Biology subjects

Saeij, J. P. J.

Publications and source records attributed to Saeij, J. P. J..

8 recordsLinked to original sources

bioRxiv2026

Toxoplasma gondii GRA66 prevents premature egress driven by the host phospholipase RARRES3, independently of RNF213

Toxoplasma gondii replicates inside a host-derived parasitophorous vacuole (PV), and interferon-gamma (IFN{gamma}) induces host restriction factors that target this compartment. Previous CRISPR screens identified the dense granule protein GRA66 and the GRA57/GRA70/GRA71 complex as required for parasite fitness in IFN{gamma}-stimulated human cells, but the host pathways they oppose were unknown. Here, we show that GRA66, a PV membrane (PVM)-associated protein predicted to be an N-acylphosphatidylethanolamine (NAPE)-hydrolyzing phospholipase D, is required to prevent premature egress driven by the host phospholipase and acyltransferase RARRES3. Loss of GRA66 caused premature parasite egress, host cell death, and impaired replication in human cells. These phenotypes persisted in cells lacking RNF213, the E3 ubiquitin ligase that dominates IFN{gamma}-dependent Toxoplasma restriction in human cells, and{Delta} gra66 vacuoles recruited less RNF213 and ubiquitin than wild type, indicating an RNF213-independent mechanism rather than an exaggerated RNF213 response. Complementation with a catalytic-site mutant of the GRA66 zinc-binding motif failed to restore any of these phenotypes, indicating a requirement for its predicted enzymatic activity. Deleting RARRES3 rescued the premature egress of {Delta}gra66 and catalytic-mutant parasites but not of{Delta} gra70 parasites, whereas the replication defect persisted, revealing a second, RARRES3-independent consequence of GRA66 loss. RARRES3 was recruited to the PVM and intravacuolar network after IFN{gamma} stimulation, and structural modeling supported assignment of GRA66 to the NAPE-phospholipase D family with an intact di-zinc active site. These findings define a lipid-centered, RNF213-independent arm of human cell-autonomous immunity and identify the parasite effector required to withstand it.

microbiology↗

bioRxiv2025

Toxoplasma effector TgWIP hijacks dendritic cell actin and motility via Nck1/Grb2 and the WAVE complex

The intracellular parasite Toxoplasma gondii enhances its dissemination to distant organs by hijacking infected leukocytes via a Trojan Horse mechanism. Upon infecting dendritic cells (DCs), Toxoplasma induces a hypermigratory phenotype characterized by podosome dissolution and formation of F-actin stress fibers. We previously showed that these cytoskeletal changes depend on the effector protein Toxoplasma WAVE complex-interacting protein (TgWIP) secreted from parasites to infected leukocytes. Here, we identify the host adaptor proteins Non-catalytic region of tyrosine kinase adaptor protein 1 and 2 (Nck1/2) and Growth factor receptor-bound protein 2 (Grb2) as direct TgWIP interactors. TgWIP mainly uses two distinct proline-rich regions (PRRs) to interact with Nck1 and Grb2. Mutating these PRRs abrogates TgWIP binding to Nck1 and Grb2 and diminishes podosome dissolution and DC hypermotility. Furthermore, we show that TgWIP directly interacts with the actin nucleation promoting factor WAVE Regulatory Complex (WRC) via a WRC-interacting receptor sequence (WIRS). Disrupting this interaction also influences actin cytoskeletal remodeling and DC hypermotility. Collectively, our data reveal that TgWIP directly interacts with multiple actin regulators, including Nck1, Grb2, and the WRC, to remodel the actin cytoskeleton of the host cells, elucidating a key mechanism that Toxoplasma exploits to enhance host cell migration and dissemination.

microbiology↗

bioRxiv2025

The early human interferon gamma response to Toxoplasma gondii is driven by Vγ9Vδ2 T-cell sensing of host phosphoantigens and subsequent NK-cell activation

