The Toxoplasma gondii effector GRA83 modulates the host's innate immune response to regulate parasite infection.

Toxoplasma gondii's propensity to infect its host and cause disease is highly dependent on its ability to modulate host cell functions. One of the strategies the parasite uses to accomplish this is via the export of effector proteins from the secretory dense granules. Dense granule (GRA) proteins are known to play roles in nutrient acquisition, host cell cycle manipulation, and immune regulation. Here, we characterize a novel dense granule protein named GRA83, which localizes to the parasitophorous vacuole (PV) in tachyzoites and bradyzoites. Disruption of GRA83 results in increased virulence, weight loss, and parasitemia during the acute infection, as well as a marked increase in the cyst burden during the chronic infection. This increased parasitemia was associated with an accumulation of inflammatory infiltrates in tissues in both acute and chronic infections. Murine macrophages infected with ∆gra83 tachyzoites produced less interleukin-12 (IL-12) in vitro, which was confirmed with reduced IL-12 and interferon-gamma in vivo. This dysregulation of cytokines correlates with reduced nuclear translocation of the p65 subunit of the nuclear factor-κB (NF-κB) complex. While GRA15 similarly regulates NF-κB, infection with ∆gra83/∆gra15 parasites did not further reduce p65 translocation to the host cell nucleus, suggesting these GRAs function in converging pathways. We also used proximity labeling experiments to reveal candidate GRA83 interacting T. gondii-derived partners. Taken together, this work reveals a novel effector that stimulates the innate immune response, enabling the host to limit the parasite burden. Importance Toxoplasma gondii poses a significant public health concern as it is recognized as one of the leading foodborne pathogens in the United States. Infection with the parasite can cause congenital defects in neonates, life-threatening complications in immunosuppressed patients, and ocular disease. Specialized secretory organelles, including the dense granules, play an important role in the parasite's ability to efficiently invade and regulate components of the host's infection response machinery to limit parasite clearance and establish an acute infection. Toxoplasma's ability to avoid early clearance, while also successfully infecting the host long enough to establish a persistent chronic infection, is crucial in allowing for its transmission to a new host. While multiple GRAs directly modulate host signaling pathways, they do so in various ways highlighting the parasite's diverse arsenal of effectors that govern infection. Understanding how parasite-derived effectors harness host functions to evade defenses yet ensure a robust infection is important for understanding the complexity of the pathogen's tightly regulated infection. In this study, we characterize a novel secreted protein named GRA83 that stimulates the host cell's response to limit infection.

The Toxoplasma gondii effector GRA83 modulates the host's innate immune response to regulate parasite infection.

Toxoplasma gondii 's propensity to infect its host and cause disease is highly dependent on its ability to modulate host cell functions. One of the strategies the parasite uses to accomplish this is via the export of effector proteins from the secretory dense granules. Dense granule (GRA) proteins are known to play roles in nutrient acquisition, host cell cycle manipulation, and immune regulation. Here, we characterize a novel dense granule protein named GRA83, which localizes to the parasitophorous vacuole in tachyzoites and bradyzoites. Disruption of GRA83 results in increased virulence, weight loss, and parasitemia during the acute infection, as well as a marked increase in the cyst burden during the chronic infection. This increased parasitemia was associated with an accumulation of inflammatory infiltrates in tissues in both the acute and chronic infection. Murine macrophages infected with Δ gra83 tachyzoites produced less interleukin-12 (IL-12) in vitro , which was confirmed with reduced IL-12 and interferon gamma (IFN-γ) in vivo . This dysregulation of cytokines correlates with reduced nuclear translocation of the p65 subunit of the NF-κB complex. While GRA15 similarly regulates NF-κB, infection with Δ gra83/ Δ gra15 parasites did not further reduce p65 translocation to the host cell nucleus, suggesting these GRAs function in converging pathways. We also used proximity labelling experiments to reveal candidate GRA83 interacting T. gondii derived partners. Taken together, this work reveals a novel effector that stimulates the innate immune response, enabling the host to limit parasite burden. Importance: Toxoplasma gondii poses a significant public health concern as it is recognized as one of the leading foodborne pathogens in the United States. Infection with the parasite can cause congenital defects in neonates, life-threatening complications in immunosuppressed patients, and ocular disease. Specialized secretory organelles, including the dense granules, play an important role in the parasite's ability to efficiently invade and regulate components of the host's infection response machinery to limit parasite clearance and establish an acute infection. Toxoplasma' s ability to avoid early clearance, while also successfully infecting the host long enough to establish a persistent chronic infection, is crucial in allowing for its transmission to a new host. While multiple GRAs directly modulate host signaling pathways, they do so in various ways highlighting the parasite's diverse arsenal of effectors that govern infection. Understanding how parasite-derived effectors harness host functions to evade defenses yet ensure a robust infection are important for understanding the complexity of the pathogen's tightly regulated infection. In this study, we characterize a novel secreted protein named GRA83 that stimulates the host cell's response to limit infection.

In Vivo Biotinylation of the Toxoplasma Parasitophorous Vacuole Reveals Novel Dense Granule Proteins Important for Parasite Growth and Pathogenesis.

