Toxoplasma gondii sporozoites form a transient parasitophorous vacuole that is impermeable and contains only a subset of dense-granule proteins.

Toxoplasma gondii sporozoites form two parasitophorous vacuoles during development within host cells, the first (PV1) during host cell invasion and the second (PV2) 18 to 24 h postinoculation. PV1 is structurally distinctive due to its large size, yet it lacks a tubulovesicular network (C. A. Speer, M. Tilley, M. Temple, J. A. Blixt, J. P. Dubey, and M. W. White, Mol. Biochem. Parasitol. 75:75-86, 1995). Confirming the finding that sporozoites have a different electron-dense-granule composition, we have now found that sporozoites within oocysts lack the mRNAs encoding the 5' nucleoside triphosphate hydrolases (NTPase). NTPase first appears 12 h postinfection. Other tachyzoite dense-granule proteins, GRA1, GRA2, GRA4, GRA5, and GRA6, were detected in oocyst extracts, and antibodies against these proteins stained granules in the sporozoite cytoplasm. In contrast to tachyzoite invasion of host cells, however, sporozoites did not exocytose the dense-granule proteins GRA1, GRA2, or GRA4 during PV1 formation. Even after NTPase induction, these proteins were retained within cytoplasmic granules rather than being secreted into PV1. Only GRA5 was secreted by the sporozoite during host cell invasion, becoming associated with the membrane surrounding PV1. Microinjection of sporozoite-infected cells with fluorescent dyes showed that PV1 is impermeable to fluorescent dyes with molecular masses as small as 330 Da, indicating that PV1 lacks channels through which molecules can pass from the host cytoplasm into the vacuole. By contrast, lucifer yellow rapidly diffused into PV2, demonstrating the presence of molecular channels. These studies indicate that PV1 and PV2 are morphologically, immunologically, and functionally distinct, and that PV2 appears to be identical to the tachyzoite vacuole. The inaccessibility of PV1 to host cell nutrients may explain why parasite replication does not occur in this vacuole.     

Exocytosis ofToxoplasma gondii dense granules into the parasitophorous vacuole after host cell invasion

Tachyzoites ofToxoplasma gondii have been shown to exocytose the contents of dense granules into the parasitophorous vacuole after host cell invasion. A monoclonal antibody specific for a 27-kDa protein was used to locate the dense granules by immunoelectron microscopy. The same antibody also reacted with the tubular network found in the parasitophorous vacuole, which confirmed that the dense granules were exocytosed by tachyzoites.     

Class I major histocompatibility complex presentation of antigens that escape from the parasitophorous vacuole of Toxoplasma gondii

The intracellular parasite Toxoplasma gondii, the causative agent of toxoplasmosis, induces a protective CD8 T-cell response in its host; however, the mechanisms by which T. gondii proteins are presented by the class I major histocompatibility complex remain largely unexplored. T. gondii resides within a specialized compartment, the parasitophorous vacuole, that sequesters the parasite and its secreted proteins from the host cell cytoplasm, suggesting that an alternative cross-priming pathway might be necessary for class I presentation of T. gondii antigens. Here we used a strain of T. gondii expressing yellow fluorescent protein and a secreted version of the model antigen ovalbumin to investigate this question. We found that presentation of ovalbumin secreted by the parasite requires the peptide transporter TAP (transporter associated with antigen processing) and occurs primarily in actively infected cells rather than bystander cells. We also found that dendritic cells are a major target of T. gondii infection in vivo and account for much of the antigen-presenting activity in the spleen. Finally, we obtained evidence that Cre protein secreted by T. gondii can mediate recombination in the nucleus of the host cell. Together, these results indicate that Toxoplasma proteins can escape from the parasitophorous vacuole into the host cytoplasm and be presented by the endogenous class I pathway, leading to direct recognition of infected cells by CD8 T cells.     

Differential membrane targeting of the secretory proteins GRA4 and GRA6 within the parasitophorous vacuole formed by Toxoplasma gondii.

