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Background: The transmission of parasites, including Babesia, plasmodia, and Trypanosoma cruzi, via transfusions is an important public health concern. INACTINE technology is a pathogen-reduction process that utilizes PEN110, an electrophilic agent that inac-tivates a wide range of pathogens by disrupting nucleic acid replication. The present study investigated the effect of PEN110 treatment on the viability of protozoa in RBCs.Study Design and Methods: B. microti-parasitized RBCs from infected hamsters were treated with PEN110 and inoculated to naïve animals. Parasitemia was detected by blood smears and PCR. Human RBCs infected with P. falciparum were treated with PEN110 and incubated with fresh RBCs. P. falciparum multiplication was detected by blood smears. Human RBCs spiked with T. cruzi and treated with PEN110 were analyzed for the presence of live parasites using in-vitro infectivity assay or by inoculating susceptible mice.Results: Treatment of RBCs infected with B. microti or P. falciparum with 0.01 to 0.1 percent (vol/vol) PEN110 resulted in parasite inactivation to below the limit of detection during 24 hours. T. cruzi inoculated into human RBCs was inactivated below the limit of detection by 0.1 percent PEN110 after 3 hours.Conclusion: The study demonstrates that treatment of blood with PEN110 is highly effective in eradicating transfusion-transmitted protozoan parasites. [ABSTRACT FROM AUTHOR]

As a model organism in the study of immunity to infection, Toxoplasma gondii has been instrumental in establishing key principles of host anti-microbial defense and its regulation. Here, we employed an attenuated uracil auxotroph strain of Type I Toxoplasma designated OMP to further untangle the early immune response to this parasitic pathogen. Experiments using αβ T cell-deficient Tcrb -/- mice unexpectedly revealed that an intact αβ T lymphocyte compartment was essential to survive infection with OMP. Subsequent antibody depletion and knockout mouse experiments demonstrated contributions from CD4 + T cells and most predominantly CD8 + T cells in resistance. Using transgenic knockout mice, we found only a partial requirement for IFN-γ and a lack of requirement for Toll-like receptor (TLR) adaptor MyD88 in resistance. In contrast to other studies on Toxoplasma , the ability to survive OMP infection did not require IL-12p40. Surprisingly, T cell-dependent IL-10 was found to be critical for survival, and deficiency of this cytokine triggered an abnormally high systemic inflammatory response. We also found that parasite molecule GRA24, a dense granule protein that triggers TLR-independent IL-12 production, acts as a virulence factor contributing to death of OMP-infected Tcrb -/- and IL-10 -/- mice. Furthermore, resistance against OMP was restored in Tcrb -/- mice via monoclonal depletion of IL-12p40, suggesting that GRA24-induced IL-12 underlies the fatal immunopathology observed. Collectively, our studies provide insight into a novel and rapidly arising T lymphocyte-dependent anti-inflammatory response to T. gondii which operates independently of MyD88 and IL-12 and that depends on the function of parasite-dense granule protein GRA24.IMPORTANCEAs a model infectious microbe and an important human pathogen, the apicomplexan Toxoplasma gondii has provided many important insights into innate and adaptive immunity to infection. We show here that a low virulence uracil auxotrophic Toxoplasma strain emerges as a virulent parasite in the absence of an intact T cell compartment. Both CD4 + and CD8 + T lymphocytes are required for optimal protection, in line with previous findings in other models of Toxoplasma infection. Nevertheless, several novel aspects of the response were identified in our study. Protection occurs independently of IL-12 and MyD88 and only partially requires IFN-γ. This is noteworthy particularly because the cytokines IL-12 and IFN-γ have previously been regarded as essential for protective immunity to T. gondii . Instead, we identified the anti-inflammatory effects of T cell-dependent IL-10 as the critical factor enabling host survival. The parasite dense granule protein GRA24, a host-directed mitogen-activated protein kinase activator, was identified as a major virulence factor in T cell-deficient hosts. Collectively, our results provide new and unexpected insights into host resistance to Toxoplasma ., Competing Interests: The authors declare no conflict of interest.

Toxoplasma , an important intracellular parasite of humans and animals, causes life-threatening toxoplasmosis in immunocompromised individuals. Although Toxoplasma secretory proteins during acute infection (tachyzoite, which divides rapidly and causes inflammation) have been extensively characterized, those involved in chronic infection (bradyzoite, which divides slowly and is surrounded by a cyst wall) remain uncertain. Regulation of the cyst wall is essential to the parasite life cycle, and polysaccharides, such as chitin, in the cyst wall are necessary to sustain latent infection. Toxoplasma secretory proteins during the bradyzoite stage may have important roles in regulating the cyst wall via polysaccharides. Here, we focused on characterizing the hypothetical T. gondii chitinase, chitinase-like protein 1 (TgCLP1). We found that the chitinase-like domain containing TgCLP1 is partially present in the bradyzoite microneme and confirmed, albeit partially, its previous identification in the tachyzoite microneme. Furthermore, although parasites lacking TgCLP1 could convert from tachyzoites to bradyzoites and make an intact cyst wall, they failed to convert from bradyzoites to tachyzoites, indicating that TgCLP1 is necessary for bradyzoite reactivation. Taken together, our findings deepen our understanding of the molecular basis of recrudescence and could contribute to the development of novel strategies for the control of toxoplasmosis., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 Bando, Murata, Han, Sugi, Fukuda, Bzik, Fox and Kato.)

Toxoplasma gondii is a pervasive protozoan parasite that is responsible for significant zoonoses. A wide array of vaccines using different effector molecules of T. gondii have been studied worldwide to control toxoplasmosis. None of the existing vaccines are sufficiently effective to confer protective immunity. Among the different Toxoplasma-derived effector molecules, T. gondii dense granule protein 15 from the type II strain (GRA15 (II)) was recently characterized as an immunomodulatory molecule that induced host immunity via NF-κB. Therefore, we assessed the immunostimulatory and protective efficacy of recombinant GRA15 (II) (rGRA15) against T. gondii infection in a C57BL/6 mouse model. We observed that rGRA15 treatment increased the production of IL-12p40 from mouse peritoneal macrophages in vitro. Immunization of mice with rGRA15 induced the production of anti-TgGRA15-specific IgG, IgG1 and IgG2c antibodies. The rGRA15-sensitized spleen cells from mice inoculated with the same antigen strongly promoted spleen cell proliferation and IFN-γ production. Immunization with rGRA15 significantly enhanced the survival rate of mice and dramatically decreased parasite burden in mice challenged with the Pru (type II) strain. These results suggested that rGRA15 triggered humoral and cellular immune responses to control infection. However, all of the immunized mice died when challenged with the GRA15-deficient Pru strain or the RH (type I) strain. These results suggest that GRA15 (II)-dependent immunity plays a crucial role in protection against challenge infection with the type II strain of T. gondii. This study is the first report to show GRA15 (II) as a recombinant vaccine antigen against Toxoplasma infection., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

Toxoplasma gondii causes morbidity, mortality, and disseminates widely via cat sexual stages. Here, we find T. gondii ornithine aminotransferase (OAT) is conserved across phyla. We solve Tg O/GABA-AT structures with bound inactivators at 1.55 Å and identify an inactivator selective for Tg O/GABA-AT over human OAT and GABA-AT. However, abrogating Tg O/GABA-AT genetically does not diminish replication, virulence, cyst-formation, or eliminate cat's oocyst shedding. Increased sporozoite/merozoite Tg O/GABA-AT expression led to our study of a mutagenized clone with oocyst formation blocked, arresting after forming male and female gametes, with "Rosetta stone"-like mutations in genes expressed in merozoites. Mutations are similar to those in organisms from plants to mammals, causing defects in conception and zygote formation, affecting merozoite capacitation, pH/ionicity/sodium-GABA concentrations, drawing attention to cyclic AMP/PKA, and genes enhancing energy or substrate formation in Tg O/GABA-AT-related-pathways. These candidates potentially influence merozoite's capacity to make gametes that fuse to become zygotes, thereby contaminating environments and causing disease., Competing Interests: H.V.L. is currently a program officer at the National Institute on Drug Abuse at the National Institutes of Health. H.A.L. is currently a scientist working at National Institute of Diabetes, Digestive and Kidney Disease (NIDDK) at the National Institutes of Health. The findings and conclusions of this article are those of the authors and do not necessarily reflect the views of the National Institute on Drug Abuse, National Institute of Diabetes, Digestive and Kidney Diseases (NIDDK), the National Institutes of Health, nor the US Department of Health and Human Services. R.B.S. and R.L.M., with H.V.L., submitted Patent Number: US 10,632,088 B2 INACTIVATORS OF TOXOPLASMA GONDII ORNITHINE AMINOTRANSFERASE FOR TREATING TOXOPLASMOSIS AND MALARIA through their Technology Transfer offices. The authors declare no competing financial interest., (© 2023 The Authors.)

