Intravacuolar Membranes Regulate CD8 T Cell Recognition of Membrane-Bound Toxoplasma gondii Protective Antigen (original) (raw)
Frontiers in Immunology, 2019
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.
Journal of Experimental Medicine, 2009
In strong contrast, no association between hER and phagosomes or Ag presentation activity was observed in DCs containing phagocytosed live or dead parasites. Importantly, cross-presentation of parasite-derived Ag in actively infected cells was blocked when hER retrotranslocation was inhibited, indicating that the hER serves as a conduit for the transport of Ag between the PV and host cytosol. Collectively, these fi ndings demonstrate that pathogen-driven hER -PV interaction can serve as an important mechanism for Ag entry into the MHC class I pathway and CD8 + T cell cross-priming.
PLoS ONE, 2011
Effective control of the intracellular protozoan parasite Toxoplasma gondii depends on the activation of antigen-specific CD8 + T-cells that manage acute disease and prevent recrudescence during chronic infection. T-cell activation in turn, requires presentation of parasite antigens by MHC-I molecules on the surface of antigen presenting cells. CD8 + T-cell epitopes have been defined for several T. gondii proteins, but it is unclear how these antigens enter into the presentation pathway. We have exploited the well-characterized model antigen ovalbumin (OVA) to investigate the ability of parasite proteins to enter the MHC-I presentation pathway, by engineering recombinant expression in various organelles. CD8 + Tcell activation was assayed using 'B3Z' reporter cells in vitro, or adoptively-transferred OVA-specific 'OT-I' CD8 + T-cells in vivo. As expected, OVA secreted into the parasitophorous vacuole strongly stimulated antigen-presenting cells. Lower levels of activation were observed using glycophosphatidyl inositol (GPI) anchored OVA associated with (or shed from) the parasite surface. Little CD8 + T-cell activation was detected using parasites expressing intracellular OVA in the cytosol, mitochondrion, or inner membrane complex (IMC). These results indicate that effective presentation of parasite proteins to CD8 + T-cells is a consequence of active protein secretion by T. gondii and escape from the parasitophorous vacuole, rather than degradation of phagocytosed parasites or parasite products.
Experimental Parasitology, 1999
3Ј-3-3Ј-tetramethylindocarbocyanine; DMEM, Dulbecco's modified minimal essential me-Toxoplasma gondii resides in a vacuole that avoids fusion with host cell dium; EM, electron microscopy; Hepes, N-[2-hydroxyethyl] piperaendocytic and exocytic vesicular trafficking pathways. Experimental zine-N Ј-[4-butanesulfonic acid]; HF, human fibroblasts; IF, Parasitology 92, 87-99. Toxoplasma gondii actively penetrates its immunofluorescence; LAMP1, lysosome-associated membrane protein vertebrate host cell to establish a nonfusigenic compartment called the 1; MEM, minimal essential medium; MHC, major histocompatability parasitophorous vacuole (PV) that has previously been characterized complex; C 6-NBD ceramide, N-[6-[(7-nitro-benz-2-oxa-l,3-diazol-4primarily in phagocytic cells. To determine the fate of this unique yl)amino]caproyl] sphingosine; NBD, nitrobenzadiazole; NSF, N-ethyl compartment in nonphagocytic cells, we examined the trafficking of maleamide sensitive factor; PBS, phosphate-buffered saline; PV, parasihost cell proteins and lipids in Toxoplasma-infected fibroblasts using tophorous vacuole; TfR, transferrin receptor; TGN, trans-Golgi netquantitative immunofluorescence and immunoelectron microscopy. work. Toxoplasma-containing vacuoles remained segregated from all levels of the endocytic pathway, as shown by the absence of delivery of transferrin receptors, mannose phosphate receptors, and the lysosomalassociated protein LAMP1 to the vacuole. The PV was also inaccessible to lipids (DiIC 16 , and GM1) that were internalized from the plasma membrane via the endocytic system. In contrast, vacuoles containing