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 treatment with denaturing agents established that the association of GRA4 with the network membranes was mediated by strong protein-protein interactions. In contrast, GRA6 was predominantly influenced by hydrophobic interactions, and a phosphorylated form of this protein present within the vacuole showed increased association with the network membranes. Cross-linking studies established that GRA4 and GRA6 specifically interact with GRA2 to form a multimeric complex that is stably associated with the intravacuolar network. Formation of this protein complex, which is based on both protein-protein and hydrophobic interactions, may participate in nutrient or protein transport within the vacuole.     

Antigen presentation by Leishmania mexicana-infected macrophages: activation of helper T cells by a model parasite antigen secreted into the parasitophorous vacuole or expressed on the amastigote surface

Leishmania are protozoan parasites which invade mammalian macrophages and multiply as amastigotes in phagolysosomes (parasitophorous vacuoles). Using L. mexicana and bone marrow-derived macrophages (BMM), the question is addressed whether infected BMM induced to express major histocompatibility complex class II molecules can present defined antigens to specific T helper type 1 cells. As a model antigen, a membrane-bound acid phosphatase (MAP), a minor protein associated with intracellular vesicles in amastigotes, was either overexpressed at the surface of the parasites or overexpressed in a soluble form leading to antigen secretion into the parasitophorous vacuole. Presentation of MAP epitopes by these three types of amastigotes was then compared for macrophages containing live parasites or amastigotes inactivated by drug treatment. It is shown that surface-exposed and secreted MAP can be efficiently presented to T cells by macrophages harboring live amastigotes. Therefore, the parasitophorous vacuole communicates by vesicular membrane traffic with the plasmalemma of the host cell. The intracellular MAP of wild-type cells or the abundant lysosomal cysteine proteinases are not or only inefficiently presented, respectively. After killing of the parasites, abundant proteins such as overexpressed MAP and the cysteine proteinases efficiently stimulate T cells, while wild-type MAP levels are not effective. We conclude that intracellular proteins of intact amastigotes are not available for presentation, while after parasite inactivation, presentation depends on antigen abundance and possibly stability. The cell biological and possible immunological consequences of these results are discussed.     

Plasmodium falciparum merozoite surface protein 8 is a ring-stage membrane protein that localizes to the parasitophorous vacuole of infected erythrocytes

To date, the following seven glycosylphosphatidylinositol (GPI)-anchored merozoite antigens have been described in Plasmodium falciparum: merozoite-associated surface protein 1 (MSP-1), MSP-2, MSP-4, MSP-5, MSP-8, MSP-10, and the rhoptry-associated membrane antigen. Of these, MSP-1, MSP-8, and MSP-10 possess a double epidermal growth factor (EGF)-like domain at the C terminus, and these modules are considered potential targets of protective immunity. In this study, we found that surprisingly, P. falciparum MSP-8 is transcribed and translated in the ring stage and is absent from the surface of merozoites. MSP-8 is the only GPI-anchored protein known to be expressed at this time. It is synthesized as a mature 80-kDa protein which is rapidly processed to a C-terminal 17-kDa species that contains the double EGF module. As determined by a combination of immunofluorescence and membrane purification approaches, it appears likely that MSP-8 initially localizes to the parasite plasma membrane in the ring stage. Although the C-terminal 17-kDa fragment is present in more mature stages, at these times it is found in the food vacuole. We successfully disrupted the MSP-8 gene in P. falciparum, a process that validated the specificity of the antibodies used in this study and also demonstrated that MSP-8 does not play a role essential to maintenance of the erythrocyte cycle. This finding, together with the observation that MSP-8 is exclusively intracellular, casts doubt over the viability of this antigen as a vaccine. However, it is still possible that MSP-8 is involved in an early parasitophorous vacuole function that is significant for pathogenesis in the human host.     

