Disruption of the Plasmodium falciparum Life Cycle through Transcriptional Reprogramming by Inhibitors of Jumonji Demethylases.

Little is known about the role of the three Jumonji C (JmjC) enzymes in Plasmodium falciparum (Pf). Here, we show that JIB-04 and other established inhibitors of mammalian JmjC histone demethylases kill asexual blood stage parasites and are even more potent at blocking gametocyte development and gamete formation. In late stage parasites, JIB-04 increased levels of trimethylated lysine residues on histones, suggesting the inhibition of P. falciparum Jumonji demethylase activity. These epigenetic defects coincide with deregulation of invasion, cell motor, and sexual development gene programs, including gene targets coregulated by the PfAP2-I transcription factor and chromatin-binding factor, PfBDP1. Mechanistically, we demonstrate that PfJmj3 converts 2-oxoglutarate to succinate in an iron-dependent manner consistent with mammalian Jumonji enzymes, and this catalytic activity is inhibited by JIB-04 and other Jumonji inhibitors. Our pharmacological studies of Jumonji activity in the malaria parasite provide evidence that inhibition of these enzymatic activities is detrimental to the parasite.

A mevalonate bypass system facilitates elucidation of plastid biology in malaria parasites.

Malaria parasites rely on a plastid organelle for survival during the blood stages of infection. However, the entire organelle is dispensable as long as the isoprenoid precursor, isopentenyl pyrophosphate (IPP), is supplemented in the culture medium. We engineered parasites to produce isoprenoid precursors from a mevalonate-dependent pathway, creating a parasite line that replicates normally after the loss of the apicoplast organelle. We show that carbon-labeled mevalonate is specifically incorporated into isoprenoid products, opening new avenues for researching this essential class of metabolites in malaria parasites. We also show that essential apicoplast proteins, such as the enzyme target of the drug fosmidomycin, can be deleted in this mevalonate bypass parasite line, providing a new method to determine the roles of other important apicoplast-resident proteins. Several antibacterial drugs kill malaria parasites by targeting basic processes, such as transcription, in the organelle. We used metabolomic and transcriptomic methods to characterize parasite metabolism after azithromycin treatment triggered loss of the apicoplast and found that parasite metabolism and the production of apicoplast proteins is largely unaltered. These results provide insight into the effects of apicoplast-disrupting drugs, several of which have been used to treat malaria infections in humans. Overall, the mevalonate bypass system provides a way to probe essential aspects of apicoplast biology and study the effects of drugs that target apicoplast processes.

Host biotin is required for liver stage development in malaria parasites.

Acetyl-CoA carboxylase (ACC) is a biotin-dependent enzyme that is the target of several classes of herbicides. Malaria parasites contain a plant-like ACC, and this is the only protein predicted to be biotinylated in the parasite. We found that ACC is expressed in the apicoplast organelle in liver- and blood-stage malaria parasites; however, it is activated through biotinylation only in the liver stages. Consistent with this observation, deletion of the biotin ligase responsible for ACC biotinylation does not impede blood-stage growth, but results in late liver-stage developmental defects. Biotin depletion increases the severity of the developmental defects, demonstrating that parasite and host biotin metabolism are required for normal liver-stage progression. This finding may link the development of liver-stage malaria parasites to the nutritional status of the host, as neither the parasite nor the human host can synthesize biotin.

A novel lipoate attachment enzyme is shared by Plasmodium and Chlamydia species.

Lipoate is an essential cofactor for enzymes that are important for central metabolism and other processes. In malaria parasites, scavenged lipoate from the human host is required for survival. The Plasmodium falciparum mitochondrion contains two enzymes (PfLipL1 and PfLipL2) that are responsible for activating mitochondrial proteins through the covalent attachment of lipoate (lipoylation). Lipoylation occurs via a novel redox-gated mechanism that remains poorly understood. We show that PfLipL1 functions as a redox switch that determines which downstream proteins will be activated. Based on the lipoate redox state, PfLipL1 either functions as a canonical lipoate ligase or as a lipoate activating enzyme which works in conjunction with PfLipL2. We demonstrate that PfLipL2 is a lipoyltransferase and is a member of a novel clade of lipoate attachment enzymes. We show that a LipL2 enzyme from Chlamydia trachomatis has similar activity, demonstrating conservation between intracellular pathogens from different phylogenetic kingdoms and supporting the hypothesis that an early ancestor of malaria parasites once contained a chlamydial endosymbiont. Redox-dependent lipoylation may regulate processes such as central metabolism and oxidative defense pathways.

Redox-dependent lipoylation of mitochondrial proteins in Plasmodium falciparum.

