Traffic jams: protein transport in Plasmodium falciparum

Protein targeting in malaria parasites is a complex process, involving several cellular compartments that distinguish these cells from more familiar systems, such as yeast or mammals. At least a dozen distinct protein destinations are known. The best studied of these is the vestigial chloroplast (the apicoplast), but new tools promise rapid progress in understanding how Plasmodium falciparum and related apicomplexan parasites traffic proteins to their invasion-related organelles, and how they modify the host by trafficking proteins into its cytoplasm and plasma membrane. Here, Giel van Dooren and colleagues discuss recent insights into protein targeting via the secretory pathway in this fascinating and important system. This topic emerged as a major theme at the Molecular Approaches to Malaria conference, Lorne, Australia, 2-5 February 2000.     

Interactions of Plasmodium falciparum erythrocyte membrane protein 3 with the red blood cell membrane skeleton

Plasmodium falciparum parasites express and traffick numerous proteins into the red blood cell (RBC), where some associate specifically with the membrane skeleton. Importantly, these interactions underlie the major alterations to the modified structural and functional properties of the parasite-infected RBC. P. falciparum Erythrocyte Membrane Protein 3 (PfEMP3) is one such parasite protein that is found in association with the membrane skeleton. Using recombinant PfEMP3 proteins in vitro, we have identified the region of PfEMP3 that binds to the RBC membrane skeleton, specifically to spectrin and actin. Kinetic studies revealed that residues 38-97 of PfEMP3 bound to purified spectrin with moderately high affinity (K(D(kin))=8.5 x 10(-8) M). Subsequent deletion mapping analysis further defined the binding domain to a 14-residue sequence (IFEIRLKRSLAQVL; K(D(kin))=3.8 x 10(-7) M). Interestingly, this same domain also bound to F-actin in a specific and saturable manner. These interactions are of physiological relevance as evidenced by the binding of this region to the membrane skeleton of inside-out RBCs and when introduced into resealed RBCs. Identification of a 14-residue region of PfEMP3 that binds to both spectrin and actin provides insight into the potential function of PfEMP3 in P. falciparum-infected RBCs.     

Export of Plasmodium falciparum calcium-dependent protein kinase 1 to the parasitophorous vacuole is dependent on three N-terminal membrane anchor motifs

Calcium-dependent protein kinases play a pivotal role in calcium signalling in plants and some protozoa, including the malaria parasites. They are found in various subcellular locations, suggesting an involvement in multiple signal transduction pathways. Recently, Plasmodium falciparum calcium-dependent protein kinase 1 (PfCDPK1) has been found in the membrane and organelle fraction of the parasite. The kinase contains three motifs for membrane binding at its N-terminus, a consensus sequence for myristoylation, a putative palmitoylation site and a basic motif. Endogenous PfCDPK1 and the in vitro translated kinase were both shown to be myristoylated. The supposed membrane attachment function of the basic cluster was experimentally verified and shown to participate together with N-myristoylation in membrane anchoring of the kinase. Using immunogold electron microscopy, the protein was detected in the parasitophorous vacuole and the tubovesicular system of the parasite. Mutagenesis of the predicted acylated residues and the basic motif confirmed that dual acylation and the basic cluster are required for correct targeting of Aequorea victoria green fluorescent protein to the parasitophorous vacuole, suggesting that PfCDPK1 as the leishmanial hydrophilic acylated surface protein B is a representative of a novel class of proteins whose export is dependent on a 'non-classical' pathway involving N-myristoylation/palmitoylation.     

A Plasmodium falciparum exo-antigen alters erythrocyte membrane deformability

Here we describe a reduced membrane deformability of human erythrocytes when aspirated into 0.6 microns diameter in polycarbonate sieves, after exposure of uninfected cells to spent parasite-culture supernatant. This, taken in concert with a previous observation that intra-erythrocytic development of the parasite P. falciparum decreases host localised membrane deformability, may indicate a biological role for such parasite-induced changes in the rheological properties of the erythrocyte.     

A database for Plasmodium falciparum protein models

There is an urgent need for developing alternate strategies to combat Malaria caused by Plasmodium falciparum (P. falciparum) because of growing drug resistance and increased incidents of infection in humans. 3D models of P. falciparum annotated proteins using molecular modeling techniques will enhance our understanding about the mechanism of host parasite interactions for the identification of drug targets and malarial vaccine design. Potential structural templates for P. falciparum annotated proteins were selected from PDB (protein databank) using BLASTP (basic local alignment search tool for proteins). This exercise identified 476 Plasmodium proteins with one or more known structural templates (>or= 40 % identity) for further modeling. The pair-wise sequence alignments generated for protein modeling were manually checked for error. The models were then constructed using MODELLER (a comparative protein modelling program for modelling protein structures) followed by energy minimization in AMBER force field and checked for error using PROCHECK. AVAILABILITY: http://bioinfo.icgeb.res.in/codes/model.html.     

