Characterisation of the Plasmodium falciparum export complex

Plasmodium parasites extensively remodel the mammalian host cells they infect, namely the erythrocytes and hepatocytes. This is achieved through exporting hundreds of parasite proteins into the host where they play many virulence related roles including altering membrane permeability to acquire nutrients for rapid growth and for increasing immune evasion. Proteins are secreted from the parasite into the vacuole that surrounds them, where they must then pass across the parasitophorous vacuole membrane (PVM) to gain access to the host cytoplasm. It has previously been shown that a protein complex, the Plasmodium Translocon of EXported proteins (PTEX), is found on the PVM and is most likely the protein translocon responsible for this export process. To validate and further dissect PTEX function, a conditional expression system that utilizes the glmS riboswitch was employed to conditionally knockdown the levels of PTEX150 within parasites. Using this approach, even a relatively low level of PTEX150 knockdown lead to a significant decrease in the ability of the parasites to export proteins across the PVM. This failure of protein export across the PVM arrested growth and eventually caused parasite death. Interestingly, the export of all classes of protein cargoes tested, including PfEMP1, were blocked upon PTEX150 knockdown suggesting PTEX may serve as a single portal for export. This demonstrates the importance of PTEX to the malaria parasite and validates it as a potent drug target, since blocking it would prevent hundreds of exported proteins from reaching their functional destinations. To further investigate the specific function of PTEX150 we performed C-terminal truncations in order to generate a parasite line with a partially defective protein. These lines showed destabilisation of the PTEX complex, suggesting that the C-terminus of PTEX150, while not essential for PTEX function or the absolute binding of the other components, is required for stabilising the PTEX complex. Interestingly, these parasites did not show a defect in either their ability to grow or export proteins. To further investigate the effect of PTEX150 knockdown on parasite health the new permeability pathways (NPPs) were investigated. To do this a novel method was developed to investigate NPP function using luciferase. This method is highly sensitive, reducing the amount of parasites required as well as removing the need to purify parasites from culture. The effect of PTEX150 knockdown on NPPs was unclear using this method. Due to the high sensitivity and ease of this method it was developed as a HTS to discover NPP inhibitors. Using this method the malaria box of 400 compounds was screened. Importantly, two compounds, MMV020439and MMV007571, showed inhibitory activity significantly higher than the control compound NPPB.