Elucidating the transport mechanisms in the parasitophorous vacuole of Plasmodium

Clemson University, Clemson SC

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Abstract

Malaria infections culminate with the death of ~600,000 people each year which is caused, in part, by wide-spread antimalarial resistance and current ineffective vaccines. This highlights the need to continue understanding the factors involved in establishing malaria infection. To aid in this fight, this proposal aims to understand the mechanism used by the deadliest of the malaria-causing parasites, Plasmodium falciparum, to remodel human red blood cells (RBCs) for survival. Specifically, this proposal will focus on a vacuolar compartment generated by the parasite during invasion called the parasitophorous vacuole (PV) which is the main interphase of host-pathogen interactions. As the membrane of the PV (PVM) presents a physical barrier between the parasite and its host, it contains parasite-derived mechanisms for solute exchange and protein translocation. Nutrient transport occurs by EXP2 which forms a nonselective, heptameric pore at the PVM. This pore is then further functionalized by two other proteins, HSP101 and PTEX150, to form a protein-conducting pore called PTEX that translocates secreted proteins across the PVM and into the RBC host. While the function of these proteins is known, it is not currently known how these proteins are established at the PVM. We hypothesize that EXP2’s membrane insertion occurs by the interplay between one of its domains and the lipid content of the PVM. Then, once in the membrane, its function in protein export through PTEX is established via interaction with other vacuolar proteins. This hypothesis will first be explored by elucidating the mechanism by which EXP2, which traffics in a soluble state similar to bacterial toxins, is inserted into the PV membrane. Specifically, we will first explore the domains that guard the shielding of the EXP2 transmembrane domain as well as understand the lipid composition required for successful membrane insertion. To understand how PTEX is assembled once EXP2 is at the membrane, we will explore the role of a vacuolar protein called RON3 whose role has been recently tied to nutrient and protein translocation across the PVM. Thus, using a generated RON3 knockdown line, we will explore the effect on RON3 knockdown in PTEX localization and assembly. We will then explore the important domains required for RON3 function followed by proximity labeling experiments to identify the interacting partners of RON3. Ultimately, unveiling the mechanism used to establish transport functions across the PVM is a critical step for the development of novel anti-malarial compounds.