Lipoate scavenging from the human host is essential for malaria parasite survival. Scavenged lipoate is covalently attached to three parasite proteins: the H-protein and the E2 subunits of branched chain amino acid dehydrogenase (BCDH) and α-ketoglutarate dehydrogenase (KDH). We show mitochondrial localization for the E2 subunits of BCDH and KDH, similar to previously localized H-protein, demonstrating that all three lipoylated proteins reside in the parasite mitochondrion. The lipoate ligase 1, LipL1, has been shown to reside in the mitochondrion and it catalyses the lipoylation of the H-protein; however, we show that LipL1 alone cannot lipoylate BCDH or KDH. A second mitochondrial protein with homology to lipoate ligases, LipL2, does not show ligase activity and is not capable of lipoylating any of the mitochondrial substrates. Instead, BCDH and KDH are lipoylated through a novel mechanism requiring both LipL1 and LipL2. This mechanism is sensitive to redox conditions where BCDH and KDH are exclusively lipoylated under strong reducing conditions in contrast to the H-protein which is preferentially lipoylated under less reducing conditions. Thus, malaria parasites contain two different routes of mitochondrial lipoylation, an arrangement that has not been described for any other organism.

The suf iron-sulfur cluster synthesis pathway is required for apicoplast maintenance in malaria parasites.

The apicoplast organelle of the malaria parasite Plasmodium falciparum contains metabolic pathways critical for liver-stage and blood-stage development. During the blood stages, parasites lacking an apicoplast can grow in the presence of isopentenyl pyrophosphate (IPP), demonstrating that isoprenoids are the only metabolites produced in the apicoplast which are needed outside of the organelle. Two of the isoprenoid biosynthesis enzymes are predicted to rely on iron-sulfur (FeS) cluster cofactors, however, little is known about FeS cluster synthesis in the parasite or the roles that FeS cluster proteins play in parasite biology. We investigated two putative FeS cluster synthesis pathways (Isc and Suf) focusing on the initial step of sulfur acquisition. In other eukaryotes, these proteins can be located in multiple subcellular compartments, raising the possibility of cross-talk between the pathways or redundant functions. In P. falciparum, SufS and its partner SufE were found exclusively the apicoplast and SufS was shown to have cysteine desulfurase activity in a complementation assay. IscS and its effector Isd11 were solely mitochondrial, suggesting that the Isc pathway cannot contribute to apicoplast FeS cluster synthesis. The Suf pathway was disrupted with a dominant negative mutant resulting in parasites that were only viable when supplemented with IPP. These parasites lacked the apicoplast organelle and its organellar genome--a phenotype not observed when isoprenoid biosynthesis was specifically inhibited with fosmidomycin. Taken together, these results demonstrate that the Suf pathway is essential for parasite survival and has a fundamental role in maintaining the apicoplast organelle in addition to any role in isoprenoid biosynthesis.

A key role for lipoic acid synthesis during Plasmodium liver stage development.

The successful navigation of malaria parasites through their life cycle, which alternates between vertebrate hosts and mosquito vectors, requires a complex interplay of metabolite synthesis and salvage pathways. Using the rodent parasite Plasmodium berghei, we have explored the synthesis and scavenging pathways for lipoic acid, a short-chain fatty acid derivative that regulates the activity of α-ketoacid dehydrogenases including pyruvate dehydrogenase. In Plasmodium, lipoic acid is either synthesized de novo in the apicoplast or is scavenged from the host into the mitochondrion. Our data show that sporozoites lacking the apicoplast lipoic acid protein ligase LipB are markedly attenuated in their infectivity for mice, and in vitro studies document a very late liver stage arrest shortly before the final phase of intra-hepaticparasite maturation. LipB-deficient asexual blood stage parasites show unimpaired rates of growth in normal in vitro or in vivo conditions. However, these parasites showed reduced growth in lipid-restricted conditions induced by treatment with the lipoic acid analogue 8-bromo-octanoate or with the lipid-reducing agent clofibrate. This finding has implications for understanding Plasmodium pathogenesis in malnourished children that bear the brunt of malarial disease. This study also highlights the potential of exploiting lipid metabolism pathways for the design of genetically attenuated sporozoite vaccines.

Plasmodium falciparum apicoplast transit peptides are unstructured in vitro and during apicoplast import.

Trafficking of soluble proteins to the apicoplast in Plasmodium falciparum is determined by an N-terminal transit peptide (TP) which is necessary and sufficient for apicoplast import. Apicoplast precursor proteins are synthesized at the rough endoplasmic reticulum, but are then specifically sorted from other proteins in the secretory pathway. The mechanism of TP recognition is presently unknown. Apicoplast TPs do not contain a conserved sequence motif; therefore, we asked whether they contain an essential structural motif. Using nuclear magnetic resonance to study a model TP from acyl carrier protein, we found a short, low-occupancy helix, but the TP was otherwise disordered. Using an in vivo localization assay, we blocked TP secondary structure by proline mutagenesis, but found robust apicoplast localization. Alternatively, we increased the helical content of the TP through mutation while maintaining established TP characteristics. Apicoplast import was disrupted in a helical mutant TP, but import was then restored by the further addition of a single proline. We conclude that structure in the TP interferes with apicoplast import, and therefore TPs are functionally disordered. These results provide an explanation for the amino acid bias observed in apicoplast TPs.

