Theileria parva: expression of a sporozoite surface coat antigen

A monoclonal antibody specific for the Theileria parva sporozoite, which recognizes a determinant on the surface coat and blocks sporozoite infectivity, was used to investigate the presence of the determinant on other stages of the parasite lifecycle. Immunofluorescence techniques did not demonstrate this determinant on the kinete, schizont, merozoite, or piroplasm stages of the parasite. Immunoautoradiography, using a tritiated form of the monoclonal antibody, on sections of infected salivary glands collected from ticks that had fed for 0, 1, 2, 3, or 4 days revealed that the determinant recognized was synthesized predominantly during sporogony, between 2 to 3 days after the tick started feeding. Immunoelectron microscopy was performed on ultrathin frozen sections of infected tick salivary glands incubated with the monoclonal antibody followed by Protein-A--colloidal gold. The antigen or its precursor could be detected in the developing parasite. In ticks fed 2 days, the sporoblast was labeled, both in the cytoplasm and on parasite membranes, often including the nuclear envelope. In sections from ticks fed 4 days, the sporozoite surface membrane was labeled, as were membrane-bounded sporozoite organelles identified as micronemes. Observation by immunofluorescence, on sporozoites incubated with bovine peripheral blood lymphocytes, suggested that the antigen recognized by the monoclonal antibody does not enter the lymphocyte during sporozoite endocytosis. We conclude that synthesis of the antigen or its precursor(s) occurs during sporogony in the feeding tick, at the time of maximal parasite proliferation, and precedes the formation of morphologically mature sporozoites; the antigen's role in the parasite life cycle also appears to be limited to events associated with the sporozoite entry process.     

The entry of Theileria parva sporozoites into bovine lymphocytes: evidence for MHC class I involvement

We have examined the process of Theileria parva sporozoite entry into susceptible bovine lymphocytes and have begun to identify one of the possible molecular interactions involved in the process. The entry process involves a defined series of events and we have used a number of experimental procedures in combination with a method of quantitation to examine various aspects of this process. T. parva sporozoites are nonmotile organisms and the initial sporozoite-lymphocyte interaction is a chance event which can occur at 0-2 degrees C. All subsequent stages in the process are temperature dependent, require the participation of live intact sporozoites and host cells, and involve some cytochalasin-inhibitable rearrangement of the host cell surface membrane or cytoskeleton. Sporozoite entry can be inhibited by antibodies (mAbs) reactive with major histocompatibility complex (MHC) class I molecules (IL-A 19, IL-A 88) and with beta 2 microglobulin (B1G6), whereas mAbs reactive with MHC class II molecules (IL-A 21, J 11), and a common panleucocyte surface antigen, (IL-A 87; a bovine equivalent of CD 11a) have no effect. These results indicate that MHC class I molecules play a role in the process of T. parva sporozoite entry into bovine lymphocytes although as yet the precise role has not been determined. Once internalized within the lymphocyte, a process that takes less than 3 min at 37 degrees C, the sporozoite rapidly escapes from the encapsulating host cell membrane; a process which occurs concurrently with the discharge of the contents of the sporozoite rhoptries and microspheres. The intracytoplasmic parasite is covered by a layer of sporozoite-derived fuzzy material to which host cell microtubules rapidly become associated.     

Plasmodium malariae: distribution of circumsporozoite protein in midgut oocysts and salivary gland sporozoites

The distribution of the circumsporozoite protein within developing Plasmodium malariae oocysts and salivary gland sporozoites was examined by immunoelectron microscopy using protein A-gold and a monoclonal antibody specific for the CS protein of P. malariae. Gold particles were found along the capsule of immature oocysts but rarely within the cytoplasm. Gold label was detected on the inner surface of peripheral vacuoles during oocyst maturation and the plasma membrane of the sporoblast. Salivary gland sporozoites and budding sporozoites in mature oocysts were labeled uniformly on the outer surface of their plasma membranes. The surface of sporozoites that ruptured into midgut epithelial cells were entirely covered with gold particles. No label was seen on the surface of sporozoites which ruptured into the midgut lumen. In addition, a rabbit polyclonal antibody against repeat a region of P. brasilianum CS protein reacted with P. malariae sporozoites.     

