title: 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 full-length 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.
authors.affiliation: Department of Parasitology, Biomedical Primate Research Centre, Lange Kleiweg 157, 2280 GJ Rijswijk, The Netherlands.
authors.forename: C H
authors.initials: CH
authors.lastname: Kocken
authors.forename: A M
authors.initials: AM
authors.lastname: van der Wel
authors.forename: M A
authors.initials: MA
authors.lastname: Dubbeld
authors.forename: D L
authors.initials: DL
authors.lastname: Narum
authors.forename: F M
authors.initials: FM
authors.lastname: van de Rijke
authors.forename: G J
authors.initials: GJ
authors.lastname: van Gemert
authors.forename: X
authors.initials: X
authors.lastname: van der Linde
authors.forename: L H
authors.initials: LH
authors.lastname: Bannister
authors.forename: C
authors.initials: C
authors.lastname: Janse
authors.forename: A P
authors.initials: AP
authors.lastname: Waters
authors.forename: A W
authors.initials: AW
authors.lastname: Thomas
mesh_terms: D000818:Animals; D000953:Antigens, Protozoan / Q000276:immunology; D000954:Antigens, Surface / Q000276:immunology; D005455:Fluorescent Antibody Technique; D005786:Gene Expression Regulation / Q000235:genetics; D007114:Immunization; D008288:Malaria / Q000503:physiopathology; D008565:Membrane Proteins / Q000737:chemistry* / Q000235:genetics; D016253:Microscopy, Immunoelectron; D010962:Plasmodium berghei / Q000235:genetics*; D010963:Plasmodium falciparum / Q000235:genetics*; D011401:Promoter Regions, Genetic / Q000235:genetics; D011499:Protein Processing, Post-Translational / Q000502:physiology; D015800:Protozoan Proteins / Q000737:chemistry* / Q000235:genetics; D012333:RNA, Messenger / Q000378:metabolism; D012377:Rodentia; D014162:Transfection / Q000235:genetics; D019076:Transgenes / Q000235:genetics*
chemical_list: D000953:Antigens, Protozoan; D000954:Antigens, Surface; D008565:Membrane Proteins; D015800:Protozoan Proteins; D012333:RNA, Messenger; C090077:apical membrane antigen I, Plasmodium