Activation of sterol regulatory element binding proteins in the absence of Scap in Drosophila melanogaster.

The escort factor Scap is essential in mammalian cells for regulated activation of sterol regulatory element binding proteins (SREBPs). SREBPs are membrane-bound transcription factors. Cells lacking Scap cannot activate SREBP. They are therefore deficient in the transcription of numerous genes involved in lipid synthesis and uptake; they cannot survive in the absence of exogenous lipid. Here we report that, in contrast to mammalian cells, Drosophila completely lacking dscap are viable. Flies lacking dscap emerge at approximately 70% of the expected rate and readily survive as homozygous stocks. These animals continue to cleave dSREBP in some tissues. Transcription of dSREBP target genes in dscap mutant larvae is reduced compared to wild type. It is greater than in mutants lacking dSREBP and remains responsive to dietary lipids in dscap mutants. Flies lacking dscap do not require the caspase Drice to activate dSREBP. This contrasts with ds2p mutants. ds2p encodes a protease that releases the transcription factor domain of dSREBP from the membrane. Larvae doubly mutant for dscap and ds2p exhibit phenotypes similar to those of ds2p single mutants. Thus, dScap and dS2P, essential components of the SREBP activation machinery in mammalian cells, are dispensable in Drosophila owing to different compensatory mechanisms.

Activation of sterol regulatory element-binding protein by the caspase Drice in Drosophila larvae.

During larval development in Drosophila melanogaster, transcriptional activation of target genes by sterol regulatory element-binding protein (dSREBP) is essential for survival. In all cases studied to date, activation of SREBPs requires sequential proteolysis of the membrane-bound precursor by site-1 protease (S1P) and site-2 protease (S2P). Cleavage by S2P, within the first membrane-spanning helix of SREBP, releases the transcription factor. In contrast to flies lacking dSREBP, flies lacking dS2P are viable. The Drosophila effector caspase Drice cleaves dSREBP, and cleavage requires an Asp residue at position 386, in the cytoplasmic juxtamembrane stalk. The initiator caspase Dronc does not cleave dSREBP, but animals lacking dS2P require both drice and dronc to complete development. They do not require Dcp1, although this effector caspase also can cleave dSREBP in vitro. Cleavage of dSREBP by Drice releases the amino-terminal transcription factor domain of dSREBP to travel to the nucleus where it mediates the increased transcription of target genes needed for lipid synthesis and uptake. Drice-dependent activation of dSREBP explains why flies lacking dS2P are viable, and flies lacking dSREBP itself are not.

Alternative processing of sterol regulatory element binding protein during larval development in Drosophila melanogaster.

Sterol regulatory element binding protein (SREBP) is a major transcriptional regulator of lipid metabolism. Nuclear Drosophila SREBP (dSREBP) is essential for larval development in Drosophila melanogaster but dispensable in adults. dSREBP(-) larvae die at second instar owing to loss of dSREBP-mediated transcription but survive to adulthood when fed fatty acids. Activation of SREBP requires two separate cleavages. Site-1 protease (S1P) cleaves in the luminal loop of the membrane-bound SREBP precursor, cutting it in two. The NH(2)- and COOH-terminal domains remain membrane bound owing to their single membrane-spanning helices. The NH(2)-terminal cleavage product is the substrate for site-2 protease (S2P), which cleaves within its membrane-spanning helix to release the transcription factor. In mice, loss of S1P is lethal but the consequences of loss of S2P in animals remain undefined. All known functions of SREBP require its cleavage by S2P. We isolated Drosophila mutants that eliminate all dS2P function (dS2P(-)). Unexpectedly, larvae lacking dS2P are viable. They are deficient in transcription of some dSREBP target genes but less so than larvae lacking dSREBP. Despite loss of dS2P, dSREBP is processed in mutant larvae. Therefore, larvae have an alternative cleavage mechanism for producing transcriptionally active dSREBP, and this permits survival of dS2P mutants.

Fatty acid auxotrophy in Drosophila larvae lacking SREBP.

SREBPs are membrane bound transcription factors that are crucial for normal lipid synthesis in animal cells. Here, we show that Drosophila lacking dSREBP die before the third larval instar. Mutant larvae exhibit pronounced growth defects prior to lethality, along with substantial deficits in the transcription of genes required for fatty acid synthesis. Compared to wild-type larvae, mutants contain markedly less fatty acid, although its composition is unaltered. Dietary supplementation with fatty acids rescues mutants to adulthood. The most effective fatty acid, oleate, rescues 80% of homozygotes. Rescue by dSREBP requires expression only in fat body and gut. Larvae expressing dSREBP prior to pupariation complete development and are viable as adults even when dSREBP expression is subsequently extinguished. The role, if any, of dSREBP in adults is not yet apparent. These data indicate that dSREBP deficiency renders Drosophila larvae auxotrophic for fatty acids.