Caveola-vesicle Complexes Research Articles

An Erythrocyte Membrane-Associated Antigen, PvTRAg-26 of Plasmodium vivax: A Study of Its Antigenicity and Immunogenicity.

Background: Plasmodium tryptophan-rich (TR) proteins have been proposed as potential vaccine candidate antigens. Among them, P. vivax tryptophan-rich antigens (PvTR-Ags), which have positionally conserved tryptophan residues in a TR domain, are highly antigenic in humans. Several of these antigens, including PvTRAg-26, have exhibited erythrocyte-binding activities.Methods: Subclasses of IgG antibodies against PvTRAg-26 were detected by enzyme-linked immunosorbent assay in 35 P. vivax infected patients and mice immunized with the recombinant antigen to characterize its antigenicity and immunogenicity. Moreover, the antigen-specific immune responses and Th1/Th2-type cytokine patterns of splenocytes from the immunized animals were determined in vitro. The subcellular localization of PvTRAg-26 in ring-stage parasites was also detected by indirect immunofluorescence assay.Results: The IgG1 and IgG3 levels in P. vivax-infected patients were significantly higher than those in uninfected individuals. In the PvTRAg-26-immunized mice, elevated levels of antigen-specific IgG antibodies were observed, dominated by the IgG1 subclass, and Th1-type cytokines were remarkably increased compared with Th2-type cytokines. Additionally, the subcellular location of the PvTRAg-26 protein was closely associated with the caveola-vesicle complex on the infected-erythrocyte membrane in the early ring stage of P. vivax.Conclusions: PvTRAg-26, a P. vivax TR antigen, with high antigenicity and immunogenicity, induces Th1-cytokine response and increases production of IgG1 antibodies. This immune profiling study provided a substantial evidence that PvTRAg-26 may be a potential candidate for P. vivax vaccine development.

Characterization of Caveola-Vesicle Complexes (CVCs) Protein, PHIST/CVC-8195 in Plasmodium vivax.

Plasmodium vivax produces numerous caveola-vesicle complex (CVC) structures beneath the membrane of infected erythrocytes. Recently, a member helical interspersed subtelomeric (PHIST) superfamily protein, PcyPHIST/CVC-8195, was identified as CVCs-associated protein in Plasmodium cynomolgi and essential for survival of this parasite. Very little information has been documented to date about PHIST/CVC-8195 protein in P. vivax. In this study, the recombinant PvPHIST/CVC-8195 N and C termini were expressed, and immunoreactivity was assessed using confirmed vivax malaria patients sera by protein microarray. The subcellular localization of PvPHIST/CVC-8195 N and C termini in blood stage parasites was also determined. The antigenicity of recombinant PvPHIST/CVC-8195 N and C terminal proteins were analyzed by using serum samples from the Republic of Korea. The results showed that immunoreactivities to these proteins had 61% and 43% sensitivity and 96.9% and 93.8% specificity, respectively. The N terminal of PvPHIST/CVC-8195 which contains transmembrane domain and export motif (PEXEL; RxLxE/Q/D) produced CVCs location throughout the erythrocytic-stage parasites. However, no fluorescence was detected with antibodies against C terminal fragment of PvPHIST/CVC-8195. These results suggest that the PvPHIST/CVC-8195 is localized on the CVCs and may be immunogenic in natural infection of P. vivax.

Host cell remodeling by pathogens: the exomembrane system inPlasmodium-infected erythrocytes

Malaria is caused by infection of erythrocytes by parasites of the genus Plasmodium. To survive inside erythrocytes, these parasites induce sweeping changes within the host cell, one of the most dramatic of which is the formation of multiple membranous compartments, collectively referred to as the exomembrane system. As an uninfected mammalian erythrocyte is devoid of internal membranes, the parasite must be the force and the source behind the formation of these compartments. Even though the first evidence of the presence these of internal compartments was obtained over a century ago, their functions remain mostly unclear, and in some cases completely unknown, and the mechanisms underlying their formation are still mysterious. In this review, we provide an overview of the different parts of the exomembrane system, describing the parasitophorous vacuole, the tubovesicular network, Maurer's clefts, the caveola-vesicle complex, J dots and other mobile compartments, and the small vesicles that have been observed in Plasmodium-infected cells. Finally, we combine the data into a simplified view of the exomembrane system and its relation to the alterations of the host erythrocyte.

