Immunobiology of Plasmodium in liver and brain.

Malaria remains one of the most serious health problems globally, but our understanding of the biology of the parasite and the pathogenesis of severe disease is still limited. Multiple cellular effector mechanisms that mediate parasite elimination from the liver have been described, but how effector cells use classical granule-mediated cytotoxicity to attack infected hepatocytes and how cytokines and chemokines spread via the unique fluid pathways of the liver to reach the parasites over considerable distances remains unknown. Similarly, a wealth of information on cerebral malaria (CM), one of the most severe manifestations of the disease, was gained from post-mortem analyses of human brain and murine disease models, but the cellular processes that ultimately cause disease are not fully understood. Here, we discuss how imaging of the local dynamics of parasite infection and host response as well as consideration of anatomical and physiological features of liver and brain can provide a better understanding of the initial asymptomatic hepatic phase of the infection and the cascade of events leading to CM. Given the increasing drug resistance of both parasite and vector and the unavailability of a protective vaccine, the urgency to reduce the tremendous morbidity and mortality associated with severe malaria is obvious.

Dual interaction of the malaria circumsporozoite protein with the low density lipoprotein receptor-related protein (LRP) and heparan sulfate proteoglycans.

Speed and selectivity of hepatocyte invasion by malaria sporozoites have suggested a receptor-mediated mechanism and the specific interaction of the circumsporozoite (CS) protein with liver-specific heparan sulfate proteoglycans (HSPGs) has been implicated in the targeting to the liver. Here we show that the CS protein interacts not only with cell surface heparan sulfate, but also with the low density lipoprotein receptor-related protein (LRP). Binding of 125I-CS protein to purified LRP occurs with a Kd of 4.9 nM 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 results in more than 90% 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 reduces Plasmodium berghei invasion by 60-70%. Parasite development in heparinase-pretreated HepG2 cells is inhibited by 65% when RAP is present during sporozoite 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 invasion.

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.

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.

Proteasome activity is required for the stage-specific transformation of a protozoan parasite.

A prominent feature of the life cycle of intracellular parasites is the profound morphological changes they undergo during development in the vertebrate and invertebrate hosts. In eukaryotic cells, most cytoplasmic proteins are degraded in proteasomes. Here, we show that the transformation in axenic medium of trypomastigotes of Trypanosoma cruzi into amastigote-like organisms, and the intracellular development of the parasite from amastigotes into trypomastigotes, are prevented by lactacystin, or by a peptide aldehyde that inhibits proteasome function. Clasto-lactacystin, an inactive analogue of lactacystin, and cell-permeant peptide aldehyde inhibitors of T. cruzi cysteine proteinases have no effect. We have also identified the 20S proteasomes from T. cruzi as a target of lactacystin in vivo. Our results document the essential role of proteasomes in the stage-specific transformation of a protozoan.

Migration of Plasmodium sporozoites through cells before infection.

Intracellular bacteria and parasites typically invade host cells through the formation of an internalization vacuole around the invading pathogen. Plasmodium sporozoites, the infective stage of the malaria parasite transmitted by mosquitoes, have an alternative mechanism to enter cells. We observed breaching of the plasma membrane of the host cell followed by rapid repair. This mode of entry did not result in the formation of a vacuole around the sporozoite, and was followed by exit of the parasite from the host cell. Sporozoites traversed the cytosol of several cells before invading a hepatocyte by formation of a parasitophorous vacuole, in which they developed into the next infective stage. Sporozoite migration through several cells in the mammalian host appears to be essential for the completion of the life cycle.

Trypanosome lytic factors: novel mediators of human innate immunity.

A novel trypanosome lytic factor (TLF) has been characterized that protects humans from infection by Trypanosoma brucei brucei. The mechanism of trypanolysis is unknown; contrary to one hypothesis, TLF does not kill trypanosomes by generating oxygen radicals. However, these trypanosomes become human-infective when they express a serum-resistance-associated gene.

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.

Release of malaria circumsporozoite protein into the host cell cytoplasm and interaction with ribosomes.

To date, the circumsporozoite (CS) protein has been implicated in guiding malaria sporozoites to the liver [Cerami et al., Cell 70, 1992, 1021-1033]. Here we show that shortly after invasion, P. berghei and P. yoelii sporozoites lie free in the invaded cell and release considerable amounts of CS protein into the cytoplasm. The intracytoplasmic deposition of CS protein begins during the attachment of the sporozoite to the host cell surface and reaches its peak during the first 4-6 h after invasion. Initially, the CS protein spreads over the entire cytoplasm of the infected cell where it interacts with cytosolic as well as endoplasmic reticulum-associated ribosomes. During the subsequent development of the parasites to exoerythrocytic forms, the CS protein binding becomes gradually restricted to ribosomes lining the outer membrane of the nuclear envelope of the host cell. The distribution pattern of the parasite-released CS protein in the host cell cytoplasm is independent of the permissiveness of the host cell for the development of the parasites to exoerythrocytic forms. It requires neither the host cell metabolism nor does it involve the endocytotic machinery. Recombinant P. falciparum CS protein interacts with RNAse-sensitive sites on endoplasmic reticulum-associated ribosomes as shown by microinjection and immunoelectron microscopy. The generalized interaction of the CS protein with host cell ribosomes suggests that the CS protein has an intracellular function during the hepatic phase in the life cycle of Plasmodium and may also explain the generation of a CD8+ T cell response in the course of rodent malaria infections.

