Precise Timing of Expression of a Plasmodium falciparum- derived Transgene in Plasmodium berghei Is a Critical Determinant of Subsequent Subcellular Localization

Abstract

The development of transfection technology for malaria parasites holds significant promise for a more detailed characterization of molecules targeted by vaccines or drugs. One asexual blood stage vaccine candidate, apical membrane antigen-1 (AMA-1) of merozoite rhoptries has been shown to be the target of inhibitory, protective antibodies in both in vitro and in vivo studies. We have investigated heterologous (trans-species) expression of the human malaria Plasmodium falciparum AMA-1 (PF83/AMA-1) in the rodent parasite Plasmodium berghei. Transfected P. berghei expressed correctly folded and processed PF83/AMA-1 under control of both pb66/ama-1 and dhfr-ts promoters. Timing of expression was highly promoter-dependent and was critical for subsequent subcellular localization. Under control of pb66/ama-1, PF83/AMA-1 expression and localization in P. berghei was limited to the rhoptries of mature schizonts, similar to that observed for PF83/ AMA-1 in P. falciparum. In contrast the dhfr-ts promoter permitted PF83/AMA-1 expression throughout schizogony as well as in gametocytes and gametes. Localization was aberrant and included direct expression at the merozoite and gamete surface. Processing from the fulllength 83-kDa protein to a 66-kDa protein was observed not only in schizonts but also in gametocytes, indicating that processing could be mediated outside of rhoptries by a common protease. Trans-species expressed PF83/ AMA-1 was highly immunogenic in mice, resulting in a response against a functionally critical domain of the molecule.The protozoan parasite Plasmodium falciparum is a causative agent of malaria, one of the major human infectious diseases. In the search for new methods to combat the disease, the advent of transfection technology for Plasmodium species is critical, because it offers the opportunity to relate genotype to phenotype, and this will permit a more rational design of vaccines and drugs. To date, stable episomal maintenance of plasmid DNA introduced into Plasmodium has been reported (1-3) as well as site-directed integration of DNA into the parasite genome (4 -8). This technology also offers the possibility to dissect the thus far poorly characterized Plasmodium promoter function (9 -11) and study the relation between the tightly controlled timing of expression and the subcellular trafficking and localization of stage-specific proteins. Trans-species expression of malarial antigens will allow targeted development of attenuated parasite vaccines and opens possibilities for complementation of otherwise detrimental integration into essential genes. Apical membrane antigen-1 (AMA-1)1 is an attractive candidate for such studies, because it appears to be intimately involved in red cell invasion (12). Expression and post-translational N-terminal proteolytic cleavage of AMA-1 are restricted to the final stages of schizogony (13), during which the protein is localized within the neck of the rhoptry, an apical secretory organelle of the merozoite involved in red cell invasion (14). AMA-1 is a major cand...

trans Expression of a Plasmodium falciparum Histidine-rich Protein II (HRPII) Reveals Sorting of Soluble Proteins in the Periphery of the Host Erythrocyte and Disrupts Transport to the Malarial Food Vacuole

“…The trans gene is maximally expressed and exported at the trophozoite and schizont stages, whereas the endogenous protein is exported at the ring stage. This suggests that unlike transport to apical organelles (32), protein secretion to the red cell is not temporally regulated. This argues against a "secondary ER" that has been proposed to exist as a specialized compartment in Plasmodium dedicated to ring stage export to the erythrocyte (33).…”

Structure of AMA1 from Plasmodium falciparum reveals a clustering of polymorphisms that surround a conserved hydrophobic pocket

“…Both are bound by inhibitory antibodies in vitro, and the clustering of polymorphisms suggests that they are both targets of protective antibody responses in humans (21,(26)(27)(28)(29). Domain I is conserved among Plasmodium species and in other apicomplexan parasites.…”

EDiP: the Epitope Dilution Phenomenon. Lessons learnt from a malaria vaccine antigen and its applicability to polymorphic antigens

“…AMA1 has a unique feature which becomes apparent when projecting the variable positions on the crystal structure; it has a polymorphic and a conserved face ( Figure 1) [13][14][15]. The conserved face has a conformational epitope that is recognized by the 4G2 monoclonal antibody [5,[13][14][15]. This monoclonal antibody neutralizes all strains hitherto tested and this conserved (functional) site may potentially also be exploited for an AMA1-based vaccine.…”

“…In Plasmodium falciparum, it is a 622-amino-acid-long, type 1 transmembrane protein, initially expressed as an 83-kDa precursor protein in merozoite micronemes and later processed to a 66-kDa protein by the removal of the N-terminal prosequence [2][3][4][5]. The AMA1 ectodomain is an important vaccine target since antibodies against the ectodomain have been shown to prevent red cell invasion in vitro [6][7][8][9], and this effect requires immunization with correctly folded AMA1 [10,11].…”