Toxoplasma gondii establishes chronic infection by forming tissue cysts, and this chronic infection is one of the most common parasitic infections in humans. Our recent studies revealed that whereas CD8 + T cells of genetically resistant BALB/c mice have the capability to remove the tissue cysts of the parasite through their perforin-mediated activities, small portions of the cysts are capable of persisting in the presence of the anti-cyst CD8 + T cells. It is currently unknown how those small portions of the cysts resist or escape the T-cell immunity and persist in the hosts. In the present study, we discovered that the cysts, which persisted in the presence of the perforin-mediated CD8 + T-cell immunity, have significantly greater mRNA levels for four dense granule proteins, GRA1, GRA2, GRA3, and GRA7, and one rhoptry protein, ROP35, than the total population of the cysts present in the absence of the T cells. In addition, increased levels of mRNA for GRA1, GRA3, and ROP35 in the cysts significantly correlated with their successful persistence through the condition in which greater degrees of reduction of the cyst burden occurred through anti-cyst CD8 + T cells. In addition, GRA3-deficient T. gondii displayed significantly enhanced elimination of the cysts by anti-cyst CD8 + T cells when compared to the wild-type parasite. These results indicate that GRA3 is a key molecule that mediates in the capability of T. gondii cysts to persist by resisting or evading the anti-cyst activity of CD8 + T cells during the later stage of infection., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Mani, Abdelaziz, Ochiai, Sa, Fox, Bzik and Suzuki.)

Nutrient acquisition by apicomplexan parasites is essential to drive their intracellular replication, yet the mechanisms that underpin essential nutrient acquisition are not defined. Using the apicomplexan model Toxoplasma gondii , we show that host cell proteins including the transferrin receptor 1, transferrin, ferritin heavy and light chains, and clathrin light chain are robustly taken up by tachyzoites. Tachyzoite acquisition of host cell protein was not related to host cell type or parasite virulence phenotypes. Bradyzoites possessed little capacity to acquire host cell proteins consistent with the cyst wall representing a barrier to host cell protein cargo. Increased trafficking of host cell transferrin receptor 1 and transferrin to endolysosomes boosted tachyzoite acquisition of host proteins and growth rate. Theft of host transferrin 1 and transferrin did not significantly affect iron levels in the tachyzoite. This study provides insight into essential functions associated with parasite theft of host iron sequestration and storage proteins.

Resistance and tolerance are vital for survivability of the host-pathogen relationship. Virulence during Toxoplasma infection in mice is mediated by parasite kinase-dependent antagonism of IFN-γ-induced host resistance. Whether avirulence requires expression of parasite factors that induce host tolerance mechanisms or is a default status reflecting the absence of resistance-interfering factors is not known. In this study, we present evidence that avirulence in Toxoplasma requires parasite engagement of the scavenger receptor CD36. CD36 promotes macrophage tropism but is dispensable for the development of resistance mechanisms. Instead CD36 is critical for re-establishing tissue homeostasis and survival following the acute phase of infection. The CD36-binding capacity of T. gondii strains is negatively controlled by the virulence factor, ROP18. Thus, the absence of resistance-interfering virulence factors and the presence of tolerance-inducing avirulence factors are both required for long-term host-pathogen survival., (Copyright © 2021 by The American Association of Immunologists, Inc.)

Toxoplasma gondii causes a chronic infection that renders the immunocompromised human host susceptible to toxoplasmic encephalitis triggered by cyst reactivation in the central nervous system. The dense granule protein GRA12 is a major parasite virulence factor required for parasite survival during acute infection. Here, we characterized the role of four GRA12-related genes in acute and chronic stages of infection. While GRA12A, GRA12B, and GRA12D were highly expressed in asexual stage tachyzoites and bradyzoites, expression of GRA12C appeared to be restricted to the sexual stages. In contrast to deletion of GRA12 (Δ gra12 ), no major defects in acute virulence were observed in Δ gra12A , Δ gra12B , or Δg ra12D parasites, though Δ gra12B parasites exhibited an increased tachyzoite replication rate. Bradyzoites secreted GRA12A, GRA12B, and GRA12D and incorporated these molecules into the developing cyst wall, as well as the cyst matrix in distinct patterns. Similar to GRA12, GRA12A, GRA12B, and GRA12D colocalized with the dense granules in extracellular tachyzoites, with GRA2 and the intravacuolar network in the tachyzoite stage parasitophorous vacuole and with GRA2 in the cyst matrix and cyst wall. Chronic stage cyst burdens were decreased in mice infected with Δ gra12A parasites and were increased in mice infected with Δ gra12B parasites. However, Δ gra12B cysts were not efficiently maintained in vivo Δ gra12A , Δ gra12B , and Δ gra12D in vitro cysts displayed a reduced reactivation efficiency, and reactivation of Δ gra12A cysts was delayed. Collectively, our results suggest that a family of genes related to GRA12 play significant roles in the formation, maintenance, and reactivation of chronic stage cysts. IMPORTANCE If host immunity weakens, Toxoplasma gondii cysts recrudesce in the central nervous system and cause a severe toxoplasmic encephalitis. Current therapies target acute stage infection but do not eliminate chronic cysts. Parasite molecules that mediate the development and persistence of chronic infection are poorly characterized. Dense granule (GRA) proteins such as GRA12 are key virulence factors during acute infection. Here, we investigated four GRA12-related genes. GRA12-related genes were not major virulence factors during acute infection. Instead, GRA12-related proteins localized at the cyst wall and cyst matrix and played significant roles in cyst development, persistence, and reactivation during chronic infection. Similar to GRA12, the GRA12-related proteins selectively associated with the intravacuolar network of membranes inside the vacuole. Collectively, our results support the hypothesis that GRA12 proteins associated with the intravacuolar membrane system support parasite virulence during acute infection and cyst development, persistence, and reactivation during chronic infection., (Copyright © 2021 Guevara et al.)

The apicomplexan Toxoplasma gondii induces strong protective immunity dependent upon recognition by Toll-like receptors (TLR)11 and 12 operating in conjunction with MyD88 in the murine host. However, TLR11 and 12 proteins are not present in humans, inspiring us to investigate MyD88-independent pathways of resistance. Using bicistronic IL-12-YFP reporter mice on MyD88+/+ and MyD88-/- genetic backgrounds, we show that CD11c+MHCII+F4/80- dendritic cells, F4/80+ macrophages, and Ly6G+ neutrophils were the dominant cellular sources of IL-12 in both wild type and MyD88 deficient mice after parasite challenge. Parasite dense granule protein GRA24 induces p38 MAPK activation and subsequent IL-12 production in host macrophages. We show that Toxoplasma triggers an early and late p38 MAPK phosphorylation response in MyD88+/+ and MyD88-/- bone marrow-derived macrophages. Using the uracil auxotrophic Type I T. gondii strain cps1-1, we demonstrate that the late response does not require active parasite proliferation, but strictly depends upon GRA24. By i. p. inoculation with cps1-1 and cps1-1:Δgra24, we identified unique subsets of chemokines and cytokines that were up and downregulated by GRA24. Finally, we demonstrate that cps1-1 triggers a strong host-protective GRA24-dependent Th1 response in the absence of MyD88. Our data identify GRA24 as a major mediator of p38 MAPK activation, IL-12 induction and protective immunity that operates independently of the TLR/MyD88 cascade., Competing Interests: The authors have declared that no competing interests exist.

Determination of the optimum pH in a coupled enzyme assay poses significant challenges because altering the pH of the reaction mixture can affect the performance of both enzymes. Here, we demonstrate a simple and reliable method to determine the pH optimum for pyruvate kinase using the pyruvate kinase/lactate dehydrogenase coupled enzyme assay. This simple and reliable method can be broadly adapted to determine the pH optimum for various enzymes that are assayed using a coupled enzyme assay.