UNLABELLED: Toxoplasma gondii is an obligate intracellular parasite that invades host cells and replicates within a unique parasitophorous vacuole. To maintain this intracellular niche, the parasite secretes an array of dense granule proteins (GRAs) into the nascent parasitophorous vacuole. These GRAs are believed to play key roles in vacuolar remodeling, nutrient uptake, and immune evasion while the parasite is replicating within the host cell. Despite the central role of GRAs in the Toxoplasma life cycle, only a subset of these proteins have been identified, and many of their roles have not been fully elucidated. In this report, we utilize the promiscuous biotin ligase BirA* to biotinylate GRA proteins secreted into the vacuole and then identify those proteins by affinity purification and mass spectrometry. Using GRA-BirA* fusion proteins as bait, we have identified a large number of known and candidate GRAs and verified localization of 13 novel GRA proteins by endogenous gene tagging. We proceeded to functionally characterize three related GRAs from this group (GRA38, GRA39, and GRA40) by gene knockout. While Δgra38 and Δgra40 parasites showed no altered phenotype, disruption of GRA39 results in slow-growing parasites that contain striking lipid deposits in the parasitophorous vacuole, suggesting a role in lipid regulation that is important for parasite growth. In addition, parasites lacking GRA39 showed dramatically reduced virulence and a lower tissue cyst burden in vivo Together, the findings from this work reveal a partial vacuolar proteome of T. gondii and identify a novel GRA that plays a key role in parasite replication and pathogenesis. IMPORTANCE: Most intracellular pathogens reside inside a membrane-bound vacuole within their host cell that is extensively modified by the pathogen to optimize intracellular growth and avoid host defenses. In Toxoplasma, this vacuole is modified by a host of secretory GRA proteins, many of which remain unidentified. Here we demonstrate that in vivo biotinylation of proximal and interacting proteins using the promiscuous biotin ligase BirA* is a powerful approach to rapidly identify vacuolar GRA proteins. We further demonstrate that one factor identified by this approach, GRA39, plays an important role in the ability of the parasite to replicate within its host cell and cause disease.

Small-molecule inhibition of a depalmitoylase enhances Toxoplasma host-cell invasion.

Although there have been numerous advances in our understanding of how apicomplexan parasites such as Toxoplasma gondii enter host cells, many of the signaling pathways and enzymes involved in the organization of invasion mediators remain poorly defined. We recently performed a forward chemical-genetic screen in T. gondii and identified compounds that markedly enhanced infectivity. Although molecular dissection of invasion has benefited from the use of small-molecule inhibitors, the mechanisms underlying induction of invasion by small-molecule enhancers have never been described. Here we identify the Toxoplasma ortholog of human APT1, palmitoyl protein thioesterase-1 (TgPPT1), as the target of one class of small-molecule enhancers. Inhibition of this uncharacterized thioesterase triggered secretion of invasion-associated organelles, increased motility and enhanced the invasive capacity of tachyzoites. We demonstrate that TgPPT1 is a bona fide depalmitoylase, thereby establishing an important role for dynamic and reversible palmitoylation in host-cell invasion by T. gondii.

Cytoskeleton assembly in Toxoplasma gondii cell division.

Cell division across members of the protozoan parasite phylum Apicomplexa displays a surprising diversity between different species as well as between different life stages of the same parasite. In most cases, infection of a host cell by a single parasite results in the formation of a polyploid cell from which individual daughters bud in a process dependent on a final round of mitosis. Unlike other apicomplexans, Toxoplasma gondii divides by a binary process consisting of internal budding that results in only two daughter cells per round of division. Since T. gondii is experimentally accessible and displays the simplest division mode, it has manifested itself as a model for apicomplexan daughter formation. Here, we review newly emerging insights in the prominent role that assembly of the cortical cytoskeletal scaffold plays in the process of daughter parasite formation.

Identification of potential serodiagnostic and subunit vaccine antigens by antibody profiling of toxoplasmosis cases in Turkey.

Toxoplasmosis, caused by infection of the protozoan parasite Toxoplasma gondii, is associated with mild disease in healthy individuals, whereas individuals with depressed immunity may develop encephalitis, neurologic disorders, and other organ diseases. Women who develop acute toxoplasmosis during pregnancy are at risk of transmitting the infection to the fetus, which may lead to fetal damage. A diagnosis is usually confirmed by measuring IgG, or IgM where it is important to determine the onset of infection. A negative IgM result essentially excludes acute infection, whereas a positive IgM test is largely uninterpretable because IgM can persist for up to 18 months after infection. To identify antigens for improved diagnosis of acute infection, we probed protein microarrays displaying the polypeptide products of 1357 Toxoplasma exons with well-characterized sera from Turkey. The sera were classified according to conventional assays into (1) seronegative individuals with no history of T. gondii infection; (2) acute infections defined by clinical symptoms, high IgM titers, and low avidity IgG; (3) chronic/convalescent cases with high avidity IgG but persisting IgM; (iv) true chronic infections, defined by high avidity IgG and no IgM. We have identified 38 IgG target antigens and 108 IgM target antigens that can discriminate infected patients from healthy controls, one or more of which could form the basis of a 'tier-1' test to determine current or previous exposure. Of these, three IgG antigens and five IgM antigens have the potential to discriminate chronic/IgM persisting or true chronics from recent acutely infected patients (a 'tier-2' test). Our analysis of the antigens revealed several enriched features relative to the whole proteome, which include transmembrane domains, signal peptides, or predicted localization at the outer membrane. This is the first protein microarray survey of the antibody response to T. gondii, and will help in the development of improved serodiagnostics and vaccines.