Malaria parasites utilize two essential plasma membrane fusogens for gamete fertilization.

Cell fusion of female and male gametes is the climax of sexual reproduction. In many organisms, the Hapless 2 (HAP2) family of proteins play a critical role in gamete fusion. We find that Plasmodium falciparum, the causative agent of human malaria, expresses two HAP2 proteins: PfHAP2 and PfHAP2p. These proteins are present in stage V gametocytes and localize throughout the flagellum of male gametes. Gene deletion analysis and genetic crosses show that PfHAP2 and PfHAP2p individually are essential for male fertility and thereby, parasite transmission to the mosquito. Using a cell fusion assay, we demonstrate that PfHAP2 and PfHAP2p are both authentic plasma membrane fusogens. Our results establish nonredundant essential roles for PfHAP2 and PfHAP2p in mediating gamete fusion in Plasmodium and suggest avenues in the design of novel strategies to prevent malaria parasite transmission from humans to mosquitoes.

Plasmodium yoelii S4/CelTOS is important for sporozoite gliding motility and cell traversal.

Gliding motility and cell traversal by the Plasmodium ookinete and sporozoite invasive stages allow penetration of cellular barriers to establish infection of the mosquito vector and mammalian host, respectively. Motility and traversal are not observed in red cell infectious merozoites, and we have previously classified genes that are expressed in sporozoites but not merozoites (S genes) in order to identify proteins involved in these processes. The S4 gene has been described as criticaly involved in Cell Traversal for Ookinetes and Sporozoites (CelTOS), yet knockout parasites (s4/celtos¯) do not generate robust salivary gland sporozoite numbers, precluding a thorough analysis of S4/CelTOS function during host infection. We show here that a failure of oocysts to develop or survive in the midgut contributes to the poor mosquito infection by Plasmodium yoelii (Py) s4/celtos¯ rodent malaria parasites. We rescued this phenotype by expressing S4/CelTOS under the ookinete-specific circumsporozoite protein and thrombospondin-related anonymous protein-related protein (CTRP) promoter (S4/CelTOSCTRP ), generating robust numbers of salivary gland sporozoites lacking S4/CelTOS that were suitable for phenotypic analysis. Py S4/CelTOSCTRP sporozoites showed reduced infectivity in BALB/c mice when compared to wild-type sporozoites, although they appeared more infectious than sporozoites deficient in the related traversal protein PLP1/SPECT2 (Py plp1/spect2¯). Using in vitro assays, we substantiate the role of S4/CelTOS in sporozoite cell traversal, but also uncover a previously unappreciated role for this protein for sporozoite gliding motility.

Plasmodium falciparum genetic crosses in a humanized mouse model.

Genetic crosses of phenotypically distinct strains of the human malaria parasite Plasmodium falciparum are a powerful tool for identifying genes controlling drug resistance and other key phenotypes. Previous studies relied on the isolation of recombinant parasites from splenectomized chimpanzees, a research avenue that is no longer available. Here we demonstrate that human-liver chimeric mice support recovery of recombinant progeny for the identification of genetic determinants of parasite traits and adaptations.

A next-generation genetically attenuated Plasmodium falciparum parasite created by triple gene deletion.

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 to >200 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.

Type II fatty acid biosynthesis is essential for Plasmodium falciparum sporozoite development in the midgut of Anopheles mosquitoes.

The prodigious rate at which malaria parasites proliferate during asexual blood-stage replication, midgut sporozoite production, and intrahepatic development creates a substantial requirement for essential nutrients, including fatty acids that likely are necessary for parasite membrane formation. Plasmodium parasites obtain fatty acids either by scavenging from the vertebrate host and mosquito vector or by producing fatty acids de novo via the type two fatty acid biosynthesis pathway (FAS-II). Here, we study the FAS-II pathway in Plasmodium falciparum, the species responsible for the most lethal form of human malaria. Using antibodies, we find that the FAS-II enzyme FabI is expressed in mosquito midgut oocysts and sporozoites as well as liver-stage parasites but not during the blood stages. As expected, FabI colocalizes with the apicoplast-targeted acyl carrier protein, indicating that FabI functions in the apicoplast. We further analyze the FAS-II pathway in Plasmodium falciparum by assessing the functional consequences of deleting fabI and fabB/F. Targeted deletion or disruption of these genes in P. falciparum did not affect asexual blood-stage replication or the generation of midgut oocysts; however, subsequent sporozoite development was abolished. We conclude that the P. falciparum FAS-II pathway is essential for sporozoite development within the midgut oocyst. These findings reveal an important distinction from the rodent Plasmodium parasites P. berghei and P. yoelii, where the FAS-II pathway is known to be required for normal parasite progression through the liver stage but is not required for oocyst development in the Anopheles mosquito midgut.

