Lipoic acid protein ligases in Plasmodium spp.
PhD thesis, University of Glasgow.
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Protozoan parasites of the genus Plasmodium are the causative agent of malaria. The four human pathogenic species infect more than 500 million people each year, causing the death of at least 1 million people. The most severe form of human malaria is caused by P. falciparum, which is responsible for 90% of the malaria deaths. A major problem in the treatment of this disease is resistance of the parasites against most of the existing chemotherapies. Therefore, there is an urgent need to identify, validate and assess potential new drug targets. The prerequisite of a potential drug target is that it should not be of significance for the human host or it should be sufficiently different from the human counterpart, so that parasite-specific inhibition is feasible. Lipoic acid metabolism in Plasmodium differs from that of mammals in some ways and therefore it might be a promising target for the development of new antimalarials. This study investigated the importance of lipoic acid ligation in P. falciparum using reverse genetic approaches, to assess whether this pathway has potential for drug design. In addition, a spectrophotometric assay system was developed that allowed the biochemical characterisation of lipoic acid ligases and can be adapted to high-throughput screening approaches of inhibitors for these enzymes.
Lipoic acid, also known as 6,8-thioctic acid, is an essential cofactor of alpha-keto acid dehydrogenase complexes (KADH) and the glycine cleavage system (GCV). The KADH include the pyruvate dehydrogenase (PDH), branched chain alpha-keto acid dehydrogenase (BCDH) and alpha-ketoglutarate dehydrogenase (KGDH), which are an integral part for any cell's metabolism. In Plasmodium spp. the lipoic acid dependent enzyme complexes are found in the apicoplast, a plastid related organelle, and in the mitochondrion and thus two organelle specific lipoylation pathways are present in these parasites. Biosynthesis of the cofactor occurs in the apicoplast. Octanoyl-[acyl carrier protein]: protein N-octanoyltransferase (LipB) catalyses the attachment of octanoyl-acyl carrier protein (octanoyl-ACP) to the PDH and lipoic acid synthase (LipA) then catalyses the insertion of two sulfurs into the octanoyl-chain to form lipoamide. In the mitochondrion, scavenged lipoic acid is ligated to the enzyme complexes by the action of lipoic acid protein ligase A (LplA1), in an ATP-dependent reaction. However, a second lipoate protein ligase A (LplA2) was identified in the genome of P. falciparum, but its subcellular localisation could not be predicted using the available prediction programs. To further analyse its localisation, parasites were generated expressing full length LplA2 in frame with green fluorescent protein (GFP). In addition, immunofluorescence analyses on wild-type parasites using LplA2 specific antibodies were performed. These studies showed that LplA2 is dually targeted to the apicoplast as well as to the mitochondrion, raising the question about potential redundancy between the ligases present in the parasites. To further analyse this possibility, knock-out studies of lplA1 and lplA2 were performed in the human and rodent malaria parasites P. falciparum and P. berghei, respectively. Knock-out studies showed that LplA1 and LplA2 are non-redundant and strongly suggested that LplA1 is crucial for intraerythrocytic development, whereas LplA2 is essential for sexual development in the mosquito. According to these results it appears that (1) a key regulator of lipoic acid metabolism in Plasmodium spp. is stage specific expression of the relevant proteins and (2) both ligases are potential drug targets as knock-out of lplA1 appeared impossible in the blood stages and knock-out of lplA2 resulted in the interruption of parasite sexual development in the mosquito, and thus transmission of the parasites would be blocked if LplA2 was inhibited.
To further analyse the biochemical properties of P. falciparum LplA1 and LplA2, a spectrophotometric assay system was developed, which is also suitable for the development of a high-throughput assay system. The spectrophotometric assay monitors the first part of the LplA reaction - the activation of lipoic acid by ATP. The released pyrophosphate is converted to phosphate which is detected by acidic ammonium molybdate. Using the Escherichia coli LplA protein as a positive control, kinetic parameters for the bacterial protein were determined that are in reasonable agreement with the published data. The results validate the assay and suggest that it might be suitable for inhibitor screening in the future.
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