Ute Frevert, DVM, PhD, Associate Professor

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Malaria sporozoite surface proteins recognize liver-specific proteoglycans

Within minutes after transmission by an infected mosquito, malaria sporozoites invade hepatocytes, their initial site of replication in the liver. Speed and selectivity of the sporozoite targeting to the liver have suggested a receptor-mediated mechanism. We initially showed that the sporozoites with their major surface proteins, the circumsporozoite protein (CSP) and the thrombospondin-related adhesive protein (TRAP), interact with heparan sulfate proteoglycans expressed on the basolateral cell surface of hepatocytes. We further demonstrated that the interaction occurs between the two conserved regions I and II-plus of CSP and heparin-like, highly sulfated oligosaccharides in heparan sulfate. Since heparan sulfate from liver differs from the heparan sulfate species from all other mammalian tissues in that it contains an unusually high (heparin-like) degree of sulfation, this preferential interaction offers an explanation for the rapid homing and selective arrest of the parasites in the liver sinusoid. Thus, malaria sporozoites appear to utilize an existing clearance mechanism of the host to home to the liver.

Fig 1. CSP is targeted to Kupffer cells and the space of Disse of the liver. Recombinant P. falciparum CSP was inoculated intravenously in a mouse. Thirty minutes later, the liver was fixed and CSP was detected by immunoperoxidase labeling. CSP (red) is found in a patchy distribution in the space of Disse and also inside lysosomes of a Kupffer cell.

Stellate cell-derived extracellular matrix proteoglycans mediate sporozoite arrest in the sinusoid

We provided evidence that the initial arrest of malaria sporozoites in the liver sinusoid is mediated by heparan sulfate proteoglycans secreted by stellate cells into the extacellular matrix in the space of Disse. We found that sporozoites as well as their major surface proteins, CSP and TRAP, recognize distinct cell type-specific surface proteoglycans from primary Kupffer cells, hepatocytes and stellate cells, but not sinusoidal endothelia. P. berghei sporozoites attach to heparan sulfate on hepatocytes and stellate cells, whereas Kupffer cell recognition involves both chondroitin sulfate and heparan sulfate proteoglycans. Recombinant P. falciparum CSP also interacts with secreted proteoglycans from stellate cells, the major producers of extracellular matrix in the liver. In situ binding studies using frozen liver sections indicate that the majority of CSP binding sites are associated with these matrix proteoglycans. Our data suggest that sporozoites are first arrested in the sinusoid by binding to extracellular matrix proteoglycans and then recognize proteoglycans on the surface of Kupffer cells, which they use to traverse the sinusoidal cell barrier.

Fig 2. An SEM micrograph of a sporozoite superimposed onto an SEM picture of sinusoidal sieve plates demonstrates clearly that extracellular matrix proteoglycans protruding through the sinusoidal fenestration provide enough of a surface for sporozoites to glide on.

Intravital imaging of Plasmodium sporozoite infection of the liver

Plasmodium sporozoite invasion of liver cells has been an extremely elusive event to study. In the prevailing model, sporozoites enter the liver by passing through Kupffer cells, but this model was based solely on incidental observations in fixed specimens and on biochemical and physiological data. To obtain direct information on the dynamics of sporozoite infection of the liver, live mice were infected with red or green fluorescent Plasmodium berghei sporozoites and monitored their behavior using intravital microscopy. Digital recordings show that sporozoites entering a liver lobule abruptly adhere to the sinusoidal cell layer, suggesting a high-affinity interaction. They glide along the sinusoid, with or against the bloodstream, to a Kupffer cell, and, by slowly pushing through a constriction, traverse across the space of Disse. Once inside the liver parenchyma, sporozoites move rapidly for many minutes, traversing several hepatocytes, until ultimately settling within a final one. Migration damage to hepatocytes was confirmed in liver sections, revealing clusters of necrotic hepatocytes adjacent to structurally intact, sporozoite-infected hepatocytes, and by elevated serum alanine aminotransferase activity. In summary, malaria sporozoites bind tightly to the sinusoidal cell layer, cross Kupffer cells, and leave behind a trail of dead hepatocytes when migrating to their final destination in the liver.

