Rapid clearance of malaria circumsporozoite protein (CS) by hepatocytes.

The circumsporozoite protein (CS) covers uniformly the plasma membrane of malaria sporozoites. In vitro, CS multimers bind specifically to regions of the hepatocyte plasma membrane that are exposed to circulating blood in the Disse space. The ligand is in the region II-plus of CS, an evolutionarily conserved stretch of the protein that has amino acid sequence homology to a cell adhesive motif of thrombospondin. We have now found that intravenously injected CS constructs bind rapidly to the basolateral surface of hepatocytes, provided that the recombinant proteins contain region II-plus, and that they are aggregated. Significant amounts of CS were not retained in any other organ. The striking parallelism between these in vitro and in vivo findings with the target specificity of malaria sporozoites, reinforces the hypothesis that the attachment of the parasites to hepatocytes is via region II-plus of CS.

Remnant lipoproteins inhibit malaria sporozoite invasion of hepatocytes.

Remnants of lipoproteins, intestinal chylomicrons, and very low density lipoprotein (VLDL), are rapidly cleared from plasma and enter hepatocytes. It has been suggested that remnant lipoproteins are initially captured in the space of Disse by heparan sulfate proteoglycans (HSPGs), and that their subsequent internalization into hepatocytes is mediated by members of the LDL-receptor gene family. Similarly to lipoprotein remnants, malaria sporozoites are removed from the blood circulation by the liver within minutes after injection by Anopheles mosquitoes. The sporozoite's surface is covered by the circumsporozoite protein (CS), and its region II-plus has been implicated in the binding of the parasites to glycosaminoglycan chains of hepatocyte HSPGs. Lactoferrin, a protein with antibacterial properties found in breast milk and neutrophil granules, is also rapidly cleared from the circulation by hepatocytes, and can inhibit the hepatic uptake of lipoprotein remnants. Here we provide evidence that sporozoites, lactoferrin, and remnant lipoproteins are cleared from the blood by similar mechanisms. CS, lactoferrin, and remnant lipoproteins compete in vitro and in vivo for binding sites on liver cells. The relevance of this binding event for sporozoite infectivity is highlighted by our demonstration that apoliprotein E-enriched beta-VLDI and lactoferrin inhibit sporozoite invasion of HepG2 cells. In addition, malaria sporozoites are less infective in LDL-receptor knockout (LDLR -/-) mice maintained on a high fat diet, as compared with littermates maintained on a normal diet. We conclude that the clearance of lipoprotein remnants and sporozoites from the blood is mediated by the same set of highly sulfated HSPGs on the hepatocyte plasma membrane.

Structural and functional properties of region II-plus of the malaria circumsporozoite protein.

During feeding, infected mosquitos inject malaria sporozoites into the host circulation. Within minutes, the parasites are found in the liver where they initiate the first stage of malaria infection. All species of malaria sporozoites are uniformly covered by the circumsporozoite protein (CS), which contains a conserved COOH-terminal sequence called region II-plus. We have previously shown that region II-plus is the parasite's hepatocyte-binding ligand and that this ligand binds to heparan sulfate proteoglycans (HSPGs) on the hepatocyte membrane. Using a series of substituted region II-plus peptides, we show here that the downstream basic amino acids as well as the interdispersed hydrophobic residues are required for binding of CS to hepatocyte HSPGs. We also show that this positively charged stretch of amino acids must be aggregated in order to bind to the receptor. On the basis of this information, we have synthesized a multiple antigen peptide that mimics the hepatocyte-binding ligand. This construct inhibits both CS binding to HepG2 cells in vitro as well as CS clearance in mice.

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.

Cell invasion by the vertebrate stages of Plasmodium.

Protozoans of the genus Plasmodium are the causative agents of malaria; they have a complex life cycle involving vertebrate and arthropod hosts and have three distinct invasive stages. Although the invasive stages probably invade cells using similar mechanisms, each stage has a different host cell specificity and utilizes different receptors to enter cells.

