Intracellular parasites scavenge nutrients from their host cell in order to grow and Plasmodium, the apicomplexan parasite that causes malaria, is no exception. The initial infection in the host is the clinically silent liver stage of the life cycle. During liver stage development, the parasite undergoes massive replication within a single hepatocyte to produce tens of thousands of exoerythrocytic merozoites, which are released and invade erythrocytes to initiate the symptomatic blood stage of the life cycle. The hepatocyte is metabolically very active and one would assume that it could supply the parasite with all the nutrients it requires for growth and replication. However, contrary to this assumption, we have recently shown that the developing liver stage has an absolute requirement for its own de novo fatty acid synthesis pathway. This is a highly significant finding as it opens up an avenue for prophylactic drug design against the liver stages of the parasite. The parasite s type II fatty acid synthetic (FAS II) machinery is located in the apicoplast, an organelle derived from the secondary endosymbiosis of an ancient cyanobacterium. Due to its ancestry, many of the proteins targeted to the apicoplast are bacterial in origin and thus constitute ideal drug targets as they are not present in the human host. FAS II is one such pathway and is divergent from the mammalian FAS I pathway. Utilizing a novel epitope-tagging methodology and gene knockout studies, we have shown that the enzymes for FAS II elongation are expressed in liver stage parasites and are essential for the completion of liver stage development. Furthermore, we have established that the parasite s sole apicoplast-targeted pyruvate dehydrogenase complex (PDH) is also essential only for late liver stage development. We thus hypothesize that the Plasmodium liver stage completely depends on fatty acids synthesized by FAS II during late stage development and that fatty acids are further processed and incorporated into membrane phospholipids required for complete liver stage development.
All aims are designed to test our hypothesis. Experiments will utilize innovative techniques we and our prospective collaborators have developed to study parasite protein localization and expression by epitope tagging as well as the effects of the knockout or conditional knockdown of genes involved in fatty acid modification on liver stage development. In addition we have developed an in vitro assay to study the effect of FAS II inhibitors on liver stage growth. Our research will also extend to the human parasite P. falciparum to determine the effects of FAS II deletion on its liver stage development. To do this, we will collaborate with investigators who have developed a unique mouse model with a humanized liver, which we will use to examine the fatty acid synthesis requirements of P. falciparum liver stage development.
A bacterial type II fatty acid synthesis (FAS II) pathway is encoded in the Plasmodium genome. We provide data that the FAS II pathway and the pyruvate dehydrogenase complex which fuels FAS II are essential only for the complete development of the liver stage of the parasite life cycle. This proposal will extensively examine the FAS II pathway and the enzymes which contribute to fatty acid modification necessary for membrane production in the developing liver stage parasite.
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