A novel copper-binding fold for the periplasmic copper resistance protein CusF

We have determined the crystal structure of apo-CusF, a periplasmic protein involved in copper and silver resistance in Escherichia coli. The protein forms a five-stranded beta-barrel, classified as an OB-fold, which is a unique topology for a copper-binding protein. NMR chemical shift mapping experiments suggest that Cu(I) is bound by conserved residues H36, M47, and M49 located in beta-strands 2 and 3. These residues are clustered at one end of the beta-barrel, and their side chains are oriented toward the interior of the barrel. Cu(I) can be modeled into the apo-CusF structure with only minimal structural changes using H36, M47, and M49 as ligands. The unique structure and metal binding site of CusF are distinct from those of previously characterized copper-binding proteins.     

Plasmodium falciparum erythrocyte membrane protein 3 (PfEMP3) destabilizes erythrocyte membrane skeleton

Plasmodium falciparum erythrocyte membrane protein 3 (PfEMP3) is a parasite-derived protein that appears on the cytoplasmic surface of the host cell membrane in the later stages of the parasite's development where it associates with membrane skeleton. We have recently demonstrated that a 60-residue fragment (FIa1, residues 38-97) of PfEMP3 bound to spectrin. Here we show that this polypeptide binds specifically to a site near the C terminus of alpha-spectrin at the point that spectrin attaches to actin and protein 4.1R in forming the junctions of the membrane skeletal network. We further show that this polypeptide disrupts formation of the ternary spectrin-actin-4.1R complex in solution. Importantly, when incorporated into the cell, the PfEMP3 fragment causes extensive reduction in shear resistance of the cell. We conjecture that the loss of mechanical cohesion of the membrane may facilitate the exit of the mature merozoites from the cell.     

Integral membrane protein located in the apical complex of Plasmodium falciparum.

We describe the cloning of a novel antigen of Plasmodium falciparum which contains a hydrophobic domain typical of an integral membrane protein. This antigen is designated apical membrane antigen 1 because it appears to be located in the apical complex. Apical membrane antigen 1 appears to be transported to the merozoite surface near the time of schizont rupture.     

A genomic perspective of protein kinases in Plasmodium falciparum

Protein kinases are central to regulation of cellular signaling in the eukaryotes. Well-conserved and lineage-specific protein kinases have previously been identified from various completely sequenced genomes of eukaryotes. The current work describes a genome-wide analysis for protein kinases encoded in the Plasmodium falciparum genome. Using a few different profile matching methods, we have identified 99 protein kinases or related proteins in the parasite genome. We have classified these kinases into subfamilies and analyzed them in the context of noncatalytic domains that occur in these catalytic kinase domain-containing proteins. Compared to most eukaryotic protein kinases, these sequences vary significantly in terms of their lengths, inserts in catalytic domains, and co-occurring domains. Catalytic and noncatalytic domains contain long stretches of repeats of positively charged and other polar amino acids. Various components of the cell cycle, including 4 cyclin-dependent kinase (CDK) homologues, 2 cyclins, 1 CDK regulatory subunit, and 1 kinase-associated phosphatase, are identified. Identification of putative mitogen-activated protein (MAP) Kinase and MAP Kinase Kinase of P. falciparum suggests a new paradigm in the highly conserved signaling pathway of eukaryotes. The calcium-dependent kinase family, well represented in P. falciparum, shows varying domain combinations with EF-hands and pleckstrin homology domains. The analysis reveals a new subfamily of protein kinases having limited sequence similarity with previously known subfamilies. A new transmembrane kinase with 6 membrane-spanning regions is identified. Putative apicoplast targeting sequences have been detected in some of these protein kinases, suggesting their export to the apicoplast.     

Optimized expression of Plasmodium falciparum erythrocyte membrane protein 1 domains in Escherichia coli