International Journal for Parasitology, 2004
Important components of the parasitophorous vacuole in which the intracellular protozoan parasite Toxoplasma gondii develops, comprise proteins secreted from apicomplexan specific secretory organelles named the dense granules. Here, we confirm by immunofluorescence and by cryo-electron microscopy that the recently isolated B10 protein (318 amino acids, 41 kDa) is a new dense granule protein that should now be referred to as GRA9. Within the vacuolar compartment, GRA9, like GRA2, GRA4 and GRA6, associates with the network of tubular membranes connected to the parasitophorous vacuole delimiting membrane. Like the other GRA proteins, GRA9 is secreted into the vacuole from the anterior end of the parasite. However, unlike GRA2 or GRA6, GRA9 does not transit by the posterior invaginated pocket of the parasite where the network first assembles. Within the dense granules, GRA9 exists in both a soluble and an insoluble state. Like the other GRA proteins, GRA9 is secreted as a soluble form only and like most of the GRA proteins, two forms of GRA9 of the similar molecular weight are detected within the vacuolar space: a soluble form and a membrane associated form. The dual properties of GRA9 are not only ascribed by the presence of amphipathic and hydrophobic alpha-helices but also by the fact that the protein is mainly hydrophilic. q
Parasitology International, 2013
Toxoplasma gondii is an intracellular protozoan parasite, which relies on a specialized compartment, the parasitophorous vacuole (PV), to survive within host cells. Dense granules within the parasite release a large variety of proteins to maintain the integrity of the vacuole structure. Here, we identified a novel dense granule protein in T. gondii, TgGRA23, which is a homolog of the Sarcocystis muris dense granule protein, SmDG32. Recombinant TgGRA23 (rTgGRA23) expressed in Escherichia coli as a glutathione S-transferase (GST) fusion protein was used to raise antisera in mice and rabbits. Immunoblotting showed that antisera from the immunized mice and rabbits reacted with parasite lysates to yield a 21-kDa native protein. In addition, immuno-electron microscopic examination showed that TgGRA23 resides in the dense granules, PV membrane and intravacuolar network of the parasite. To confirm the precise subcellular localization of TgGRA23 in T. gondii, an immunofluorescent antibody test was performed using dense granule markers. Notably, TgGRA23 co-localized with other dense granule proteins including TgGRA4 and TgGRA7, in the extracellular-stage parasites. Biochemical experiments indicated that TgGRA23 is insoluble and may form an electrostatic complex that is resistant to non-ionic detergents. Furthermore, specific antibodies to TgGRA23 were detected during the chronic stage of Toxoplasma infection in mice. Our results suggest that TgGRA23 is an as yet unknown member of the T. gondii dense granule proteins, and that it may be involved in remodeling or maintenance of the PV.
Journal of Experimental Medicine, 1999
The protozoan parasite Toxoplasma gondii actively penetrates its host cell by squeezing through a moving junction that forms between the host cell plasma membrane and the parasite. During invasion, this junction selectively controls internalization of host cell plasma membrane components into the parasite-containing vacuole. Membrane lipids flowed past the junction, as shown by the presence of the glycosphingolipid GM1 and the cationic lipid label 1.1′-dihexadecyl-3-3′-3-3′-tetramethylindocarbocyanine (DiIC16). Glycosylphosphatidylinositol (GPI)-anchored surface proteins, such as Sca-1 and CD55, were also readily incorporated into the parasitophorous vacuole (PV). In contrast, host cell transmembrane proteins, including CD44, Na+/K+ ATPase, and β1-integrin, were excluded from the vacuole. To eliminate potential differences in sorting due to the extracellular domains, parasite invasion was examined in host cells transfected with recombinant forms of intercellular adhesion molecule 1 ...