Immunoprofiling of the Tryptophan-Rich Antigen Family in Plasmodium vivax

Tryptophan-rich antigens (TRAgs) are an antigen family that has been identified in human and rodent malaria parasites. TRAgs have been proposed as candidate antigens for potential vaccines. The Plasmodium vivax TRAg (PvTRAg) family includes 36 members. Each PvTRAg contains a tryptophan-rich (TR) domain in the C-terminal region. In this study, we recombinantly expressed all 36 PvTRAgs using a cell-free expression system, and, for the first time, profiled the IgG antibody responses against all PvTRAgs in the sera from 96 vivax malaria patients and 40 healthy individuals using protein microarray technology. The mean seropositive rate for all PvTRAgs was 60.3%. Among them, nine PvTRAgs were newly identified in this study and showed a seropositive rate of >50%. Five of them, PvTRAg_13, PvTRAg_15, PvTRAg_16, PvTRAg_26, and PvTRAg_29, produced higher levels of IgG antibody, even in low-endemicity countries. In addition, the results of an immunofluorescence analysis suggest that PvTRAgs are, at least in part, associated with caveola-vesicle complexes, a unique structure of P. vivax-infected erythrocytes. The mechanism of formation and the function of these abundant membrane structures are not known. Further investigation aimed at determining the functions of these proteins would lead to a better understanding of the blood-stage biology of P. vivax.

A 95 kDa protein of Plasmodium vivax and P. cynomolgi visualized by three‐dimensional tomography in the caveola–vesicle complexes (Schüffner's dots) of infected erythrocytes is a member of the PHIST family

Plasmodium vivax and P. cynomolgi produce numerous caveola-vesicle complex (CVC) structures within the surface of the infected erythrocyte membrane. These contrast with the electron-dense knob protrusions expressed at the surface of Plasmodium falciparum-infected erythrocytes. Here we investigate the three-dimensional (3-D) structure of the CVCs and the identity of a predominantly expressed 95 kDa CVC protein. Liquid chromatography - tandem mass spectrometry analysis of immunoprecipitates by monoclonal antibodies from P. cynomolgi extracts identified this protein as a member of the Plasmodium helical interspersed subtelomeric (PHIST) superfamily with a calculated mass of 81 kDa. We named the orthologous proteins PvPHIST/CVC-81(95) and PcyPHIST/CVC-81(95) , analysed their structural features, including a PEXEL motif, repeated sequences and a C-terminal PHIST domain, and show that PHIST/CVC-81(95) is most highly expressed in trophozoites. We generated images of CVCs in 3-D using electron tomography (ET), and used immuno-ET to show PHIST/CVC-81(95) localizes to the cytoplasmic side of the CVC tubular extensions. Targeted gene disruptions were attempted in vivo. The pcyphist/cvc-81(95) gene was not disrupted, but parasites containing episomes with the tgdhfr selection cassette were retrieved by selection with pyrimethamine. This suggests that PHIST/CVC-81(95) is essential for survival of these malaria parasites.

Caveolins and flotillin-2 are present in the blood stages of Plasmodium vivax

Blood stages of Plasmodium vivax induce the development of caveolae and caveola-vesicle complexes (CVC) in the membrane of their host erythrocyte. Caveolae are found in almost all types of cells and are involved in endogenous processes as calcium and cholesterol homeostasis, cell signalling, transporting, ligand internalization and transcytosis of serum components. Major structural components of caveolae are the proteins caveolins and flotillins. The functional role of caveolae in the P. vivax-infected erythrocyte is not properly understood. As these organelles have been shown to contain malaria antigens, it has been suggested that they are involved in the transport and release of specific parasite antigens from the infected erythrocyte and in the uptake of plasma proteins. Using specific antibodies to classical caveolae proteins and an immunolocalization approach, we found caveolin-2, caveolin-3, and flotillin-2 in the vesicle profiles and some CVC of P. vivax-infected erythrocytes. Caveolin-1-3 were not found in uninfected erythrocytes. This is the first report of identification and localization of caveolins in the CVC present in erythrocytes infected with P. vivax, thereby providing evidence of the role of this particular organelle in the protein-trafficking pathway that connect parasite-encoded proteins with the erythrocyte cytoplasm and the cell surface throughout the asexual blood cycle of vivax malaria parasite.

Antigenic characterization of Plasmodium vivax with monoclonal antibodies.