A scalable assessment of Plasmodium falciparum transmission in the standard membrane-feeding assay, using transgenic parasites expressing green fluorescent protein-luciferase.

BACKGROUND: The development of drugs and vaccines to reduce malaria transmission is an important part of eradication plans. The transmission-reducing activity (TRA) of these agents is currently determined in the standard membrane-feeding assay (SMFA), based on subjective microscopy-based readouts and with limitations in upscaling and throughput. METHODS: Using a Plasmodium falciparum strain expressing the firefly luciferase protein, we present a luminescence-based approach to SMFA evaluation that eliminates the requirement for mosquito dissections in favor of a simple approach in which whole mosquitoes are homogenized and examined directly for luciferase activity. RESULTS: Analysis of 6860 Anopheles stephensi mosquitoes across 68 experimental feeds shows that the luminescence assay was as sensitive as microscopy for infection detection. The mean luminescence intensity of individual and pooled mosquitoes accurately quantifies mean oocyst intensity and generates comparable TRA estimates. The luminescence assay presented here could increase SMFA throughput so that 10-30 experimental feeds could be evaluated in a single 96-well plate. CONCLUSIONS: This new method of assessing Plasmodium infection and transmission intensity could expedite the screening of novel drug compounds, vaccine candidates, and sera from malaria-exposed individuals for TRA. Luminescence-based estimates of oocyst intensity in individual mosquitoes should be interpreted with caution.

Kinetic flux profiling elucidates two independent acetyl-CoA biosynthetic pathways in Plasmodium falciparum.

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.

Malaria blood stage infection suppresses liver stage infection via host-induced interferons but not hepcidin.

Malaria-causing Plasmodium parasites first replicate as liver stages (LS), which then seed symptomatic blood stage (BS) infection. Emerging evidence suggests that these stages impact each other via perturbation of host responses, and this influences the outcome of natural infection. We sought to understand whether the parasite stage interplay would affect live-attenuated whole parasite vaccination, since the efficacy of whole parasite vaccines strongly correlates with their extend of development in the liver. We thus investigated the impact of BS infection on LS development of genetically attenuated and wildtype parasites in female rodent malaria models and observed that for both, LS infection suffered severe suppression during concurrent BS infection. Strikingly and in contrast to previously published studies, we find that the BS-induced iron-regulating hormone hepcidin is not mediating suppression of LS development. Instead, we demonstrate that BS-induced host interferons are the main mediators of LS developmental suppression. The type of interferon involved depended on the BS-causing parasite species. Our study provides important mechanistic insights into the BS-mediated suppression of LS development. This has direct implications for understanding the outcomes of live-attenuated Plasmodium parasite vaccination in malaria-endemic areas and might impact the epidemiology of natural malaria infection.

A replication competent Plasmodium falciparum parasite completely attenuated by dual gene deletion.

Vaccination with infectious Plasmodium falciparum (Pf) sporozoites (SPZ) administered with antimalarial drugs (PfSPZ-CVac), confers superior sterilizing protection against infection when compared to vaccination with replication-deficient, radiation-attenuated PfSPZ. However, the requirement for drug administration constitutes a major limitation for PfSPZ-CVac. To obviate this limitation, we generated late liver stage-arresting replication competent (LARC) parasites by deletion of the Mei2 and LINUP genes (mei2-/linup- or LARC2). We show that Plasmodium yoelii (Py) LARC2 sporozoites did not cause breakthrough blood stage infections and engendered durable sterilizing immunity against various infectious sporozoite challenges in diverse strains of mice. We next genetically engineered a PfLARC2 parasite strain that was devoid of extraneous DNA and produced cryopreserved PfSPZ-LARC2. PfSPZ-LARC2 liver stages replicated robustly in liver-humanized mice but displayed severe defects in late liver stage differentiation and did not form liver stage merozoites. This resulted in complete abrogation of parasite transition to viable blood stage infection. Therefore, PfSPZ-LARC2 is the next-generation vaccine strain expected to unite the safety profile of radiation-attenuated PfSPZ with the superior protective efficacy of PfSPZ-CVac.

Plasmodium microtubule-binding protein EB1 is critical for partitioning of nuclei in male gametogenesis.

