Plasmodium falciparum, the causative agent of malaria, is responsible for 600,000 deaths every year and causes clinical illness in 300-500 million more. Although several drugs are currently used to treat malaria, the emergence and spread of drug-resistant parasites is a major threat to effective disease control. Novel antimalarial therapies targeting essential and original parasitic processes are urgently needed. Central to the capacity of this microorganism to grow inside red blood cells and to thrive inside the blood stream is its ability to export about 5-8% of its proteins beyond an encasing vacuole and into the cytosol of its host cell. The intracellular survival of Plasmodium falciparum within human red blood cells is dependent on export of parasite proteins that remodel the infected host cell to support its virulence and parasitic lifestyle. To do so, the parasite installs a large protein complex, the Plasmodium Translocon of EXported proteins) (PTEX), in the membrane of the encasing vacuole that enables it to export these hundreds of proteins into the red blood cell. PTEX is composed of five parasite proteins: the EXported Protein 2 (EXP2) that assembles into a trans-membrane pore to conduct the cargo proteins, the ATPase HSP101 that provides the energy driving the export process by unfolding the cargo proteins and threading them through the EXP2 channel and, proteins PTEX150, PTEX88 and TRX2 that recognize, prepare and deliver the translocated proteins to the `channel-engine' complex. The essential subunits EXP2, HSP101 and PTEX150 tightly associate into a core complex. As a common portal for numerous crucial biological processes, PTEX represents an Achilles heel in Plasmodium's life cycle. However, to this day, the structure and detailed mechanism of action of this complex protein export machine remain unknown. To this aim, we have already purified subunits TRX2, EXP2, and HSP101 expressed from recombinant sources and solved the crystal structures of the TRX2 subunit and the N-terminal domain of the HSP101 ATPase. Through the combination of cryo-electron microscopy and X-ray crystallography, we will pursue the structure of the ATPase HSP101, a key regulator of protein trafficking in Plasmodium. We also seek to complete the reconstitution of PTEX150. As a consequence of the high AT codon bias of Plasmodium's genome, PTEX150 contains several asparagine-rich repeats, sequences with a high propensity for disorder and aggregation. To overcome this obstacle, we propose to explore the use of the Plasmodium HSP110, a chaperone shown to stabilize the asparagine-rich parasitic proteins, as a coexpression partner to enable and/or improve expression of PTEX150 and its reconstitution into a functional PTEX ternary core. Using yeast or insect cells as heterologous expression systems, we will use synthetic genes to coexpress PTEX subunits in presence of this specialized chaperone and reconstitute higher order PTEX complexes.
Malaria is the world's most important disease caused by a parasite; however the emergence of drug resistance to existing antimalarial therapies threatens current efforts to control and eradicate the disease. To survive in its human host, the parasite relies on a unique protein export system, a translocon, used to hijack its host cells. The goal of this exploratory research is to reconstitute and purify this parasitic protein export molecular machine to enable the study of its three-dimensional structure and ultimately guide the rational design of novel classes of potent antimalarial drugs inhibiting its function.
|Ho, Chi-Min; Beck, Josh R; Lai, Mason et al. (2018) Malaria parasite translocon structure and mechanism of effector export. Nature 561:70-75|