Identification of a sporozoite-specific member of the Toxoplasma SAG superfamily via genetic complementation

Toxoplasma gondii sporozoites possess an array of stage-specific antigens that are localized to the membrane and internal cellular space, as well as secreted into the primary parasitophorous vacuole. Specific labelling of viable sporozoites excysted from oocysts reveals a complex admixture of surface proteins partially shared with tachyzoites. SAG1, SRS3 and SAG3 were detected on sporozoites as well as numerous minor antigens. In contrast, tachyzoite SAG2A and B were completely absent whereas a dominant 25 kDa protein was unique to the sporozoite surface. The sporozoite gene encoding this protein was identified in tachyzoites genetically complemented with a sporozoite cDNA library and cloned via site-specific recombination into a bacterial shuttle vector. The sporozoite cDNA identified in these experiments encoded a protein with conserved structural features of the prototypical T. gondii SAG1 (P30) and shared sequence identity with surface proteins from Sarcocystis spp. This new member of the SAG superfamily was designated SporoSAG. Expression of SporoSAG in tachyzoites conferred enhanced invasion on transgenic parasites suggesting a role for this protein in oocyst/sporozoite transmission to susceptible hosts.     

Neutralization-sensitive epitopes are exposed on the surface of infectious Cryptosporidium parvum sporozoites

Cryptosporidiosis is a diarrheal disease of humans, calves, and other mammals caused by the coccidian parasite Cryptosporidium parvum. Immune bovine serum and two surface-reactive antisporozoite mAb with neutralizing activity were used to identify sporozoite surface Ag by radioimmunoprecipitation/SDS-PAGE and immunoblotting. When isolated sporozoites were incubated with mAb 18.44, 12 to 25 times the ID50 for mice was completely neutralized. This mAb binds diffusely to the sporozoite surface and recognizes a sporozoite surface Ag that eluted in the void volume of a Bio Gel A column with an exclusion limit of 500,000 daltons. The Ag recognized by mAb 18.44 was not radiolabeled with 125I or [35S] methionine, migrated with the dye front in SDS-PAGE, and was insensitive to proteinase K digestion, suggesting a non-protein composition. mAb 17.41 significantly neutralized 25 times the ID50 of sporozoites for mice. This mAb binds multifocally to the sporozoite surface and recognizes [35S] methionine-labeled sporozoite surface Ag of 28,000 m.w., 55,000 m.w., and 98,000 m.w. Immune bovine serum immunoprecipitated [35S] methionine- or 125I-labeled sporozoite Ag ranging from less than 14,300 m.w. to greater than 200,000 m.w., including surface Ag of 28,000 m.w. and 55,000 m.w. The results indicate that two different molecules capable of inducing neutralizing antibody are exposed on the surface of C. parvum sporozoites.     

Identification of antigens of Sarcocystis cruzi sporozoites, merozoites, and bradyzoites with monoclonal antibodies

Sporozoites and culture-derived merozoites of Sarcocystis cruzi were used to elicit monoclonal antibodies (MAb's) in mice. Some of these antibodies reacted with the surface of live sporozoites and merozoites as determined by immunofluorescence. An array of similar antigens was identified in Western blots of sporozoites by both anti-merozoite MAb's and an anti-sporozoite MAb. At least 1 antigen in blots of bradyzoites was identified by anti-merozoite MAb's and a cluster of antigens was identified by an anti-sporozoite antibody. These results indicate that several surface epitopes of sporozoites and merozoites are shared with molecules of bradyzoites and that antigen patterns of molecules bearing these epitopes in 3 stages of Sarcocystis may be either distinct or similar.     