Fig 3. Using live transgenic mice that express GFP in Kupffer cells, we observed fluorescent P. berghei sporozoites through the intact liver capsule with an intravital microscope. Digital movies show that parasites entering the liver lobule were arrested abruptly by binding to the sinusoidal cell layer. After a short break, the parasites glided along the sinusoidal cell layer (A), either with or against the bloodstream. When they encountered a Kupffer cell (B), they bound to it facing it with their apical cell pole. Then they traversed the phagocyte and migrated in the liver tissue for many minutes (C) before eventually settling down in a final hepatocyte for liver stage development.

Kupffer cells are obligatory for Plasmodium sporozoite infection of the liver

Homozygous op/op mice lack macrophage colony stimulating factor 1 (CSF-1), which is required for macrophage maturation due to a deactivating point mutation in the osteopetrosis gene. These mice have 77% fewer Kupffer cells and exhibit reduced clearance of colloidal carbon particles compared with heterozygous phenotypically normal littermates. Using a novel quantitative reverse transcription polymerase chain reaction assay for P. yoelii 18S rRNA, we found liver infection of op/op mice to be decreased by 84% compared with controls. However, after elimination of Kupffer cells by treatment with liposome encapsulated clodronate, infection of normal mice was enhanced seven- to 15-fold. This was explained by electron microscopy showing temporary gaps in the sinusoidal cell layer caused by the synchronous apoptotic death and expulsion from the sinusoidal cell layer of millions of Kupffer cells. Thus, Kupffer cell deficiency in op/op mice decreases sporozoite infection by reducing the number of portals to the liver parenchyma, whereas clodronate increases sporozoite infection by opening portals and providing direct access to hepatocytes. Together these data provide strong support for the hypothesis that Kupffer cells are the portal for sporozoites to hepatocytes and critical for the onset of a malaria infection.

Fig. 4. Role of Kupffer cells in Plasmodium sporozoite entry into the liver. (A) Sporozoite infection of the normal, intact liver occurs by Kupffer cell passage (B) Sporozoite entry into the op/op mouse liver is impaired due to a drastic reduction in the number of Kupffer cells.(C) Kupffer cell removal by clodronate facilitates sporozoite entry into the liver by creating openings in the sinusoidal cell layer.

Sporozoites invade Kuppfer cells actively and enter a vacuole that does not acidify or fuse with lysosomes

In vitro studies showed that Plasmodium sporozoites attach to and enter Kupffer cells, but not sinusoidal endothelia. Inhibition of phagocytosis with gadolinium chloride had no effect on Kupffer cell invasion. After Kupffer cell entry, the sporozoites become enclosed in a vacuole that does not colocalize with markers for acidified organelles. Thus, malaria sporozoites selectively recognize and actively invade Kupffer cells and prevent acidification of the vacuole, and safely passage through these phagocytes to reach hepatocytes.

A	B
C	D

Fig 5.The vacuole harboring malaria sporozoites in Kupffer cells does not fuse with lysosomes. Kupffer cells were isolated from rat liver and loaded over night with dextran-rhodamine (red). P. berghei sporozoites (A and B; green) or FITC-labeled zymosan (C; green) were added for 2 h. Neither elongated (A) nor coiled (B) sporozoites colocalize with the lysosomal marker. In contrast, dextran-rhodamine has entered the phagosome containing yeast cells (D).

CSP binds with high affinity to a scavengoer receptor

In addition to heparan sulfate proteoglycans, sporozoites and CSP also interact with high affinity with the low density lipoprotein receptor-related protein (LRP), a multifunctional scavenger receptor of the liver. The binding of CSP to purified LRP occurs in the nanomolar range and can be inhibited by the receptor-associated protein (RAP). Blockage of LRP by RAP or anti-LRP antibodies on heparan sulfate-deficient CHO cells causes inhibition of binding and endocytosis of recombinant CS protein. Conversely, blockage or enzymatic removal of the cell surface heparan sulfate from LRP-deficient embryonic mouse fibroblasts yields the same degree of inhibition. Heparinase-pretreatment of LRP-deficient fibroblasts or blockage of LRP on heparan sulfate-deficient CHO cells by RAP, lactoferrin or anti-LRP antibodies inhibits P. berghei invasion. These findings suggest that malaria sporozoites utilize the interaction of the CS protein with HSPGs and LRP as the major mechanism for host cell recognition.