Toxoplasma gondii is a globally prevalent intracellular parasite that infects [~]40 million Americans. The murine immune response to Toxoplasma relies on both toll-like receptor (TLR) 11/12 and immunity related GTPase-mediated (IRGs) responses, which humans lack, making it unclear how the human immune response detects and responds to the parasite. We investigated whether human V{gamma}9V{delta}2 T cells, which detect phosphoantigens through the BTN3A1 receptor, shape the early immune response to the parasite. Using primary human peripheral blood mononuclear cells (PBMCs), we show that V{gamma}9V{delta}2 T cells are activated by Toxoplasma-infected cells in a BTN3A1- dependent manner leading to secretion of interferon gamma (IFN{gamma}) and tumor necrosis factor-alpha (TNF). Additionally, these T cells potentiate IFN{gamma} production by natural killer (NK) cells, likely via TNF and interleukin (IL)-12 produced during infection. Active parasite invasion is required to stimulate the IFN{gamma} response, and inhibition of the host mevalonate pathway, which limits the synthesis of the phosphoantigen isopentenyl pyrophosphate (IPP), attenuates the cytokine response, indicating Toxoplasma infection increases host phosphoantigens leading to V{gamma}9V{delta}2 T cell activation. Our findings identify V{gamma}9V{delta}2 T cells as key effectors that potentiate NK cells in the early human immune response to Toxoplasma, bridging innate and adaptive immunity in the absence of TLR11/12 signaling.

immunology↗

bioRxiv2023

Host E3 ubiquitin ligase ITCH mediates Toxoplasma gondii effector GRA35-triggered NLRP1 inflammasome activation and cell-autonomous immunity.

Toxoplasma gondii is an intracellular parasite that can activate the NLRP1 inflammasome leading to macrophage pyroptosis in Lewis rats, but the underlying mechanism is not well understood. In this study, we performed a genome-wide CRISPR screen and identified the dense granule proteins GRA35, GRA42, and GRA43 as the Toxoplasma effectors mediating cell death in Lewis rat macrophages. GRA35 localizes on the parasitophorous vacuole membrane, where it interacts with the host E3 ubiquitin ligase ITCH. Inhibition of proteasome activity or ITCH knockout prevented pyroptosis in Toxoplasma-infected Lewis rat macrophages, consistent with the "NLRP1 functional degradation model". However, there was no evidence that ITCH directly ubiquitinates or interacts with rat NLRP1. We also found that GRA35-ITCH interaction affected Toxoplasma fitness in IFN{gamma}-activated human fibroblasts, likely due to ITCHs role in recruiting ubiquitin and the parasite-restriction factor RNF213 to the parasitophorous vacuole membrane. These findings identify a new role of host E3 ubiquitin ligase ITCH in mediating effector-triggered immunity, a critical concept that involves recognizing intracellular pathogens and initiating host innate immune responses. IMPORTANCEEffector-triggered immunity represents an innate immune defense mechanism that plays a crucial role in sensing and controlling intracellular pathogen infection. The NLRP1 inflammasome in the Lewis rats can detect Toxoplasma infection, which triggers proptosis in infected macrophages and eliminates the parasites replication niche. The work reported here revealed that host E3 ubiquitin ligase ITCH is able to recognize and interact with Toxoplasma effector protein GRA35 localized on the parasite-host interface, leading to NLRP1 inflammasome activation in Lewis rat macrophages. Furthermore, ITCH-GRA35 interaction contributes to the restriction of Toxoplasma in human fibroblasts stimulated by IFN{gamma}. Thus, this research provides valuable insights into understanding pathogen recognition and restriction mediated by host E3 ubiquitin ligase.

immunology↗

bioRxiv2023

GRA47 and GRA72 are Toxoplasma gondii pore-forming proteins that influence small molecule permeability of the parasitophorous vacuole