The parasitophorous vacuole of Encephalitozoon cuniculi: biogenesis and characteristics of the host cell-pathogen interface

Microsporidia are obligate intracellular fungal pathogens of increasing importance in immunocompromised patients. They have developed a unique invasion mechanism, which is based on the explosive discharge of a hollow tubulus, the so-called polar tube. The infectious sporoplasm is subsequently extruded through this flexible tube and injected into the host cell. The model microsporidium Encephalitozoon cuniculi is a paradigm of a fungus with an extreme host cell dependency. This human pathogen possesses one of the smallest eukaryotic genomes (<3MB) identified so far and has reduced its own biosynthetic pathways to a minimum, thus depending on an efficient supply of metabolites from the host cell. E. cuniculi spends its entire intracellular life cycle inside a parasitophorous vacuole (PV), which is formed during invasion. We have provided here an overview of the biogenesis and characteristics of this important host cell-pathogen interface and suggest in this context a modified model for E. cuniculi invasion. According to the model, the host cell plasma membrane is not pierced by the polar tube, but is pushed at the contact site into the cell interior by the mechanical force of the expelled polar tube. This results in a channel-like invagination of the plasma membrane, from which finally the parasitophorous vacuole is pinched-off.     

Branching network of proteinaceous filaments within the parasitophorous vacuole of Encephalitozoon cuniculi and Encephalitozoon hellem

The microsporidia are a diverse phylum of obligate intracellular parasites that infect all major animal groups and have been recognized as emerging human pathogens for which few chemotherapeutic options currently exist. These organisms infect every tissue and organ system, causing significant pathology, especially in immune-compromised populations. The microsporidian spore employs a unique infection strategy in which its contents are delivered into a host cell via the polar tube, an organelle that lies coiled within the resting spore but erupts with a force sufficient to pierce the plasma membrane of its host cell. Using biochemical and molecular approaches, we have previously identified components of the polar tube and spore wall of the Encephalitozoonidae. In this study, we employed a shotgun proteomic strategy to identify novel structural components of these organelles in Encephalitozoon cuniculi. As a result, a new component of the E. cuniculi developing spore wall was identified. Surprisingly, using the same approach, a heretofore undescribed filamentous network within the lumen of the parasitophorous vacuole was discovered. This network was also present in the parasitophorous vacuole of Encephalitozoon hellem. Thus, in addition to further elucidating the molecular composition of seminal organelles and revealing novel diagnostic and therapeutic targets, proteomic analysis-driven approaches exploring the spore may also uncover unknown facets of microsporidian biology.     

Identification of the pro-mature processing site of Toxoplasma ROP1 by mass spectrometry.

The rhoptries are specialized secretory organelles that function during host cell invasion in the obligate intracellular parasite Toxoplasma gondii. All T. gondii rhoptry proteins studied to date are synthesized as pro-proteins that are then processed to their mature forms. To understand the role of the pro region in rhoptry protein function, we have precisely defined the processing site of the pro-region of the rhoptry protein ROP1. Efforts to determine such processing sites have been prevented by blocked N-termini of mature proteins isolated from T. gondii. To overcome this problem, we have used an engineered form of ROP1 and mass spectrometry to demonstrate that proROP1 is processed to its mature form between the glutamic acid at position 83 and alanine at position 84. These data also show that mature ROP1 lacks substantial post-translational modifications, a result which has important implications for targeting of rhoptry proteins.     

The Toxoplasma gondii dense granule protein GRA7 is phosphorylated upon invasion and forms an unexpected association with the rhoptry proteins ROP2 and ROP4

The obligate intracellular parasite Toxoplasma gondii infects warm-blooded animals throughout the world and is an opportunistic pathogen of humans. As it invades a host cell, Toxoplasma forms a novel organelle, the parasitophorous vacuole, in which it resides during its intracellular development. The parasite modifies the parasitophorous vacuole and its host cell with numerous proteins delivered from rhoptries and dense granules, which are secretory organelles unique to the phylum Apicomplexa. For the majority of these proteins, little is known other than their localization. Here we show that the dense granule protein GRA7 is phosphorylated but only in the presence of host cells. Within 10 min of invasion, GRA7 is present in strand-like structures in the host cytosol that contain rhoptry proteins. GRA7 strands also contain GRA1 and GRA3. Independently of its phosphorylation state, GRA7 associates with the rhoptry proteins ROP2 and ROP4 in infected host cells. This is the first report of interactions between proteins secreted from rhoptries and dense granules.     