Fig 6. (A and B) CSP binds specifically to purified LRP. Iodinated recombinant P. falciparum CSP was incubated at 4°C with LRP- or BSA-coated wells in the presence of unlabeled CSP (A) or RAP (B). The curves represent the best-fit to a single class of binding sites with a Kd of 4.9 nM for CSP and a Ki of 9.5 for RAP. (C) and (D) CSP binds to LRP on the surface of HepG2 cells. HepG2 cells were incubated with iodinated CSP in the presence of unlabeled CSP (A) or RAP (B). Radiolabeled CSP is displaced by cold ligand with an apparent Kd of 0.4 nM and by RAP with an apparent Ki of 0.8 nM.

Plasmodium sporozoites and recombinant P. falciparum CSP suppress the respiratory burst in Kupffer cells

Sporozoites and CSP increased the intracellular concentration of cyclic adenosyl monophosphate (cAMP) and inositol 1,4,5-triphosphate in Kupffer cells, but not in hepatocytes or liver endothelia. Preincubation with cAMP analogues or inhibition of phosphodiesterase also inhibited the respiratory burst. By contrast, adenylyl cyclase inhibition abrogated the suppressive effect of sporozoites. Selective protein kinase A (PKA) inhibitors failed to reverse the CSP-mediated blockage and stimulation of the exchange protein directly activated by cAMP (EPAC), but not PKA inhibited the formation of reactive oxygen species (ROS). Both blockage of the low-density lipoprotein receptor-related protein (LRP-1) with receptor-associated protein and elimination of cell surface proteoglycans inhibited the cAMP increase in Kupffer cells. We propose that by binding of CSP to LRP and cell surface proteoglycans, malaria sporozoites induce a cAMP/EPAC-dependent, but PKA-independent signal transduction pathway that suppresses defense mechanisms in Kupffer cells. This allows the sporozoites to safely pass through these professional phagocytes and to develop inside neighboring hepatocytes.

Fig. 7. Model for the CSP-mediated inhibition of ROS formation in Kupffer cells. Plasmodium sporozoite adhesion to Kupffer cells is mediated by a multivalent, high-avidity interaction between the major sporozoite surface protein CSP and chondroitin and heparan sulfate chains from syndecans. This interaction facilitates engagement of some CSP molecules in a specific, high affinity interaction with LRP-1. The ensuing activation of a Gαs protein stimulates adenylyl cyclase leading to the formation of a local pool of cAMP. LRP-1 ligation also rapidly increases the IP3 level, which triggers the release of calcium from intracellular stores thus contributing to adenylyl cyclase activation. Phosphodiesterases control the cAMP level by converting it to AMP. The increase in the intracellular cAMP concentration induces EPAC activation, which prevents ROS formation. Because CSP inhibits the respiratory burst also after direct stimulation of PKC with PMA, EPAC may prevent the PKC-mediated phosphorylation of p47phox. The resulting block in the assembly of the hetero-hexameric NADPH oxidase prevents the formation of ROS.

Ribotoxic properties of CSP

CSP appears to have another function in the mammalian host. Upon cell contact, malaria sporozoites release considerable amounts of CSP into the cytosol of mammalian cells. Sporozoite-derived native CSP as well as recombinant CSP introduced into the cytoplasm by liposome fusion or transient transfection, all inhibit protein synthesis in mammalian cells. Furthermore, recombinant CSP and peptides representing the conserved regions I and II-plus of CSP block protein synthesis in eukaryotic in vitro translation systems. It is conceivable that Plasmodium sporozoites use this ribotoxic action of the CS protein to selectively eliminate those Kupffer cells they have traversed, because activation of these phagocytes by secondary stimuli must be expected to be detrimental for parasites developing in neighboring hepatocytes. Inhibition of translation may therefore represent an additional immune evasion mechanism of Plasmodium.

