Viruslike particles containing knob-associated histidine-rich protein are secreted into the culture medium of Plasmodium falciparum in vitro cultures

This report describes the isolation of a viruslike particle from in vitro cultures of the human malaria parasite P. falciparum. Electronmicroscopic observations suggest that the particles are liberated into the culture medium by budding from the erythrocyte membrane. The density of the free particles is 1.16, they contain nucleic acid and two distinct molecular species of the knob-associated Histidine-rich protein. Proteins of the particles are recognized by sera from malaria patients. The previously described knobs may correspond to viral coats inserted in the membrane.     

Heme binding to the histidine-rich protein II from Plasmodium falciparum

The histidine-rich protein II (HRP II) from Plasmodium falciparum has been implicated in the formation of hemozoin, a detoxified, crystalline form of ferric protoporphyrin IX (Fe(3+)-PPIX) produced by the parasite. Fe(3+)-PPIX titrations coupled with quantitative amino acid analysis showed that HRP II binds 15 Fe(3+)-PPIX molecules per 30 kDa monomer. Circular dichroism spectroscopy was used to probe the secondary structure of HRP II with and without bound Fe(3+)-PPIX. These studies have revealed large changes in the secondary structure with Fe(3+)-PPIX binding, changing from a random coil in the absence of Fe(3+)-PPIX to a more ordered helical structure in the presence of Fe(3+)-PPIX. The Fe(3+)-PPIX-bound HRP II structure most closely resembles a 3(10)-helix. Coincident with this structural change caused by Fe(3+)-PPIX binding, the formation of an intermolecular disulfide bond occurs between HRP II monomers. In vitro pull-down assays show an interaction between monomers that is dependent on the presence of Fe(3+)-PPIX. One model that best fits with the data reported here requires formation of 15 intermolecular bishistidyl ligated Fe(3+)-PPIX molecules arranged in a head to head fashion, which would then allow for the formation of an intermolecular disulfide bond. The structure best able to accommodate these requirements is a 3(10)-helix.     

Malaria and Leishmania parasites share the knob-associated histidine-rich protein gene sequences

1. The main coding region of the knob-associated histidine-rich protein (KAHRP) gene of Plasmodium falciparum hydridized with genomic DNA of Leishmania donovani. 2. A total of five EcoRI fragments of various sizes (7.5, 5.5, 3.2, 0.75 and 0.56 Kb) were recognized by this probe, under lower stringent conditions. However, under a higher stringency of washing, two of the smallest fragments were washed away. 3. Out of these EcoRI fragments, the 5.5 Kb band showed a maximum homology with the probe which contains the histidine-rich coding sequences, whereas the 3.2 Kb band showed none. Thus there is a possibility that the Leishmania parasite also contains a KAHRP-like gene.     

Structural and functional studies of interaction between Plasmodium falciparum knob-associated histidine-rich protein (KAHRP) and erythrocyte spectrin

Plasmodium falciparum dramatically modifies the structure and function of the membrane of the parasitized host erythrocyte. Altered membrane properties are the consequence of the interaction of a group of exported malaria proteins with host cell membrane proteins. KAHRP (the knob-associated histidine-rich protein), a member of this group, has been shown to interact with erythrocyte membrane skeletal protein spectrin. However, the molecular basis for this interaction has yet to be defined. In the present study, we defined the binding motifs in both KAHRP and spectrin and identified a functional role for this interaction. We showed that spectrin bound to a 72-amino-acid KAHRP fragment (residues 370-441). Among nine-spectrin fragments, which encompass the entire alpha and beta spectrin molecules (four alpha spectrin and five beta spectrin fragments), KAHRP bound only to one, the alpha N-5 fragment. The KAHRP-binding site within the alpha N-5 fragment was localized uniquely to repeat 4. The interaction of full-length spectrin dimer to KAHRP was inhibited by repeat 4 of alpha spectrin. Importantly, resealing of this repeat peptide into erythrocytes mislocalized KAHRP in the parasitized cells. We concluded that the interaction of KAHRP with spectrin is critical for appropriate membrane localization of KAHRP in parasitized erythrocytes. As the presence of KAHRP at the erythrocyte membrane is necessary for cytoadherence in vivo, our findings have implications for the development of new therapies for mitigating the severity of malaria infection.     

