Malaria sporozoite-hepatocyte interactions.
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Amino Acid Sequence↗
Biomedical subjects
U Frevert
Publications and source records attributed to U Frevert.
Malaria circumsporozoite protein binds to heparan sulfate proteoglycans associated with the surface membrane of hepatocytes.
During feeding by infected mosquitoes, malaria sporozoites are injected into the host's bloodstream and enter hepatocytes within minutes. The remarkable target cell specificity of this parasite may be explained by the presence of receptors for the region II-plus of the circumsporozoite protein (CS) on the basolateral domain of the plasma membrane of hepatocytes. We have now identified these receptors as heparan sulfate proteoglycans (HSPG). The binding of CS to the receptors is abolished by heparitinase treatment, indicating that the recognition of region II-plus is via the glycosaminoglycan chains. We have purified and partially characterized the CS-binding HSPGs from HepG2 cells. They have a molecular weight of 400,000-700,000, are tightly associated with the plasma membrane, and are released from the cell surface by very mild trypsinization, a property which the CS receptors share with the syndecan family of proteoglycans.
Amino Acid Sequence↗
The basolateral domain of the hepatocyte plasma membrane bears receptors for the circumsporozoite protein of Plasmodium falciparum sporozoites.
Minutes after injection into the circulation, malaria sporozoites enter hepatocytes. The speed and specificity of the invasion process suggest that it is receptor mediated. We show here that recombinant Plasmodium falciparum circumsporozoite protein (CS) binds specifically to regions of the plasma membrane of hepatocytes exposed to circulating blood in the Disse space. No binding has been detected in other organs, or even in other regions of the hepatocyte membrane. The interaction of CS with hepatocytes, as well as sporozoite invasion of HepG2 cells, is inhibited by synthetic peptides representing the evolutionarily conserved region II of CS. We conclude that region II is a sporozoite ligand for hepatocyte receptors localized to the basolateral domain of the plasma membrane. Our findings provide a rational explanation for the target cell specificity of malaria sporozoites.
Amino Acid Sequence↗
Stage-specific expression and intracellular shedding of the cell surface trans-sialidase of Trypanosoma cruzi.
We have used antibodies to the Trypanosoma cruzi trans-sialidase and to its product, the host cell invasion-related Ssp-3 epitope, to study the expression of the corresponding antigens during the intracellular development of the parasite and in the extracellular trypomastigotes. As soon as 2 h after host cell invasion, trans-sialidase was no longer detected, whereas the Ssp-3 epitope was still present on intracellular parasites. The amastigotes which subsequently developed remained nonreactive with the antibodies. Expression of enzymatically active T. cruzi trans-sialidase started again only after transformation of the amastigotes into trypomastigotes 72 h after host cell invasion. trans-Sialidase was shed from the trypanosomes into the host cell cytoplasm, where the enzyme accumulated until release of the parasites. All released trypomastigotes expressed trans-sialidase on their surfaces and in the flagellar pockets, but stumpy trypomastigotes were stained more intensely than slender trypomastigotes. Ssp-3, the sialylated reaction product of trans-sialidase, was assembled only after rupture of the host cell membrane and was detected on the plasma membranes and in the flagellar pockets of all trypomastigotes.
Animals↗
Trypanosoma congolense bloodstream forms evade complement lysis in vitro by shedding of immune complexes.
In the presence of antibodies against the variant surface glycoprotein (VSG) and guinea pig complement, Trypanosoma congolense bloodstream forms were lysed. Parasites, which had been preincubated with antibodies at 37 degrees C before addition of complement, escaped from complement lysis in a time- and temperature-dependent process. Preincubation caused removal of the antibodies from the cell surface by formation of filopodia and accumulation of the immune complexes between aggregated cells. Addition of secondary antibodies or of complement component C1q did not enhance this effect. In order to eliminate effects due to cell aggregation, single living trypanosomes, which had been immobilized by attachment to formvar-coated glass slides, were incubated under equivalent conditions. Immunofluorescence showed that in these experiments, anti-VSG antibodies were neither capped nor shed from the surface unless coincubation with secondary antibodies or C1q was performed. Fixation of the cells after incubation with anti-VSG prevented patching and capping of the antibodies. Removal of immune complexes apparently required no secondary cross-linker: removal from the surface of T. congolense obviously occurred during cell aggregation. This mechanism could therefore be of significance also in vivo.
Animals↗
Biogenesis of bacteriorhodopsin.
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Amino Acids↗
Formation of filopodia in Trypanosoma congolense by crosslinking the variant surface antigen.
Trypanosoma congolense was exposed to various substances binding to the variant surface antigen (VSG). All methods of crosslinking VSG molecules caused the rapid accumulation of ligands along the line of flagellar attachment and their shedding by formation of coat-covered vesicles and filopodia. This phenomenon was observed after treatment of the parasites with concanavalin A (Con A), anti-VSG-IgG plus protein A-gold, attachment of the cells to surfaces coated with poly-L-lysine and Con A and to Formvar films before negative staining. Moreover, trypanosomes aggregated by primary antibodies formed vesicles and filopodia at the points of contact. Those antibodies bound to the remaining cell surface, however, remained distributed uniformly. This indicates that primary antibodies alone do not cause crosslinking of VSG on the surface of T.congolense.
Animals↗
Endocytosis and intracellular occurrence of the variant surface glycoprotein in Trypanosoma congolense.
