Functional, biochemical and structural analyses of Plasmodium falciparum pyruvate dehydrogenase complex

The apicomplexan parasite Plasmodium is the causative agent of the devastating tropical disease, malaria. The World Health Organisation reported that in 2010 there were an estimated 219 million malaria cases and about 660,000 deaths. Sub-Saharan Africa is the worst affected, with 80% of malaria deaths and 90% of cases occurring in this area of the world. P. falciparum causes the most severe form of the illness and accounts for the majority of malaria deaths. Parasite resistance to antimalarials including the most effective drug, artemisinin, is becoming an increasing problem, thus research into new drug targets is vital. The pyruvate dehydrogenase complex (PDC) is one of the alpha-ketoacid dehydrogenase complexes, which are involved in energy and amino acid metabolism. The PDC catalyses the transfer of the acetyl group from pyruvate to coenzyme A (CoA) to form acetyl-CoA. The complex comprises three enzymes; pyruvate dehydrogenase (E1), dihydrolipoamide acetyltransferase (E2) and dihydrolipoamide dehydrogenase (E3). E2 has a multi-domain structure where the C-terminal catalytic domains (CD) of several E2 subunits interact to form the core of the complex. This core is an icosahedral 60-mer in mammals, plants and Gram-positive bacteria and an octahedral 24-mer in Gram-negative bacteria. The N- terminal part of E2 consists of the sub-unit binding domain (SBD) and 1 to 3 lipoyl- domains (LD) creating the ‘swinging arm’ of the PDC, which facilitates substrate channelling. E1 (a heterotetramer) and E3 (a homodimer) bind to the SBD to form the functional PDC. In humans, PDC converts pyruvate to acetyl-CoA, which leads into the citric acid cycle located in the mitochondrion. P. falciparum PDC, however, is found only in the apicoplast and produces acetyl-CoA for fatty acid biosynthesis. Plasmodium PDC has recently been shown to be important for parasite progression from the asymptomatic liver stage to the symptomatic erythrocytic stage. Thus, inhibiting PDC could prevent development of malaria. This study focuses on the role of the PDC in P. falciparum blood stages and the identification and characterisation of structural and biochemical differences between parasite and human PDC that may be ultimately exploited for drug or vaccine development. I have optimised the expression and purification of soluble recombinant mature-length P. falciparum (Pf) E2 (His-rPfE2m), truncated PfE2 consisting of the SBD and CD (His- rPfE2bc) and mature-length apicoplast PfE3 (His-rPfaE3) to obtain mg amounts of protein for biochemical and structural analyses. Each of the recombinant proteins was catalytically active. Analytical ultracentrifugation (AUC) showed that His-rPfE2m forms the typical trimer building blocks required for the large E2 core formation. Sedimentation velocity (SV) experiments showed a main species with a sedimentation coefficient of 6.6 ± 0.1 S, which corresponded to a species consistent with the PfE2 trimer size observed in sedimentation equilibrium (SE) studies. However, no 24-mer or 60-mer core species were detected in SV experiments. SE analyses did show some larger molecular mass species (> 1.5 MDa), however, whether these represent a PfE2 core complex was inconclusive due to interference by aggregated protein in the sample. My conclusion from these data is that PfE2 may form a large core structure but that this is very unstable compared with E2 multimers from other organisms, where the core complex is readily formed and maintained. Similar results were obtained from SV and SE analyses of His-rPfE2bc; only trimers are present. Small angle X-ray scattering (SAXS) was used to further analyse the trimer structure. The solution structure obtained for His-rPfE2bc revealed the linker between the SBD and the CD is extended and partially flexible. This conformation, which would allow SBD interaction with E1 and E3, is supported by cryo-electron microscopy images obtained by others for E2 from other organisms. AUC on His-rPfaE3 showed a main species with a sedimentation coefficient of 6.1 ± 0.1 S and SE analyses of the protein confirmed it to be a dimer as expected. These findings were further corroborated with the solution structure obtained using SAXS. Deletion of the PfE2 gene from P. falciparum was unsuccessful, as the correct gene locus was not targeted. This could be due to the gene being required for parasite survival in the blood stages. However, it could also be due to the difficulty of P. falciparum gene manipulation and hence, further attempts to delete PfE2 using different approaches will be required. The PfaE3 gene was successfully deleted, thus as shown in previous studies with murine malaria species, the gene is not essential in the human malaria parasite, P. falciparum. The PfaE3 deletion mutants did not show increased susceptibility to oxidative stressors compared with the wild type strain, suggesting that PfaE3 does not play a role in defence against oxidative stress. However, the mutants were less susceptible to triclosan, an inhibitor of the fatty acid biosynthesis enzyme FabI. In addition, the mutant parasites maintained synchronicity over more than four replication cycles as opposed to WT parasites, which gradually lost synchronicity over this period of in vitro culture. Further work will need to be carried out to fully characterise the role of PfaE3 in P. falciparum.