Kinetic tracking of Plasmodium falciparum antigen presentation reveals determinants of protein export and membrane insertion

Abstract

Intracellular malaria parasites export many proteins into their host cell, inserting several into the erythrocyte plasma membrane to enable interactions with their external environment. While static techniques have identified some surface-exposed proteins, other candidates have eluded definitive localization and membrane topology determination. Moreover, both export kinetics and the mechanisms of membrane insertion remain largely unexplored. We introduce Reporter of Insertion and Surface Exposure (RISE), a method for continuous nondestructive tracking of antigen exposure on infected cells. RISE utilizes a small 11 aa NanoLuc fragment inserted into a target protein and detects surface exposure through high-affinity complementation. We tracked insertion of CLAG3, a malaria parasite protein linked to nutrient uptake, throughout the P. falciparum cycle in human erythrocytes. Our approach also revealed key determinants of trafficking and surface exposure. Removal of a C-terminal transmembrane domain aborted export. Unexpectedly, certain increases in the exposed reporter size improved surface exposure by up to 50-fold, revealing that both size and charge of the extracellular epitope influence membrane insertion. Insertion of parasite proteins at the host cell surface and antigen accessibility is regulated by multiple factors, enabling intracellular parasite survival and immune evasion under a broad range of conditions.

The delayed bloodstream clearance of Plasmodium falciparum parasites after M5717 treatment is attributable to the inability to modify their red blood cell hosts

“…NPPs are thought to be formed by the RhopH protein complex, following the export of these proteins from the parasite to the iRBC membrane (Nguitragool et al, 2011;Counihan et al, 2017;Ito et al, 2017;Sherling et al, 2017). If export of the RhopH proteins and formation of the RhopH complex at the iRBC membrane is inhibited by M5717, this could reduce the iRBC's immunological profile, allowing these parasites to avoid clearance from the bloodstream (Shao et al, 2022). As iRBCs persisted in infected volunteers treated with M5717 we therefore measured the kinetics of NPP activity using a sorbitol lysis assay.…”