The glycosylated mucin domain of the Toxoplasma gondii cyst wall glycoprotein CST1 is heavily stained by Dolichos biflorus agglutinin, a lectin that binds to N -acetylgalactosamine. The cyst wall is also heavily stained by the chitin binding lectin succinylated wheat germ agglutinin (s-WGA), which selectively binds to N- acetylglucosamine-decorated structures. Here, we tracked the localization of N- acetylglucosamine-decorated structures that bind to s-WGA in immature and mature in vitro cysts. s-WGA localization was observed at the cyst periphery 6 h after the differentiation of the tachyzoite-stage parasitophorous vacuole. By day 1 and at all later times after differentiation, s-WGA was localized in a continuous staining pattern at the cyst wall. Coinciding with the maturation of the cyst matrix by day 3 of cyst development, s-WGA also localized in a continuous matrix pattern inside the cyst. s-WGA localized in both the outer and inner layer regions of the cyst wall and in a continuous matrix pattern inside mature 7- and 10-day-old cysts. In addition, s-WGA colocalized in the cyst wall with CST1, suggesting that N -acetylglucosamine- and N -acetylgalactosamine-decorated molecules colocalized in the cyst wall. In contrast to CST1, GRA4, and GRA6, the relative accumulation of the molecules that bind s-WGA in the cyst wall was not dependent on the expression of GRA2. Our results suggest that GRA2-dependent and GRA2-independent mechanisms regulate the trafficking and accumulation of glycosylated molecules that colocalize in the cyst wall. IMPORTANCE Chronic Toxoplasma gondii infection is maintained in the central nervous system by thick-walled cysts. If host immunity wanes, cysts recrudesce and cause severe and often lethal toxoplasmic encephalitis. Currently, there are no therapies to eliminate cysts, and little biological information is available regarding cyst structure(s). Here, we investigated cyst wall molecules recognized by succinylated wheat germ agglutinin (s-WGA), a lectin that specifically binds to N- acetylglucosamine-decorated structures. N- Acetylglucosamine regulates cell signaling and plays structural roles at the cell surface in many organisms. The cyst wall and cyst matrix were heavily stained by s-WGA in mature cysts and were differentially stained during cyst development. The relative accumulation of molecules that bind to s-WGA in the cyst wall was not dependent on the expression of GRA2. Our findings suggest that glycosylated cyst wall molecules gain access to the cyst wall via GRA2-dependent and GRA2-independent mechanisms and colocalize in the cyst wall., (Copyright © 2020 Guevara et al.)

After differentiation is triggered, the tachyzoite-stage Toxoplasma gondii parasitophorous vacuole membrane (PVM) has been hypothesized to transition into the cyst membrane that surrounds the cyst wall and encloses bradyzoites. Here, we tracked the localization of two PVM dense granule (GRA) proteins (GRA5 and GRA7) after in vitro differentiation of the tachyzoite stage parasitophorous vacuole into the mature cyst. GRA5 and GRA7 were visible at the cyst periphery at 6 h and at all later times after differentiation, suggesting that the PVM remained intact as it transitioned into the cyst membrane. By day 3 postdifferentiation, GRA5 and GRA7 were visible in a continuous pattern at the cyst periphery. In mature 7- and 10-day-old cysts permeabilized with a saponin pulse, GRA5 and GRA7 were localized to the cyst membrane and the cyst wall regions. Cysts at different stages of cyst development exhibited differential susceptibility to saponin permeabilization, and, correspondingly, saponin selectively removed GRA5 from the cyst membrane and cyst wall region in 10-day-old cysts. GRA5 and GRA7 were localized at the cyst membrane and cyst wall region at all times after differentiation of the parasitophorous vacuole, which supports a previous model proposing that the PVM develops into the cyst membrane. In addition, evaluation of Δ gra3 , Δ gra5 , Δ gra7 , Δ gra8 , and Δ gra14 mutants revealed that PVM-localized GRAs were crucial to support the normal rate of accumulation of cyst wall proteins at the cyst periphery. IMPORTANCE Toxoplasma gondii establishes chronic infection in humans by forming thick-walled cysts that persist in the brain. Once host immunity wanes, cysts reactivate to cause severe, and often lethal, toxoplasmic encephalitis. There is no available therapy to eliminate cysts or to prevent their reactivation. Furthermore, how the cyst membrane and cyst wall structures develop is poorly understood. Here, we visualized and tracked the localization of Toxoplasma parasitophorous vacuole membrane (PVM) dense granules (GRA) proteins during cyst development in vitro. PVM-localized GRA5 and GRA7 were found at the cyst membrane and cyst wall region throughout cyst development, suggesting that the PVM remains intact and develops into the cyst membrane. In addition, our results show that genetic deletion of PVM GRAs reduced the rate of accumulation of cyst wall cargo at the cyst periphery and suggest that PVM-localized GRAs mediate the development and maturation of the cyst wall and cyst membrane., (Copyright © 2020 Guevara et al.)

Little is known regarding how the chronic Toxoplasma gondii cyst develops. Here, we investigated intravacuolar-network-associated dense granule (GRA) proteins GRA1, GRA2, GRA4, GRA6, GRA9, and GRA12 during cyst development in vitro after differentiation of the tachyzoite-stage parasitophorous vacuole. By day 1 postdifferentiation, GRA1, GRA4, GRA6, GRA9, and GRA12 colocalized with Dolichos biflorus agglutinin stain at the cyst periphery. In contrast, GRA2 remained in the cyst matrix. By day 2 postdifferentiation, coinciding with localization of GRA2 to the cyst periphery, GRA1, GRA4, GRA6, and GRA9 established a continuous matrix pattern in the cyst. In contrast, GRA2 and GRA12 were colocalized in prominent cyst matrix puncta throughout cyst development. While GRA2, GRA6, and GRA12 localized in outer and inner layers of the cyst wall, GRA1, GRA4, and GRA9 localized predominantly in the inner layers of the cyst wall. GRA2 and GRA12 were colocalized in the cyst wall by day 7 postdifferentiation. However, by day 10 postdifferentiation, GRA12 was relocalized from the cyst wall to puncta in the cyst matrix. Differentiation of Δ gra2 parasites revealed a defect in the ability to establish a normal cyst matrix. In addition, the deletion of any intravacuolar-network-associated GRA protein, except GRA1, reduced the rate of accumulation of cyst wall proteins at the cyst periphery relative to the cyst interior. Our findings reveal dynamic patterns of GRA protein localization during cyst development and suggest that intravacuolar-network-associated GRA proteins regulate the formation and maturation of the cyst matrix and cyst wall structures. IMPORTANCE Toxoplasma gondii establishes chronic infection in humans by forming thick-walled cysts that persist in the brain. If host immunity wanes, cysts reactivate to cause severe, and often lethal, toxoplasmic encephalitis. There is no available therapy to eliminate cysts or to prevent their reactivation. Moreover, how the vital and characteristic cyst matrix and cyst wall structures develop is poorly understood. Here, we visualized and tracked the localization of Toxoplasma intravacuolar-network-associated dense granule (GRA) proteins during cyst development in vitro Intravacuolar-network GRAs were present within the cyst matrix and at the cyst wall in developing cysts, and genetic deletion of intravacuolar-network-associated GRAs reduced the rate of accumulation of cyst wall material at the cyst periphery. Our results show that intravacuolar-network-associated GRAs, particularly GRA2 and GRA12, play dynamic and essential roles in the development and maturation of the cyst matrix and the cyst wall structures., (Copyright © 2019 Guevara et al.)

Toxoplasma gondii secretes rhoptry (ROP) and dense granule (GRA) effector proteins to evade host immune clearance mediated by interferon gamma (IFN-γ), immunity-related GTPase (IRG) effectors, and CD8 + T cells. Here, we investigated the role of parasite-secreted effectors in regulating host access to parasitophorous vacuole (PV) localized parasite antigens and their presentation to CD8 + T cells by the major histocompatibility class I (MHC-I) pathway. Antigen presentation of PV localized parasite antigens by MHC-I was significantly increased in macrophages and/or dendritic cells infected with mutant parasites that lacked expression of secreted GRA (GRA2, GRA3, GRA4, GRA5, GRA7, GRA12) or ROP (ROP5, ROP18) effectors. The ability of various secreted GRA or ROP effectors to suppress antigen presentation by MHC-I was dependent on cell type, expression of IFN-γ, or host IRG effectors. The suppression of antigen presentation by ROP5, ROP18, and GRA7 correlated with a role for these molecules in preventing PV disruption by IFN-γ-activated host IRG effectors. However, GRA2 mediated suppression of antigen presentation was not correlated with PV disruption. In addition, the GRA2 antigen presentation phenotypes were strictly co-dependent on the expression of the GRA6 protein. These results show that MHC-I antigen presentation of PV localized parasite antigens was controlled by mechanisms that were dependent or independent of IRG effector mediated PV disruption. Our findings suggest that the GRA6 protein underpins an important mechanism that enhances CD8 + T cell recognition of parasite-infected cells with damaged or ruptured PV membranes. However, in intact PVs, parasite secreted effector proteins that associate with the PV membrane or the intravacuolar network membranes play important roles to actively suppress antigen presentation by MHC-I to reduce CD8 + T cell recognition and clearance of Toxoplasma gondii infected host cells.