A	B
C	D

Fig 8 . (A and B) HepG2 cells containing native, sporozoite-released CSP in their cytoplasm are inhibited in protein synthesis. (A) Double immunofluorescence labeling of a P. berghei-infected HepG2 cell culture shows a cell that contains Texas red-labeled CSP in the cytoplasm (red). An extracellular sporozoite is labeled with FITC (green), indicating its attachment to the outer surface of the HepG2 cell. (B) Autoradiographic exposure of the same field shows that in contrast to the neighboring control cells, the two cells that contain CS protein (red) have not incorporated [3H]leucine as demonstrated by the lack of silver grains (green) in the photographic emulsion. (C) and (D) CHO cells expressing P. falciparum CSP in their cytoplasm are inhibited in protein synthesis. The cells were transfected, cultivated for 24 h, and metabolically labeled with tritiated leucine. CSP was labeled with mAb 2A10 and GAM–FITC (green), the cells were counterstained with Evans blue (red) and exposed for microautoradiography. (C) The small cell in the center, whose nucleus is surrounded only by a narrow rim of strongly CSP-positive cytoplasm, is obviously degenerate. (D) The distribution of the silver grains in the photographic emulsion shows that the degenerate cell has not incorporated any tritiated leucine, as shown by the lack of silver grains in the photographic emulsion.

A	B
C	Fig 9. Two conserved motifs of the CS protein are involved in RNA binding. Native P. falciparum CS protein inhibits protein synthesis in in vitro translation systems in the nanomolar range (A), while micromolar concentrations of the conserved cell-adhesive thrombospondin-like motif region II-plus (C) and the conserved region I (B) are required to achieve the same effect.

Intravital imaging of Plasmodium merozoite release

Plasmodium undergoes one round of multiplication in the liver prior to invading erythrocytes and initiating the symptomatic blood phase of the malaria infection. Productive hepatocyte infection by sporozoites leads to the generation of thousands of merozoites capable of erythrocyte invasion. Intravital microscopy of GFPexpressing P. yoelii parasites showed that merozoites are released from infected hepatocytes as extrusomes, packets of hundreds of parasites surrounded by host cell membrane. The majority of extrusomes exit the liver intact suggesting that Plasmodium uses this mechanism to ensure an effective transition from the liver to the blood phase of the malaria infection.

Fig 10. Merozoite release is by extrusome formation. (A) Ex vivo confocal analysis of a mouse liver showing extrusomes formed by GFP-expressing P. yoelii parasites. Bar = 20 µm. (B) Extrusomes can measure hundreds of micrometers and extend from the liver schizont (or exoerythrocytic form, EEF) into various sinusoids. Bar = 100 µm.

Current Model

Malaria sporozoites (S) are initially arrested by binding to extracellular matrix proteoglycans that protrude from the space of Disse through the endothelial fenestration into the sinusoidal lumen. The arrested sporozoites glide along the sinusoidal endothelium (EC) until they encounter a Kupffer cell (KC), a resident macrophage of the liver, which they actively invade and safely traverse. After migration through several hepatocytes (H), the parasites eventually settle down in a final one. The parasites then begin to grow inside a parasitophorous vacuole (PV) to a size larger than its original host cell. Schizogonic division results in the formation of thousands of erythrocyte-infective merozoites (*). During the final stage of differentiation, the PVM dissolves and allows the parasites to mix with the remaining host cell organelles. Eventually, the plasma membrane of the infected hepatocyte bulges out and forms extrusomes (Ex) thus releasing merozoites, remnant bodies (RB), and host cell mitochondria (Mi) into the sinusoidal lumen. Camouflaged by host cell membrane, extrusomes are not recognized by Kupffer cells and are shuttled out of the liver. Infiltration of the remains of the infected host cell by mononuclear phagocytes (MÆ) and neutrophil granulocytes gives rise to the formation of a small granuloma.