A merozoite-specific 22-kDa rhoptry protein of the coccidium Eimeria nieschulzi (Sporozoa,Coccidia) is exocytosed in the parasitophorous vacuole upon host cell invasion

A monoclonal antibody (Mab D12A4) was used to follow the genesis and fate of rhoptries from early first-generation merogony through second-generation merozoites of the rat coccidium Eimeria nieschulzi. The epitope recognized by Mab D12A4 belongs to a 22-kDa protein which can be localized first in developing meronts 25 h post-infection. Early rhoptries appear as distinct granules in the cytoplasm of developing meronts and elongate into mature organelles before merozoite release. The 22-kDa protein is found in the parasitophorous vacuole after host cell invasion. Western blotting and immunofluorescence showed that the 22-kDa rhoptry protein is expressed in schizonts and merozoites but not in sporozoites. Received: 9 August 1997 / Accepted: 14 October 1997     

Localization, biosynthesis, processing and isolation of a major 126 kDa antigen of the parasitophorous vacuole of Plasmodium falciparum

Monoclonal antibodies prepared against a 50 kDa antigen found in Plasmodium falciparum culture supernatants identify a 126 kDa polypeptide which can be localized by immunofluorescence and immunoelectronmicroscopy at the periphery of the schizonts. This polypeptide is released from the infected erythrocytes by mild saponin lysis and is probably a component of the parasitophorous vacuole. Pulse chase kinetic analysis demonstrated its disappearance from the parasitized red blood cell from 6 to 10 h after being synthesized and the concomitant appearance of the 50 kDa molecule in the culture supernatant. Purification of metabolically labeled, schizont infected cells demonstrated that spontaneous release of merozoites is needed for the processing of the 126 to the 50 kDa whereas reinvasion is not. Polyclonal antibodies were raised in rabbit against affinity purified 126 kDa protein. These antibodies, together with another 126 kDa specific monoclonal antibody have enabled us to characterize two other cleavage products of the 126 kDa antigen in culture supernatants, namely 47 and 18 kDa polypeptides. We believe that the processing of the 126 kDa protein into low molecular weight fragments reflects a proteolytic event which may participate in merozoite release.     

Characterization of the parasitophorous vacuole membrane from Plasmodium chabaudi and implications about its role in the export of parasite proteins

Little is known about how the malaria parasite transports and targets proteins into the host erythrocyte. Parasite proteins exported into the host cell not only have to cross the parasite plasma membrane but also must traverse the parasitophorous vacuolar membrane (PVM) that surrounds the parasite. The PVM of Plasmodium chabaudi-infected erythrocytes was analyzed by immunofluorescence using an antibody against a known PVM protein, a fluorescent lipid probe, and electron microscopy. These analyses reveal qualitatively different membranous projections from the PVM. Some PVM projections are uniformly labeled with the antibody and with lipid probes and probably correspond to the Maurer's clefts. In contrast to this uniform labeling of the PVM and projections, a 93-kDa P. chabaudi erythrocyte membrane-associated protein is occasionally detected in vesicle-like structures adjacent to the parasite. These vesicle-like structures are found only coincident with protein synthesis and are located at discrete sites on the PVM. These observations suggest that the 93-kDa protein does not move along the membranous projections of the PVM toward the erythrocyte membrane. It is proposed that the 93-kDa protein is secreted directly into the erythrocyte cytoplasm at discrete PVM domains and then binds to the cytoplasmic face of the erythrocyte membrane. Supplementary material: Additional documentary material has been deposited in electronic form and can be obtained from http://link.springer.de/link/service/journals/00436/index.htm Received: 27 July 1998 / Accepted: 4 November 1998     

Induction of Protective Immune Responses by a Multiantigenic DNA Vaccine Encoding GRA7 and ROP1 of Toxoplasma gondii

