replication

ABSTRACT The protozoan parasite Toxoplasma gondii resides within a nonfusogenic vacuole during intracellular replication. Although the limiting membrane of this vacuole provides a protective barrier to acidification and degradation by lysosomal hydrolases, it also physically segregates the parasite from the host cytosol. Accordingly, it has been suggested that T. gondii acquires material from the host via membrane channels or transporters. The ability of the parasite to internalize macromolecules via endocytosis during intracellular replication has not been tested. Here, we show that Toxoplasma ingests host cytosolic proteins and digests them using cathepsin L and other proteases within its endolysosomal system. Ingestion was reduced in mutant parasites lacking an intravacuolar network of tubular membranes, implicating this apparatus as a possible conduit for trafficking to the parasite. Genetic ablation of proteins involved in the pathway is associated with diminished parasite replication and virulence attenuation. We show that both virulent type I and avirulent type II strain parasites ingest and digest host-derived protein, indicating that the pathway is not restricted to highly virulent strains. The findings provide the first definitive evidence that T. gondii internalizes proteins from the host during intracellular residence and suggest that protein digestion within the endolysosomal system of the parasite contributes to toxoplasmosis. IMPORTANCE Toxoplasma gondii causes significant disease in individuals with weak immune systems. Treatment options for this infection have drawbacks, creating a need to understand how this parasite survives within the cells it infects as a prelude to interrupting its survival strategies. This study reveals that T. gondii internalizes proteins from the cytoplasm of the cells it infects and degrades such proteins within a digestive compartment within the parasite. Disruption of proteins involved in the pathway reduced parasite replication and lessened disease severity. The identification of a novel parasite ingestion pathway opens opportunities to interfere with this process and improve the outcome of infection.

scholarly journals Innate Immune Responses of Bat and Human Cells to Filoviruses: Commonalities and Distinctions

2017 ◽  
Vol 91 (8) ◽  
Author(s):  
Ivan V. Kuzmin ◽  
Toni M. Schwarz ◽  
Philipp A. Ilinykh ◽  
Ingo Jordan ◽  
Thomas G. Ksiazek ◽  
...  
Keyword(s):  
Immune Response ◽  
Immune Responses ◽  
Innate Immune Response ◽  
Antiviral Effect ◽  
Human Cells ◽  
Innate Immune ◽  
Innate Immune Responses ◽  
Symptomatic Disease ◽  
Ifn Γ

ABSTRACT Marburg (MARV) and Ebola (EBOV) viruses are zoonotic pathogens that cause severe hemorrhagic fever in humans. The natural reservoir of MARV is the Egyptian rousette bat (Rousettus aegyptiacus); that of EBOV is unknown but believed to be another bat species. The Egyptian rousette develops subclinical productive infection with MARV but is refractory to EBOV. Interaction of filoviruses with hosts is greatly affected by the viral interferon (IFN)-inhibiting domains (IID). Our study was aimed at characterization of innate immune responses to filoviruses and the role of filovirus IID in bat and human cells. The study demonstrated that EBOV and MARV replicate to similar levels in all tested cell lines, indicating that permissiveness for EBOV at cell and organism levels do not necessarily correlate. Filoviruses, particularly MARV, induced a potent innate immune response in rousette cells, which was generally stronger than that in human cells. Both EBOV VP35 and VP24 IID were found to suppress the innate immune response in rousette cells, but only VP35 IID appeared to promote virus replication. Along with IFN-α and IFN-β, IFN-γ was demonstrated to control filovirus infection in bat cells but not in human cells, suggesting host species specificity of the antiviral effect. The antiviral effects of bat IFNs appeared not to correlate with induction of IFN-stimulated genes 54 and 56, which were detected in human cells ectopically expressing bat IFN-α and IFN-β. As bat IFN-γ induced the type I IFN pathway, its antiviral effect is likely to be partially induced via cross talk. IMPORTANCE Bats serve as reservoirs for multiple emerging viruses, including filoviruses, henipaviruses, lyssaviruses, and zoonotic coronaviruses. Although there is no evidence for symptomatic disease caused by either Marburg or Ebola viruses in bats, spillover of these viruses into human populations causes deadly outbreaks. The reason for the lack of symptomatic disease in bats infected with filoviruses remains unknown. The outcome of a virus-host interaction depends on the ability of the host immune system to suppress viral replication and the ability of a virus to counteract the host defenses. Our study is a comparative analysis of the host innate immune response to either MARV or EBOV infection in bat and human cells and the role of viral interferon-inhibiting domains in the host innate immune responses. The data are useful for understanding the interactions of filoviruses with natural and accidental hosts and for identification of factors that influence filovirus evolution.

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

mBio ◽  
2021 ◽  
Vol 12 (2) ◽  
Author(s):  
Joshua A. Kochanowsky ◽  
Kaitlin K. Thomas ◽  
Anita A. Koshy
Keyword(s):  
Reactive Oxygen Species ◽  
Toxoplasma Gondii ◽  
Host Cell ◽  
Parasite Growth ◽  
Effector Proteins ◽  
Oxygen Species ◽  
Content Type ◽  
Growth And Survival ◽  
Type Iii ◽  
Ifn Γ

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.

Strain-specific disruption of interferon-stimulated N-myc and STAT interactor (NMI) function by Toxoplasma gondii type I ROP18 in human cells

Parasitology ◽  
2020 ◽  
Vol 147 (13) ◽  
pp. 1433-1442
Author(s):  
Jing Xia ◽  
Matthew L. Blank ◽  
Li-Juan Zhou ◽  
Shui-Zhen Wu ◽  
Hong-Juan Peng ◽  
...  
Keyword(s):  
Toxoplasma Gondii ◽  
Mammalian Cells ◽  
Gene Promoter ◽  
Kinase Domain ◽  
Human Cells ◽  
Type I ◽  
Intermediate Hosts ◽  
New Host ◽  
Allele Specific ◽  
Ifn Γ

AbstractToxoplasma gondii rhoptry protein TgROP18 is a polymorphic virulence effector that targets immunity-related GTPases (IRGs) in rodents. Given that IRGs are uniquely diversified in rodents and not in other T. gondii intermediate hosts, the role of TgROP18 in manipulating non-rodent cells is unclear. Here we show that in human cells TgROP18I interacts with the interferon-gamma-inducible protein N-myc and STAT interactor (NMI) and that this is a property that is unique to the type I TgROP18 allele. Specifically, when expressed ectopically in mammalian cells only TgROP18I co-immunoprecipitates with NMI in IFN-γ-treated cells, while TgROP18II does not. In parasites expressing TgROP18I or TgROP18II, NMI only co-immunoprecipitates with TgROP18I and this is associated with allele-specific immunolocalization of NMI on the parasitophorous vacuolar membrane (PVM). We also found that TgROP18I reduces NMI association with IFN-γ-activated sequences (GAS) in the IRF1 gene promoter. Finally, we determined that polymorphisms in the C-terminal kinase domain of TgROP18I are required for allele-specific effects on NMI. Together, these data further define new host pathway targeted by TgROP18I and provide the first function driven by allelic differences in the highly polymorphic ROP18 locus.

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