Construction of Translection Vectors to target Plasmodium berghei Apical Membrane Antigen 1 and Autophagy related 18 Genes and Characterization of recombinant Parasites

Abstract

Apical membrane antigen 1 (AMA1) protein of Plasmodium is involved in host-pathogen interactions leading to invasion of erythrocytes by merozoites. It is conserved in all Apicomplexan parasites, and is an essential protein for Plasmodium development. AMA1 is also considered a potential vaccine candidate. Towards understanding the effect of AMA1 knockdown on parasite development and induction of immune responses, attempts were made to generate AMA1 knockdown Plasmodium berghei parasites, which would have the potential as a whole parasite vaccine in mice. Similarly, Autophagy related 18 (ATG18) protein has key roles in the formation of autophagosomes and vesicle biogenesis in yeast model. It was investigated for its localization and functional characterization in Plasmodium. Transfection plasmids were constructed for homology-based double crossover integration (homologous recombination) at the AMA1 and ATG18 genes loci of P. berghei ANKA strain. After successful recombination in AMA1 gene locus, AMA1 would be followed by a reporter gene Green Fluorescent Protein (GFP) only in knockin parasites (AMA1 KI) and another Destabilizing Domain (cDD) addition to GFP in knockdown parasites (AMA1 KD). As AMA1 is an essential protein, parasites with these AMA1-GFP or AMA1-GFP-cDD fusion proteins could not invade host cell and undergo schizogony. Thus, modification in AMA1 did not result in enough parasitemia of recombinant parasites in Balb/c mice suggesting highly conserved structure of AMA1 protein and lack of function upon modification. However, alternate strategies are required to generate AMA1 knockdown parasites. Similarly, in ATG18 locus, ATG18 would be replaced by GFP in knockout parasites (ATG18 KO) while ATG18 would be followed by GFP and cDD in knockdown parasites (ATG18 KD). ATG18 KO parasites could not maintain cellular homeostatis and develop further, thus enough parasitemia was not achieved in mice while generation of ATG18 KD parasites with ATG18-GFP-cDD fusion protein was successful, suggesting that ATG18 is essential for parasite development. The genomic (Polymerase Chain Reaction- PCR), cellular (fluorescent microscopy) and proteomic (western blotting) analyses of knockdown parasites confirmed the presence of ATG18-GFP-cDD in ATG18 locus of recombinant parasite genome and also the fusion protein expression. Food vacuolar localization of GFP fluorescence in ATG18 KD parasites suggests a role of ATG18 in the biogenesis of food vacuole and/or food vacuole-associated activities. These ATG18 KD parasites will be invaluable for identification of the specific functions of Plasmodium ATG18 during parasite development, which need to be explored. Keywords: AMA1, ATG18, autophagy, host-pathogen interaction, apicomplexan, fusion protein, homologous recombination, knock in, knockdown, knockout