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Effective malaria control and elimination in hyperendemic areas of the world will require treatment of the Plasmodium falciparum (Pf) blood stage that causes disease as well as the gametocyte stage that is required for transmission from humans to the mosquito vector. Most currently used therapies do not kill gametocytes, a highly specialized, non-replicating sexual parasite stage. Further confounding next generation drug development against Pf is the unknown metabolic state of the gametocyte and the lack of known biochemical activity for most parasite gene products in general. Here, we take a systematic activity-based proteomics approach to survey the activity of the large and druggable ATPase family in replicating blood stage asexual parasites and transmissible, non-replicating sexual gametocytes. ATPase activity broadly changes during the transition from asexual schizonts to sexual gametocytes, indicating altered metabolism and regulatory roles of ATPases specific for each lifecycle stage. We further experimentally confirm existing annotation and predict ATPase function for 38 uncharacterized proteins. By mapping the activity of ATPases associated with gametocytogenesis, we assign biochemical activity to a large number of uncharacterized proteins and identify new candidate transmission blocking targets.

Plasmodium vivax malaria is characterized by periodic relapses of symptomatic blood stage parasite infections likely initiated by activation of dormant liver stage parasites -hypnozoites. The lack of tractable animal models for P. vivax constitutes a severe obstacle to investigate this unique aspect of its biology and to test drug efficacy against liver stages. We show that the FRG KO huHep liver-humanized mice support P. vivax sporozoite infection, development of liver stages, and the formation of small non-replicating hypnozoites. Cellular characterization of P. vivax liver stage development in vivo demonstrates complete maturation into infectious exo-erythrocytic merozoites and continuing persistence of hypnozoites. Primaquine prophylaxis or treatment prevents and eliminates liver stage infection. Thus, the P. vivax/FRG KO huHep mouse infection model constitutes an important new tool to investigate the biology of liver stage development and dormancy and might aid in the discovery of new drugs for the prevention of relapsing malaria.

Immunization with live-attenuated Plasmodium sporozoites completely protects against malaria infection. Genetic engineering offers a versatile platform to create live-attenuated sporozoite vaccine candidates. We previously generated a genetically attenuated parasite (GAP) by deleting the P52 and P36 genes in the NF54 wild-type (WT) strain of Plasmodium falciparum (Pf p52(-)/p36(-) GAP). Preclinical assessment of p52(-)/p36(-) GAP in a humanized mouse model indicated an early and severe liver stage growth defect. However, human exposure to200 Pf p52(-)/p36(-) GAP-infected mosquito bites in a safety trial resulted in peripheral parasitemia in one of six volunteers, revealing that this GAP was incompletely attenuated. We have now created a triple gene deleted GAP by additionally removing the SAP1 gene (Pf p52(-)/p36(-)/sap1(-) GAP) and employed flippase (FLP)/flippase recognition target (FRT) recombination for drug selectable marker cassette removal. This next-generation GAP was indistinguishable from WT parasites in blood stage and mosquito stage development. Using an improved humanized mouse model transplanted with human hepatocytes and human red blood cells, we show that despite a high-dose sporozoite challenge, Pf p52(-)/p36(-)/sap1(-) GAP did not transition to blood stage infection and appeared to be completely attenuated. Thus, clinical testing of Pf p52(-)/p36(-)/sap1(-) GAP assessing safety, immunogenicity, and efficacy against sporozoite challenge is warranted.

The malaria parasite Plasmodium falciparum depends on glucose to meet its energy requirements during blood-stage development. Although glycolysis is one of the best understood pathways in the parasite, it is unclear if glucose metabolism appreciably contributes to the acetyl-CoA pools required for tricarboxylic acid metabolism (TCA) cycle and fatty acid biosynthesis. P. falciparum possesses a pyruvate dehydrogenase (PDH) complex that is localized to the apicoplast, a specialized quadruple membrane organelle, suggesting that separate acetyl-CoA pools are likely. Herein, we analyze PDH-deficient parasites using rapid stable-isotope labeling and show that PDH does not appreciably contribute to acetyl-CoA synthesis, tricarboxylic acid metabolism, or fatty acid synthesis in blood stage parasites. Rather, we find that acetyl-CoA demands are supplied through a "PDH-like" enzyme and provide evidence that the branched-chain keto acid dehydrogenase (BCKDH) complex is performing this function. We also show that acetyl-CoA synthetase can be a significant contributor to acetyl-CoA biosynthesis. Interestingly, the PDH-like pathway contributes glucose-derived acetyl-CoA to the TCA cycle in a stage-independent process, whereas anapleurotic carbon enters the TCA cycle via a stage-dependent phosphoenolpyruvate carboxylase/phosphoenolpyruvate carboxykinase process that decreases as the parasite matures. Although PDH-deficient parasites have no blood-stage growth defect, they are unable to progress beyond the oocyst phase of the parasite mosquito stage.