Toxoplasma gondii, a medically important intracellular parasite, uses GRA proteins, secreted from dense granule organelles, to mediate nutrient flux across the parasitophorous vacuole membrane (PVM). GRA17 and GRA23 are known pore-forming proteins on the PVM involved in this process, but the roles of additional proteins have remained largely uncharacterized. We recently identified GRA72 as synthetically lethal with GRA17. Deleting GRA72 produced similar phenotypes to{Delta} gra17 parasites, and computational predictions suggested it forms a pore. To understand how GRA72 functions we performed immunoprecipitation experiments and identified GRA47 as an interactor of GRA72. Deletion of GRA47 resulted in an aberrant bubble vacuole morphology with reduced small molecule permeability, mirroring the phenotype observed in GRA17 and GRA72 knockouts. Structural predictions indicated that GRA47 and GRA72 form heptameric and hexameric pores, respectively, with conserved histidine residues lining the pore. Mutational analysis highlighted the critical role of these histidines for protein functionality. Validation through electrophysiology confirmed alterations in membrane conductance, corroborating their pore-forming capabilities. Furthermore, {Delta}gra47 parasites and parasites expressing GRA47 with a histidine mutation had reduced in vitro proliferation and attenuated virulence in mice. Our findings show the important roles of GRA47 and GRA72 in regulating PVM permeability, thereby expanding the repertoire of potential therapeutic targets against Toxoplasma infections. IMPORTANCEToxoplasma gondii is a parasite that poses significant health risks to those with impaired immunity. It replicates inside host cells shielded by the parasitophorous vacuole membrane (PVM), which controls nutrient and waste exchange with the host. GRA72, previously identified as essential in the absence of the GRA17 nutrient channel, is implicated in forming an alternative nutrient channel. Here we found that GRA47 associates with GRA72 and is also important for the PVMs permeability to small molecules. Removal of GRA47 leads to distorted vacuoles and impairs small molecule transport across the PVM, resembling the effects of GRA17 and GRA72 deletions. Structural models suggest GRA47 and GRA72 form distinct pore structures, with a pore-lining histidine critical to their function. Toxoplasma strains lacking GRA47, or those with a histidine mutation, have impaired growth and reduced virulence in mice, highlighting these proteins as potential targets for new treatments against Toxoplasmosis.

microbiology↗

bioRxiv2020

Naïve CD8 T cell IFNγ responses to a vacuolar antigen are regulated by an inflammasome-independent NLRP3 pathway and Toxoplasma gondii ROP5

Host resistance to Toxoplasma gondii relies on CD8 T cell IFN{gamma} responses, which if modulated by the host or parasite could influence chronic infection and parasite transmission between hosts. Since host-parasite interactions that govern this response are not fully elucidated, we investigated requirements for eliciting naive CD8 T cell IFN{gamma} responses to a vacuolar resident antigen of T. gondii, TGD057. Naive TGD057 antigen-specific CD8 T cells (T57) were isolated from transnuclear mice and responded to parasite-infected bone marrow-derived macrophages (BMDMs) in an antigen-dependent manner, first by producing IL-2 and then IFN{gamma}. T57 IFN{gamma} responses to TGD057 were independent of the parasites protein export machinery ASP5 and MYR1. Instead, host immunity pathways downstream of the regulatory Immunity-Related GTPases (IRG), including partial dependence on Guanylate-Binding Proteins, are required. Multiple T. gondii ROP5 isoforms and allele types, including avirulent ROP5A from clade A and D parasite strains, were able to suppress CD8 T cell IFN{gamma} responses to parasite-infected BMDMs. Phenotypic variance between clades B, C, D, F, and A strains suggest T57 IFN{gamma} differentiation occurs independently of parasite virulence or any known IRG-ROP5 interaction. Consistent with this, removal of ROP5 is not enough to elicit maximal CD8 T cell IFN{gamma} production to parasite-infected cells. Instead, macrophage expression of the pathogen sensors, NLRP3 and to a large extent NLRP1, were absolute requirements. Other members of the conventional inflammasome cascade are only partially required, as revealed by decreased but not abrogated T57 IFN{gamma} responses to parasite-infected ASC, caspase-1/11, and gasdermin D deficient cells. Moreover, IFN{gamma} production was only partially reduced in the absence of IL-12, IL-18 or IL-1R signaling. In summary, T. gondii effectors and host machinery that modulate parasitophorous vacuolar membranes, as well as NLR-dependent but inflammasome-independent pathways, determine the full commitment of CD8 T cells IFN{gamma} responses to a vacuolar antigen. AUTHOR SUMMARYParasites are excellent "students" of our immune system as they can deflect, antagonize and confuse the immune response making it difficult to vaccinate against these pathogens. In this report, we analyzed how a widespread parasite of mammals, Toxoplasma gondii, manipulates an immune cell needed for immunity to many intracellular pathogens, the CD8 T cell. Host pathways that govern CD8 T cell production of the immune protective cytokine, IFN{gamma}, were also explored. We hypothesized the secreted Toxoplasma virulence factor, ROP5, work to inhibit the MHC 1 antigen presentation pathway therefore making it difficult for CD8 T cells to see T. gondii antigens sequestered inside a parasitophorous vacuole. However, manipulation through T. gondii ROP5 does not fully explain how CD8 T cells commit to making IFN{gamma} in response to infection. Importantly, CD8 T cell IFN{gamma} responses to T. gondii require the pathogen sensor NLRP3 to be expressed in the infected cell. Other proteins associated with NLRP3 activation, including members of the conventional inflammasome activation cascade pathway, are only partially involved. Our results identify a novel pathway by which NLRP3 regulates T cell function and underscore the need for inflammasome-activating adjuvants in vaccines aimed at inducing CD8 T cell IFN{gamma} responses to parasites.