Monoclonal antibodies were produced against Plasmodium vivax obtained from patients living in southeastern Mexico, where P. vivax malaria is endemic. Nine hybridomas specific for this parasite were obtained. By an indirect immunofluorescence assay, seven antibodies were found to react with epitopes present in the cytoplasm of the infected erythrocyte and two with the parasite itself. By immunoblotting, five monoclonal antibodies reacted with a 17-kD protein band, three with an 85-kD band, and two with one of 45 kD. By immunogold electron microscopy, two antibodies that reacted with the cytoplasm of infected erythrocytes by immunofluorescence also labeled cytoplasmic clefts, and one, in addition, recognized caveola-vesicle complexes and the parasite matrix. These results demonstrate the value of monoclonal antibodies in identifying P. vivax antigens and disclosing their subcellular distribution.

Ultrastructure of erythrocytes fromAotus trivirgatus andSaimiri sciureus monkeys infected byPlasmodium vivax

Erythrocytes from Aotus and Saimiri monkeys parasitized by Plasmodium vivax show dramatic changes starting during the early stages of parasite development. Invaginations of the erythrocyte membrane, caveolae, are found during all parasite development stages. Up to six vesicles can be fused with one caveola, forming a caveola-vesicle complex. As the parasite grows, large accumulations of these vesicles can be seen within the erythrocyte cytoplasm. In addition to these caveolae-vesicle complexes, knob-like structures appear on the erythrocyte surface that are similar to those seen on the host-cell surface of P. falciparum-infected red cells. Extensive membrane-bound clefts spread throughout the erythrocytic cytoplasm, sometimes forming stacks or large whorls. The density of the red cell cytoplasm begins to decrease at an early stage of parasite development. All of these changes may be responsible for an increased fragility of the P. vivax-infected red cell from Aotus or Saimiri monkeys. Moreover, the large amount of parasite material that is released during rupture of the red cell may account for the high fever paroxysms that are characteristic of P. vivax malaria infection.

Plasmodium vivax: Malarial proteins associated with the membrane-bound caveola-vesicle complexes and cytoplasmic cleft structures of infected erythrocytes

The identification of antigens of parasite origin associated with the altered membrane of Plasmodium vivax-infected erythrocytes was undertaken in this study. The 125I-lactoperoxidase catalyzed surface radiolabeling of trophozoite-infected erythrocytes revealed new bands of 95 and 70 kDa not labeled in normal erythrocytes. Erythrocyte membrane-enriched preparations from [ 35S]methionine biosynthetically labeled-infected erythrocytes also indicated that in addition to bands at 95 and 70 kDa, several other parasite proteins were possibly membrane associated. Five monoclonal antibodies (Mabs) reactive with P. vivax produced an immunofluorescent pattern of numerous small dots scattered over the entire infected erythrocyte. This pattern mimics that of Schuffner's stippling; small red dots seen in Giemsa-stained P. vivax-infected erythrocytes, which represent accumulations of dye in caveola-vesicle complexes (CVC). Four of the monoclonal antibodies immunoprecipitated a Triton X-100 detergent-insoluble 95-kDa parasite protein which was localized by immunofluorescent assay and immunoelectron microscopy exclusively to the CVC. Two of these Mabs were immunofluorescence reactive with the surface of intact infected erythrocytes in suspension. The fifth Mab, which also localized exclusively to the CVC structures, immunoprecipitated a Triton X-100 extractable protein of 70 kDa. Two other monoclonal antibodies reacted exclusively with the numerous membranous cleft structures found in the cytoplasm of infected erythrocytes. This cleft-associated parasite antigen was 28 kDa in size. Some of these Mabs recognize epitopes and produce similar IFA patterns on erythrocytes infected with P. cynomotgi, P. knowlesi, and P. ovale parasites, but not with P. falciparum- or P. brasilianum-infected erythrocytes.

Comparative morphology of human and animal malaria parasites

Human and animal malaria parasites (Plasmodium falciparum, P. malariae, P. vivax, P. berghei, P. gallinaceum) were studied using special fixation and standardized methods, with special attention to their effects on host cells. Morphological alterations induced by the parasites in infected erythrocytes included knobs, invaginations, and caveola-vesicle complexes on the surface of the host cell and clefts, microvesicles, and small vesicles in the cytoplasm of the infected erythrocytes. For P. malariae, the ultrastructural study revealed invaginations with associated microvesicles, but knobs did not occur on the surface of infected erythrocytes. The development of invaginations and microvesicles in P. malariae-infected erythrocytes corresponded to the morphological alterations induced by P. vivax. A new hypothesis concerning the origin of Schüffner's dots is discussed.