Selected Publications

1. Usynin, I., Klotz, C., Frevert, U. Malaria circumsporozoite protein inhibits the respiratory burst in Kupffer cells. Cell. Microbiol. 2007 Jun 15; [Epub ahead of print]
2. Baer, K., Roosevelt, M., Van Rooijen, N., Clarkson Jr., A.B., Frevert, U. Kuppfer cells are obigatory for Plasmodium sporozoite infection of the liver. Cell. Microbiol. 2007;9:397-412.
3. Frevert, U., Usynin, I., Baer, K., Klotz, C. Plasmodium sporozoite passage across the sinusoidal layer. In: Molecular Mechanisms of Parasite Invasion. Soldati, D., Burleigh, B. (Eds). Landis Bioscience. Georgetown. 2006.
4. Frevert, U., Usynin, I., Baer, K., Klotz, C. Nomadic or sessile: can Kupffer cells function as portals for malaria sporozoites to the liver? Cell. Microbiol. 2006;8:1537-1546.
5. Tarun, A.S., Baer, K., Dumpit, R.F., Gray, S., Lejarcegui, N., Frevert, U., Kappe, SHI. Quantitative isolation and in vitro imaging of malaria parasite liver stages. Int. J. Parasitol. 2006;36:1283-1293.
6. Frevert U. Response to Heussler and Doering: In vivo imaging enters parasitology. Trends Parasitol. 2006 April;22: 195-196.
7. Krochina, S., Barreau, C., Pradel, G., Jefferey, E., Li, J., Natarajan, R., Shabanowitz, J., Hunt, D., Frevert, U., Vernick, KD. A mosquito-specific protein family includes the candidate receptors for malaria sporozoite invasion of salivary glands. Cell. Microbiol. 2006;8:163-175.
8. Frevert, U., Engelman, S., Zougbede, S., Strange, J., Ng, B., Matuschewski, K., Liebes, L., Yee, H. Intravital observation of Plasmodium berghei sporozoite infection of the liver. PLoS Biol. 2005;3:e192.
9. Frevert, U., Nardin, E. Arrest in the liver - a gentically defined malaria vaccine? N. Eng. J. Med. 2005;352:1600-1602.
10. Mueller, A.K., Camargo, N., Kaiser, K., Andorfer, C., Frevert, U., Matuschewski, K., Kappe, SHI. Plasmodium liver stage developmental arrest by depletion of a protein at the parasite-host interface. Proc. Nat. Acad. Sci. USA. 2005;102:3022-27.
11. Frevert, U. Sneaking in Through the Back Entrance: The Biology of Malaria Liver Stages. Trends Parasitol. 2004;20: 417-424. (#J71303)
12. Vanderberg, J.P., Frevert, U. Intravital microscopy demonstrating antibody-mediated immobilisation of Plasmodium berghei sporozoites injected into skin by mosquitoes. Int J Parasitol. 2004;34, 991-996.
13. Pradel, G., Garapaty, S., Frevert, U. Kupffer and stellate cell proteoglycans mediate malaria sporozoite targeting to the liver. Molec. Microbiol. 2004;45, 637-651.
14. Bhanot, P., Frevert, U., Nussenzweig, V., Persson, C. Defective surface localization of the thrombospondin-related adhesive protein (TRAP) affects Plasmodium infectivity. Mol. Biochem. Parasitol. 2003;126: 263-273. (#J53338)
15. Pradel, G., Garapaty, S., Frevert, U. Proteoglycans mediate malaria sporozoite targeting to the liver. Comp. Hepatol. 2003;3: S47-49.
16. Pradel, G., Frevert, U. Plasmodium sporozoites actively enter and pass through Kupffer cell prior to hepatocyte invasion. Hepatology 2001;33: 1154-1165. (#J22601)
17. Mota, M., Pradel, G., Vanderberg, J.P., Hafalla, J., Frevert, U., Nussenzweig, R.S., Nussenzweig, V., Rodriguez, A. A novel mechanism of host cell invasion by Plasmodium sporozoites. Science 2001;291: 141-144.