Understanding and Targeting Host Processes Essential to Plasmodium Infection

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Project Summary Malaria is an ongoing global health burden, and the spread of drug resistance threatens progress made to eradicate the disease. The liver stage of the Plasmodium lifecycle is a promising target for drug and vaccine development as its inhibition would prevent disease manifestation and transmission. Previously, we mapped transcriptional changes throughout Plasmodium infection of hepatocytes and identified host processes critical to parasite viability. The chemical inhibition and genetic disruption of specific host proteins, such as aquaporin 3 (AQP3), were found to hinder liver stage parasite development. In mammalian cells, AQP3 transports water, glycerol, and other small solutes across cell membranes. Interestingly, we demonstrated that AQP3 localizes to the parasitophorous vacuolar membrane (PVM), the interface between the host and pathogen, in multiple Plasmodium species and stages. Our goal is to better understand host-parasite dynamics that support infection by determining how and why Plasmodium repurposes AQP3. In Aim 1, we plan to elucidate the recruitment of AQP3 to the host-pathogen interface using live-cell imaging with chemical and genetic tools. We will delineate the dynamics of AQP3 interactions with the tubulovesicular network, a membranous system that extends from the PVM. We will further probe the role of known trafficking motifs and the host endomembrane system to understand how AQP3 associates to the PVM and observe this association at an ultrastructural level with immuno-electron microscopy. In Aim 2, we will investigate AQP3 function during Plasmodium infection using a suite of imaging tools combined with AQP3 mutants to identify molecules affected by the host protein. In Aim 3, we will develop AQP3-targeting chemical probes to explore protein dynamics in the Plasmodium liver stages, including human-infective P. vivax and P. falciparum, where genetic approaches are currently unavailable. Fragment-based probe discovery will be used to identify covalent AQP3-binding molecules to label and study AQP3 in cells. Together, this work will provide insights into AQP3 recruitment and function during Plasmodium infection, thereby uncovering mechanisms that may be ubiquitously used by Plasmodium parasites to hijack host proteins. Our small molecule approach offers a route to complete fundamental biological studies probing host- parasite dynamics throughout different stages of the Plasmodium lifecycle and lays a foundation for future host- targeting compounds to address malaria infections.