Immunoelectron microscopic localization of vivax malaria antigens to the clefts and caveola-vesicle complexes of infected erythrocytes.

Erythrocytes infected with Plasmodium vivax show unique ultrastructural changes which include membranous structures in the host cell cytosol, called clefts, and caveola-vesicle complexes (CVC) in the infected erythrocyte membrane. It has been suggested that the latter structures correspond with the Schuffner's dots observed on Giemsastained thin films. The subcellular localization of a 28 kDa and a 95 kDa antigen of the erythrocytic stages of P. vivax was determined by post-embedding immunoelectron microscopy. Four monoclonal antibodies (MAbs) (2H12.B4,2H8.E10, 1H4.B6, and 4C12.G4) against the 95 kDa protein reacted with the vesicles of CVC and vesicles scattered in the cytoplasm of the infected erythrocytes. Two other MAbs (4C12.B10 and 4D7.B1) against a 28 kDa protein reacted with the cytoplasmic clefts and were also reactive with the vesicles and electron dense materials in parasitophorous vacuole. These parasite-induced structures make a contribution to the movement of some malaria proteins from the parasite to the erythrocyte surface.

Morphological changes in erythrocytes induced by malarial parasites.

Host cell alterations induced by Plasmodium falciparum, P. brasilianum, P. vivax and P. malariae were described by electron microscopy and post-embedding immunoelectron microscopy. P. falciparum infection induces knobs, electron-dense material and clefts in the erythrocyte. Clefts are involved in exporting P. falciparum antigen from the parasite to the erythrocyte membrane. P. falciparum antigen is present in knobs which adhere to endothelial cells causing the blockage of cerebral capillaries and ensuing pathological changes in cerebral tissues. P. brasilianum infection induces knobs, short and long clefts and electron-dense material. These structures appear to contain different P. brasilianum antigens. This indicates that each structure functions independently in trafficking P. brasilianum protein to the erythrocyte surface. P. vivax infection induces caveola-vesicle complexes and clefts in the erythrocyte. These structures are also involved in trafficking P. vivax protein from the parasite to the erythrocyte membrane. P. malariae induces caveolae, electron-dense material, vesicles, clefts and knobs in the erythrocyte. Although vesicles and caveolae are seen in the erythrocyte cytoplasm, they do not form caveola-vesicle complexes as seen in P. vivax-infected erythrocytes. They also appear to be involved in trafficking of malaria antigens. These studies, therefore, indicate that host cell changes occur in order to facilitate the transport of malarial antigens to the host cell membrane. The significance of these phenomena is still not clear.

Ultrastructure of Human Erythrocytes Infected with Plasmodium Ovale

Ultrastructural changes of the human erythrocytes infected with Plasmodium ovale were studied, and differences of P. ovale from other human malarial parasites were discussed. Four characteristic morphological alterations were observed on the host cells: caveola-vesicle complex, excrescence, nodule, and cytoplasmic cleft. Caveola-vesicle complexes consisted of caveolae surrounded by vesicles in an alveolar fashion and were formed along the host cell plasmalemma. Similar complexes have been reported previously in P. vivax but not in P. falciparum and P. malariae. This complex probably corresponds to a Schüffner's dot. Excrescences similar to those observed by others on the host cell membrane in P. falciparum and P. malariae were observed also in P. ovale. The excrescences in P. ovale, as in P. falciparum, were limited to the erythrocytes infected by asexual parasites, whereas the excrescences in P. malariae have been demonstrated on erythrocytes with asexual parasites and gametocytes. Nodules were observed on the erythrocytes infected with asexual parasites of P. ovale. Structures such as the nodule have not been described previously in the erythrocytes infected with any other species of malarial parasite. Clefts within the cytoplasm of the host erythrocytes were present in P. ovale, as in all other malarial parasites.

