Abbreviations: aa, amino acid(s); Ab, antibody; AMA1, apical membrane antigen 1; GST, Glutathione S transferase; PBS, phosphate-buffered saline; PCR, polymerase chain reaction; RON, rhoptry neck protein. AbstractErythrocyte invasion is an essential step in the establishment of host infection by malaria parasites, and is a major target of intervention strategies that attempt to control the disease.Recent proteome analysis of the closely-related apicomplexan parasite, Toxoplasma gondii, falciparum, suggesting that co-operative function of the rhoptry and microneme proteins is a common mechanism in apicomplexan parasites during host cell invasion. PfRON2 possesses a region displaying homology with the rhoptry body protein PfRhopH1/Clag, a component of the RhopH complex. However, here we present co-immunoprecipitation studies which suggest that PfRON2 is not a component of the RhopH complex and has an independent role. Nucleotide polymorphism analysis suggested that PfRON2 was under diversifying selective pressure. This evidence suggests that RON2 appears to have a fundamental role in host cell invasion by apicomplexan parasites, and is a potential target for malaria intervention strategies.

The major virulence determinant of the rodent malaria parasite, Plasmodium yoelii, has remained unresolved since the discovery of the lethal line in the 1970s. Because virulence in this parasite correlates with the ability to invade different types of erythrocytes, we evaluated the potential role of the parasite erythrocyte binding ligand, PyEBL. We found 1 amino acid substitution in a domain responsible for intracellular trafficking between the lethal and nonlethal parasite lines and, furthermore, that the intracellular localization of PyEBL was distinct between these lines. Genetic modification showed that this substitution was responsible not only for PyEBL localization but also the erythrocyte-type invasion preference of the parasite and subsequently its virulence in mice. This previously unrecognized mechanism for altering an invasion phenotype indicates that subtle alterations of a malaria parasite ligand can dramatically affect host-pathogen interactions and malaria virulence.dense granule ͉ invasion ͉ malaria ͉ microneme ͉ transfection T he rodent malaria parasite Plasmodium yoelii yoelii has been widely studied to understand the interactions between the malaria parasite and the host cell (1). The nonlethal 17X line mainly infects young erythrocytes (reticulocytes), whereas the lethal 17XL and YM lines infect a wide range of erythrocytes. These lines have previously been studied to identify the genetic determinants of virulence (2, 3). These differences in erythrocyte invasion preference suggest the possible involvement of a parasite ligand that recognizes erythrocyte surface receptors; however, the actual molecular basis of the observed invasion preference differences remains unclear.Erythrocyte invasion by the malaria merozoite is a multistep process, initiated by reversible binding to the erythrocyte surface, followed by the establishment of a tight junction between the apical end of the merozoite and erythrocyte surface and the subsequent movement of the merozoite into the nascent parasitophorous vacuole. Each step involves specific interactions between parasite ligands and erythrocyte receptors. Among the ligands of malaria parasites, the best characterized is a type I integral transmembrane protein encoded by the ebl (erythrocytebinding-like) gene family. Upon release from the micronemes, EBL proteins recognize erythrocyte receptors and initiate the formation of the tight junction. The importance of EBL in malaria virulence is exemplified in the human malaria parasite Plasmodium vivax, which uses an EBL orthologue, PvDBP, to recognize the Duffy antigen on the erythrocyte surface. Because the parasite is apparently unable to use an alternative invasion pathway, individuals in whom the Duffy antigen is not expressed on the erythrocyte surface are completely resistant to P. vivax (4,5). Because of this dramatic association between the disruption of a host-pathogen interaction and protection against a malaria parasite, PvDBP and the Plasmodium falciparum EBL orthologue, EBA-175, have been targeted for ...