Little is known about whether pathogen invasion of neural tissue is affected by immune-based mechanisms in endothelial cells. We examined the effects of endothelial cell CD40 on Toxoplasma gondii invasion of the retina and brain, organs seeded hematogenously. T. gondii circulates in the bloodstream within infected leukocytes (including monocytes and dendritic cells) and as extracellular tachyzoites. After T. gondii infection, mice that expressed CD40 restricted to endothelial cells exhibited diminished parasite loads and histopathology in the retina and brain. These mice also had lower parasite loads in the retina and brain after intravenous (i.v.) injection of infected monocytes or dendritic cells. The protective effect of endothelial cell CD40 was not explained by changes in cellular or humoral immunity, reduced transmigration of leukocytes into neural tissue, or reduced invasion by extracellular parasites. Circulating T. gondii -infected leukocytes (dendritic cells used as a model) led to infection of neural endothelial cells. The number of foci of infection in these cells were reduced if endothelial cells expressed CD40. Infected dendritic cells and macrophages expressed membrane-associated inducible Hsp70. Infected leukocytes triggered Hsp70-dependent autophagy in CD40 + endothelial cells and anti- T. gondii activity dependent on ULK1 and beclin 1. Reduced parasite load in the retina and brain not only required CD40 expression in endothelial cells but was also dependent on beclin 1 and the expression of inducible Hsp70 in dendritic cells. These studies suggest that during endothelial cell-leukocyte interaction, CD40 restricts T. gondii invasion of neural tissue through a mechanism that appears mediated by endothelial cell anti-parasitic activity stimulated by Hsp70., (Copyright © 2019 American Society for Microbiology.)

Toxoplasma gondii evades host immunity to establish a chronic infection. Here, we assessed the role of parasitophorous vacuole (PV) membrane (PVM)- and intravacuolar network (IVN) membrane-localized dense granule (GRA) proteins in the development of acute and chronic Toxoplasma infection. Deletion of PVM-associated GRA3, GRA7, GRA8, and GRA14 or IVN membrane-associated GRA2, GRA9, and GRA12 in the low-virulence type II Prugniaud (Pru) strain induced severe defects in the development of chronic-stage cysts in vivo without affecting the parasite growth rate or the ability to differentiate into cysts in vitro Acute virulence of the PruΔ gra2 , PruΔ gra3 , and PruΔ gra4 mutants was reduced but not abolished. In contrast, the PruΔ gra12 mutant was avirulent in mice and PruΔ gra12 parasites failed to establish a chronic infection. High-virulence type I strain RHΔ gra12 parasites also exhibited a major defect in acute virulence. In gamma interferon (IFN-γ)-activated macrophages, type I RHΔ gra12 and type II PruΔ gra12 parasites resisted the coating of the PVM with host immunity-related GTPases as effectively as the parental type I RHΔ ku80 and type II PruΔ ku80 strains, respectively. Despite this resistance, Δ gra12 PVs ultimately succumbed to IFN-γ-activated host cell innate immunity. Our findings uncover a key role for GRA12 in mediating resistance to host IFN-γ and reveal that many other IVN membrane-associated GRA proteins, as well as PVM-localized GRA proteins, play important roles in establishing chronic infection. IMPORTANCE Toxoplasma gondii cysts reactivate during immune deficiency and cause fatal encephalitis. Parasite molecules that coordinate the development of acute and chronic infection are poorly characterized. Here, we show that many intravacuolar network membrane and parasitophorous vacuole membrane-associated dense granule (GRA) proteins orchestrate the development of chronic cysts in vivo A subset of these GRA proteins also modulate acute virulence, and one protein that associates with the intravacuolar network membranes, namely GRA12, was identified as a major virulence factor required for parasite resistance to host gamma interferon (IFN-γ). Our results revealed that many parasitophorous vacuole membrane and intravacuolar network membrane-associated GRA proteins are essential for successful chronic infection., (Copyright © 2019 Fox et al.)

After host cell invasion, Toxoplasma secretes a variety of dense granule proteins (GRA proteins) from its secretory dense granules, which are involved in the biogenesis of the parasitophorous vacuole (PV). TgGRA8 I is predicted to contain proline-rich domains, which are structural features of some cytoskeleton-related proteins. In agreement with this observation, previous proteomic analyses revealed the presence of TgGRA8 I in the Toxoplasma sub-pellicular cytoskeleton. In the present study, we show (1) by docking analyses that TgGRA8 I may interact with both Toxoplasma β-tubulin and actin; (2) by immunoelectron microscopy, proteomic, biochemical, and cellular approaches that TgGRA8 I associates with sub-pellicular microtubules and actin at the parasite sub-pellicular cytoskeleton; (3) that type I parasites (RH strain) lacking the GRA8 gene (RHΔku80Δgra8) exhibit loss of conoid extrusion, diminished cell infection, and egress capabilities, and that these motility impairments were likely due to important alterations in their sub-pellicular cytoskeleton, in particular their sub-pellicular microtubules and meshwork. Parasites lacking the GRA4 gene (RHΔku80Δgra4) did not show modifications in the organization of the sub-pellicular cytoskeleton. Collectively, these results demonstrated that TgGRA8 I is a dense granule protein that, besides its role in the formation of the PV, contributes to the organization of the parasite sub-pellicular cytoskeleton and motility. This is the first proline-rich protein described in the Toxoplasma cytoskeleton, which is a key organelle for both the parasite motility and the invasion process. Knowledge about the function of cytoskeleton components in Toxoplasma is fundamental to understand the motility process and the host cell invasion mechanism. Refining this knowledge should lead to the design of novel pharmacological strategies for the treatment against toxoplasmosis.

Toxoplasma gondii can grow and replicate using either glucose or glutamine as the major carbon source. Here, we have studied the essentiality of glycolysis in the tachyzoite and bradyzoite stages of T. gondii, using transgenic parasites that lack a functional hexokinase gene (Δhk) in RH (Type-1) and Prugniaud (Type-II) strain parasites. Tachyzoite stage Δhk parasites exhibit a fitness defect similar to that reported previously for the major glucose transporter mutant, and remain virulent in mice. However, although Prugniaud strain Δhk tachyzoites were capable of transforming into bradyzoites in vitro, they were severely compromised in their ability to make mature bradyzoite cysts in the brain tissue of mice. Isotopic labelling studies reveal that glucose-deprived tacyzoites utilise glutamine to replenish glycolytic and pentose phosphate pathway intermediates via gluconeogenesis. Interestingly, while glutamine-deprived intracellular Δhk tachyzoites continued to replicate, extracellular parasites were unable to efficiently invade host cells. Further, studies on mutant tachyzoites lacking a functional phosphoenolpyruvate carboxykinase (Δpepck1) revealed that glutaminolysis is the sole source of gluconeogenic flux in glucose-deprived parasites. In addition, glutaminolysis is essential for sustaining oxidative phosphorylation in Δhk parasites, while wild type (wt) and Δpepck1 parasites can obtain ATP from either glycolysis or oxidative phosphorylation. This study provides insights into the role of nutrient metabolism during asexual propagation and development of T. gondii, and validates the versatile nature of central carbon and energy metabolism in this parasite., (Copyright © 2018 Australian Society for Parasitology. Published by Elsevier Ltd. All rights reserved.)

In the asexual stages, Toxoplasma gondii stage converts between acute phase rapidly replicating tachyzoites and chronic phase slowly dividing bradyzoites. Correspondingly, T. gondii differentially expresses two distinct genes and isoforms of the lactate dehydrogenase enzyme, expressing LDH1 exclusively in the tachyzoite stage and LDH2 preferentially in the bradyzoite stage. LDH catalyzes the interconversion of pyruvate and lactate in anaerobic growth conditions and is utilized for energy supply, however, the precise role of LDH1 and LDH2 in parasite biology in the asexual stages is still unclear. Here, we investigated the biological role of LDH1 and LDH2 in the asexual stages, and the vaccine strain potential of deletion mutants lacking LDH1, LDH2, or both genes (Δldh1, Δldh2 and Δldh1/2). Deletion of LDH1 reduced acute parasite virulence, impaired bradyzoite differentiation in vitro, and markedly reduced chronic stage cyst burdens in vivo. In contrast, deletion of LDH2 impaired chronic stage cyst burdens without affecting virulence or bradyzoite differentiation. Deletion of both LDH1 and LDH2 induced a more severe defect in chronic stage cyst burdens. These LDH mutant phenotypes were not associated with any growth defect. Vaccination of mice with a low dose of mutants deleted for LDH elicited effective protective immunity to lethal challenge infection, demonstrating the vaccine potential of LDH deletion mutants. These results suggest that lactate dehydrogenase in T. gondii controls virulence, bradyzoite differentiation, and chronic infection and reveals the potential of LDH mutants as vaccine strains.

Dihydroorotate dehydrogenase (DHODH) mediates the fourth step of de novo pyrimidine biosynthesis and is a proven drug target for inducing immunosuppression in therapy of human disease as well as a rapidly emerging drug target for treatment of malaria. In Toxoplasma gondii, disruption of the first, fifth, or sixth step of de novo pyrimidine biosynthesis induced uracil auxotrophy. However, previous attempts to generate uracil auxotrophy by genetically deleting the mitochondrion-associated DHODH of T. gondii (TgDHODH) failed. To further address the essentiality of TgDHODH, mutant gene alleles deficient in TgDHODH activity were designed to ablate the enzyme activity. Replacement of the endogenous DHODH gene with catalytically deficient DHODH gene alleles induced uracil auxotrophy. Catalytically deficient TgDHODH localized to the mitochondria, and parasites retained mitochondrial membrane potential. These results show that TgDHODH is essential for the synthesis of pyrimidines and suggest that TgDHODH is required for a second essential function independent of its role in pyrimidine biosynthesis., (Copyright © 2016, American Society for Microbiology. All Rights Reserved.)