Toxoplasma gondii is distributed worldwide and infects most species of warm-blooded animals, including humans. The heavy incidence and severe or lethal damage caused by T. gondii infection clearly indicates the need for the development of a vaccine. To evaluate the protective efficacy of a multiantigenic DNA vaccine expressing GRA7 and ROP1 of T. gondii with or without a plasmid encoding murine interleukin-12 (pIL12), we constructed DNA vaccines using the eukaryotic plasmids pGRA7, pROP1, and pGRA7-ROP1. Mice immunized with pGRA7, pROP1, or pGRA7-ROP1 showed significantly increased serum IgG2a titers; production of gamma interferon (IFN-γ), IL-10, and tumor necrosis factor alpha (TNF-α); in vitro T cell proliferation; and survival, as well as decreased cyst burdens in the brain, compared to mice immunized with either the empty plasmid, pIL12, or vector with pIL12 (vector+pIL12). Moreover, mice immunized with the multiantigenic DNA vaccine pGRA7-ROP1 had higher IgG2a titers, production of IFN-γ and TNF-α, survival time, and cyst reduction rate compared to those of mice vaccinated with either pGRA7 or pROP1 alone. Furthermore, mice immunized with either a pGRA7-ROP1+pIL12 or a single-gene vaccine combined with pIL12 showed greater Th1 immune response and protective efficacy than the single-gene-vaccinated groups. Our data suggest that the multiantigenic DNA antigen pGRA7-ROP1 was more effective in stimulating host protective immune responses than separately injected single antigens, and that IL-12 serves as a good DNA adjuvant.     

The Toxoplasma homolog of Plasmodium apical membrane antigen-1 (AMA-1) is a microneme protein secreted in response to elevated intracellular calcium levels

A monoclonal antibody (MAb) has been generated against a novel 63 kDa surface/apical antigen of Toxoplasma gondii tachyzoites which is identified here as TgAMA-1, the Toxoplasma homolog of Plasmodium apical membrane antigen-1 (AMA-1). Sequence analysis, phase partitioning in Triton X-114, and labeling of TgAMA-1 with iodonaphthalene azide all suggest that TgAMA-1 is a type I transmembrane protein. There is a high degree of sequence similarity between TgAMA-1 and Plasmodium AMA-1, most notably in the position of conserved cysteine residues within the protein's predicted extracellular domain. In contrast to full length Plasmodium AMA-1, which has previously been localized to the rhoptries, it is shown here by immunofluorescence and immunoelectron microscopy that intracellular TgAMA-1 is found in the micronemes. A 53 kDa N-terminal proteolytic fragment of TgAMA-1 is constitutively secreted from the parasite at 37 degrees C. As is the case with other microneme proteins, the proteolytic processing and secretion of TgAMA-1 is dramatically enhanced in response to treatments which increase intracellular calcium levels.     

CD52 is synthesized in cumulus cells and secreted into the cumulus matrix during ovulation

Problem Early studies have shown that an antibody to male reproductive tissue CD52 is a pathogenic factor of infertility. The molecule contains a unique carbohydrate antigen that induces antibodies interfering with sperm function. However, the characteristic properties of CD52 in female reproductive tissues are not known. We examined the expression and localization of CD52 in mature expanded cumulus masses. Method of study Mouse cumulus oocyte complexes were collected from [C57B1/6; DBA/2] F1 female mice having a superovulation treatment. Human cumulus cells were obtained from infertile patients taking in vitro fertilization-embryo transfer treatment under informed consent. CD52 messenger RNA (mRNA) and protein were detected using RT-PCR, quantitative PCR, western blotting and immunohistochemical methods. Results CD52 mRNA was found both in the human and mouse cumulus cells. Mouse CD52 mRNA was detected in cumulus cells but not oocytes and significantly increased after ovulation. The expression of the molecule was also confirmed at the protein level. Immunostaining with anti CD52 peptide antibody revealed that CD52 is present in cumulus cells and the extracellular matrix. Conclusion We first showed the expression of CD52 in human cumulus cells. CD52 has some functional roles around fertilization in females as well as in males.