Differential sorting and trafficking of Plasmodium falciparum secreted proteins. - Project Summary: Malaria continues to pose a significant global health threat, with severe cases primarily caused by P. falciparum leading to diverse clinical complications. In the symptomatic stage of malaria, the parasite undergoes asexual replication within a parasitophorous vacuole (PV) formed inside the human Red Blood Cell (RBC). Hundreds of parasite-made proteins are secreted into the PV, and a subset of these proteins is further exported to the RBC through the Plasmodium Translocon of Exported Protein (PTEX) complex located at the PV membrane. These exported proteins play a crucial role in modifying the host cell, creating a conducive niche for parasite growth, and significantly influencing the severity of the disease. The process of how exported proteins are differentially sorted from PV-resident proteins and targeted to the PTEX complex remains to be determined. Recent findings indicate that mature N-terminal sequence (NTS) of secreted proteins, exposed after processing in the endoplasmic reticulum, encodes a promiscuous signal directing secreted proteins either to the export pathway or to retention within the PV. This proposal aims to unravel the mechanisms underlying this differential sorting and trafficking. Two proposed models are under consideration. The first involves accessory proteins recognizing and binding export-competent NTSs of export-destined proteins, guiding them to the PTEX complex. Conversely, accessory proteins in this model may bind to export-incompetent NTSs of PV-resident proteins, inhibiting further trafficking. The second model posits that the chaperone HSP101, a component of the PTEX complex, discerns between export-competent and -incompetent NTSs, thereby controlling selectivity. Given the diverse nature of mature NTSs in both varieties of secreted proteins, the central hypothesis of this proposal is that both models are partially correct; secreted proteins undergo differential sorting with the assistance of multiple sorting proteins and HSP101 in a stepwise manner. In Aim 1, sorting proteins in the PV will be identified through proximity labeling and co-immunoprecipitation, using exported-destined and PV-resident reporters. A refined cell fractionation strategy will be used to isolate the PV compartment, and global protein export will be blocked by genetically manipulating the parasite for the experiments. The top 5 hits will undergo rigorous characterization to understand their role in secreted protein trafficking pathways. Aim 2 focuses on investigating the role of HSP101 in secreted protein sorting. Reciprocal immunoprecipitations of secreted reporters and HSP101 will be conducted. Structure- guided modifications of HSP101 will be tested for their potential role in cargo sorting. The applicant aspires to be an expert in protein trafficking pathways of apicomplexan parasites. Elucidating the Plasmodium protein export pathway using cutting-edge methodologies under the mentorship of pioneering molecular parasitologist Dr. Dan Goldberg will greatly enhance the candidate's prospects for an impactful research career.