Toxoplasma gondii actively invades host cells and establishes a parasitophorous vacuole (PV) that accumulates many proteins secreted by the dense granules (GRA proteins). To date, at least 23 GRA proteins have been reported, though the function(s) of most of these proteins still remains unknown. We targeted gene knockouts at ten GRA gene loci (GRA1-10) to investigate the cellular roles and essentiality of these classical GRA proteins during acute infection in the virulent type I RH strain. While eight of these genes (GRA2-9) were successfully knocked out, targeted knockouts at the GRA1 and GRA10 loci were not obtained, suggesting these GRA proteins may be essential. As expected, the Δgra2 and Δgra6 knockouts failed to form an intravacuolar network (IVN). Surprisingly, Δgra7 exhibited hyper-formation of the IVN in both normal and lipid-free growth conditions. No morphological alterations were identified in parasite or PV structures in the Δgra3, Δgra4, Δgra5, Δgra8, or Δgra9 knockouts. With the exception of the Δgra3 and Δgra8 knockouts, all of the GRA knockouts exhibited defects in their infection rate in vitro. While the single GRA knockouts did not exhibit reduced replication rates in vitro, replication rate defects were observed in three double GRA knockout strains (Δgra4Δgra6, Δgra3Δgra5 and Δgra3Δgra7). However, the virulence of single or double GRA knockout strains in CD1 mice was not affected. Collectively, our results suggest that while the eight individual GRA proteins investigated in this study (GRA2-9) are not essential, several GRA proteins may provide redundant and potentially important functions during acute infection.

Nonreplicating type I uracil auxotrophic mutants of Toxoplasma gondii possess a potent ability to activate therapeutic immunity to established solid tumors by reversing immune suppression in the tumor microenvironment. Here we engineered targeted deletions of parasite secreted effector proteins using a genetically tractable Δku80 vaccine strain to show that the secretion of specific rhoptry (ROP) and dense granule (GRA) proteins by uracil auxotrophic mutants of T. gondii in conjunction with host cell invasion activates antitumor immunity through host responses involving CD8α+ dendritic cells, the IL-12/interferon-gamma (IFN-γ) TH1 axis, as well as CD4+ and CD8+ T cells. Deletion of parasitophorous vacuole membrane (PVM) associated proteins ROP5, ROP17, ROP18, ROP35 or ROP38, intravacuolar network associated dense granule proteins GRA2 or GRA12, and GRA24 which traffics past the PVM to the host cell nucleus severely abrogated the antitumor response. In contrast, deletion of other secreted effector molecules such as GRA15, GRA16, or ROP16 that manipulate host cell signaling and transcriptional pathways, or deletion of PVM associated ROP21 or GRA3 molecules did not affect the antitumor activity. Association of ROP18 with the PVM was found to be essential for the development of the antitumor responses. Surprisingly, the ROP18 kinase activity required for resistance to IFN-γ activated host innate immunity related GTPases and virulence was not essential for the antitumor response. These data show that PVM functions of parasite secreted effector molecules, including ROP18, manipulate host cell responses through ROP18 kinase virulence independent mechanisms to activate potent antitumor responses. Our results demonstrate that PVM associated rhoptry effector proteins secreted prior to host cell invasion and dense granule effector proteins localized to the intravacuolar network and host nucleus that are secreted after host cell invasion coordinately control the development of host immune responses that provide effective antitumor immunity against established ovarian cancer.

Unlabelled: Ingestion of the obligate intracellular protozoan parasite Toxoplasma gondii causes an acute infection that leads to chronic infection of the host. To facilitate the acute phase of the infection, T. gondii manipulates the host response by secreting rhoptry organelle proteins (ROPs) into host cells during its invasion. A few key ROP proteins with signatures of kinases or pseudokinases (ROPKs) act as virulence factors that enhance parasite survival against host gamma interferon-stimulated innate immunity. However, the roles of these and other ROPK proteins in establishing chronic infection have not been tested. Here, we deleted 26 ROPK gene loci encoding 31 unique ROPK proteins of type II T. gondii and show that numerous ROPK proteins influence the development of chronic infection. Cyst burdens were increased in the Δrop16 knockout strain or moderately reduced in 11 ROPK knockout strains. In contrast, deletion of ROP5, ROP17, ROP18, ROP35, or ROP38/29/19 (ROP38, ROP29, and ROP19) severely reduced cyst burdens. Δrop5 and Δrop18 knockout strains were less resistant to host immunity-related GTPases (IRGs) and exhibited >100-fold-reduced virulence. ROP18 kinase activity and association with the parasitophorous vacuole membrane were necessary for resistance to host IRGs. The Δrop17 strain exhibited a >12-fold defect in virulence; however, virulence was not affected in the Δrop35 or Δrop38/29/19 strain. Resistance to host IRGs was not affected in the Δrop17, Δrop35, or Δrop38/29/19 strain. Collectively, these findings provide the first definitive evidence that the type II T. gondii ROPK proteome functions as virulence factors and facilitates additional mechanisms of host manipulation that are essential for chronic infection and transmission of T. gondii, Importance: Reactivation of chronic Toxoplasma gondii infection in individuals with weakened immune systems causes severe toxoplasmosis. Existing treatments for toxoplasmosis are complicated by adverse reactions to chemotherapy. Understanding key parasite molecules required for chronic infection provides new insights into potential mechanisms that can interrupt parasite survival or persistence in the host. This study reveals that key secreted rhoptry molecules are used by the parasite to establish chronic infection of the host. Certain rhoptry proteins were found to be critical virulence factors that resist innate immunity, while other rhoptry proteins were found to influence chronic infection without affecting virulence. This study reveals that rhoptry proteins utilize multiple mechanisms of host manipulation to establish chronic infection of the host. Targeted disruption of parasite rhoptry proteins involved in these biological processes opens new avenues to interfere with chronic infection with the goal to either eliminate chronic infection or to prevent recrudescent infections., (Copyright © 2016 Fox et al.)

Calcium ion signaling regulates central aspects of the biology controlling stage and life cycle transitions of apicomplexan parasites. In the current issue of Infection and Immunity, Long and coworkers (S. Long, Q. Wang, and L. D. Sibley, Infect Immun 84:1262-1273, 2016, http://dx.doi.org/10.1128/IAI.01173-15) describe a powerful genetic system enabling reliable serial genetic dissection of a large gene family encoding novel calcium-dependent protein kinases (CDPKs) that provides new insights into the roles of CDPKs during Toxoplasma gondii infection., (Copyright © 2016, American Society for Microbiology. All Rights Reserved.)

An important function of the blood-brain barrier is to exclude pathogens from the central nervous system, but some microorganisms benefit from the ability to enter this site. It has been proposed that Toxoplasma gondii can cross biological barriers as a motile extracellular form that uses transcellular or paracellular migration, or by infecting a host cell that then crosses the blood-brain barrier. Unexpectedly, analysis of acutely infected mice revealed significant numbers of free parasites in the blood and the presence of infected endothelial cells in the brain vasculature. The use of diverse transgenic parasites combined with reporter mice and intravital imaging demonstrated that replication in and lysis of endothelial cells precedes invasion of the central nervous system, and highlight a novel mechanism for parasite entry to the central nervous system., Competing Interests: The authors declare no competing financial interests.

Apicomplexa parasites such as Toxoplasma gondii target effectors to and across the boundary of their parasitophorous vacuole (PV), resulting in host cell subversion and potential presentation by MHC class I molecules for CD8 T cell recognition. The host-parasite interface comprises the PV limiting membrane and a highly curved, membranous intravacuolar network (IVN) of uncertain function. Here, using a cell-free minimal system, we dissect how membrane tubules are shaped by the parasite effectors GRA2 and GRA6. We show that membrane association regulates access of the GRA6 protective antigen to the MHC I pathway in infected cells. Although insertion of GRA6 in the PV membrane is key for immunogenicity, association of GRA6 with the IVN limits presentation and curtails GRA6-specific CD8 responses in mice. Thus, membrane deformations of the PV regulate access of antigens to the MHC class I pathway, and the IVN may play a role in immune modulation., (Copyright © 2015 The Authors. Published by Elsevier Inc. All rights reserved.)