Sexual reproduction of the malaria parasites is critical for their transmission to a mosquito vector. Several signaling molecules, such as kinases and phosphatases, are known to regulate this process. We previously demonstrated that Plasmodium falciparum (Pf) Ca2+-dependent protein kinase 4 (CDPK4) and serine/arginine-rich protein kinase 1 (SRPK1) are critical for axoneme formation during male gametogenesis, with genetic deletion of either gene causing a complete block in parasite transmission to the mosquito. A comparative phospho-proteome analysis of Pfcdpk4- and RNA-seq analysis of Pfsrpk1- gametocytes showed that these kinases regulate similar biological processes linked to both microtubule (MT) dynamics and cell motility. One of these proteins was a nuclear MT-associated End Binding protein 1 (EB1), which was hypophosphorylated in Pfcdpk4- gametocytes. To study the functional relevance of EB1, we created gene deletion parasites for EB1. We further demonstrate that Pfeb1- parasites like WT NF54 parasites proliferate normally as asexuals and undergo gametocytogenesis and gametogenesis. Strikingly, these parasites suffer a severe defect in nuclear segregation and partitioning of nuclei into emerging microgametes. Further genetic crosses utilizing male- and female-sterile parasites revealed that Pfeb1- parasites only suffer a male fertility defect. Overall, our study reveals an essential function for PfEB1 in male gamete nuclear segregation and suggests a potential therapeutic avenue in the design of transmission-blocking drugs to prevent malaria transmission from humans to mosquito. IMPORTANCE Gametogenesis and subsequent gamete fusion are central to successful transmission of the malaria parasites to a female Anopheles mosquito vector and completion of the sexual phase of the parasite life cycle. Male gametogenesis involves the formation of axonemes inside male gametes from male gametocytes via active cytoskeleton remodeling. The tubulin and tubulin-binding proteins are, thus, attractive anti-malarial drug targets. In the present study, we demonstrate that a microtubule-binding protein PfEB1 is essential for male gamete fertility, specifically for the inheritance of nuclei from activated male gametocytes. Targeting PfEB1 function may provide new avenues into designing interventions to prevent malaria transmission and disease spread.

Global gene expression of human malaria parasite liver stages throughout intrahepatocytic development.

Plasmodium falciparum (Pf) is causing the greatest malaria burden, yet the liver stages (LS) of this most important parasite species have remained poorly studied. Here, we used a human liver-chimeric mouse model in combination with a novel fluorescent PfNF54 parasite line (PfNF54cspGFP) to isolate PfLS-infected hepatocytes and generate transcriptomes that cover the major LS developmental phases in human hepatocytes. RNA-seq analysis of early Pf LS trophozoites two days after infection, revealed a central role of translational regulation in the transformation of the extracellular invasive sporozoite into intracellular LS. The developmental time course gene expression analysis indicated that fatty acid biosynthesis, isoprenoid biosynthesis and iron metabolism are sustaining LS development along with amino acid metabolism and biosynthesis. Countering oxidative stress appears to play an important role during intrahepatic LS development. Furthermore, we observed expression of the variant PfEMP1 antigen-encoding var genes, and we confirmed expression of PfEMP1 protein during LS development. Transcriptome comparison of the late Pf liver stage schizonts with P. vivax (Pv) late liver stages revealed highly conserved gene expression profiles among orthologous genes. A notable difference however was the expression of genes regulating sexual stage commitment. While Pv schizonts expressed markers of sexual commitment, the Pf LS parasites were not sexually committed and showed expression of gametocytogenesis repression factors. Our results provide the first comprehensive gene expression profile of the human malaria parasite Pf LS isolated during in vivo intrahepatocytic development. This data will inform biological studies and the search for effective intervention strategies that can prevent infection.

Disruption of the Plasmodium falciparum liver-stage antigen-1 locus causes a differentiation defect in late liver-stage parasites.

The malaria parasite Plasmodium falciparum infects humans and first targets the liver where liver-stage parasites undergo pre-erythrocytic replication. Liver-stage antigen-1 (LSA-1) is currently the only identified P. falciparum protein for which expression is restricted to liver stages. Yet, the importance of LSA-1 for liver-stage parasite development remains unknown. Here we deleted LSA-1 in the NF54 strain of P. falciparum and analysed the lsa-1(-) parasites throughout their life cycle. lsa-1(-) sporozoites had normal gliding motility and invasion into hepatocytes. Six days after infection of a hepatocytic cell line, lsa-1(-) parasites exhibited a moderate phenotype with an ~50% reduction of late liver-stage forms when compared with wild type. Strikingly, lsa-1(-) parasites growing in SCID/Alb-uPA mice with humanized livers showed a severe defect in late liver-stage differentiation and exo-erythrocytic merozoite formation 7 days after infection, a time point when wild-type parasites develop into mature merozoites. The lsa-1(-) parasites also showed aberrant liver-stage expression of key parasite proteins apical membrane antigen-1 and circumsporozoite protein. Our data show that LSA-1 plays a critical role during late liver-stage schizogony and is thus important in the parasite transition from the liver to blood. LSA-1 is the first P. falciparum protein identified to be required for this transitional stage of the parasite life cycle.

