Precise timing of expression of a Plasmodium falciparum-derived transgene in Plasmodium berghei is a critical determinant of subsequent subcellular localization.
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.
Kocken
C H
CH
Department of Parasitology, Biomedical Primate Research Centre, Lange Kleiweg 157, 2280 GJ Rijswijk, The Netherlands.
van der Wel
A M
AM
Dubbeld
M A
MA
Narum
D L
DL
van de Rijke
F M
FM
van Gemert
G J
GJ
van der Linde
X
X
Bannister
L H
LH
Janse
C
C
Waters
A P
AP
Thomas
A W
AW
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*
D000953:Antigens, Protozoan; D000954:Antigens, Surface; D008565:Membrane Proteins; D015800:Protozoan Proteins; D012333:RNA, Messenger; C090077:apical membrane antigen I, Plasmodium