Recombinant antibodies specific for the Plasmodium falciparum histidine-rich protein 2

Early diagnosis and appropriate treatment are key elements of malaria control programs in endemic areas. A major step forward in recent years has been the production and use of rapid diagnostic tests (RDTs) in settings where microscopy is impracticable. Many current RDTs target the Plasmodium falciparum histidine-rich protein 2 (PfHRP2) released in the plasma of infected individuals. These RDTs have had an indisputably positive effect on malaria management, but still present several limitations, including the poor characterization of the commercial monoclonal antibodies (mAbs) used for PfHRP2 detection, variable sensitivity and specificity and high costs. RDT use is further limited by impaired stability caused by temperature fluctuations during transport and uncontrolled storage in field-based facilities. To circumvent such drawbacks, an alternative could be the development of well-characterized, stabilized recombinant antibodies, with high binding affinity and specificity. Here, we report the characterization of the cDNA sequences encoding the Fab fragment of F1110 and F1546, two novel anti-PfHRP2 mAbs. FabF1546 was produced in the Escherichia coli periplasm. Its properties of binding to the parasite and to a recombinant PfHRP-2 antigen were similar to those of the parental mAb. As the affinity and stability of recombinant antibodies can be improved by protein engineering, our results open a novel approach for the development of an improved RDT for malaria diagnosis.Key words: Plasmodium falciparum, malaria, histidine-rich protein, monoclonal antibodies, recombinant Fab, rapid diagnostic test     

Recombinant antibodies specific for the Plasmodium falciparum histidine-rich protein 2

Early diagnosis and appropriate treatment are key elements of malaria control programs in endemic areas. A major step forward in recent years has been the production and use of rapid diagnostic tests (RDTs) in settings where microscopy is impracticable. Many current RDTs target the Plasmodium falciparum histidine-rich protein 2 (PfHRP2) released in the plasma of infected individuals. These RDTs have had an indisputably positive effect on malaria management, but still present several limitations, including the poor characterization of the commercial monoclonal antibodies (mAbs) used for PfHRP2 detection, variable sensitivity and specificity, and high costs. RDT use is further limited by impaired stability caused by temperature fluctuations during transport and uncontrolled storage in field-based facilities. To circumvent such drawbacks, an alternative could be the development of well-characterized, stabilized recombinant antibodies, with high binding affinity and specificity. Here, we report the characterization of the cDNA sequences encoding the Fab fragment of F1110 and F1546, two novels anti-PfHRP2 mAbs. FabF1546 was produced in the Escherichia coli periplasm. Its properties of binding to the parasite and to a recombinant PfHRP-2 antigen were similar to those of the parental mAb. As the affinity and stability of recombinant antibodies can be improved by protein engineering, our results open a novel approach for the development of an improved RDT for malaria diagnosis.     

Mapping the binding domains involved in the interaction between the Plasmodium falciparum knob-associated histidine-rich protein (KAHRP) and the cytoadherence ligand P. falciparum erythrocyte membrane protein 1 (PfEMP1).

Plasmodium falciparum erythrocyte membrane protein 1 (PfEMP1) clusters at electron-dense knob-like structures on the surface of malaria-infected red blood cells and mediates their adhesion to the vascular endothelium. In parasites lacking knobs, vascular adhesion is less efficient, and infected red cells are not able to immobilize successfully under hemodynamic flow conditions even though PfEMP1 is still present on the exterior of the infected red cell. We examined the interaction between the knob-associated histidine-rich protein (KAHRP), the parasite protein upon which knob formation is dependent, and PfEMP1, and we show evidence of a direct interaction between KAHRP and the cytoplasmic region of PfEMP1 (VARC). We have identified three fragments of KAHRP which bind VARC. Two of these KAHRP fragments (K1A and K2A) interact with VARC with binding affinities (K(D(kin))) of 1 x 10(-7) M and 3.3 x 10(-6) M respectively, values comparable to those reported previously for protein-protein interactions in normal and infected red cells. Further experiments localized the high affinity binding regions of KAHRP to the 63-residue histidine-rich and 70-residue 5' repeats. Deletion of these two regions from the KAHRP fragments abolished their ability to bind to VARC. Identification of the critical domains involved in interaction between KAHRP and PfEMP1 may aid development of new therapies to prevent serious complications of P. falciparum malaria.     

Neutralization of toxic heme by Plasmodium falciparum histidine-rich protein 2

Plasmodium falciparum histidine-rich protein 2 (PfHRP2) has been suggested to be an initiator of the polymerization of heme, which is produced as by-product on the digestion of hemoglobin, and a promoter of the H(2)O(2)-induced degradation of heme in food vacuoles of the malarial parasite. In this work, we have designed PfHRP2 model peptides, R18 and R27 (18 and 27 residues, respectively), and used them for optical and electron spin resonance spectroscopic measurements to confirm that the axial ligands of the heme-PfHRP2 complex are the nitrogenous donors derived from the imidazole moieties of histidine residues of PfHRP2. In addition, we revealed that the affinities of R18 and R27 for heme (K(d) = 2.21 x 10(-6) M and 0.71 x 10(-6) M, respectively) might be as high as that of PfHRP2 (K(d) = 0.94 x 10(-6) M). The R27 peptide can remove heme from membrane-intercalated heme and inhibit heme-induced hemolysis. Therefore, we suggest another function of PfHRP2: it may play an important role in the neutralization of toxic heme in the parasite cytoplasm and infected erythrocytes by removing heme from heme-bound membranes or reducing heme-induced hemolysis.     

