Malaria is a devastating parasitic disease that affects more than 200 million people annually, resulting in nearly 500,000 deaths each year. As of 2018, the World Health Organization estimates that 3.8 billion people, roughly half the world's population, are at risk of contracting malaria, and the rise of drug-resistant parasites has created a desperate need for new anti-malarial drugs. While most intracellular pathogens export a limited repertoire of effector proteins to co-opt existing host-cell metabolic machineries, the malaria-causing parasite Plasmodium falciparum exports more than 10% of its proteome into its host, the human red blood cell, during the blood stages of its life cycle. The hundreds of proteins in the P. falciparum exportome extensively remodel host erythrocytes, creating the infrastructure needed to import nutrients, export waste, and evade the host immune system. The export of these hundreds of proteins is complicated by the fact that the malaria parasite conceals itself inside a parasitophorous vacuole (PV) derived from invagination of the host cell plasma membrane during invasion. Following secretion into the PV, proteins destined for export must be unfolded and transported across the PV membrane (PVM) into the host cell in an ATP-dependent process. The export pathway is essential for parasite survival, making members of the pathway attractive potential drug targets. The complexity and breadth of its host-cell remodeling machinery make P. falciparum a rich and exciting system for the study of host-pathogen interactions. However, many of the molecular mechanisms underlying this parasite's ability to hijack human red blood cells remain enigmatic, as much of the P. falciparum proteome has proven recalcitrant to structural and biochemical characterization using traditional recombinant approaches. The goal of the proposed work is to leverage and build upon the latest advances in single-particle cryo electron microscopy and cryo focused ion beam-enabled in situ cryo electron tomography to elucidate the molecular mechanisms underlying effector protein export in P. falciparum and to identify promising targets for structure-based design of new anti-malarial therapeutics.
Three aims are proposed to accomplish these goals: 1) Establish an in vitro translocation activity assay for the Plasmodium Translocon of Exported Proteins (PTEX), a novel and essential membrane protein complex, through which all exported effector proteins must pass in order to reach the host cell cytosol. The established assay will enable biochemical characterization of the molecular mechanism of protein translocation and screening of inhibitors obtained via structure-guided design of PTEX inhibitors. 2) Structure determination of novel protein complexes of the P. falciparum exportome. 3) Direct visualization of the supramolecular effector protein export machinery in situ at the host-pathogen interface in P. falciparum-infected erythrocytes. The proposed work will provide insight into the pathogenesis of this deadly disease, identify new malarial drug targets, and enable structure-guided design of novel anti-malarial therapeutics.
The complexity and breadth of its host-cell remodeling machinery make the malaria-causing parasite P. falciparum a rich and exciting system for the study of host-pathogen interactions. However, many of the molecular mechanisms underlying this parasite's ability to hijack human red blood cells remain enigmatic, as much of the P. falciparum proteome has proven recalcitrant to structural and biochemical characterization using traditional recombinant approaches. The proposed research will build upon our previous studies in structural microbiology of the malaria parasite and leverage recent advances in cryoelectron microscopy to directly visualize how protein complexes mediate host-pathogen interactions in malaria parasite-infected red blood cells, providing insight into malaria pathogenesis and informing structure-guided design of novel anti-malarial therapeutics.