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.

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.

Imaging murine NALT following intranasal immunization with flagellin-modified circumsporozoite protein malaria vaccines.

Intranasal (IN) immunization with a Plasmodium circumsporozoite (CS) protein conjugated to flagellin, a Toll-like receptor 5 agonist, was found to elicit antibody-mediated protective immunity in our previous murine studies. To better understand IN-elicited immune responses, we examined the nasopharynx-associated lymphoid tissue (NALT) in immunized mice and the interaction of flagellin-modified CS with murine dendritic cells (DCs) in vitro. NALT of immunized mice contained a predominance of germinal center (GC) B cells and increased numbers of CD11c+ DCs localized beneath the epithelium and within the GC T-cell area. We detected microfold cells distributed throughout the NALT epithelial cell layer and DC dendrites extending into the nasal cavity, which could potentially function in luminal CS antigen uptake. Flagellin-modified CS taken up by DCs in vitro was initially localized within intracellular vesicles followed by a cytosolic distribution. Vaccine modifications to enhance delivery to the NALT and specifically target NALT antigen-presenting cell populations will advance development of an efficacious needle-free vaccine for the 40% of the world's population at risk of malaria.

Metabolomics in drug discovery and development.

Metabolomics technology is being utilized across the spectrum of drug discovery and development; from the assessment of unanticipated biochemical sequelae of target engagement in transgenic models to monitoring media content to improve the efficiency of the manufacture of biologics, the impact of the technology is expanding dramatically. Applications critical for the pharmaceutical industry include translational medicine, biomarker discovery, and patient stratification. Technological innovation and cultural acceptance will be necessary to optimally use this powerful tool.

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.

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.

Cell surface interactions between Trypanosoma congolense and macrophages during phagocytosis in vitro.

Trypanosoma congolense bloodstream forms preincubated with a high titer of anti-variant surface antigen (VSG)-specific antibody, a low amount of anti-VSG plus complement-active mouse serum (MS), MS alone, and trypsin were cocultivated with mouse peritoneal macrophages in vitro. Immunofluorescence as well as transmission and scanning electron microscopy revealed that upon attachment to the macrophages' surface, trypanosomes opsonized with anti-VSG/MS formed opsonized filopodia, which were rapidly internalized by the phagocytes. Although these cells attached as frequently as anti-VSG or trypsin-pretreated parasites, the rate of phagocytosis of anti-VSG/MS pretreated trypanosomes was reduced significantly. Trypanosomes pretreated with high antibody titers alone were lysed on the surface of the macrophages before phagocytosis was completed. Parasites opsonized with complement alone adhered only occasionally and were rarely phagocytosed. Trypsin-treated trypanosomes, which served as positive control cells, rapidly attached and remained intact until ingulfment by the macrophages was completed. Untreated control parasites did not attach to the macrophages and were not phagocytosed. Cocultivation of macrophages with anti-VSG/MS-opsonized trypanosomes caused internalization of the flagellum by membrane fusion. Filopodia formation by T. congolense is thus correlated with a marked reduction in phagocytosis even in the presence of only a sublytic antibody titer.

Malaria sporozoites actively enter and pass through rat Kupffer cells prior to hepatocyte invasion.

Malaria sporozoites have to cross the layer of sinusoidal liver cells to reach their initial site of multiplication in the mammalian host, the hepatocytes. To determine the sinusoidal cell type sporozoites use for extravasation, endothelia or Kupffer cells, we quantified sporozoite adhesion to and invasion of sinusoidal cells isolated from rat liver. In vitro invasion assays reveal that Plasmodium berghei and P. yoelii sporozoites attach to and enter Kupffer cells, but not sinusoidal endothelia. Unlike hepatocytes and other nonphagocytic cells, which are invaded in vitro only within the first hour of parasite exposure, the number of intracellular sporozoites in Kupffer cells increases for up to 12 hours. By confocal and electron microscopy, sporozoites are enclosed in a vacuole that does not colocalize with lysosomal markers. Inhibition of phagocytosis with gadolinium chloride has no effect on Kupffer cell invasion, but abolishes phagocytosis of inactivated sporozoites. Furthermore, sporozoites traverse in vitro from Kupffer cells to hepatocytes where they eventually develop into exoerythrocytic schizonts. Thus, malaria sporozoites selectively recognize and actively invade Kupffer cells, avoid phagosomal acidification, and safely passage through these phagocytes.

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.

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.