Localization of globoside and Forssman glycolipids on erythrocyte membranes

Using the freeze-etch technique, the membrane localization of globoside, a principal glycolipid in human erythrocytes, and Forssman antigen, the chief glycolipid in sheep erythrocytes was evaluated using ferritin and colloidal gold as morphological markers for rabbit antibodies prepared against these glycolipids. Brief trypsinization of human red cell ghosts markedly aggregated intramembranous particles and permitted labeling of globoside, which appeared in a clustered arrangement. The aggregates of ferritin-anti-globoside differed from those of ferritin-wheat germ agglutinin, a label for glycophorin, which corresponded with the aggregates of intramembranous particles. Double-labeling of human trypsinized ghosts with anti-globoside/ Staphylococcal protein A-colloidal gold and ferritin-wheat germ agglutinin indicated that the patterns of labeling were different and that the aggregates of globoside did not bear a direct relationship to the intramembranous particles, which represent transmembrane proteins. Resealed sheep erythrocyte ghosts labeled with ferritin-conjugated rabbit anti-Forssman showed small clusters of Forssman glycolipid on the erythrocyte surface, which could be markedly aggregated with a second goat anti-rabbit antibody, indicating relative mobility of the small glycolipid domains. The distribution of ferritin-anti-Forssman label in sheep ghosts treated at pH 5.5 to aggregate intramembranous particles also did not show definite correspondence between intramembranous particles and the clusters of ferritin-anti-Forssman.     

A role for apical membrane antigen 1 during invasion of hepatocytes by Plasmodium falciparum sporozoites

Plasmodium sporozoites are transmitted through the bite of infected mosquitoes and invade hepatocytes as a first and obligatory step of the parasite life cycle in man. Hepatocyte invasion involves proteins secreted from parasite vesicles called micronemes, the most characterized being the thrombospondin-related adhesive protein (TRAP). Here we investigated the expression and function of another microneme protein recently identified in Plasmodium falciparum sporozoites, apical membrane antigen 1 (AMA-1). P. falciparum AMA-1 is expressed in sporozoites and is lost after invasion of hepatocytes, and anti-AMA-1 antibodies inhibit sporozoite invasion, suggesting that the protein is involved during invasion of hepatocytes. As observed with TRAP, AMA-1 is initially mostly sequestered within the sporozoite. Upon microneme exocytosis, AMA-1 and TRAP relocate to the sporozoite surface, where they are proteolytically cleaved, resulting in the shedding of soluble fragments. A subset of serine protease inhibitors blocks the processing and shedding of both AMA-1 and TRAP and inhibits sporozoite infectivity, suggesting that interfering with sporozoite proteolytic processing may constitute a valuable strategy to prevent hepatocyte infection.     

Hepatocyte CD81 is required for Plasmodium falciparum and Plasmodium yoelii sporozoite infectivity

Plasmodium sporozoites are transmitted through the bite of infected mosquitoes and first invade the liver of the mammalian host, as an obligatory step of the life cycle of the malaria parasite. Within hepatocytes, Plasmodium sporozoites reside in a membrane-bound vacuole, where they differentiate into exoerythrocytic forms and merozoites that subsequently infect erythrocytes and cause the malaria disease. Plasmodium sporozoite targeting to the liver is mediated by the specific binding of major sporozoite surface proteins, the circumsporozoite protein and the thrombospondin-related anonymous protein, to glycosaminoglycans on the hepatocyte surface. Still, the molecular mechanisms underlying sporozoite entry and differentiation within hepatocytes are largely unknown. Here we show that the tetraspanin CD81, a putative receptor for hepatitis C virus, is required on hepatocytes for human Plasmodium falciparum and rodent Plasmodium yoelii sporozoite infectivity. P. yoelii sporozoites fail to infect CD81-deficient mouse hepatocytes, in vivo and in vitro, and antibodies against mouse and human CD81 inhibit in vitro the hepatic development of P. yoelii and P. falciparum, respectively. We further demonstrate that the requirement for CD81 is linked to sporozoite entry into hepatocytes by formation of a parasitophorous vacuole, which is essential for parasite differentiation into exoerythrocytic forms.     

