title: Functional analysis of the leading malaria vaccine candidate AMA-1 reveals an essential role for the cytoplasmic domain in the invasion process.
abstract: A key process in the lifecycle of the malaria parasite Plasmodium falciparum is the fast invasion of human erythrocytes. Entry into the host cell requires the apical membrane antigen 1 (AMA-1), a type I transmembrane protein located in the micronemes of the merozoite. Although AMA-1 is evolving into the leading blood-stage malaria vaccine candidate, its precise role in invasion is still unclear. We investigate AMA-1 function using live video microscopy in the absence and presence of an AMA-1 inhibitory peptide. This data reveals a crucial function of AMA-1 during the primary contact period upstream of the entry process at around the time of moving junction formation. We generate a Plasmodium falciparum cell line that expresses a functional GFP-tagged AMA-1. This allows the visualization of the dynamics of AMA-1 in live parasites. We functionally validate the ectopically expressed AMA-1 by establishing a complementation assay based on strain-specific inhibition. This method provides the basis for the functional analysis of essential genes that are refractory to any genetic manipulation. Using the complementation assay, we show that the cytoplasmic domain of AMA-1 is not required for correct trafficking and surface translocation but is essential for AMA-1 function. Although this function can be mimicked by the highly conserved cytoplasmic domains of P. vivax and P. berghei, the exchange with the heterologous domain of the microneme protein EBA-175 or the rhoptry protein Rh2b leads to a loss of function. We identify several residues in the cytoplasmic tail that are essential for AMA-1 function. We validate this data using additional transgenic parasite lines expressing AMA-1 mutants with TY1 epitopes. We show that the cytoplasmic domain of AMA-1 is phosphorylated. Mutational analysis suggests an important role for the phosphorylation in the invasion process, which might translate into novel therapeutic strategies.
authors.affiliation: Department of Molecular Parasitology, Bernhard-Nocht-Institute for Tropical Medicine, Hamburg, Germany.
authors.forename: Moritz
authors.initials: M
authors.lastname: Treeck
authors.forename: Sonja
authors.initials: S
authors.lastname: Zacherl
authors.forename: Susann
authors.initials: S
authors.lastname: Herrmann
authors.forename: Ana
authors.initials: A
authors.lastname: Cabrera
authors.forename: Maya
authors.initials: M
authors.lastname: Kono
authors.forename: Nicole S
authors.initials: NS
authors.lastname: Struck
authors.forename: Klemens
authors.initials: K
authors.lastname: Engelberg
authors.forename: Silvia
authors.initials: S
authors.lastname: Haase
authors.forename: Friedrich
authors.initials: F
authors.lastname: Frischknecht
authors.forename: Kota
authors.initials: K
authors.lastname: Miura
authors.forename: Tobias
authors.initials: T
authors.lastname: Spielmann
authors.forename: Tim W
authors.initials: TW
authors.lastname: Gilberger
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D000953:Antigens, Protozoan / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D015153:Blotting, Western; D003593:Cytoplasm / Q000378:metabolism; D005455:Fluorescent Antibody Technique; D006801:Humans; D015151:Immunoblotting; D047468:Immunoprecipitation; D017780:Malaria Vaccines / Q000235:genetics / Q000378:metabolism*; D008565:Membrane Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D018613:Microscopy, Confocal; D008969:Molecular Sequence Data; D010766:Phosphorylation; D010963:Plasmodium falciparum / Q000472:pathogenicity*; D016133:Polymerase Chain Reaction; D015800:Protozoan Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D019076:Transgenes
chemical_list: D000953:Antigens, Protozoan; D017780:Malaria Vaccines; D008565:Membrane Proteins; D015800:Protozoan Proteins; C090077:apical membrane antigen I, Plasmodium