Towards functional assignment of Plasmodium membrane transport proteins: an experimental genetics study on four diverse proteins

Abstract

Membrane transport proteins (MTPs) transfer nutrients, metabolic products and inorganic ions across membranes. Many MTPs are essential for Plasmodium blood infection and gain importance as candidate drug targets in malaria therapy, whereas the physiological functions of many MTPs still remain enigmatic. In this thesis, we applied experimental genetics to determine key characteristics of four selected Plasmodium MTPs, including spatio-temporal expression and phenotypical analysis of knockout mutants. We employed the murine malaria model parasite Plasmodium berghei and in vitro blood cultures of the human malaria parasite Plasmodium falciparum. For this study, we selected one conserved MTP called FT2, which was previously shown to transport folate, a P-type ATPase that is specific for P. falciparum as well as two essential MTPs, CRT and ATP4, with important roles in anti-malarial therapy. These targets exemplify the range of druggable candidates and illustrate the potential and limitations of reverse genetics to decipher their physiological roles for Plasmodium life cycle progression. A combination of transgenic and knockout strategies was applied to the P. berghei folate transporter 2 (FT2). We show that endogenously tagged FT2 localises to the apicoplast membranes, and is broadly expressed throughout the parasite’s life cycle. Strikingly, in two stages, schizonts and sporozoites, FT2 displays a dual localisation. Analysis of FT2-deficient parasites revealed a severe sporulation defect in the vector; the vast majority of ft2– oocysts form large intracellular vesicles which displace the cytoplasm. Accordingly, very few sporozoites are generated and these are non-infectious to the mammalian host, resulting in a complete arrest of Plasmodium transmission. A candidate aminophospholipid P-type ATPase, that is encoded by human malaria parasites, but not P. berghei and related Vinckeia parasites, was assessed by a CRISPR/Cas9-mediated gene disruption. Compared to many vital P-type ATPases this gene is dispensable for asexual blood replication. Two MTPs, ATP4 and CRT are prime targets for antimalarial therapies. A comprehensive spatio-temporal expression analysis of transgenic parasites expressing mCherry tagged proteins revealed expression beyond blood infection, indicative of functions in additional parasite stages. Together, the findings of this study contribute towards a better understanding of the roles of the four MTPs based on localisation, expression and functional deletion.