Spectroscopic characterization of the heme-binding sites in Plasmodium falciparum histidine-rich protein 2

Proteolysis of hemoglobin provides an essential nutrient source for the malaria parasite Plasmodium falciparum during the intraerythrocytic stage of the parasite's lifecycle. Detoxification of the liberated heme occurs through a unique heme polymerization pathway, leading to the formation of hemozoin. Heme polymerization has been demonstrated in the presence of P. falciparum histidine-rich protein 2 (PfHRP2) [Sullivan, D. J., Gluzman, I. Y., and Goldberg, D. E. (1996) Science 271, 219-221]; however, the molecular role that PfHRP2 plays in this polymerization is currently unknown. PfHRP2 is a 30 kDa protein composed of several His-His-Ala-His-His-Ala-Ala-Asp repeats and is present in the parasite food vacuole, the site of hemoglobin degradation and heme polymerization. We found that, at pH 7.0, PfHRP2 forms a saturable complex with heme, with a PfHRP2 to heme stoichiometry of 1:50. Spectroscopic characterization of heme binding by electronic absorption, resonance Raman, and EPR has shown that bound hemes share remarkably similar heme environments as >95% of all bound hemes are six-coordinate, low-spin, and bis-histidyl ligated. The PfHRP2-ferric heme complex at pH 5.5 (pH of the food vacuole) has the same heme spin state and coordination as observed at pH 7.0; however, polymerization occurs as heme saturation is approached. Therefore, formation of a PfHRP2-heme complex appears to be a requisite step in the formation of hemozoin.     

A histidine-rich protein gene marks a linkage group favored strongly in a genetic cross of Plasmodium falciparum

Two histidine-rich protein genes in Plasmodium falciparum are related by an ancestral duplication and interchromosomal transposition. We have followed the inheritance of these genes in a cross between two clones of P. falciparum. Examination of progeny shows that one gene, encoding the protein HRP-II, behaves as expected and may be inherited from either parent. The other gene, encoding HRP-III, has been found to derive from one parent in all progeny examined. We conclude the linkage group marked by HRP-III is favored strongly in the cross. This linkage group spans a region at one end of chromosome 13. Growth studies suggest the favored inheritance is explained by rapid expansion of progeny possessing the HRP-III marker relative to slower growth of progeny without it.     

The malaria genome sequencing project: complete sequence of Plasmodium falciparum chromosome 2

An international consortium has been formed to sequence the entire genome of the human malaria parasite Plasmodium falciparum. We sequenced chromosome 2 of clone 3D7 using a shotgun sequencing strategy. Chromosome 2 is 947 kb in length, has a base composition of 80.2% A + T, and contains 210 predicted genes. In comparison to the Saccharomyces cerevisiae genome, chromosome 2 has a lower gene density, a greater proportion of genes containing introns, and nearly twice as many proteins containing predicted non-globular domains. A group of putative surface proteins was identified, rifins, which are encoded by a gene family comprising up to 7% of the protein-encoding gene in the genome. The rifins exhibit considerable sequence diversity and may play an important role in antigenic variation. Sixteen genes encoded on chromosome 2 showed signs of a plastid or mitochondrial origin, including several genes involved in fatty acid biosynthesis. Completion of the chromosome 2 sequence demonstrated that the A + T-rich genome of P. falciparum can be sequenced by the shotgun approach. Within 2-3 years, the sequence of almost all P. falciparum genes will have been determined, paving the way for genetic, biochemical, and immunological research aimed at developing new drugs and vaccines against malaria.     

The gene encoding DNA polymerase alpha from Plasmodium falciparum.

The gene encoding DNA polymerase alpha from the human malaria parasite Plasmodium falciparum has been sequenced and characterised. The deduced amino acid sequence possesses the seven sequence motifs which characterise eukaryotic replicative DNA polymerases (I-VII) and four of five motifs (A-E) identified in alpha DNA polymerases. The predicted protein also contains sequences which are reminiscent of Plasmodium proteins but absent from other DNA polymerases. These include four blocks of additional amino acids interspersed with the conserved motifs of the DNA polymerases, four asparagine rich sequences and a novel carboxy-terminal extension. Repetitive sequences similar to those found in other malarial proteins are also present. cDNA-directed PCR was used to establish the presence of these features in the approximately 7kb mRNA. The coding sequence contains a single intron. The gene for DNAPol alpha is located on chromosome 4 and is transcribed in both asexual and sexual erythrocytic stages of the parasite.     

Amino acid substitution and modification resulting from Escherichia coli expression of recombinant Plasmodium falciparum histidine-rich protein II

The histidine-rich protein II (HRP II) from Plasmodium falciparum is an unusual protein composed of 40% alanine, 36% histidine, and 11% aspartate residues. Expression of HRP II in Escherichia coli results in the isolation of a heterogeneous protein. Mass spectrometry reveals a reduction in mass by multiples of 9 Da from the expected molecular mass that can be attributed to the substitution of glutamine for some histidine residues in the sequence. The extent of the glutamine for histidine substitution can be reduced by slowing the expression rate. Mass spectral analysis of HRP II also revealed alpha-amino methylation of the N-terminal alanine residue of HRP II.