Characterization of a Membrane-associated Rhoptry Protein of Plasmodium falciparum

Abstract

Invasive forms of apicomplexan parasites contain secretory organelles called rhoptries that are essential for entry into host cells. We present a detailed characterization of an unusual rhoptry protein of the human malaria parasite Plasmodium falciparum, the rhoptryassociated membrane antigen (RAMA) that appears to have roles in both rhoptry biogenesis and host cell invasion. RAMA is synthesized as a 170-kDa protein in early trophozoites, several hours before rhoptry formation and is transiently localized within the endoplasmic reticulum and Golgi within lipid-rich microdomains. Regions of the Golgi membrane containing RAMA bud to form vesicles that later mature into rhoptries in a process that is inhibitable by brefeldin A. Other rhoptry proteins such as RhopH3 and RAP1 are found in close apposition with RAMA suggesting direct protein-protein interactions. We suggest that RAMA is involved in trafficking of these proteins into rhoptries. In rhoptries, RAMA is proteolytically processed to give a 60-kDa form that is anchored in the inner face of the rhoptry membrane by means of the glycosylphosphatidylinositol anchor. The p60 RAMA form is discharged from the rhoptries of free merozoites and binds to the red blood cell membrane by its most C-terminal region. In early ring stages RAMA is found in association with the parasitophorous vacuole.Plasmodium falciparum malaria is one of the most important infectious diseases of humans, accounting for ϳ2 million deaths each year. The stages of the parasite that grow and multiply in red blood cells (RBCs) 1 cause all the pathological effects associated with the disease, and accordingly invasion of red blood cells is one of the most important steps in the parasite life cycle. Three sets of secretory organelles, the rhoptries, micronemes, and dense granules are involved in and are essential for the invasion process. The understanding of the role of these organelles provides important knowledge about the basic biology of malaria and potential therapeutical targets.Rhoptries of Plasmodium parasites are paired club-shaped organelles located at the apical end of merozoites, the form of the parasite that invades RBCs. Following the attachment of merozoites to the RBC surface, rhoptries discharge their contents onto the RBC membrane (1). Rhoptry organelles disappear after internalization of merozoites and thus are formed de novo with each erythrocytic cycle. Rhoptry formation occurs late in the erythrocytic stages of the parasite, and elucidation of rhoptry biogenesis of malaria parasites has been hindered by the lack of early organelle markers. Most of our knowledge is based on microscopic examinations, which suggest that rhoptry biogenesis follows the secretory pathway route, with rhoptry organelles being formed by sequential fusion of post-Golgi vesicles (2, 3), although why particular vesicles are selected is unclear.Rhoptry contents include both protein and lipid components, which assemble to form membrane-like structures. Protein constituents of the rhoptry contents are stil...

Associations between Responses to the Rhoptry-Associated Membrane Antigen of Plasmodium falciparum and Immunity to Malaria Infection

“…Monoclonal antibodies directed against epitopes within Ag44 showed growth-inhibitory properties in in vitro assays (6,7). As in vitro RBC-binding assays indicated that Ag44 harbors an RBC-binding domain (21), there appears to be a link between protection and responses to RBC-binding domains for both RAMA and RhopH3.…”

“…Antibodies of the IgG3 isotype directed to fragment E are strongly associated with resistance to infection in our study population. Fragment E contains the RBC-binding domain and may be involved in the process of merozoite entry and the formation of the parasitophorous vacuole (21). As such, RAMA-E might have potential as a candidate for inclusion in an antimalarial vaccine.…”

“…The target antigens for measuring antibody responses were five recombinant RAMA fragments and the C-terminal region of RhopH3 (Ag44) (5), expressed as glutathione S-transferase (GST) fusion proteins in Escherichia coli as described previously (21). An extreme N-terminal fragment of RAMA (RAMA-A) was expressed as a GST fusion protein as before (21), using primers p869 (5Ј-gctaggatccACATATTTAGAACAAATAAAAAATGGT-3Ј) and p1005 (5Ј-tagcgtcgacGTCATTTTCCAACTTATTTATTTC-3Ј) for PCR amplification from a cDNA template (added extensions are shown in lowercase letters and introduced restriction sites are underlined).…”

“…An extreme N-terminal fragment of RAMA (RAMA-A) was expressed as a GST fusion protein as before (21), using primers p869 (5Ј-gctaggatccACATATTTAGAACAAATAAAAAATGGT-3Ј) and p1005 (5Ј-tagcgtcgacGTCATTTTCCAACTTATTTATTTC-3Ј) for PCR amplification from a cDNA template (added extensions are shown in lowercase letters and introduced restriction sites are underlined). GST was used as a negative control to determine antibody responses specific to the RAMA or Ag44 portions of fusion proteins.…”

“…Recently, the rhoptry-associated membrane antigen (RAMA) of P. falciparum was characterized (21). RAMA is expressed as a 170-kDa protein (p170) that contains three distinct repeat regions (R1, R2, and R3) within the N-terminal half.…”