Plasmodium yoelii YM MAEBL protein is coexpressed and colocalizes with rhoptry proteins.

We have previously cloned genes from multiple rodent malaria species exhibiting characteristics of the genes encoding Duffy binding like-erythrocyte binding proteins (DBL-EBP). Homology is seen in the intron/exon structure of the genes and in the carboxyl terminal region (including the deduced carboxyl cysteine-rich domain) of the proteins they encode. However, the amino termini of these proteins are not homologous to the DBL-EBP but contain tandem cysteine-rich regions that are similar to the cysteine-rich region of AMA-1 (apical membrane antigen-1), a rhoptry protein. This new family of proteins has been termed MAEBL and these are paralogues of both AMA-1 and the DBL-EBP. Serum against the carboxyl cysteine-rich region of the Plasmodium yoelii YM MAEBL reacted to parasites with a punctate fluorescence pattern characteristic of apical organelle proteins and also localized MAEBL to the surface of merozoites within schizonts. This antiserum immunoprecipitated a protein doublet (120/128 kDa) that was unexpectedly insoluble when compared to members of the DBL-EBP. Characterization of MAEBL was extended through colocalization studies comparing the P. yoelii YM MAEBL to other parasite proteins. This protein appeared to be located in the rhoptry organelles as it colocalized with both AMA-1 and the P. yoelii 235 kDa rhoptry proteins within parasites. In addition, MAEBL is expressed relatively early in schizont development and appears on the merozoite surface after segmentation. Both the pattern and time of expression of the P. yoelii YM MAEBL are consistent with a rhoptry rather than a microneme protein.

Spatial and temporal dynamics of the secretory pathway during differentiation of the Plasmodium yoelii schizont.

A specialized complex of apical organelles facilitates Plasmodium merozoite invasion into the erythrocyte. Even though the apical organelles are crucial to the invasion process, relatively little is known about how they function or their biosynthesis during asexual replication. MAEBL is an erythrocyte binding protein located in the rhoptries and on the surface of mature merozoites and is expressed at the beginning of schizogony before the first nuclear division. Therefore, we have characterized MAEBL as a marker for the biosynthetic pathway of the rhoptry apical organelle during the final phase of intraerythrocytic development and as a marker for the nascent rhoptry vesicle in the immature schizont. An extensive proliferation of the endoplasmic reticulum occurred at the onset of schizogony and was seen as a complex but transient tubule array near the parasite surface. Both the rhoptry protein MAEBL and surface protein MSP-1 appeared to be present in this tubular reticular network together with endoplasmic reticulum markers. MAEBL then transits through Golgi bodies positioned near the parasite plasma membrane, directly adjacent to the network. Rhoptry organelle precursors are seen at the three to four nuclei stage of schizont development, remaining near the plasma membrane throughout schizogony. These studies constitute the first direct evidence that proteins of the rhoptry organelles transit through compartments of the 'classical' secretory pathway.

Erythrocyte binding protein homologues of rodent malaria parasites.

Erythrocyte invasion by malaria parasites requires specific molecular interactions between the merozoite and erythrocyte surface receptors. A well-conserved, functionally important family of erythrocyte binding proteins is the EBP family. The EBP family includes the Plasmodium vivax, P. knowlesi Duffy binding protein (DBP) family and the P. falciparum erythrocyte binding antigen-175 (EBA-175). The EBP are transmembrane proteins, characterized by two conserved cysteine-rich domains, expressed in the micronemes of invasive merozoites. Oligonucleotide primers matching the region encoding the carboxyl cysteine-rich domain of the EBA-175 were used in a polymerase chain reaction to identify homologous genes in P. berghei and P. yoelii yoelii, leading to the isolation of a P. berghei partial genomic clone. This clone contained a 323 bp region that had high deduced amino acid sequence similarity to the amino acid sequences of the carboxyl cysteine-rich domains of the DBP family and EBA-175. The P. berghei carboxyl cysteine-rich domain was followed by a putative transmembrane domain and a cytoplasmic domain, demonstrating an exon-intron structure at the 3' end homologous to P. vivax dbp and P. falciparum eba-175. The carboxyl cysteine-rich domain is also highly conserved among P. berghei, P. y. yoelii, P. chabaudi and P. vinckei and is encoded by a single copy gene. Antisera prepared against the carboxyl cysteine-rich domain of the rodent malaria EBP homologues reacted with a 120 and 128 kDa protein doublet on Western blots of P. berghei parasite antigen and showed an apical localization pattern within merozoites by indirect immunofluorescence assays.

A family of chimeric erythrocyte binding proteins of malaria parasites.

Proteins sequestered within organelles of the apical complex of malaria merozoites are involved in erythrocyte invasion, but few of these proteins and their interaction with the host erythrocyte have been characterized. In this report we describe MAEBL, a family of erythrocyte binding proteins identified in the rodent malaria parasites Plasmodium yoelii yoelii and Plasmodium berghei. MAEBL has a chimeric character, uniting domains from two distinct apical organelle protein families within one protein. MAEBL has a molecular structure homologous to the Duffy binding-like family of erythrocyte binding proteins located in the micronemes of merozoites. However, the amino cysteine-rich domain of MAEBL has no similarity to the consensus Duffy binding-like amino cysteine-rich ligand domain, but instead is similar to the 44-kDa ectodomain fragment of the apical membrane antigen 1 (AMA-1) rhoptry protein family. MAEBL has a tandem duplication of this AMA-1-like domain, and both of these cysteine-rich domains bound erythrocytes when expressed in vitro. Differential transcription and splicing of the maebl locus occurred in the YM clone of P. yoelii yoelii. The apical distribution of MAEBL suggested localization within the rhoptry organelles of the apical complex. We propose that MAEBL is a member of a highly conserved family of erythrocyte binding proteins of Plasmodium involved in host cell invasion.

Expression and serologic activity of a soluble recombinant Plasmodium vivax Duffy binding protein.

Plasmodium vivax Duffy binding protein (DBP) is a conserved functionally important protein. P. vivax DBP is an asexual blood-stage malaria vaccine candidate because adhesion of P. vivax DBP to its erythrocyte receptor is essential for the parasite to continue development in human blood. We developed a soluble recombinant protein of P. vivax DBP (rDBP) and examined serologic activity to it in residents of a region of high endemicity. This soluble rDBP product contained the cysteine-rich ligand domain and most of the contiguous proline-rich hydrophilic region. rDBP was expressed as a glutathione S-transferase (GST) fusion protein and was isolated from GST by thrombin treatment of the purified fusion protein bound on glutathione agarose beads. P. vivax rDBP was immunogenic in rabbits and induced antibodies that reacted with P. vivax and Plasmodium knowlesi merozoites. Human sera from adult residents of a region of Papua New Guinea where malaria is highly endemic or P. vivax-infected North American residents reacted with rDBP in an immunoblot and an enzyme-linked immunosorbent assay. The reactivity to reduced, denatured P. vivax rDBP and the cross-reactivity with P. knowlesi indicated the presence of immunogenic conserved linear B-cell epitopes. A more extensive serologic survey of Papua New Guinea residents showed that antibody response to P. vivax DBP is common and increases with age, suggesting a possible boosting of the antibody response in some by repeated exposure to P. vivax. A positive humoral response to P. vivax DBP correlated with a significantly higher response to P. vivax MSP-1(19). The natural immunogenicity of this DBP should strengthen its usefulness as a vaccine.