Elucidating the essential role of PTEX during the asexual blood stage of Plasmodium falciparum

Abstract

The human malaria parasite Plasmodium falciparum is renowned for its ability to modify its host red blood cell (RBC), during the asexual blood stage of its life cycle. Many host cell modifications are involved in immune evasion and are therefore of clinical importance. These modifications are established by parasite effector proteins, exported across a vacuole membrane that envelops the intracellular parasite. Protein export is performed by an ATP-driven translocon termed the Plasmodium translocon of exported proteins (PTEX). PTEX is a parasite-derived protein complex and constitutes the sole pore for these exported effector proteins to gain access into the RBC compartment. Importantly, PTEX has been found to be essential for blood stage growth, likely as a result of it exporting essential effector proteins into the RBC. The parasite is predicted to export around 500 effector proteins into the RBC, however, most of these proteins have yet to be studied or allocated specific roles, and therefore a great deal remains to be discovered about these proteins in Plasmodium species.

In this thesis I employed three approaches to study why PTEX is essential. In the first Aim, I attempted to derive the functions of 13 exported proteins previously shown to physically associate with the essential RhopH complex that the parasite installs in the RBC membrane to import vital nutrients from the blood plasma. Although none of the 13 proteins appeared important for nutrient uptake, five of the proteins studied interacted with the RhopH complex, potentially to help traffic or stabilise the complex to the iRBC membrane. I also confirmed the localisation of six new exported proteins and the interactome of 10 of the 13 proteins studied and the complexes they form.

In my second Aim, I defined the essential exportome in P. falciparum and studied five proteins identified in my analysis and characterised them further. Three of the five proteins showed reduced growth when they were conditionally depleted indicating they might be important for blood stage growth. One of the proteins was not exported- but identified here as a novel protein of the parasite vacuole membrane.

In my third and final Aim, I investigated metabolic changes in parasites following knockdown of PTEX, where preliminary data had indicated that knocking down PTEX disrupts the haemoglobin digestion pathway. To investigate this further I studied the association of PTEX with two haemoglobin proteases, plasmepsin II (PMII) and falcipain 2a (FP2a). I found that knocking down PTEX affected the trafficking of these two proteases. Furthermore, through the use of protein-binding and inducible folding assays, I determined that the FP2a reporter protein directly associated with a subunit of PTEX, HSP101, which unfolds protein cargo prior to export. These results indicate that PTEX may not only be required to help deliver important proteins into the RBC compartment but also to correctly traffic proteins at the parasite surface that are important for intracellular functions such as haemoglobin digestion.

Taken together, this research greatly expands our knowledge of the diverse roles that both PTEX and its various cargo proteins perform in different compartments of the infected RBC.