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...
Rhoptry proteins participate in the invasion of red blood cells by merozoites during the malaria parasite's asexual-stage cycle. Interference with the rhoptry protein function has been shown to prevent invasion, and three rhoptry proteins have been suggested as potential components of a vaccine against malaria. Rhoptryassociated membrane antigen (RAMA) is a 170-kDa protein of Plasmodium falciparum which is processed to a 60-kDa mature form in the rhoptries. p60/RAMA is discharged from rhoptries of free merozoites and binds to the red-cell membrane before being internalized to form part of the parasitophorous vacuole of the newly developing ring. We examined the range of anti-RAMA responses in individuals living in an area of endemicity for malaria and determined its association with clinical immunity. RAMA is immunogenic during infections, and at least three epitopes within RAMA are recognized by hyperimmune sera in immunoblots. Sera from individuals living in a region of Vietnam where malaria is endemic possessed strong antibody responses toward two C-terminal regions of RAMA. Cytophilic antibody isotypes (immunoglobulin G1 [IgG1] and IgG3) predominated in humoral responses to both C-terminal epitopes. Acute episodes of P. falciparum infection result in significant boosting of levels of antibody to an epitope at the extreme C terminus of RAMA that harbors the red-cell-binding domain. Immunity to P. falciparum infection was linked to elevated levels of IgG3 responses to this functional domain of RAMA, suggesting that the region may contain a protective epitope useful for inclusion in a multiepitope vaccine against malaria.Plasmodium falciparum malaria is responsible for Ͼ2 million deaths each year. With the increasing resistance of Plasmodium parasites to antimalarial drugs and of the Anopheles mosquito vector to commonly available insecticides, an effective vaccine that would protect nonimmune individuals from death would be valuable. The life cycle of malaria parasites is quite complex and provides a number of potential vaccine targets. Several proteins expressed by P. falciparum during the erythrocytic stages are being investigated as candidates for a subunit vaccine, among them rhoptry-associated proteins. The most important immune response for immunity to asexual-stage infection is believed to be humoral, although cell-mediated responses may also contribute to immunity.Rhoptries are intracellular organelles of malaria parasites involved in the invasion of red blood cells (RBCs) by Plasmodium merozoites. Although their contents are only transiently accessible to antibodies, seroepidemiological studies have demonstrated the development of antibody responses to the rhoptry proteins RhopH3, RAP1, and RAP2 following infection (8,12,20,23). In vitro growth inhibition assays have indicated that antibodies directed against the RAP1 and RAP2 proteins have inhibitory effects on P. falciparum growth in in vitro culture (6,9,14,18). Moreover, immunization of Saimiri monkeys with RAP1 protected the animals from a lethal...
BackgroundBacteria of the suborder Corynebacterineae include significant human pathogens such as Mycobacterium tuberculosis and M. leprae. Drug resistance in mycobacteria is increasingly common making identification of new antimicrobials a priority. Mycobacteria replicate intracellularly, most commonly within the phagosomes of macrophages, and bacterial proteins essential for intracellular survival and persistence are particularly attractive targets for intervention with new generations of anti-mycobacterial drugs.Methodology/Principal FindingsWe have identified a novel gene that, when inactivated, leads to accelerated death of M. smegmatis within a macrophage cell line in the first eight hours following infection. Complementation of the mutant with an intact copy of the gene restored survival to near wild type levels. Gene disruption did not affect growth compared to wild type M. smegmatis in axenic culture or in the presence of low pH or reactive oxygen intermediates, suggesting the growth defect is not related to increased susceptibility to these stresses. The disrupted gene, MSMEG_5817, is conserved in all mycobacteria for which genome sequence information is available, and designated Rv0807 in M. tuberculosis. Although homology searches suggest that MSMEG_5817 is similar to the serine:pyruvate aminotransferase of Brevibacterium linens suggesting a possible role in glyoxylate metabolism, enzymatic assays comparing activity in wild type and mutant strains demonstrated no differences in the capacity to metabolize glyoxylate.Conclusions/Significance MSMEG_5817 is a previously uncharacterized gene that facilitates intracellular survival of mycobacteria. Interference with the function of MSMEG_5817 may provide a novel therapeutic approach for control of mycobacterial pathogens by assisting the host immune system in clearance of persistent intracellular bacteria.
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