An immunoradiometric assay for the quantification of Plasmodium sporozoite invasion of HepG2 cells.

Malaria infection in a vertebrate host is initiated when Plasmodium sporozoites invade hepatocytes after injection by an infected mosquito. In vitro, the parasites invade and develop in HepG2 cells and these cells have been used to study target cell invasion by sporozoites. Previously described in vitro invasion assays involve staining and counting of intracellular sporozoites or exoerythrocytic forms of the parasite. Here we describe an immunoradiometric assay that can quantify sporozoite invasion of HepG2 cells in vitro. The assay relies on the differential detection of intracellular and extracellular circumsporozoite protein (CS; the major surface protein of the sporozoite) which can then be used to calculate the efficiency of invasion. Since this assay can be performed more rapidly than the current assays in which parasites must be counted under a microscope, it enables investigators to more rapidly screen inhibitors of sporozoite invasion.

Anopheles stephensi salivary glands bear receptors for region I of the circumsporozoite protein of Plasmodium falciparum.

In the mosquito, Plasmodium sporozoites rupture from oocysts found on the midgut wall, circulate in the hemolymph and invade salivary glands where they wait to be injected into a vertebrate host during a bloodmeal. The mechanisms by which sporozoites specifically attach to and invade salivary glands are not known but evidence suggests that it is a receptor-mediated process. Here we show that the major surface protein of sporozoites, the circumsporozoite protein (CS), binds preferentially to salivary glands when compared to other organs exposed to the circulating hemolymph. In addition, we show that a peptide encompassing region I, a highly conserved sequence found in all rodent and primate Plasmodium CS proteins, inhibits binding of CS to mosquito salivary glands.

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.

The malaria sporozoite's journey into the liver.

Perhaps the most challenging event of the malaria parasite's lifecycle is the sporozoite's journey to the hepatocyte. Because few parasites are injected by the mosquito, they must be efficiently and rapidly targeted to hepatocytes, where they will invade and develop into merozoites, the form of the parasite infective for red blood cells. Little is known about how sporozoites make their way to the liver and subsequently invade hepatocytes. Some evidence suggests that they are initially trapped by Kupffer cells and then transported to hepatocytes. Other findings support the hypothesis that sporozoites home to hepatocytes directly. We have found that the major surface protein of malaria sporozoites, the CS protein, binds to the basolateral domain of hepatocytes and, when injected intravenously into mice, is rapidly cleared from the circulation by the liver. Whether sporozoites are arrested in the liver by the same mechanisms as CS protein is not known, although preliminary data suggests this may be the case. Other sporozoite proteins are also likely to be involved in hepatocyte invasion. TRAP or SSP2, found on the parasite surface and in micronemes, binds to hepatocytes in a similar pattern as CS protein. There is evidence demonstrating its involvement in invasion, although it is not known whether it functions in the initial sequestration of the parasites by the liver or in subsequent invasion events.

Long-range restriction maps of Plasmodium falciparum chromosomes: crossingover and size variation among geographically distant isolates.

Homologous chromosomes from the human malaria parasite Plasmodium falciparum exhibit striking size polymorphism from isolate to isolate. To examine the structural basis for these variations, we have determined full-length restriction maps of chromosome 4 from three P. falciparum clones. Two clones, HB3 and 3D7, are derived from geographically distant strains, while the third, XP5, is the product of an HB3/3D7 cross. The restriction maps show that, while the overall structure and organization of chromosome 4 from each clone are similar, large-scale variations occur within a few hundred kilobase pairs of the chromosome ends. An apparent crossover between the 3D7 and the HB3 parent chromosomes accounts for a chromosome of intermediate size in clone XP5. Similar restriction studies extended to other parasite chromosomes will ultimately yield a long-range physical map of the P. falciparum genome.