Theileria parva: sporozoite entry into bovine lymphocytes is not dependent on the parasite cytoskeleton.

The main conclusion from the present study is that T. parva sporozoite entry is dependent on a functional host cell actin cytoskeleton and is not driven by the parasite. Treating lymphocytes with cytochalasin D resulted in a dose-dependent reduction in the levels of host cell infection. However, the primary effect was to block sporozoite binding and only at the highest concentration (20 microM) was sporozoite internalization significantly reduced. In fact at lower concentrations (1-10 microM) cytochalasin treatment lead to a relative increase in sporozoite internalization. The results are consistent with sporozoite entry being primarily a passive process and with a functional host cell actin cytoskeleton that is required only to maintain the molecular integrity of the surface membrane. Thus T. parva sporozoite entry differs from the process in other apicomplexans, although the results are consistent with a number of features of sporozoite biology. Treatment of lymphocytes with either the microtubule-destabilizing agent, nocodazole, or taxol, which induces microtubule polymerization, had no significant effect on sporozoite binding or entry. As both reagents had the expected effects on the lymphocyte microtubule system, it is unlikely that host cell microtubules are essential for successful sporozoite invasion or establishment.     

Imaging of immunogold labelled antigens on capping thymocytes by confocal reflection contrast scanning optical microscopy

Confocal laser scanning optical microscopy (CLSM) in the reflection contrast mode has been used to image single 40 nm gold particles, and to study changes in the distribution of gold label associated with capping of the leukocyte sialoglycoprotein (LSGP) antigen on the surface of fixed rat thymocytes, labelled with the mouse monoclonal antibody W3/13 and a goat anti-mouse IgG immunogold conjugate. This imaging method has also been applied to live thymocytes labelled with gold-conjugated antibodies, to study the dynamics of the capping process.     

Molecular interactions of cultured turkey kidney cells with specific antigens of Eimeria adenoeides sporozoites

Earlier studies suggested that specific communication between the parasite and the host cell may play a role in cellular invasion by sporozoites of species of avian Eimeria. In this study, quantification of cellular invasion and modified Western blot analysis were used to explore the possibility that parasite receptors for interaction with the host cell might be involved in the sporozoite-host cell communication. Invasion in cultured cells treated with a homogenate of Eimeria adenoeides sporozoites was approximately 50% lower than that in untreated cultures. When the sporozoite homogenate was solubilized in sodium dodecyl sulfate and electrophoretically separated, components of the cultured host cells bound consistently to sporozoite bands having Mr of 23 and 40 kDa. Biotinylation of intact sporozoites revealed at least 14 biotin-labeled bands, including bands at 23 and 40 kDa, that were considered to be surface molecules. If the sporozoites were incubated in trypsin after they were biotinylated, only two biotinylated bands at 18 and 23 kDa remained; the 40-kDa biotinylated band was not detected. Despite the removal of the majority of the surface molecules, the cell homogenate still bound to the trypsin-treated sporozoites; the intensity of the label was similar to that resulting from the binding of cell homogenate to untreated sporozoites. The data show specific interactions between 23- and 40-kDa sporozoite bands and host cell components, and provide evidence that the 23-kDa molecule may be located on the sporozoite surface and the 40-kDa molecule located intracellularly.     

AMA1 and MAEBL are important for Plasmodium falciparum sporozoite infection of the liver

The malaria sporozoite injected by a mosquito migrates to the liver by traversing host cells. The sporozoite also traverses hepatocytes before invading a terminal hepatocyte and developing into exoerythrocytic forms. Hepatocyte infection is critical for parasite development into merozoites that infect erythrocytes, and the sporozoite is thus an important target for antimalarial intervention. Here, we investigated two abundant sporozoite proteins of the most virulent malaria parasite Plasmodium falciparum and show that they play important roles during cell traversal and invasion of human hepatocytes. Incubation of P. falciparum sporozoites with R1 peptide, an inhibitor of apical merozoite antigen 1 (AMA1) that blocks merozoite invasion of erythrocytes, strongly reduced cell traversal activity. Consistent with its inhibitory effect on merozoites, R1 peptide also reduced sporozoite entry into human hepatocytes. The strong but incomplete inhibition prompted us to study the AMA‐like protein, merozoite apical erythrocyte‐binding ligand (MAEBL). MAEBL‐deficient P. falciparum sporozoites were severely attenuated for cell traversal activity and hepatocyte entry in vitro and for liver infection in humanized chimeric liver mice. This study shows that AMA1 and MAEBL are important for P. falciparum sporozoites to perform typical functions necessary for infection of human hepatocytes. These two proteins therefore have important roles during infection at distinct points in the life cycle, including the blood, mosquito, and liver stages.     