Electron Microscopy of Knobs in Plasmodium falciparum-Infected Erythrocytes

Erythrocytes infected by certain species of malarial parasites show two types of alterations of erythrocyte membranes such as electron-dense protrusions called knobs and caveola-vesicle complexes (Aikawa et al., 1975, Am. J. Path. 79: 285; Aikawa, 1977, Bull. WHO 55:2). The knobs are found on the membrane of erythrocytes infected with falciparum-type malaria parasites. These knobs form focal junctions with the endothelial cell membranes or with the knobs of other erythrocytes, resulting in the sequestration of these infected erythrocytes along the vascular endothelium (Trager et al., 1966, Bull. WHO 35: 883; Luse and Miller, 1971, Am. J. Trop. Med. Hyg. 20: 655; Aikawa et al., 1972, Z. Zellforsch. 124: 72; Udeinya et al., 1981, Science 213: 555). The erythrocyte membrane covering the knobs is immunologically different from the rest of the erythrocyte membrane (Kilejian et al., 1977, Exp. Parasitol. 42: 157; Langreth et al., 1979, J. Exp. Med. 150:1241; Chulay et al., 1981, Am. J. Trop. Med. Hyg. 30: 12). The erythrocytes infected with small trophozoites show few knobs, but they increase in number as the parasite grows within the erythrocyte. Because the morphology of these knobs is still not fully understood, we have attempted to characterize them by transmission and scanning electron microscopy together with freeze-fracture and etch techniques. Plasmodiumfalciparum (Camp strain) in Aotus erythrocytes and P. falciparum (Thailand and Liberian strains) in human erythrocytes were obtained from culture (Udeinya et al., 1981, Science 213: 555). The infected erythrocytes were fixed in 2% glutaraldehyde in 0.1 M cacodylate buffer (pH 7.4) containing 4% sucrose, washed in 0.1 M cacodylate buffer and postfixed for 1 hr in 1% osmium tetroxide. After fixation, the samples were centrifuged and then processed for thin section transmission and scanning electron microscopy. Freeze-fracturing and etching were performed on P. falciparum in human RBC's. Fracturing was carried out at -120 C under a vacuum of 2 X 10-7 Torr. Etching was performed at -100 C, under a vacuum of 2 X 10-7 Torr for 2 min. The surfaces obtained were replicated with platinum and carbon. Replicas were cleaned as described previously (Aikawa et al., 1981, J. Cell Biol. 91: 55). Transmission electron microscopy was performed with a JEOL 100CX electron microscope. Scanning electron microscopy was performed with a JEOL 100CX electron microscope with a scanning unit at 20 kV. Thin-section electron microscopy showed many knobs on the erythrocytes infected with P. falciparum (Fig. 1). Each knob is a cone-shaped, electron-dense structure measuring 30 to 40 nm in height and 9 to 100 nm in width and is covered by the erythrocyte membrane. The base is not sharply demarcated, but gradually merges into the erythrocyte cytoplasm. Scanning electron microscopy showed numerous cone-shaped knobs evenly distributed over the entire erythrocyte surface (Fig. 2). The distribution pattern of the knobs was more easily detectable by scanning electron microscopy than by thin-section transmission electron microscopy. Therefore, scanning electron microscopy appears to be a useful and easy method for the study of knob distribution. Freeze-fracture and etching demonstrated that the knobs were protruded structures and were evenly distributed over the erythrocyte membrane. The P face of the infected, human RBC membrane showed evenly distributed intramembrane particles (IMP) and no aggregation or depletion of IMP could be observed over the erythrocyte membrane covering the knobs (Fig.

Structural alteration of the erythrocyte membrane during malarial parasite invasion and intraerythrocytic development.

Erythrocyte entry by malarial merozoites causes structural alteration of the erythrocyte membrane. First, entry into erythrocytes by merozoites requires the formation of a junction between the erythrocyte membrane and the apical end of the merozoite. Secondly, migration of the junction parallel to the long axis of the merozoite brings the merozoite into an invagination of the erythrocyte membrane. Freeze-fracture studies show that the junction consists of a narrow band of rhomboidally arrayed intramembrane particles (IMP) on the P face of the erythrocyte membrane and matching rhomboidally arrayed pits on the E face. IMP on the P face of the erythrocyte membrane disappear beyond this junction, resulting in the absence of IMP from the P face of the parasitophorous vacuole membrane which originated from the erythrocyte membrane. The erythrocyte membrane is sealed off by fusion of the junction at the posterior end of the merozoite in the fashion of an iris diaphragm. After completing its entry into the erythrocyte the merozoite is surrounded by a parasitophorous vacuole membrane which is different in its molecular organization from the original erythrocyte membrane. In addition, two types of erythrocyte membrane modification are induced by intraerythrocytic parasites. They include electron-dense protrusions called knobs and caveola-vesicle complexes along the erythrocyte membrane.