Molecular and Biochemical Parasitology, 1995
This work describes the molecular characterization of GRA6, a novel Toxoplasma gondii dense granule antigen of 32 kDa. cDNA clones encoding this protein were isolated using a rat serum directed against an HPLC fraction enriched in the protein GRAS. Cross-reactivity between GRAS and GRA6 was demonstrated by production of sera against the recombinant GRAS protein. A serum against a recombinant fragment of GRA6 which does not react with GRAS allowed the localization of this antigen at the subcellular level. GRA6 is detected in the dense granules of tachyzoites, and in the parasitophorous vacuole, closely associated to the network. The gene encoding GRA6 and its flanking regions were completely sequenced from cDNA and genomic inserts. Primer extension experiments demonstrated that the cap site of the GRA6 gene was located 37 bp upstream of the 5' end of the longest cDNA insert (1600 bp). The GRA6 gene potentially encodes a 230-amino-acid polypeptide, does not contain any introns and seems to be present as a single copy in the genome of T. gondii. The deduced polypeptide contains two hydrophobic regions with the characteristics of transmembrane domains. The N-terminal domain does not fit the classical feature of a signal peptide. The central hydrophobic domain is flanked by two hydrophilic domains which contain four blocks of amino acids homologous to the GRAS protein. The C-terminal hydrophilic region comprises 24% of glycine residues, which may indicate a structural role for GRA6 in the network. 0166-6851/95/$09.50 0 1995 Elsevier Science B.V. Ah rights reserved SSDI 0166-6851(95)00010-O to dramatic consequences in the case of immunodeficiency or pregnancy. Inside host cells, the dividing forms of the parasite or tachyzoites are surrounded by a parasitophorous vacuole (PV) which does not fuse with lyzosomes [l]. The PV is composed of a membranous network and a delimiting membrane devoid of host cell membrane-specific markers [2].
BioMed Research International, 2014
The intracellular parasiteToxoplasma gondiican penetrate any warm-blooded animal cell. Conserved molecular assemblies of host cell plasma membranes should be involved in the parasite-host cell recognition. Lipid rafts are well-conserved membrane microdomains that contain high concentrations of cholesterol, sphingolipids, glycosylphosphatidylinositol, GPI-anchored proteins, and dually acylated proteins such as members of the Src family of tyrosine kinases. Disturbing lipid rafts of mouse peritoneal macrophages and epithelial cells of the lineage LLC-MK2 with methyl-beta cyclodextrin (MβCD) and filipin, which interfere with cholesterol or lidocaine, significantly inhibited internalization ofT. gondiiin both cell types, although adhesion remained unaffected in macrophages and decreased only in LLC-MK2 cells. Scanning and transmission electron microscopy confirmed these observations. Results are discussed in terms of the original role of macrophages as professional phagocytes versus the...
Brazilian Journal of Development, 2021
Toxoplasma gondii is a protozoan parasite responsible for toxoplasmosis, and may be causing any problems in different systems. Some of these complications are associated with the change of intercellular communication mediated by Junctions Communicators that allows direct communication between tissues. However, there are still systems that are not fully consistent with the junctional communication, including the innate immune system, represented by Macrophages. Thus, we used J774-G8 macrophage cell line culture infected by the RH strain of Toxoplasma in its tachyzoite form. The results revealed that in J774-G8 cells The Cx43 and Phalloidin proteins interact in the plasma membrane of the J774-G8 lineage, and they undergo a sensitive reduction in the membrane after 72 hours of infection with the parasite Toxoplasma gondii. The evaluation of the Cx43 protein expression by immunoelectrophoretic transfer has been shown to be altered (elevated) in J774-G8 macrophage cells infected with the parasite Toxoplasma gondii 24 and 48 hours compared to uninfected cells.
Proceedings of the National Academy of Sciences, 1996
Most intracellular pathogens avoid lysing their host cells during invasion by wrapping themselves in a vacuolar membrane. This parasitophorous vacuole membrane (PVM) is often retained, serving as a critical transport interface between the parasite and the host cell cytoplasm. To test whether the PVM formed by the parasite Toxoplasma gondii is derived from host cell membrane or from lipids secreted by the parasite, we used time-resolved capacitance measurements and video microscopy to assay host cell surface area during invasion. We observed no significant change in host cell surface area during PVM formation, demonstrating that the PVM consists primarily of invaginated host cell membrane. Pinching off of the PVM from the host cell membrane occurred after an unexpected delay (34-305 sec) and was seen as a 0.219 ± 0.006 pF drop in capacitance, which corresponds well to the predicted surface area of the entire PVM (30-33 Lim2). The formation and closure ofa fission pore
Structure and Function of the Parasitophorous Vacuole Membrane Surrounding Toxoplasma gondii
Annals of the New York Academy of Sciences, 1994
Toxoplusma gondii is growing in popularity as a model for studying intracellular parasitism.' The parasite has also gained more attention recently as the most common cause of focal central nervous system infections in patients with the acquired immunodeficiency syndrome,* and it continues to cause more than 3,000 cases per year of congenital birth defects in the United States alone. T. gondii infects nearly all animals and most birds and is one of the most widely distributed of all intracellular parasites. In vitro, tachyzoites of T. gondii can invade and replicate within essentially all nucleated cells, an unusual feat for any intracellular organism. The intracellular tachyzoites reside within a vacuole that is incapable of acidifying or fusing with any membrane-bound organelle within the host cell endocytic system= and as such is effectively hidden from the host, yet the parasite replicates rapidly. The fusion incompetence of the vacuole is established at the time of cell entry and does not depend on secretion by the parasite of a soluble inhibitor of the fusion or acidification events. On the basis of these findings and in conjunction with the known morphology of the newly formed parasitophorous vacuole membrane (PVM), we hypothesized that fusion incompetence results from the absence of protein signals for vesicular fusion events. Nonetheless, the PVM surrounding T. gondii serves a critical additional function which is likely to depend on proteins, by allowing access of necessary nutrients from and exchange of metabolites with the host cell. A second potential function for the PVM, binding of host cell mitochondria and endoplasmic reticulum,7 is also likely to be protein dependent. We believe that proteins that provide or contribute to these additional functions for the PVM are likely to be derived from parasite secretory organelles, but little direct data exist to support this hypothesis.