BACKGROUND: Removal of exhaled air from total body emanations or artificially standardising carbon dioxide (CO2) outputs has previously been shown to eliminate differential attractiveness of humans to certain blackfly (Simuliidae) and mosquito (Culicidae) species. Whether or not breath contributes to between-person differences in relative attractiveness to the highly anthropophilic malaria vector Anopheles gambiae sensu stricto remains unknown and was the focus of the present study. METHODS: The contribution to and possible interaction of breath (BR) and body odours (BO) in the attraction of An. gambiae s.s. to humans was investigated by conducting dual choice tests using a recently developed olfactometer. Either one or two human subjects were used as bait. The single person experiments compared the attractiveness of a person's BR versus that person's BO or a control (empty tent with no odour). His BO and total emanations (TE = BR+BO) were also compared with a control. The two-person experiments compared the relative attractiveness of their TE, BO or BR, and the TE of each person against the BO of the other. RESULTS: Experiments with one human subject (P1) as bait found that his BO and TE collected more mosquitoes than the control (P = 0.005 and P < 0.001, respectively), as did his BO and the control versus his BR (P < 0.001 and P = 0.034, respectively). The TE of P1 attracted more mosquitoes than that of another person designated P8 (P < 0.021), whereas the BR of P8 attracted more mosquitoes than the BR of P1 (P = 0.001). The attractiveness of the BO of P1 versus the BO of P8 did not differ (P = 0.346). The BO from either individual was consistently more attractive than the TE from the other (P < 0.001). CONCLUSIONS: We demonstrated for the first time that human breath, although known to contain semiochemicals that elicit behavioural and/or electrophysiological responses (CO2, ammonia, fatty acids) in An. gambiae also contains one or more constituents with allomonal (~repellent) properties, which inhibit attraction and may serve as an important contributor to between-person differences in the relative attractiveness of humans to this important malaria vector.     

Secretion of Plasmodium falciparum rhoptry protein into the plasma membrane of host erythrocytes

The rhoptry is an organelle of the malarial merozoite which has been suggested to play a role in parasite invasion of its host cell, the erythrocyte. A monoclonal antibody selected for reactivity with this organelle identifies a parasite synthesized protein of 110 kD. From biosynthetic labeling experiments it was demonstrated that the protein is synthesized midway through the erythrocytic cycle (the trophozoite stage) but immunofluorescence indicates the protein is not localized in the organelle until the final stage (segmenter stage) of intraerythrocytic development. Immunoelectron microscopy shows that the protein is localized in the matrix of the rhoptry organelle and on membranous whorls secreted from the merozoite. mAb recognition of the protein is dithiothreitol (DTT) labile, indicating that the conformation of the epitope is dependent on a disulfide linkage. During erythrocyte reinvasion by the extracellular merozoite, immunofluorescence shows the rhoptry protein discharging from the merozoite and spreading around the surface of the erythrocyte. The protein is located in the plasma membrane of the newly invaded erythrocyte. These studies suggest that the 110-kD rhoptry protein is inserted into the membrane of the host erythrocyte during merozoite invasion.     

Deciphering the key residues in Plasmodium falciparum beta-ketoacyl acyl carrier protein reductase responsible for interactions with Plasmodium falciparum acyl carrier protein

The type II fatty acid synthase (FAS) pathway of Plasmodium falciparum is a validated unique target for developing novel antimalarials, due to its intrinsic differences from the typeI pathway operating in humans. beta-Ketoacyl acyl carrier protein (ACP) reductase (FabG) performs the NADPH-dependent reduction of beta-ketoacyl-ACP to beta-hydroxyacyl-ACP, the first reductive step in the elongation cycle of fatty acid biosynthesis. In this article, we report intensive studies on the direct interactions of Plasmodium FabG and Plasmodium ACP in solution, in the presence and absence of its cofactor, NADPH, by monitoring the change in intrinsic fluorescence of P.falciparum FabG (PfFabG) and by surface plasmon resonance. To address the issue of the importance of the residues involved in strong, specific and stoichiometric binding of PfFabG to P.falciparum ACP (PfACP), we mutated Arg187, Arg190 and Arg230 of PfFabG. The activities of the mutants were assessed using both an ACP-dependent and an ACP-independent assay. The affinities of all the PfFabG mutants for acetoacetyl-ACP (the physiological substrate) were reduced to different extents as compared to wild-type PfFabG, but were equally active in biochemical assays with the substrate analog acetoacetyl-CoA. Kinetic analysis and studies of direct binding between PfFabG and PfACP confirmed the identification of Arg187 and Arg230 as critical residues for the PfFabG-PfACP interactions. Our studies thus reveal the significance of the positively charged/hydrophobic patch located adjacent to the active site cavities of PfFabG for interactions with PfACP.     

Coordination of platinum therapeutic agents to met-rich motifs of human copper transport protein1

Platinum therapeutic agents are widely used in the treatment of several forms of cancer. Various mechanisms for the transport of the drugs have been proposed including passive diffusion across the cellular membrane and active transport via proteins. The copper transport protein Ctr1 is responsible for high affinity copper uptake but has also been implicated in the transport of cisplatin into cells. Human hCtr1 contains two methionine-rich Mets motifs on its extracellular N-terminus that are potential platinum-binding sites: the first one encompasses residues 7-14 with amino acid sequence Met-Gly-Met-Ser-Tyr-Met-Asp-Ser and the second one spans residues 39-46 with sequence Met-Met-Met-Met-Pro-Met-Thr-Phe. In these studies, we use liquid chromatography and mass spectrometry to compare the binding interactions between cisplatin, carboplatin and oxaliplatin with synthetic peptides corresponding to hCtr1 Mets motifs. The interactions of cisplatin and carboplatin with Met-rich motifs that contain three or more methionines result in removal of the carrier ligands of both platinum complexes. In contrast, oxaliplatin retains its cyclohexyldiamine ligand upon platinum coordination to the peptide.     