Plasmodium vivax:A Monoclonal Antibody Recognizes a Circumsporozoite Protein Precursor on the Sporozoite Surface

Gonzalez-Ceron, L., Rodriguez, M. H., Wirtz, R. A., Sina, B. J., Palomeque, O. L., Nettel, J. A., and Tsutsumi, V. 1998.Plasmodium vivax:A monoclonal antibody recognizes a circumsporozoite protein precursor on the sporozoite surface.Experimental Parasitology90, 203–211. The major surface circumsporozoite (CS) proteins are known to play a role in malaria sporozoite development and invasion of invertebrate and vertebrate host cells.Plasmodium vivaxCS protein processing during mosquito midgut oocyst and salivary gland sporozoite development was studied using monoclonal antibodies which recognize different CS protein epitopes. Monoclonal antibodies which react with the CS amino acid repeat sequences by ELISA recognized a 50-kDa precursor protein in immature oocyst and additional 47- and 42-kDa proteins in older oocysts. A 42-kDa CS protein was detected after initial sporozoite invasion of mosquito salivary glands and an additional 50-kDa precursor CS protein observed later in infected salivary glands. These data confirm previous results with otherPlasmodiumspecies, in which more CS protein precursors were detected in oocysts than in salivary gland sporozoites. A monoclonal antibody (PvPCS) was characterized which reacts with an epitope found only in the 50-kDa precursor CS protein. PvPCS reacted with allP. vivaxsporozoite strains tested by indirect immunofluorescent assay, homogeneously staining the sporozoite periphery with much lower intensity than that produced by anti-CS repeat antibodies. Immunoelectron microscopy using PvPCS showed that the CS protein precursor was associated with peripheral cytoplasmic vacuoles and membranes of sporoblast and budding sporozoites in development oocysts. In salivary gland sporozoites, the CS protein precursor was primarily associated with micronemes and sporozoite membranes. Our results suggest that the 50-kDa CS protein precursor is synthesized intracellularly and secreted on the membrane surface, where it is proteolytically processed to form the 42-kDa mature CS protein. These data indicate that differences in CS protein processing in oocyst and salivary gland sporozoites development may occur.     

Immunocytochemical evidence for the translocation of alpha-granule membrane glycoprotein IIb/IIIa (integrin alpha IIb beta 3) of human platelets to the surface membrane during the release reaction.

The localization of glycoprotein (GP) IIb/IIIa (integrin alpha IIb beta 3) in both resting and thrombin-activated platelets was studied immunocytochemically. By the preembedding method where only the GP IIb/IIIa molecules on the surface of platelets were immunostained, the distribution of protein A-colloidal gold label was randomly distributed along the surface membrane of resting platelets at a density of 18.0 +/- 2.7 gold particles/microns of membrane. At 15 s after stimulation by 0.1 U/ml of thrombin in an unstirred platelet suspension, the spheroid-shaped platelets with pseudopodia still had normal numbers of alpha-granules, and the density of gold particles was 19.7 +/- 3.6 particles/microns. At 5 min, the alpha-granules were no longer present because of the release reaction, and the density of gold particles significantly increased (27.0 +/- 3.7 particles/microns; p less than 0.01). In immuno-stained ultra-thin frozen sections, the gold particles were detected not only on the surface membrane, including the open canalicular system (OCS), but also on the alpha-granule membranes of resting platelets. At 30 s after thrombin stimulation the alpha-granules fused with the OCS, resulting in the formation of a swollen OCS, which still had gold particles on its membrane. At 5 min, the gold particles were detected on the membrane of the swollen OCS located near the surface membrane, while very few gold particles were present on the membrane of the OCS in the central part of the platelets. These results demonstrate that alpha-granule membrane GPIIb/IIIa translocates to the surface membrane through the membrane of the OCS.(ABSTRACT TRUNCATED AT 250 WORDS)     

