Vacuolar-type h+-pumping pyrophosphatases in the Malaria parasite, Plasmodium falciparum
Creators: name: Ramanayake, Darinee Nimeka Arundathie
Publisher: name: The Australian National University
Year: 2014
Subjects: subject: Malaria
subject: Plasmodium falciparum Genetics
subject: Acid phosphatase
subject: Biological transport, Active
subject: Antimalarials Testing

description: Vacuolar-type H+-pumping pyrophosphatases (V-H+-PPases) are primary active transporters that utilise the energy from PPi (inorganic pyrophosphate) hydrolysis to translocate H+ ions across a biological membrane. These proteins have been well characterised in plant cells where they function in parallel with the ubiquitous vacuolar-type H+-pumping ATPase (V-H+-ATPase) to generate proton gradients across the plant vacuole membrane. V-H+-PPases have also been identified on the plasma membrane and intracellular organelle membranes of several protozoan parasites responsible for causing severe disease in humans. The identification of V-H+-PPases in these disease-causing agents, and the absence of this class of proton pump in human cells, has exciting implications for its potential as an antiparasitic drug target. The P. falciparum genome encodes two genes, PfVP1 and PfVP2, that encode putative K+-sensitive (type I) and K+-independent (type II) V-H+-PPases respectively. Furthermore, V-H+-PPase activity in P. falciparum has been demonstrated in the asexual blood-stage parasite, both in terms of PPi-induced acidification of subcellular parasite compartments and PPi hydrolysis activity in parasite membrane preparations. V-H+-PPase activity in the blood-stage parasite was further characterised in this study. PPi-induced acidification of the parasite digestive vacuole (DV; a large lysosome-like organelle within the parasite cytosol) was sensitive to Ca2+, which is known to inhibit V-H+-PPase activity, but was insensitive to the V-H+-PPase type-specific inhibitor aminomethylenediphosphonate (AMDP). PPi hydrolysis in membrane-permeabilised, intact cells was also sensitive to Ca2+. This study also confirmed previous observations of K+ sensitivity of both H+ translocation and PPi hydrolysis, consistent with the presence of a K+ sensitive, type I V-H+-PPase in the intraerythrocytic parasite. This study specifically investigated the role of the putative type II V-H+-PPase, PfVP2, using PfVP2 knockout (KO) parasites. Although PfVP2 was not active on the DV membrane, it extruded H+ ions out of the parasite cytosol in the D10 strain of P. falciparum (only apparent when the parasite V-H+-ATPase was inhibited with the V-H+-ATPase inhibitor concanamycin A). PfVP2 KO parasites also displayed a small but significant decrease in PPi hydrolysis compared to wild type (WT) parasites in the D10 strain, consistent with PfVP2 hydrolysing PPi to Pi (inorganic phosphate). Despite these results supporting the presence of a functional PfVP2 protein in the intraerythrocytic-stage parasite, PfVP2 gene disruption was not associated with a fitness cost or increased sensitivity to the antiplasmodial agents concanamycin A, chloroquine and artemisinin in both the 3D7 and D10 strains. The subcellular localisation of PfVP2 was also investigated in this study. PfVP2, tagged with the green fluorescent protein (GFP) localised to punctate structures within the intraerythrocytic parasite cytosol. Disruption of PfVP2-GFP by brefeldin A (a compound known to interfere with the secretory pathway) and partial co-localisation of PfVP2-GFP with the P. falciparum Golgi Reassembly and Stacking Protein or PfGRASP, are consistent with a Golgi localisation of PfVP2.