The protein folding machinery in Plasmodium falciparum, the main causative agent of malaria disease, has been directly linked to emerging parasite drug resistance. The Chaperonin Containing TCP-1 ring (CCT) complex is the most up-regulated chaperonin in these drug resistant parasites and is therefore an attractive target to antagonize resistance in the field. Whilst there is a wealth of information about the CCT complex in higher eukaryotes, there is a paucity of information about the complex in more divergent species, such as the malaria parasite.
The work presented in this thesis explores the CCT complex in P. falciparum (PfCCT) and the folding of its substrates, including the essential and abundant protein actin. First, I present a technique for studying actin on a single molecule level. Using this system, I observe the dynamic changes in actin’s macromolecular structure, its folding and drug-binding in real-time. To understand PfCCT, I biochemically char- acterise one of its essential cofactors, PhLP2, and isolate the complex from parasite lysate. I identify the PfCCT interactome and obtain a low-resolution structure of the complex, which has a highly conserved interactome and structure.
Towards targeting the complex, I use a biosynthetic platform that expresses a puta- tive CCT-targeting compound, violacein, to conduct a high-throughput screen. I test the effect of violacein and biosynthetically-derived violacein derivatives on parasite growth. I show that violacein inhibits both wild type and drug-resistant parasite iso- lates from the field and identify a number of derivatives that are more potent than the parent compound. I show that violacein affects actin dynamics in vivo, resulting in an accumulation of actin signal indicative of incorrect protein folding, supporting the hypothesis that violacein targets the CCT complex. The work presented in this thesis provides a clear route for antimalarial drug development, ultimately towards targeting protein folding to stem the deadly disease.