Eimeria acervulina: DNA cloning and characterization of recombinant sporozoite and merozoite antigens

Genes encoding antigens of Eimeria acervulina were cloned from cDNA expression libraries prepared from the sporozoite and merozoite stages in order to examine humoral and cellular immune responses to this protozoan parasite. Two clones expressing surface antigens were characterized by DNA hybridization studies to identify homologous genomic DNA fragments. The proteins they encode were identified by 125I-labeling, immunoblotting, immunofluorescence, and T-cell activation experiments. One, designated cSZ-1, encodes a 130-kDa beta-galactosidase fusion protein which represents a portion of a p240/p160 immunodominant sporozoite surface antigen. Immunofluorescence studies using anti-cSZ-1 sera and live or 1% paraformaldehyde-fixed E. acervulina sporozoites have confirmed this surface locale. Purified cSZ-1 fusion protein, which is not recognized by sera from E. acervulina-infected chickens, induced the activation of immune T lymphocytes in vitro. Another cDNA clone, designated cMZ-8, gives rise to a 150-kDa fusion protein and encodes a portion of a p250 immunodominant merozoite surface antigen. This was established by immunoblotting of 125I-labeled merozoite proteins with anti-cMZ-8 sera and immunofluorescence staining of live and 1% paraformaldehyde-fixed E. acervulina merozoites. Purified cMZ-8 is recognized by sera from E. acervulina-infected chickens and induces a significant activation of immune T lymphocytes in vitro.     

Immunogold localization of nitrate reductase in maize leaves

Mature maize leaf tissue (Zea mays L.) was immunolabeled using a pre-embedding protocol with specific antibodies for nitrate reductase and protein A-colloidal gold. Immunogold label was found exclusively in the cytoplasm of mesophyll cells; no reaction was detected in bundle sheath cells. Chloroplasts, which were sliced open during cryosectioning, had no labeling. Thus, it appears nitrate reductase is localized exclusively in the cytoplasm of maize leaf mesophyll cells. No gold labeling was found on tissue sections embedded in L. R. White's or Lowicryl resin, indicating that previous chloroplast localization utilizing these protocols may be artifactual, resulting from shared epitopes or nonspecific antibody binding.     

Changes in the cytoplasmic elements of cultured cells infected with Eimeria vermiformis sporozoites

Epithelial-type (PK-15) and fibroblast-type (MDBK) mammalian cell cultures were inoculated with purified Eimeria vermiformis sporozoites. Matched samples from 0 to 93 h after inoculation (HAI) were processed for electron microscopy; half of the sample preparations were extracted with non-ionic detergent prior to fixation. Specimens were examined by both transmission and scanning electron microscopy. Numerous sporozoites were attached to the cultured cells from 2 to 93 HAI, usually near the cell periphery. Some host cell microvilli extended up and appeared attached to the sporozoites. Sporozoites fixed during the penetration process were markedly constricted at the site of entry; however, no noticeable changes occurred in the host cell membrane or surface microvilli during sporozoite invasion or in sporozoite-infected cells. In cells extracted with 1% Triton X-100, the host cytoskeleton was progressively reorganized about the parasites but changes were limited to the immediate area of the sporozoite. Around resident sporozoites, the cytoskeleton became less dense but also more ordered, which contrasted with adjacent cell areas. Cytoskeletal elements passed both over and under the parasites. The appearance of the cytoskeleton suggested that the host cell formed a loose, basket-like net of cytoskeletal elements about the parasite.