immunology↗

bioRxiv2019

A genome-wide loss-of-function screen identifies Toxoplasma gondii genes that determine fitness in interferon gamma-activated murine macrophages

Macrophages play an essential role in the early immune response against Toxoplasma and are the cell type preferentially infected by the parasite in vivo. Interferon gamma (IFN{gamma}) elicits a variety of anti-Toxoplasma activities in macrophages. Using a genome-wide CRISPR screen we identified [~]130 Toxoplasma genes that determine parasite fitness in naive macrophages and [~]466 genes that determine fitness in IFN{gamma}-stimulated murine macrophages, seven of which we investigated and confirmed. We show that one of these genes encodes dense granule protein GRA45, which contains a putative chaperone-like domain, and which we show is critical in preventing other GRA effectors from aggregating. Parasites lacking GRA45 mislocalize GRA effectors upon secretion, are more susceptible to IFN{gamma}-mediated growth inhibition, and have reduced virulence in mice. Our results provide a resource for the community to further explore the function of Toxoplasma genes that determine fitness in IFN{gamma}-stimulated macrophages. IMPORTANCEThe intracellular parasite Toxoplasma gondii can cause congenital infections and severe disease in immunocompromised patients. The cytokine IFN{gamma} can block parasite replication by upregulating a variety of toxoplasmacidal mechanisms in many cells, including macrophages. Toxoplasma preferentially infects macrophages. Therefore, the parasite has evolved mechanisms to survive in these cells in the presence of IFN{gamma}. Here, we generated pools of Toxoplasma mutants for every gene and determined which mutants were specifically depleted in IFN{gamma}-stimulated macrophages, thus identifying parasite genes determining fitness in these cells. We show that one of these genes encodes for a dense granule protein (GRA45) that plays an important role in preventing other GRA effectors from aggregating. Parasites without GRA45 mislocalize GRA effectors upon secretion, have enhanced susceptibility to IFN{gamma}-mediated growth inhibition, and are avirulent in mice. Thus, our screen provides a resource to the Toxoplasma community to determine the function of Toxoplasma genes that affect its fitness in IFN{gamma}-stimulated macrophages.

microbiology↗

bioRxiv2019

Identification of a master regulator of differentiation in Toxoplasma

Toxoplasma gondii chronically infects a quarter of the worlds population, and its recrudescence can cause life-threatening disease in immunocompromised individuals and recurrent ocular lesions in the immunocompetent. Chronic stages are established by differentiation of rapidly replicating tachyzoites into slow-growing bradyzoites, which form intracellular cysts resistant to immune clearance and existing therapies. Despite its central role in infection, the molecular basis of chronic differentiation is not understood. Through Cas9-mediated genetic screening and single-cell transcriptional profiling, we identify and characterize a putative transcription factor (BFD1) as necessary and sufficient for differentiation. Translation of BFD1 appears to be stress regulated, and its constitutive expression elicits differentiation in the absence of stress. As a Myb-like factor, BFD1 provides a counterpoint to the ApiAP2 factors which dominate our current view of parasite gene regulation. Overall, BFD1 provides a genetic switch to study and control Toxoplasma differentiation, and will inform prevention and treatment of chronic infection.

microbiology↗