Structural alteration of the membrane of erythrocytes infected with Plasmodium falciparum

Human erythrocytes infected with five strains of Plasmodium falciparum and Aotus erythrocytes infected with three strains of P. falciparum were studied by thin-section and freeze-fracture electron microscopy. All strains of P. falciparum we studied induced electron-dense conical knobs, measuring 30-40 nm in height and 90-100 nm in diameter on erythrocyte membranes. Freeze-fracture demonstrated that the knobs were distributed over the membrane of both human and Aotus erythrocytes. A distinct difference was seen between the intramembrane particle (IMP) distribution over the knobs of human and Aotus erythrocyte membranes. There was no change in IMP distribution in infected human erythrocyte membranes, but infected Aotus erythrocytes showed an aggregation of IMP over the P face of the knobs with a clear zone at the base. This difference in IMP distribution was related only to the host species and not to parasite strains. Biochemical analysis demonstrated that a higher proportion of band 3 was bound to the cytoskeleton of uninfected Aotus erythrocytes than uninfected human erythrocytes after Triton X-100 extraction. This may account for the different effects of P. falciparum infection on IMP distribution in the two different cell types.     

Plasmodium falciparum expresses a multidrug resistance-associated protein

Plasmodium falciparum proteins that efflux toxic metabolic products such as oxidised glutathione (GSSG) are possible targets for anti-malarial drug development. Proteins capable of transporting GSSG and glutathione conjugates include the multidrug resistance-associated transporters (MRPs). A gene, PFA0590w, encoding a MRP homologue, has been identified in P. falciparum. Here we show the presence of full-length mRNA (5.5 kb) of this PfMRP in trophozoites by RT-PCR and Northern blotting. A polyclonal anti-PfMRP antibody generated against two unique, hydrophilic peptides in the predicted sequence produced a strong immunoreactive protein band of 210-215 kDa on Western blots of schizonts of chloroquine-sensitive and chloroquine-resistant strains, confirming expression of PfMRP protein. Using confocal microscopy the protein was seen to be localised at the edge of the schizonts with no obvious staining of the food vacuole. We suggest that PfMRP may act as the GSSG transporter in the parasite plasma membrane.     

Plasmodium falciparum merozoite surface protein 1.

In addition to the major carbohydrate moieties of the glycosylphosphatidylinositol (GPI) anchor, we report that Plasmodium falciparum merozoite surface protein 1 (MSP-1) bears O-GlcNAc modifications predominantly in beta-anomeric configuration, in both the C- and N-terminal portions of the protein. Subcellular fractionation of parasitized erythrocytes in the late trophozoite/schizont stage reveals that GPI-anchored C-terminal fragments of MSP-1 are recovered in Triton X-100 resistant, low-density membrane fractions. Our results suggest that O-GlcNAc-modified MSP-1 N-terminal fragments tend to localize within the parasitophorous vacuolar membrane while GPI-anchored MSP-1 C-terminal fragments associate with low-density, Triton X-100 resistant membrane domains (rafts), redistribute in the parasitized erythrocyte and are eventually shed as membrane vesicles that also contain the endogenous, GPI-linked CD59.     

Plasmodium falciparum: glycosylation status of Plasmodium falciparum circumsporozoite protein expressed in the baculovirus system

We expressed the main surface antigen of Plasmodium falciparum sporozoites, the circumsporozoite protein (CSP), in High Five (Trichoplusia ni) insect cells using the baculovirus system. Significant amounts of the recombinant protein could be obtained, as judged by SDS-PAGE, Western blot, and immunofluorescence analysis. The cellular localization for recombinant CSP was determined by immunofluorescence. The high fluorescence signal of the permeabilized cells, relative to that of fixed nonpermeabilized cells, revealed a clear intracellular localization of this surface antigen. Analysis of possible posttranslational modifications of CSP showed that this recombinant protein is only N-glycosylated in the baculovirus system. Although DNA-sequence analysis revealed a GPI-cleavage/attachment site, no GPI anchor could be demonstrated. These analyses show that the glycosylation status of this recombinant protein may not reflect its native form in P. falciparum. The impact of these findings on vaccine development will be discussed.