Intravacuolar Membranes Regulate CD8 T Cell Recognition of Membrane-Bound Toxoplasma gondii Protective Antigen (original) (raw)
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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...