Trypanosoma congolense bloodstream forms were examined for binding sites of polyclonal anti-variant surface glycoprotein (VSG) antibodies using immunoelectron microscopy. Besides the surface, the antibodies labeled intracellular vesicles, the tubular membrane system, secondary lysosomes, and the digestive vacuole. Protein A gold (PAG), peroxidase gold (POG), anti-VSG antibodies preincubated with PAG, ferritin, concanavalin A-ferritin, and microperoxidase were examined for their suitability as endocytosis tracers in combination with immunoelectron microscopy. Endocytosis of PAG and POG was most effective and was mediated by vesicles transporting the tracer to secondary lysosomes. Gold particles eventually accumulated in the digestive vacuole. Apparantly only low amounts of VSG were internalized during endocytosis. VSG export from the cell interior to the flagellar pocket was not observed during excessive endocytosis of PAG, whereas after incubation with substances causing the formation of filopodia by binding to the surface coat, VSG-labeled vesicles were present near the flagellar pocket.
Animals↗
[How the academic physician replaced the hangman and the stone cutter. Medicine and the lower classes in the 19th century: professional interests--missions of civilization--social politics].
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Germany↗
In vitro susceptibility of different mammalian lymphocytes to sporozoites of Theileria annulata.
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Animals↗
Isolation and immunoelectromicroscopical characterization of Theileria annulata macroschizonts.
Theileria annulata macroschizonts were isolated from bovine lymphoblastoid cells grown in cell culture. To release the parasites, the cells were homogenized under hypotonic conditions. Intact host lymphocyte nuclei were lysed and the resulting chromatin precipitate was degraded by DNase. Host cell fragments were removed by ion-exchange chromatography. As revealed by electron microscopy, the preparations were free of intact host lymphocytes, lymphocyte nuclei and organelles. Antisera raised in rabbits against purified macroschizonts showed a specific reaction with the intracellular parasite in the indirect immunofluorescence test and in immuno-electron microscopy.
Animals↗
Morphological changes in Trypanosoma congolense after proteolytic removal of the surface coat.
Bloodstream forms of Trypanosoma congolense were exposed to proteases at various concentrations, and the consequences of this treatment were continuously examined by electron microscopy. Unexpectedly, proteolysis did not simply result in the removal of the surface coat, but in dramatic morphological changes characterized by membrane adhesions, subsequently leading to flagella/plasmamembrane and to plasmamembrane/plasmamembrane fusions. The resulting axonemal internalization and rearrangement of cell organelles were followed by profound changes in cell shape. The axonemal motility, however, was maintained.
Animals↗
Professional medicine and the working classes in Germany.
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Germany↗
Molecular characterization of glycosomal NAD(+)-dependent glycerol 3-phosphate dehydrogenase from Trypanosoma brucei rhodesiense.
The primary structure of a 38-kDa protein isolated from membrane preparations of African trypanosomes was determined by protein and DNA sequencing. Searching of the protein database with the trypanosome translated amino acid sequence identified glycerol 3-phosphate dehydrogenase (EC 1.1.1.8) from various prokaryotic and eukaryotic organisms as the optimal scoring protein. Surprisingly, the eukaryotic trypanosome enzyme showed the highest degree of sequence identity with the corresponding enzyme from the prokaryote Escherichia coli. The trypanosome molecule was expressed in Escherichia coli and found to be enzymatically active, thus confirming the identity of the molecule as an NAD(+)-dependent glycerol 3-phosphate dehydrogenase. A monoclonal antibody specific for the 38-kDa protein was used to localize the enzyme to glycosomes. Immunoblotting showed that the monoclonal antibody bound to a 38-kDa protein in African trypanosomes but not in T. cruzi, Leishmania or Crithidia. The enzyme has a pI of 9.1, a net charge of +17 and contains the peroxisome-like targeting tripeptide SKM at its C-terminus, all characteristic of glycosomal enzymes. Amino acids predicted to be involved in the NAD(+)-dependent glycerol 3-phosphate dehydrogenase active site have diverged from those of the mammalian enzyme. Kinetic analyses of the trypanosome GPD and GPD from rabbit muscle showed that the Km values of the two enzymes are different. The data suggest that the trypanosome protein may be a candidate target for rational drug design.
Amino Acid Sequence↗
Cell surface glycosaminoglycans are not obligatory for Plasmodium berghei sporozoite invasion in vitro.
The malaria circumsporozoite (CS) protein binds to glycosaminoglycan chains from heparan sulfate proteoglycans present on the basolateral surface of hepatocytes and hepatoma cells in vitro. When injected into mice, CS protein is rapidly cleared from the blood circulation by hepatocytes. The binding region for the HSPGs is the evolutionarily conserved region II-plus of the CS protein. Here we have asked whether the presence of glycosaminoglycans on the plasma membrane of target cells is required for sporozoite invasion in vitro. Two types of target cells were used: HepG2 cells, which are permissive for Plasmodium berghei sporozoite development into mature exoerythrocytic forms, and CHO cells, in which the intracellular development of the parasites is arrested early after penetration. The invasion of mutant CHO cells expressing undersulfated glycosaminoglycans or no glycosaminoglycans was only inhibited 41-49% or 24-32%, respectively, in comparison to invasion of CHO-K1 cells. Previous cleavage of HepG2 surface membrane glycosaminoglycans with heparinase or heparitinase had no significant inhibitory effect on subsequent P. berghei sporozoite invasion and EEF development in these cells, although the glycosaminoglycan lyase treatments removed over 80% of CS binding sites from the cell surface. These results suggest that although the presence of glycosaminoglycans on the target cell surface enhances sporozoite invasion, glycosaminoglycans are not required for sporozoite penetration or the development of exoerythrocytic forms in vitro.
Animals↗