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.

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.

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.

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.

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.

Proteasome-dependent cyst formation and stage-specific ubiquitin mRNA accumulation in Entamoeba invadens.

Proteases play an important role in the pathogenic mechanisms and differentiation events of protozoan parasites; the proteasome/ubiquitin system is essential for maintaining the differentiation state of many cell types. A single input of the specific inhibitor of proteasomes, lactacystin, prevented encystation of the protozoan parasite Entameoba invadens, whereas a cysteine protease inhibitor, E64, only delayed encystation. The ameba target of lactacystin was purified and it displayed the features typical of eukaryotic 20S proteasome complexes. In addition, transcripts encoding ubiquitin were detectable in trophozoites stage cells, disappeared immediately following transfer of amoebae to encystation induction medium, and reappeared at the same time during encystation as other encystation-specific transcripts. These results demonstrate that proteasome function is required during the conversion of the disease-causing trophozoite into the infectious cyst stage of Entamoeba parasites, and that ubiquitin transcript levels undergo an unusual decrease during the early stages of this differentiation process.

Continuous axenic cultivation of Pneumocystis carinii.

Continuous axenic culture of Pneumocystis carinii has been achieved. A culture vessel is used that allows for frequent medium exchange without disturbance of organisms that grow attached to a collagen-coated porous membrane. The growth medium is based on Minimal Essential Medium with Earle's salt supplemented with S-adenosyl-L-methionine, putrescine, ferric pyrophosphate, N-acetyl glucosamine, putrescine, p-aminobenzoic acid, L-cysteine and L-glutamine, and horse serum. Incubation is in room air at 31 degrees C. The pH of the medium begins at 8.8 and rises to approximately 9 as the cells grow. Doubling times calculated from growth curves obtained from cultures inoculated at moderate densities ranged from 35 to 65 hours. With a low-density inoculum, the doubling time is reduced to 19 hours. The morphology of cultured organisms in stained smears and in transmission electron micrographs is that of P. carinii, and P. carinii-specific mAbs label the cultured material. Cultured organisms are infective for immunosuppressed rats and can be stored frozen and used to reinitiate culture.

Malaria circumsporozoite protein inhibits protein synthesis in mammalian cells.

Native Plasmodium circumsporozoite (CS) protein, translocated by sporozoites into the cytosol of host cells, as well as recombinant CS constructs introduced into the cytoplasm by liposome fusion or transient transfection, all lead to inhibition of protein synthesis in mammalian cells. The following findings suggest that this inhibition of translation is caused by a binding of the CS protein to ribosomes. (i) The distribution of native CS protein translocated by sporozoites into the cytoplasm as well as microinjected recombinant CS protein suggests association with ribosomes. (ii) Recombinant CS protein binds to RNase-sensitive sites on rough microsomes. (iii) Synthetic peptides representing the conserved regions I and II-plus of the P.falciparum CS protein displace recombinant CS protein from rough microsomes with dissociation constants in the nanomolar range. (iv) Synthetic peptides representing region I from the P.falciparum CS protein and region II-plus from the P.falciparum, P.berghei or P.vivax CS protein inhibit in vitro translation. We propose that Plasmodium manipulates hepatocyte protein synthesis to meet the requirements of a rapidly developing schizont. Since macrophages appear to be particularly sensitive to the presence of CS protein in the cytosol, inhibition of translation may represent a novel immune evasion mechanism of Plasmodium.

TRAP is necessary for gliding motility and infectivity of plasmodium sporozoites.

Many protozoans of the phylum Apicomplexa are invasive parasites that exhibit a substrate-dependent gliding motility. Plasmodium (malaria) sporozoites, the stage of the parasite that invades the salivary glands of the mosquito vector and the liver of the vertebrate host, express a surface protein called thrombospondin-related anonymous protein (TRAP) that has homologs in other Apicomplexa. By gene targeting in a rodent Plasmodium, we demonstrate that TRAP is critical for sporozoite infection of the mosquito salivary glands and the rat liver, and is essential for sporozoite gliding motility in vitro. This suggests that in Plasmodium sporozoites, and likely in other Apicomplexa, gliding locomotion and cell invasion have a common molecular basis.

The malaria circumsporozoite protein: interaction of the conserved regions I and II-plus with heparin-like oligosaccharides in heparan sulfate.

The malaria circumsporozoite (CS) protein binds to glycosaminoglycans from heparan sulfate proteoglycans on the cell surface of hepatocytes and is specifically cleared from the bloodstream by the liver. We show here that the two conserved regions, I and II-plus, of the CS protein, in a concerted action, preferentially bind to highly sulfated heparin-like oligosaccharides in heparan sulfate. In a concentration-dependent manner, peptides representing region I and region II-plus inhibited the binding of recombinant CS protein to HepG2 cells by 62 and 84%, respectively. Furthermore, the action of endoproteinase Arg-C, which cleaves the recombinant CS constructs CS27IVC and CSFZ(Cys) predominantly at the conserved region I, was inhibited by heparin in a concentration-dependent fashion. CSFZ(Cys), which has a higher affinity to HSPGs than CS27IVC, was stabilized by heparin at a w/w ratio (CS protein:glycosaminoglycan) of 20/1, whereas full protection of CS27IVC required more heparin (5/1). Heparan sulfate provided full protection of CSFZ(Cys) only at a ratio of 1/10. Native fucoidan as well as normally sulfated fuco-oligosaccharides (0.76 mol sulfate/mol fucose) inhibited Plasmodium berghei development in HepG2 cells by 84 and 66%, respectively, in a concentration-dependent manner and sporozoite invasion into CHO cells by 80%. Desulfated fucoidan oligosaccharides were inactive. These results may explain the selective interaction between the CS protein and the unique heparan sulfate from liver, which is noted for its unusually high degree of sulfation, and may provide a plausible explanation for the selective targeting of the malaria CS protein to the liver.

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.

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.

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.

A major tyrosine-phosphorylated protein of Trypanosoma brucei is a nucleolar RNA-binding protein.

We have previously identified a set of tyrosine-phosphorylated proteins with apparent molecular masses of 44-46 kDa as some of the major tyrosine phosphorylated species in the protozoan parasite Trypanosoma brucei. We now show that these molecules, herein named Nopp44/46, are localized in the nucleolus. Using monoclonal antibodies, we have isolated Nopp44/46 cDNA clones from expression libraries. Sequence analysis reveals that the predicted amino acid sequence of the molecule is composed of an N-terminal unique region, an internal acidic region, and C-terminal repeat region. Analysis of the cDNA clones and genomic Southern analysis indicated that Nopp44/46 belongs to a multigene family in which different gene copies are very similar but vary in the number of repeats. Interestingly, the repetitive amino acid sequence motif contains multiple RGG (Arg-Gly-Gly) boxes characteristic of RNA-binding proteins. In vitro binding experiments demonstrated that Nopp44/46 is indeed capable of binding nucleic acids. Competition experiments with different RNA homopolymers demonstrated that Nopp44/46 preferentially binds to poly(U). These studies suggest that Nopp44/46 may play a role in RNA metabolism in trypanosomes and raise the possibility that tyrosine phosphorylation may regulate the process.

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.

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.