Functional Analysis of the Leading Malaria Vaccine Candidate AMA-1 Reveals an Essential Role for the Cytoplasmic Domain in the Invasion Process

PLoS Pathogens

Kota Miura¹  Sonja Zacherl²  Silvia Haase²  Klemens Engelberg²  Tobias Spielmann²  Nicole S. Struck²  Susann Herrmann²  Tim W. Gilberger²  Moritz Treeck²  Ana Cabrera²  Maya Kono²  Friedrich Frischknecht³ 

[1] Centre for Molecular and Cellular Imaging, EMBL, Heidelberg, Germany;Department of Molecular Parasitology, Bernhard-Nocht-Institute for Tropical Medicine, Hamburg, Germany;Hygiene Institut, Abteilung Parasitologie, Universitätsklinikum Heidelberg, Heidelberg, Germany

关键词: Merozoites;    Red blood cells;    Phosphorylation;    Plasmodium;    Fluorescence recovery after photobleaching;    Host cells;    Malarial parasites;    Video microscopy;   

DOI  :  10.1371/journal.ppat.1000322

学科分类：生物科学（综合）

来源: Public Library of Science

【 摘 要 】

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.

【 授权许可】

CC BY