Characterizing a serine-threonine phosphatase essential for asexual replication in Plasmodium falciparum

Significant progress have been made toward malaria eradication, further attempts are hampered by the fact that little is known about the basic cellular biology of Plasmodium falciparum, the causative agent of malaria. In the asexual stage, where the parasite replicates in red blood cells and causes malaria’s characteristic cyclical fevers, a contractile ring structure known as the basal complex is required for replication and serves to separate budding daughter cells. A member of the basal complex has been identified that is essential for parasite growth and division, PfPPPP8, and acts as a serine-threonine phosphatase. PfPPP8 is the only basal complex protein with known enzymatic activity, and It is believed PfPPP8 is essential to drive the formation and organization of the basal complex during asexual division. The project will use CRISPR-Cas9 to insert an exogenous copy of PfPPP8, with mutations in predicted key catalytic residues, into a PfPPP8 inducible knockdown line, knock down the endogenous copy, and determine the impact of each mutation on parasite growth and division. To further characterize PfPPP8’s role in division, quantitative phosphoproteomic analysis will be performed in the same inducible knockdown line and identify PfPPP8’s enzymatic substrates. These substrates will themselves be genetically modified with an epitope tag and a knockdown system so their localization throughout Plasmodium division can be visualized and the phenotypic consequences of their absence can be compared to the PfPPP8 knockdown phenotype. To identify novel basal complex proteins using PfPPP8,  immunoprecipitation was performed on PfPPP8 and another epitope tagged basal complex protein PfCINCH in the PfPPP8 inducible knockdown background. Proteins will be selected based on their absence or depletion in the PfPPP8 knockdown condition, which are likely to be uncharacterized members of the basal complex, prioritizing those which are predicted to be essential, and introduce similar genetic modifications as the putative substrates to determine if they also localize to and are required for the formation of the basal complex. similarly the project will also select and examine top candidate proteins only present in the PfPPP8 pulldown, which are likely to be specifically relevant to PfPPP8, in order to further characterize PfPPP8’s mechanism of action and role within the parasite throughout division. Because PfPPP8 is essential to Plasmodium growth and replication during the pathogenic stage and because it is conserved only among Plasmodium species, further study could prove it a useful new target for developing antimalarials in the face of rising resistance to extant drugs.

Project details

Basic Science

Feb 2022 — Jan 2025

$76,012

United States