We have recently reported that treatment of disseminated pancreatic cancer with an attenuated Toxoplasma gondii uracil auxotroph vaccine promoted antitumor CD8 + T cell responses and long-term survival. Here, we optimized the treatment strategy for disseminated pancreatic cancer and show that attenuated Toxoplasma gondii therapy stimulated effective long-term immunity to pancreatic cancer through mechanisms involving CD4 + T cells and pancreatic tumor-specific IgG. Our results suggest that cell-mediated immunity in conjunction with humoral antibody immunity may offer greater resistance to recurrence of highly aggressive tumors. Cancer immunotherapeutic strategies using attenuated Toxoplasma gondii vaccines merit further investigation as a novel strategy to reawaken immunity to primary pancreatic carcinoma and to generate long-lasting immunity to pancreatic cancer recurrences.

Toxoplasma gondii is an obligate intracellular protozoan parasite. This apicomplexan is the causative agent of toxoplasmosis, a leading cause of central nervous system disease in AIDS. It has long been known that T. gondii interferes with major histocompatibility complex class II (MHC-II) antigen presentation to attenuate CD4(+) T cell responses and establish persisting infections. Transcriptional downregulation of MHC-II genes by T. gondii was previously established, but the precise mechanisms inhibiting MHC-II function are currently unknown. Here, we show that, in addition to transcriptional regulation of MHC-II, the parasite modulates the expression of key components of the MHC-II antigen presentation pathway, namely, the MHC-II-associated invariant chain (Ii or CD74) and the peptide editor H2-DM, in professional antigen-presenting cells (pAPCs). Genetic deletion of CD74 restored the ability of infected dendritic cells to present a parasite antigen in the context of MHC-II in vitro. CD74 mRNA and protein levels were, surprisingly, elevated in infected cells, whereas MHC-II and H2-DM expression was inhibited. CD74 accumulated mainly in the endoplasmic reticulum (ER), and this phenotype required live parasites, but not active replication. Finally, we compared the impacts of genetic deletion of CD74 and H2-DM genes on parasite dissemination toward lymphoid organs in mice, as well as activation of CD4(+) T cells and interferon gamma (IFN-γ) levels during acute infection. Cyst burdens and survival during the chronic phase of infection were also evaluated in wild-type and knockout mice. These results highlight the fact that the infection is influenced by multiple levels of parasite manipulation of the MHC-II antigen presentation pathway., (Copyright © 2015, American Society for Microbiology. All Rights Reserved.)

Suppressive myeloid cells represent a significant barrier to the generation of productive antitumor immune responses to many solid tumors. Eliminating or reprogramming suppressive myeloid cells to abrogate tumor-associated immune suppression is a promising therapeutic approach. We asked whether treatment of established aggressive disseminated pancreatic cancer with the immunotherapeutic attenuated Toxoplasma gondii vaccine strain CPS would trigger tumor-associated myeloid cells to generate therapeutic antitumor immune responses. CPS treatment significantly decreased tumor-associated macrophages and markedly increased dendritic cell infiltration of the pancreatic tumor microenvironment. Tumor-resident macrophages and dendritic cells, particularly cells actively invaded by CPS, increased expression of costimulatory molecules CD80 and CD86 and concomitantly boosted their production of IL12. CPS treatment increased CD4(+) and CD8(+) T-cell infiltration into the tumor microenvironment, activated tumor-resident T cells, and increased IFNγ production by T-cell populations. CPS treatment provided a significant therapeutic benefit in pancreatic tumor-bearing mice. This therapeutic benefit depended on IL12 and IFNγ production, MyD88 signaling, and CD8(+) T-cell populations. Although CD4(+) T cells exhibited activated effector phenotypes and produced IFNγ, CD4(+) T cells as well as natural killer cells were not required for the therapeutic benefit. In addition, CD8(+) T cells isolated from CPS-treated tumor-bearing mice produced IFNγ after re-exposure to pancreatic tumor antigen, suggesting this immunotherapeutic treatment stimulated tumor cell antigen-specific CD8(+) T-cell responses. This work highlights the potency and immunotherapeutic efficacy of CPS treatment and demonstrates the significance of targeting tumor-associated myeloid cells as a mechanism to stimulate more effective immunity to pancreatic cancer., (©2015 American Association for Cancer Research.)

Live attenuated vaccine strains, such as type I nonreplicating uracil auxotroph mutants, are highly effective in eliciting lifelong immunity to virulent acute infection by Toxoplasma gondii. However, it is currently unknown whether vaccine-elicited immunity can provide protection against acute infection and also prevent chronic infection. To address this problem, we developed nonreverting, nonreplicating, live attenuated uracil auxotroph vaccine strains in the type II Δku80 genetic background by targeting the deletion of the orotidine 5'-monophosphate decarboxylase (OMPDC) and uridine phosphorylase (UP) genes. Deletion of OMPDC induced a severe uracil auxotrophy with loss of replication, loss of virulence in mice, and loss of the ability to develop cysts and chronic infection. Vaccination of mice using type II Δku80 Δompdc mutants stimulated a fully protective CD8(+) T cell-dependent immunity that prevented acute infection by type I and type II strains of T. gondii, and this vaccination also severely reduced or prevented cyst formation after type II challenge infection. Nonreverting, nonreplicating, and non-cyst-forming Δompdc mutants provide new tools to examine protective immune responses elicited by vaccination with a live attenuated type II vaccine., (Copyright © 2015, American Society for Microbiology. All Rights Reserved.)

The obligate intracellular protozoan parasite Toxoplasma gondii interferes with major histocompatibility complex class II antigen presentation to dampen host CD4(+) T cell responses. While it is known that T. gondii inhibits major histocompatibility complex class II gene transcription and expression in infected host cells, the mechanism of this host manipulation is unknown. Here, we show that soluble parasite proteins inhibit IFNγ-induced expression of major histocompatibility complex class II on the surface of the infected cell in a dose-dependent response that was abolished by protease treatment. Subcellular fractionation of T. gondii tachyzoites revealed that the major histocompatibility complex class II inhibitory activity co-partitioned with rhoptries and/or dense granules. However, parasite mutants deleted for single rhoptries or dense granules genes (ROP1, 4/7, 14, 16 and 18 or GRA 2-9 and 12 knock-out strains) retained the ability to inhibit expression of major histocompatibility complex class II. In addition, excreted/secreted antigens released by extracellular tachyzoites displayed immunomodulatory activity characterized by an inhibition of major histocompatibility complex class II expression, and reduced expression and release of TNFα by macrophages. Tandem MS analysis of parasite excreted/secreted antigens generated a list of T. gondii secreted proteins that may participate in major histocompatibility complex class II inhibition and the modulation of host immune functions., (Copyright © 2015 Australian Society for Parasitology Inc. All rights reserved.)

Apicomplexan parasites including Toxoplasma gondii have complex life cycles within different hosts and their infectivity relies on their capacity to regulate gene expression. However, little is known about the nuclear factors that regulate gene expression in these pathogens. Here, we report that T. gondii enolase TgENO2 is targeted to the nucleus of actively replicating parasites, where it specifically binds to nuclear chromatin in vivo. Using a ChIP-Seq technique, we provide evidence for TgENO2 enrichment at the 5' untranslated gene regions containing the putative promoters of 241 nuclear genes. Ectopic expression of HA-tagged TgENO1 or TgENO2 led to changes in transcript levels of numerous gene targets. Targeted disruption of TgENO1 gene results in a decrease in brain cyst burden of chronically infected mice and in changes in transcript levels of several nuclear genes. Complementation of this knockout mutant with ectopic TgENO1-HA fully restored normal transcript levels. Our findings reveal that enolase functions extend beyond glycolytic activity and include a direct role in coordinating gene regulation in T. gondii.

Unlike most intracellular pathogens that gain access into host cells through endocytic pathways, Toxoplasma gondii initiates infection at the cell surface by active penetration through a moving junction and subsequent formation of a parasitophorous vacuole. Here, we describe a noncanonical pathway for T. gondii infection of macrophages, in which parasites are initially internalized through phagocytosis, and then actively invade from within a phagosomal compartment to form a parasitophorous vacuole. This phagosome to vacuole invasion (PTVI) pathway may represent an intermediary link between the endocytic and the penetrative routes for host cell entry by intracellular pathogens. The PTVI pathway is preferentially used by avirulent strains of T. gondii and confers an infectious advantage over virulent strains for macrophage tropism.

Pro-inflammatory M1 macrophages are critical for defense against intracellular pathogens while alternatively-activated M2 macrophages mediate tissue homeostasis and repair. Whether these distinct activation programs are mutually exclusive or can co-exist within the same cell is unclear. Here, we report the co-existence of these programs in Toxoplasma gondii-elicited inflammatory macrophages. This is independent of parasite expression of the virulence factor ROP16 and host cell expression of signal transducer and activator of transcription 6 (STAT6). Furthermore, this observation was recapitulated by IFN-γ and IL-4 treated bone marrow-derived macrophages in vitro. These results highlight the multi-functionality of macrophages as they respond to diverse microbial and endogenous stimuli., (Copyright © 2013 Australian Society for Parasitology Inc. Published by Elsevier Ltd. All rights reserved.)