Infection and Immunity, 2007
Challenge with the intracellular protozoan parasite Toxoplasma gondii induces a potent CD8+ T-cell response that is required for resistance to infection, but many questions remain about the factors that regulate the presentation of major histocompatibility complex class I (MHC-I)-restricted parasite antigens and about the role of professional and nonprofessional accessory cells. In order to address these issues, transgenic parasites expressing ovalbumin (OVA), reagents that track OVA/MHC-I presentation, and OVA-specific CD8+ T cells were exploited to compare the abilities of different infected cell types to stimulate CD8+ T cells and to define the factors that contribute to antigen processing. These studies reveal that a variety of infected cell types, including hematopoietic and nonhematopoietic cells, are capable of activating an OVA-specific CD8+ T-cell hybridoma, and that this phenomenon is dependent on the transporter associated with antigen processing and requires live T. gond...
Passive Immunity and Antibody Response Induced by Toxoplasma gondii VLP Immunization
Vaccines, 2021
Passive immunity can provide immediate protection against infectious pathogens. To date, only a few studies have investigated the effect of passive immunization against Toxoplasma gondii, and the use of immune sera acquired from VLP-vaccinated mice for passive immunity assessment remains unreported. In this study, immune sera were produced by a single immunization with virus-like particles (VLPs) expressing the inner membrane complex (IMC), rhoptry protein 18 (ROP18), and microneme protein 8 (MIC8) of Toxoplasma gondii, with or without a CpG-ODN adjuvant. The passive immunization of immune sera conferred protection in mice, as indicated by their potent parasite-specific antibody response, lessened brain cyst counts, lower bodyweight loss, and enhanced survival. In order to confirm that the immune sera of the VLP-immunized mice were truly protective, the antibody responses and other immunological parameters were measured in the VLP-immunized mice. We found that VLP immunization induc...
CD8 T Cells and Toxoplasma gondii: A New Paradigm
Journal of Parasitology Research, 2011
CD8 T cells are essential for control of Toxoplasma gondii infection. Once activated they undergo differentiation into short-lived effector and memory precursor effector cells. As effector cells, CD8 T cells exert immune pressure on the parasite via production of inflammatory cytokines and through their cytolytic activity. Once immune control has been established, the parasite encysts and develops into chronic infection regulated by the memory CD8 T-cell population. Several signals are needed for this process to be initiated and for development of fully differentiated memory CD8 T cells. With newly developed tools including CD8 Tcell tetramers and TCR transgenic mice, dissecting the biology behind T. gondii-specific CD8 T-cell responses can now be more effectively addressed. In this paper, we discuss what is known about the signals required for effective T. gondii-specific CD8 T-cell development, their differentiation, and effector function.