Malaria parasite transmission to humans is initiated by the inoculation of Plasmodium sporozoites into the skin by mosquitoes. Sporozoites develop within mosquito midgut oocysts, first invade the salivary glands of mosquitoes, and finally infect hepatocytes in mammals. The apical structure of sporozoites is conserved with the infective forms of other apicomplexan parasites that have secretory organelles, such as rhoptries and micronemes. Because some rhoptry proteins are crucial for Plasmodium merozoite infection of erythrocytes, we examined the roles of rhoptry proteins in sporozoites. Here, we demonstrate that rhoptry neck protein 2 (RON2) is also localized to rhoptries in sporozoites. To elucidate RON2 function in sporozoites, we applied a promoter swapping strategy to restrict ron2 transcription to the intraerythrocytic stage in the rodent malaria parasite, Plasmodium berghei. Ron2 knockdown sporozoites were severely impaired in their ability to invade salivary glands, via decreasing the attachment capacity to the substrate. This is the first rhoptry protein demonstrated to be involved in salivary gland invasion. In addition, ron2 knockdown sporozoites showed less infectivity to hepatocytes, possibly due to decreased attachment/gliding ability, indicating that parts of the parasite invasion machinery are conserved, but their contribution might differ among infective forms. Our sporozoite stage‐specific knockdown system will help to facilitate understanding the comprehensive molecular mechanisms of parasite invasion of target cells.

f Erythrocyte invasion by merozoites is an obligatory stage of Plasmodium infection and is essential to disease progression. Proteins in the apical organelles of merozoites mediate the invasion of erythrocytes and are potential malaria vaccine candidates. Rhoptry-associated, leucine zipper-like protein 1 (RALP1) of Plasmodium falciparum was previously found to be specifically expressed in schizont stages and localized to the rhoptries of merozoites by immunofluorescence assay (IFA). Also, RALP1 has been refractory to gene knockout attempts, suggesting that it is essential for blood-stage parasite survival. These characteristics suggest that RALP1 can be a potential blood-stage vaccine candidate antigen, and here we assessed its potential in this regard. Antibodies were raised against recombinant RALP1 proteins synthesized by using the wheat germ cell-free system. Immunoelectron microscopy demonstrated for the first time that RALP1 is a rhoptry neck protein of merozoites. Moreover, our IFA data showed that RALP1 translocates from the rhoptry neck to the moving junction during merozoite invasion. Growth and invasion inhibition assays revealed that anti-RALP1 antibodies inhibit the invasion of erythrocytes by merozoites. The findings that RALP1 possesses an erythrocyte-binding epitope in the C-terminal region and that anti-RALP1 antibodies disrupt tight-junction formation, are evidence that RALP1 plays an important role during merozoite invasion of erythrocytes. In addition, human sera collected from areas in Thailand and Mali where malaria is endemic recognized this protein. Overall, our findings indicate that RALP1 is a rhoptry neck erythrocyte-binding protein and that it qualifies as a potential blood-stage vaccine candidate.

The number of malaria vaccine candidates in preclinical and clinical development is limited. To identify novel blood-stage malaria vaccine candidates, we constructed a library of 1,827P. falciparum proteins prepared using the wheat germ cell-free system (WGCFS). Also, a high-throughput AlphaScreen procedure was developed to measure antibody reactivity to the recombinant products. Purified IgGs from residents in malaria endemic areas have shown functional activity against blood-stage parasites as judged by an in vitro parasite Growth Inhibition Assay (GIA). Therefore, we evaluated the GIA activity of 51 plasma samples prepared from Malian adults living in a malaria endemic area against the WGCFS library. Using the AlphaScreen-based immunoreactivity measurements, antibody reactivity against 3 proteins was positively associated with GIA activity. Since anti-LSA3-C responses showed the strongest correlation with GIA activity, this protein was investigated further. Anti-LSA3-C-specific antibody purified from Malian adult plasmas showed GIA activity, and expression of LSA3 in blood-stage parasites was confirmed by western blotting. Taken together, we identified LSA3 as a novel blood-stage vaccine candidate, and we propose that this system will be useful for future vaccine candidate discovery.

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Four selected plants (corn, groundnut, cow pea, and mungbean) were tested for their ability to germinate and grow in an acidic soil contaminated with phenanthrene or pyrene, two typical polycyclic aromatic hydrocarbons (PAHs). The growth of corn root was the least sensitive to, but its germination rate was the lowest in the presence of, contaminants. Among the legumes, the growth of groundnut root was better than others. Corn and groundnut were selected to further test their ability to tolerate a mixture of phenanthrene and pyrene in the acidic soil. The presence of both PAHs led to a greater decrease in the lengths of shoot and root of groundnut than phenanthrene or pyrene alone, but the lengths of shoot and root of corn were decreased to a similar extent as when phenanthrene or pyrene was present alone. The growth of corn root was also better than that of groundnut root when they were grown in oil-contaminated soil. Based on these results, we conclude that corn is the most suitable to be grown in PAH-contaminated acidic soil.