INVESTIGATION OF PLASMODIUM VACUOLAR PROTEIN SORTING ASSOCIATED PROTEIN 4 THROUGH LOCALIZATION AND GENE DELETION

Abstract

In the liver, Plasmodium sporozoites experience a radical transformation to a highly replicative trophozoite as means of adaptation to exploit the nutrient-rich environment within hepatocytes. Recent microscopy studies have unveiled that the metamorphosis of the sporozoite is linked to the elimination of invasion organelles such as micronemes through an unconventional secretion process that utilizes the autophagy machinery and the endocytic pathway. We developed a hypothetical model referred to as “exophagy” from microscopic observation and analysis that suggests proteins such as autophagy related protein 8 (ATG8), Golgi reassembly and stacking protein (GRASP), and vacuolar protein sorting-associated protein 4 (VPS4), known to play a role in phagophore elongation, autophagosome tethering to MVB, and amphisome scission away from the plasma membrane and into the PV lumen, respectively, are important for the unconventional secretion of micronemes. Moreover, is it possible that the role of VPS4 in the scission of membranes could be an essential step for the clearance of micronemes and therefore the intrahepatic development of Plasmodium. Consequently, to understand the function of VPS4 as the parasite metamorphoses in the liver, we generated a PbVPS4 (Plasmodium berghei VPS4) knockout parasite by complete gene deletion, a functional PbVPS4 knockout by deleting the ATPase domain essential for the catalytic activity of the enzyme and mEmerald tagged PbVPS4 reporter protein construct. Additionally, we observed the localization of PbVPS4 at multiple timepoints post-hepatocyte infection by immunofluorescence microscopy. iv In conclusion, our immunofluorescence microscopy experiments illustrated PbVPS4 as it expands into a substantial tubule-vesicular network with partial overlap with PbATG8 by 48 hpi of hepatocytes which suggests a continuous VPS4 expression during metamorphosis and later throughout hepatic development. More importantly, it appears VPS4 is a non-essential gene as neither the complete nor partial deletion of VPS4 produced an observable lethal phenotype in a heterogeneous population of Plasmodium berghei.