We examined the efficacy of using attenuated non-replicating Toxoplasma gondii uracil auxotrophs that can be safely delivered as anticancer immunotherapeutics. This strategy exerted remarkable therapeutic activity in murine models of melanoma and ovarian carcinoma, and holds broad potential for the development of novel, highly effective anticancer vaccines.

Toxoplasma gondii is an intracellular parasite that has evolved to actively control its invaded host cells. Toxoplasma triggers then actively regulates host innate interleukin-12 (IL-12) and interferon-γ (IFN-γ) responses that elicit T cell control of infection. A live, nonreplicating avirulent uracil auxotroph vaccine strain (cps) of Toxoplasma triggers novel innate immune responses that stimulate amplified CD8(+) T cell responses and life-long immunity in vaccinated mice. Here, we review recent reports showing that intratumoral treatment with cps activated immune-mediated regression of established solid tumors in mice. We speculate that a better understanding of host-parasite interaction at the molecular level and applying improved genetic models based on Δku80 Toxoplasma strains will stimulate development of highly effective immunotherapeutic cancer vaccine strategies using engineered uracil auxotrophs., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

Targeted genetic manipulation using homologous recombination is the method of choice for functional genomic analysis to obtain a detailed view of gene function and phenotype(s). The development of mutant strains with targeted gene deletions, targeted mutations, complemented gene function, and/or tagged genes provides powerful strategies to address gene function, particularly if these genetic manipulations can be efficiently targeted to the gene locus of interest using integration mediated by double cross over homologous recombination. Due to very high rates of nonhomologous recombination, functional genomic analysis of Toxoplasma gondii has been previously limited by the absence of efficient methods for targeting gene deletions and gene replacements to specific genetic loci. Recently, we abolished the major pathway of nonhomologous recombination in type I and type II strains of T. gondii by deleting the gene encoding the KU80 protein(1,2). The Δku80 strains behave normally during tachyzoite (acute) and bradyzoite (chronic) stages in vitro and in vivo and exhibit essentially a 100% frequency of homologous recombination. The Δku80 strains make functional genomic studies feasible on the single gene as well as on the genome scale(1-4). Here, we report methods for using type I and type II Δku80Δhxgprt strains to advance gene targeting approaches in T. gondii. We outline efficient methods for generating gene deletions, gene replacements, and tagged genes by targeted insertion or deletion of the hypoxanthine-xanthine-guanine phosphoribosyltransferase (HXGPRT) selectable marker. The described gene targeting protocol can be used in a variety of ways in Δku80 strains to advance functional analysis of the parasite genome and to develop single strains that carry multiple targeted genetic manipulations. The application of this genetic method and subsequent phenotypic assays will reveal fundamental and unique aspects of the biology of T. gondii and related significant human pathogens that cause malaria (Plasmodium sp.) and cryptosporidiosis (Cryptosporidium).

Reversing tumor-associated immunosuppression seems necessary to stimulate effective therapeutic immunity against lethal epithelial tumors. Here, we show this goal can be addressed using cps, an avirulent, nonreplicating uracil auxotroph strain of the parasite Toxoplasma gondii (T. gondii), which preferentially invades immunosuppressive CD11c(+) antigen-presenting cells in the ovarian carcinoma microenvironment. Tumor-associated CD11c(+) cells invaded by cps were converted to immunostimulatory phenotypes, which expressed increased levels of the T-cell receptor costimulatory molecules CD80 and CD86. In response to cps treatment of the immunosuppressive ovarian tumor environment, CD11c(+) cells regained the ability to efficiently cross-present antigen and prime CD8(+) T-cell responses. Correspondingly, cps treatment markedly increased tumor antigen-specific responses by CD8(+) T cells. Adoptive transfer experiments showed that these antitumor T-cell responses were effective in suppressing solid tumor development. Indeed, intraperitoneal cps treatment triggered rejection of established ID8-VegfA tumors, an aggressive xenograft model of ovarian carcinoma, also conferring a survival benefit in a related aggressive model (ID8-Defb29/Vegf-A). The therapeutic benefit of cps treatment relied on expression of IL-12, but it was unexpectedly independent of MyD88 signaling as well as immune experience with T. gondii. Taken together, our results establish that cps preferentially invades tumor-associated antigen-presenting cells and restores their ability to trigger potent antitumor CD8(+) T-cell responses. Immunochemotherapeutic applications of cps might be broadly useful to reawaken natural immunity in the highly immunosuppressive microenvironment of most solid tumors., (©2013 AACR.)

Toxoplasma gondii infects up to one third of the world's population. A key to the success of T. gondii as a parasite is its ability to persist for the life of its host as bradyzoites within tissue cysts. The glycosylated cyst wall is the key structural feature that facilitates persistence and oral transmission of this parasite. Because most of the antibodies and reagents that recognize the cyst wall recognize carbohydrates, identification of the components of the cyst wall has been technically challenging. We have identified CST1 (TGME49_064660) as a 250 kDa SRS (SAG1 related sequence) domain protein with a large mucin-like domain. CST1 is responsible for the Dolichos biflorus Agglutinin (DBA) lectin binding characteristic of T. gondii cysts. Deletion of CST1 results in reduced cyst number and a fragile brain cyst phenotype characterized by a thinning and disruption of the underlying region of the cyst wall. These defects are reversed by complementation of CST1. Additional complementation experiments demonstrate that the CST1-mucin domain is necessary for the formation of a normal cyst wall structure, the ability of the cyst to resist mechanical stress, and binding of DBA to the cyst wall. RNA-seq transcriptome analysis demonstrated dysregulation of bradyzoite genes within the various cst1 mutants. These results indicate that CST1 functions as a key structural component that confers essential sturdiness to the T. gondii tissue cyst critical for persistence of bradyzoite forms.

Immune recognition of tumors can limit cancer development, but antitumor immune responses are often blocked by tumor-mediated immunosuppression. Because microbes or microbial constituents are powerful adjuvants to stimulate immune responses, we evaluated whether intratumoral administration of a highly immunogenic but attenuated parasite could induce rejection of an established poorly immunogenic tumor. We treated intradermal B16F10 murine melanoma by intratumoral injection of an attenuated strain of Toxoplasma gondii (cps) that cannot replicate in vivo and therefore is not infective. The cps treatment stimulated a strong CD8(+) T cell-mediated antitumor immune response in vivo that regressed established primary melanoma. The cps monotherapy rapidly modified the tumor microenvironment, halting tumor growth, and subsequently, as tumor-reactive T cells expanded, the tumors disappeared and rarely returned. The treatment required live cps that could invade cells and also required CD8(+) T cells and NK cells, but did not require CD4(+) T cells. Furthermore, we demonstrate that IL-12, IFN-γ, and the CXCR3-stimulating cytokines are required for full treatment efficacy. The treatment developed systemic antitumor immune activity as well as antitumor immune memory and therefore might have an impact against human metastatic disease. The approach is not specific for either B16F10 or melanoma. Direct intratumoral injection of cps has efficacy against an inducible genetic melanoma model and transplantable lung and ovarian tumors, demonstrating potential for broad clinical use. The combination of efficacy, systemic antitumor immune response, and complete attenuation with no observed host toxicity demonstrates the potential value of this novel cancer therapy.

The pyrimidine biosynthesis pathway in the protozoan pathogen Toxoplasma gondii is essential for parasite growth during infection. To investigate the properties of dihydroorotate dehydrogenase (TgDHOD), the fourth enzyme in the T. gondii pyrimidine pathway, we expressed and purified recombinant TgDHOD. TgDHOD exhibited a specific activity of 84U/mg, a k(cat) of 89s(-1), a K(m)=60μM for l-dihydroorotate, and a K(m)=29μM for decylubiquinone (Q(D)). Quinones lacking or having short isoprenoid side chains yielded lower k(cat)s than Q(D). As expected, fumarate was a poor electron acceptor for this family 2 DHOD. The IC(50)s determined for A77-1726, the active derivative of the human DHOD inhibitor leflunomide, and related compounds MD249 and MD209 were, 91μM, 96μM, and 60μM, respectively. The enzyme was not significantly affected by brequinar or TTFA, known inhibitors of human DHOD, or by atovaquone. DSM190, a known inhibitor of Plasmodium falciparum DHOD, was a poor inhibitor of TgDHOD. TgDHOD exhibits a lengthy 157-residue N-terminal extension, consistent with a potential organellar targeting signal. We constructed C-terminally c-myc tagged TgDHODs to examine subcellular localization of TgDHOD in transgenic parasites expressing the tagged protein. Using both exogenous and endogenous expression strategies, anti-myc fluorescence signal colocalized with antibodies against the mitochondrial marker ATPase. These findings demonstrate that TgDHOD is associated with the parasite's mitochondrion, revealing this organelle as the site of orotate production in T. gondii. The TgDHOD gene appears to be essential because while gene tagging was successful at the TgDHOD gene locus, attempts to delete the TgDHOD gene were not successful in the KU80 background. Collectively, our study suggests that TgDHOD is an excellent target for the development of anti-Toxoplasma drugs., (Copyright © 2012 Elsevier B.V. All rights reserved.)