The Journal of Immunology, 2010
The Toxoplasma gondii population consists of multiple strains, defined by genotype and virulence. Previous studies have established that protective immunity to this organism is mediated by IL-12, which drives T cells to produce IFN-g. Paradoxically, although type I and type II strains of T. gondii both induce IL-12 and IFN-g in the mouse, type I parasites are lethal, whereas type II strains establish chronic infection. The cellular basis for these strain-dependent differences remains unclear. To better understand these events, the CD8 + T cell and dendritic cell (DC) responses to transgenic, OVA-expressing type I RH (RH OVA) and type II Prugniuad (Pru OVA) parasites were examined. Pru OVA-infected mice developed a robust DC response at the site of infection and the draining lymph node and generated a population of endogenous OVA-specific CD8 + T cells. In contrast, RH OVA-infected mice had fewer DCs and OVA-specific CD8 + T cells. RH OVA-infected mice given preactivated OVA-specific CD8 + T cells were protected, suggesting that reduced DC-derived signals contributed to the low OVA-specific CD8 + T cell numbers observed during type I infection. Indeed, DC depletion prior to Pru OVA infection resulted in a failure to generate activated OVA-specific CD8 + T cells, and IL-12p70 treatment during RH OVA infection modestly increased the number of Ag-specific cells. Together, these data are consistent with a model of immunity to T. gondii in which strain-dependent DC responses shape the generation of Ag-specific CD8 + T cells and determine the outcome of infection.
Molecular and biochemical parasitology, 1999
Following secretion into the parasitophorous vacuole, dense granule proteins, referred to as GRA proteins, are targeted to different locations including a complex of tubular membranes that are connected with the vacuolar membrane. To further define the formation of this intravacuolar network, we have investigated the secretion, trafficking and membrane association of GRA4 and GRA6 within the parasitophorous vacuole. In extracellular parasites, GRA4 and GRA6 were found exclusively in dense secretory granules where they were packaged primarily as soluble proteins. Following release into the vacuole, GRA6 was rapidly translocated to the posterior end of the parasite where, like previously reported for GRA2, it bound to a cluster of multi-lamellar vesicles that give rise to the network. In contrast, GRA4 was distributed throughout the lumen of the vacuole and only later became associated with the mature network that is found dispersed throughout the vacuole. Cell fractionation and treat...
ABSTRACTHost resistance to Toxoplasma gondii relies on CD8 T cell IFNγ responses, which if modulated by the host or parasite could influence chronic infection and parasite transmission between hosts. Since host-parasite interactions that govern this response are not fully elucidated, we investigated requirements for eliciting naïve CD8 T cell IFNγ responses to a vacuolar resident antigen of T. gondii, TGD057. Naïve TGD057 antigen-specific CD8 T cells (T57) were isolated from transnuclear mice and responded to parasite-infected bone marrow-derived macrophages (BMDMs) in an antigen-dependent manner, first by producing IL-2 and then IFNγ. T57 IFNγ responses to TGD057 were independent of the parasite’s protein export machinery ASP5 and MYR1. Instead, host immunity pathways downstream of the regulatory Immunity-Related GTPases (IRG), including partial dependence on Guanylate-Binding Proteins, are required. Multiple T. gondii ROP5 isoforms and allele types, including ‘avirulent’ ROP5A fro...
PLoS Pathogens, 2014
During infection with the intracellular parasite Toxoplasma gondii, the presentation of parasite-derived antigens to CD4 + and CD8 + T cells is essential for long-term resistance to this pathogen. Fundamental questions remain regarding the roles of phagocytosis and active invasion in the events that lead to the processing and presentation of parasite antigens. To understand the most proximal events in this process, an attenuated non-replicating strain of T. gondii (the cpsII strain) was combined with a cytometry-based approach to distinguish active invasion from phagocytic uptake. In vivo studies revealed that T. gondii disproportionately infected dendritic cells and macrophages, and that infected dendritic cells and macrophages displayed an activated phenotype characterized by enhanced levels of CD86 compared to cells that had phagocytosed the parasite, thus suggesting a role for these cells in priming naïve T cells. Indeed, dendritic cells were required for optimal CD4 + and CD8 + T cell responses, and the phagocytosis of heat-killed or invasion-blocked parasites was not sufficient to induce T cell responses. Rather, the selective transfer of cpsII-infected dendritic cells or macrophages (but not those that had phagocytosed the parasite) to naïve mice potently induced CD4 + and CD8 + T cell responses, and conferred protection against challenge with virulent T. gondii. Collectively, these results point toward a critical role for actively infected host cells in initiating T. gondii-specific CD4 + and CD8 + T cell responses.