Genetic manipulation of Plasmodium falciparum for conditional knockdown of potassium channels

Abstract

Malaria is a devastating disease with the most severe cases caused by the Plasmodium falciparum parasite. Despite it being a preventable disease, mortality rates remain a concern due to emerging drug resistance. Ion channels have been recognized as important therapeutic targets for treating numerous illnesses, since the maintenance of the ion concentration gradient regulates a wide range of physiological processes. Studies on Na+ transporter PfATP4 have revealed its potential as an effective drug target, and disrupts the K+ gradient with an ionophore, such as salinomycin, kills the parasite throughout the intra-erythrocytic stages. K+ channels are essential to maintain the electrochemical gradient in a eukaryotic cell. In P. falciparum, two K+ channels, PF3D7_1227200 (K1) and PF3D7_1465500 (K2), have been identified. Both K+ channels are expressed throughout the asexual intra-erythrocytic stages, whereas only K2 is expressed in the sexual intra-erythrocytic stages. However, the essentiality and localisation of these channels throughout intra-erythrocytic development is unknown. In this study, it was attempted to genetically modify K1 and K2 by adding a green fluorescent protein tag and conditional knockdown mechanism to investigate localisation and prove their essentiality in P. falciparum in future studies. This was done by cloning C-terminal gene fragments into two plasmid systems that contain an inducible riboswitch which regulates the level of mRNA and ultimately leads to reduced levels of protein produced, thus knocking down the protein. An original glmS and a modified SLI glmS system was compared. The modified SLI glmS system use a double selection mechanism and thus produces modified parasites at a faster rate than the original glmS system. Recombinant parasite lines were produced for K2, but not for K1. The original glmS system proved to integrate faster into the genome compared to the modified SLI glmS system. The successful construction of these recombinant lines allows for subsequent studies to determine essentiality and localisation of K2 in P. falciparum during intra-erythrocytic development. This study provides the basis for future studies that may develop K+ homeostasis as an intra-erythrocytic drug target.