The intracellular protozoan Toxoplasma gondii is 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.

Type II Toxoplasma gondii KU80 knockouts (Δku80) deficient in nonhomologous end joining were developed to delete the dominant pathway mediating random integration of targeting episomes. Gene targeting frequency in the type II Δku80 Δhxgprt strain measured at the orotate (OPRT) and the uracil (UPRT) phosphoribosyltransferase loci was highly efficient. To assess the potential of the type II Δku80 Δhxgprt strain to examine gene function affecting cyst biology and latent stages of infection, we targeted the deletion of four parasite antigen genes (GRA4, GRA6, ROP7, and tgd057) that encode characterized CD8(+) T cell epitopes that elicit corresponding antigen-specific CD8(+) T cell populations associated with control of infection. Cyst development in these type II mutant strains was not found to be strictly dependent on antigen-specific CD8(+) T cell host responses. In contrast, a significant biological role was revealed for the dense granule proteins GRA4 and GRA6 in cyst development since brain tissue cyst burdens were drastically reduced specifically in mutant strains with GRA4 and/or GRA6 deleted. Complementation of the Δgra4 and Δgra6 mutant strains using a functional allele of the deleted GRA coding region placed under the control of the endogenous UPRT locus was found to significantly restore brain cyst burdens. These results reveal that GRA proteins play a functional role in establishing cyst burdens and latent infection. Collectively, our results suggest that a type II Δku80 Δhxgprt genetic background enables a higher-throughput functional analysis of the parasite genome to reveal fundamental aspects of parasite biology controlling virulence, pathogenesis, and transmission.

The ROP16 kinase of Toxoplasma gondii is injected into the host cell cytosol where it activates signal transducer and activator of transcription (STAT)-3 and STAT6. Here, we generated a ROP16 deletion mutant on a Type I parasite strain background, as well as a control complementation mutant with restored ROP16 expression. We investigated the biological role of the ROP16 molecule during T. gondii infection. Infection of mouse bone marrow-derived macrophages with rop16-deleted (ΔROP16) parasites resulted in increased amounts of IL-12p40 production relative to the ROP16-positive RH parental strain. High level IL-12p40 production in ΔROP16 infection was dependent on the host cell adaptor molecule MyD88, but surprisingly was independent of any previously recognized T. gondii triggered pathway linking to MyD88 (TLR2, TLR4, TLR9, TLR11, IL-1ß and IL-18). In addition, ROP16 was found to mediate the suppressive effects of Toxoplasma on LPS-induced cytokine synthesis in macrophages and on IFN-γ-induced nitric oxide production by astrocytes and microglial cells. Furthermore, ROP16 triggered synthesis of host cell arginase-1 in a STAT6-dependent manner. In fibroblasts and macrophages, failure to induce arginase-1 by ΔROP16 tachyzoites resulted in resistance to starvation conditions of limiting arginine, an essential amino acid for replication and virulence of this parasite. ΔROP16 tachyzoites that failed to induce host cell arginase-1 displayed increased replication and dissemination during in vivo infection. We conclude that encounter between Toxoplasma ROP16 and the host cell STAT signaling cascade has pleiotropic downstream effects that act in multiple and complex ways to direct the course of infection.

Macrophages are specialized to detect and destroy intracellular microbes and yet a number of pathogens have evolved to exploit this hostile niche. Here we demonstrate that the obligate intracellular parasite Toxoplasma gondii disarms macrophage innate clearance mechanisms by secreting a serine threonine kinase called ROP18, which binds to and phosphorylates immunity-related GTPases (IRGs). Substrate profiling of ROP18 revealed a preference for a conserved motif within switch region I of the GTPase domain, a modification predicted to disrupt IRG function. Consistent with this, expression of ROP18 was both necessary and sufficient to block recruitment of Irgb6, which was in turn required for parasite destruction. ROP18 phosphorylation of IRGs prevented clearance within inflammatory monocytes and IFN-γ-activated macrophages, conferring parasite survival in vivo and promoting virulence. IRGs are implicated in clearance of a variety of intracellular pathogens, suggesting that other virulence factors may similarly thwart this innate cellular defense mechanism., (Copyright © 2010 Elsevier Inc. All rights reserved.)

The orotidine-5'-monophosphate decarboxylase (OMPDC) gene, encoding the final enzyme of the de novo pyrimidine biosynthesis pathway, was deleted using Toxoplasma gondii KU80 knockouts to develop an avirulent nonreverting pyrimidine auxotroph strain. Additionally, to functionally address the role of the pyrimidine salvage pathway, the uridine phosphorylase (UP) salvage activity was knocked out and a double knockout of UP and OMPDC was also constructed. The nonreverting DeltaOMPDC, DeltaUP, and DeltaOMPDC DeltaUP knockout strains were evaluated for pyrimidine auxotrophy, for attenuation of virulence, and for their ability to elicit potent immunity to reinfection. The DeltaUP knockout strain was replication competent and virulent. In contrast, the DeltaOMPDC and DeltaOMPDC DeltaUP strains were uracil auxotrophs that rapidly lost their viability during pyrimidine starvation. Replication of the DeltaOMPDC strain but not the DeltaOMPDC DeltaUP strain was also partially rescued in vitro with uridine or cytidine supplementation. Compared to their hypervirulent parental type I strain, the DeltaOMPDC and DeltaOMPDC DeltaUP knockout strains exhibited extreme attenuation in murine virulence (approximately 8 logs). Genetic complementation of the DeltaOMPDC strain using a functional OMPDC allele restored normal replication and type I parental strain virulence phenotypes. A single immunization of mice with either the live critically attenuated DeltaOMPDC strain or the DeltaOMPDC DeltaUP knockout strain effectively induced potent protective immunity to lethal challenge infection. The avirulent nonreverting DeltaOMPDC and DeltaOMPDC DeltaUP strains provide new tools for the dissection of the host response to infection and are promising candidates for safe and effective Th1 vaccine platforms that can be easily genetically engineered.

In the inflamed retina, CD4(+) T cells can cause retinal damage when they are not properly regulated. Since tissue expression of major histocompatibility complex (MHC) class II and costimulatory molecules is a key mechanism for regulating effector T cells, we tested the hypothesis that upregulation of these proteins in the retina contributes to the regulation of CD4 T cells. Here we report that in retinas infected with the protozoan parasite Toxoplasma gondii, MHC class II is upregulated on infiltrating leukocytes as well as on resident retinal cells, including photoreceptors. Flow cytometric analysis indicated that B7 costimulatory family members (CD80, CD86, ICOS-L, and programmed death ligand 2 [PD-L2]) were not expressed on class II(+) cells. In contrast, PD-L1 (also named B7-H1 or CD274) was expressed on the majority of both hematopoietic and resident retinal MHC class II-expressing cells. Retinal cells from Toxoplasma-infected animals were able to suppress T-cell activation in a PD-L1-dependent manner. Finally, we demonstrate that the expression of MHC class II and PD-L1 was critically dependent on gamma interferon (IFN-gamma) expression. These data suggest that retinal MHC class II and PD-L1 expression is a novel mechanism by which the retina protects itself from CD4 T-cell-mediated immune damage in ocular toxoplasmosis and other types of retinal immune responses.

C57BL/6 (B6) mice are genetically highly susceptible to chronic type II Toxoplasma gondii infections that invariably cause lethal toxoplasmic encephalitis. We examined the ability of an attenuated type I vaccine strain to elicit long-term immunity to lethal acute or chronic type II infections in susceptible B6 mice. Mice immunized with the type I cps1-1 vaccine strain were not susceptible to a lethal (100-cyst) challenge with the type II strain ME49. Immunized mice challenged with 10 ME49 cysts exhibited significant reductions in brain cyst and parasite burdens compared to naive mice, regardless of the route of challenge infection. Remarkably, cps1-1 strain-immunized B6 mice chronically infected with ME49 survived for at least 12 months without succumbing to the chronic infection. Potent immunity to type II challenge infections persisted for at least 10 months after vaccination. While the cps1-1 strain-elicited immunity did not prevent the establishment of a chronic infection or clear established brain cysts, cps1-1 strain-elicited CD8(+) immune T cells significantly inhibited recrudescence of brain cysts during chronic ME49 infection. In addition, we show that uracil starvation of the cps1-1 strain induces early markers of bradyzoite differentiation. Collectively, these results suggest that more effective immune control of chronic type II infection in the genetically susceptible B6 background is established by vaccination with the nonreplicating type I uracil auxotroph cps1-1 strain.