The binding of the circumsporozoite protein to cell surface heparan sulfate proteoglycans is required for plasmodium sporozoite attachment to target cells.

The major surface protein of malaria sporozoites, the circumsporozoite protein, binds to heparan sulfate proteoglycans on the surface of hepatocytes. It has been proposed that this binding event is responsible for the rapid and specific localization of sporozoites to the liver after their injection into the skin by an infected anopheline mosquito. Previous in vitro studies performed under static conditions have failed to demonstrate a significant role for heparan sulfate proteoglycans during sporozoite invasion of cells. We performed sporozoite attachment and invasion assays under more dynamic conditions and found a dramatic decrease in sporozoite attachment to cells in the presence of heparin. In contrast to its effect on attachment, heparin does not appear to have an effect on sporozoite invasion of cells. When substituted heparins were used as competitive inhibitors of sporozoite attachment, we found that sulfation of the glycosaminoglycan chains at both the N- and O-positions was important for sporozoite adhesion to cells. We conclude that the binding of the circumsporozoite protein to hepatic heparan sulfate proteoglycans is likely to function during sporozoite attachment in the liver and that this adhesion event depends on the sulfated glycosaminoglycan chains of the proteoglycans.

Cell adhesion to a motif shared by the malaria circumsporozoite protein and thrombospondin is mediated by its glycosaminoglycan-binding region and not by CSVTCG.

The malaria circumsporozoite protein (CS), thrombospondin (TSP), and several other proteins including the terminal complement proteins and the neural adhesion molecules F-spondin and Unc-5, share a cell adhesive sequence. In CS this sequence is designated as region II-plus (EWSPCSVTCGNGIQVRIK) and in TSP it is found in the type I repeats. Previous studies aimed at fine mapping the amino acid residues required for cell adhesion have yielded discrepant results. Here we show in three different cell lines that the downstream basic residues are required for cell adhesion whereas the CSVTCG sequence is not. Using mutant Chinese hamster ovary cells selected for deficiencies in proteoglycan synthesis, we show that in wild type cells, heparan sulfate proteoglycans are the binding sites for this motif. This finding is supported by additional experiments with two other cell lines demonstrating that treatment with heparitinase but not chondroitinase abolishes cell adhesion to peptides representing this motif. Using Chinese hamster ovary cell mutants deficient in heparan sulfate proteoglycans but possessing chondroitin sulfate proteoglycans, we show that cell surface chondroitin sulfate proteoglycans can also mediate binding to this motif although higher concentrations of peptides are required for adhesion. Chondroitinase, but not heparitinase, treatment of these cells destroys cell surface-binding sites. Taken together, these results indicate that cell adhesion to this motif involves an interaction between the downstream positively-charged residues and the negatively charged glycosaminoglycan chains of heparan sulfate, or in some cases chondroitin sulfate, proteoglycans on the cell surface.

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.

Proteasome inhibitors block development of Plasmodium spp.

Proteasomes degrade most of the proteins inside eukaryotic cells, including transcription factors and regulators of cell cycle progression. Here we show that nanomolar concentrations of lactacystin, a specific irreversible inhibitor of the 20S proteasome, inhibit development of the exoerythrocytic and erythrocytic stages of the malaria parasite. Although lactacystin-treated Plasmodium berghei sporozoites are still invasive, their development into exoerythrocytic forms (EEF) is inhibited in vitro and in vivo. Erythrocytic schizogony of P. falciparum in vitro is also profoundly inhibited when drug treatment of the synchronized parasites is prior, but not subsequent, to the initiation of DNA synthesis, suggesting that the inhibitory effect of lactacystin is cell cycle specific. Lactacystin reduces P. berghei parasitemia in rats, but the therapeutic index is very low. Along with other studies showing that lactacystin inhibits stage-specific transformation in Trypanosoma and Entamoeba spp., these findings highlight the potential of proteasome inhibitors as drugs for the treatment of diseases caused by protozoan parasites.