Unravelling the intracellular interactions and signalling of Apical membrane antigen-1 (AMA1) in malaria parasites. (360G-Wellcome-210861_Z_18_Z)
Apical membrane antigen-1 (AMA1) is a type-I integral membrane protein of malaria parasites, required for host cell invasion. Invasion requires phosphorylation of the AMA1 cytoplasmic domain (CD), suggesting a signalling role. Exactly what process(es) the AMA1 CD regulates, and how it does so, is unknown. This project will examine the role of the AMA1 CD in regulating cellular events during invasion, using the recently culture-adapted malarial species Plasmodium knowlesi. A conditional mutagenesis system (DiCre) will be used to switch expression of wild-type AMA1 to mutant alleles (e.g. non-phosphorylated forms) to examine the role of the CD in invasion. The impact of mutagenesis on discharge of secretory organelles containing invasion ligands will be examined using immunofluorescence and electron microscopy. Proteins that interact with the AMA1 CD will be identified using directed biotinylation by an APEX2 tag fused to the CD, followed by pulldown and mass spectrometry. The role of interacting proteins in invasion will then be explored using DiCre-mediated knockout. A high-throughput Cas9 mutagenesis screen of the AMA1 CD will also be performed to identify crucial residues involved in mediating interaction of partner proteins with the AMA1 CD. This project will elucidate the long-hypothesised signalling role of AMA1.
£250,000 24 Apr 2018