A Plasmodium falciparum ATP-binding cassette transporter is essential for liver stage entry into schizogony.

Plasmodium sporozoites invade hepatocytes and transform into liver stages within a parasitophorous vacuole (PV). The parasites then grow and replicate their genome to form exoerythrocytic merozoites that infect red blood cells. We report that the human malaria parasite Plasmodium falciparum (Pf) expresses a C-type ATP-binding cassette transporter, Pf ABCC2, which marks the transition from invasive sporozoite to intrahepatocytic early liver stage. Using a humanized mouse infection model, we show that Pf ABCC2 localizes to the parasite plasma membrane in early and mid-liver stage parasites but is not detectable in late liver stages. Pf abcc2 - sporozoites invade hepatocytes, form a PV, and transform into liver stage trophozoites but cannot transition to exoerythrocytic schizogony and fail to transition to blood stage infection. Thus, Pf ABCC2 is an expression marker for early phases of parasite liver infection and plays an essential role in the successful initiation of liver stage replication.

Anti-TRAP/SSP2 monoclonal antibodies can inhibit sporozoite infection and may enhance protection of anti-CSP monoclonal antibodies.

Vaccine-induced sterilizing protection from infection by Plasmodium parasites, the pathogens that cause malaria, will be essential in the fight against malaria as it would prevent both malaria-related disease and transmission. Stopping the relatively small number of parasites injected by the mosquito before they can migrate from the skin to the liver is an attractive means to this goal. Antibody-eliciting vaccines have been used to pursue this objective by targeting the major parasite surface protein present during this stage, the circumsporozoite protein (CSP). While CSP-based vaccines have recently had encouraging success in disease reduction, this was only achieved with extremely high antibody titers and appeared less effective for a complete block of infection (i.e., sterile protection). While such disease reduction is important, these and other results indicate that strategies focusing on CSP alone may not achieve the high levels of sterile protection needed for malaria eradication. Here, we show that monoclonal antibodies (mAbs) recognizing another sporozoite protein, TRAP/SSP2, exhibit a range of inhibitory activity and that these mAbs may augment CSP-based protection despite conferring no sterile protection on their own. Therefore, pursuing a multivalent subunit vaccine immunization is a promising strategy for improving infection-blocking malaria vaccines.

A Malaria Parasite Cross Reveals Genetic Determinants of Plasmodium falciparum Growth in Different Culture Media.

What genes determine in vitro growth and nutrient utilization in asexual blood-stage malaria parasites? Competition experiments between NF54, clone 3D7, a lab-adapted African parasite, and a recently isolated Asian parasite (NHP4026) reveal contrasting outcomes in different media: 3D7 outcompetes NHP4026 in media containing human serum, while NHP4026 outcompetes 3D7 in media containing AlbuMAX, a commercial lipid-rich bovine serum formulation. To determine the basis for this polymorphism, we conducted parasite genetic crosses using humanized mice and compared genome-wide allele frequency changes in three independent progeny populations cultured in media containing human serum or AlbuMAX. This bulk segregant analysis detected three quantitative trait loci (QTL) regions [on chromosome (chr) 2 containing aspartate transaminase AST; chr 13 containing EBA-140; and chr 14 containing cysteine protease ATG4] linked with differential growth in serum or AlbuMAX in each of the three independent progeny pools. Selection driving differential growth was strong (s = 0.10 - 0.23 per 48-hour lifecycle). We conducted validation experiments for the strongest QTL on chr 13: competition experiments between ΔEBA-140 and 3D7 wildtype parasites showed fitness reversals in the two medium types as seen in the parental parasites, validating this locus as the causative gene. These results (i) demonstrate the effectiveness of bulk segregant analysis for dissecting fitness traits in P. falciparum genetic crosses, and (ii) reveal intimate links between red blood cell invasion and nutrient composition of growth media. Use of parasite crosses combined with bulk segregant analysis will allow systematic dissection of key nutrient acquisition/metabolism and red blood cell invasion pathways in P. falciparum.

A genetically engineered Plasmodium falciparum parasite vaccine provides protection from controlled human malaria infection.

Genetically engineered live Plasmodium falciparum sporozoites constitute a potential platform for creating consistently attenuated, genetically defined, whole-parasite vaccines against malaria through targeted gene deletions. Such genetically attenuated parasites (GAPs) do not require attenuation by irradiation or concomitant drug treatment. We previously developed a P. falciparum (Pf) GAP with deletions in P52, P36, and SAP1 genes (PfGAP3KO) and demonstrated its safety and immunogenicity in humans. Here, we further assessed safety, tolerability, and immunogenicity of the PfGAP3KO vaccine and tested its efficacy against controlled human malaria infection (CHMI) in malaria-naïve subjects. The vaccine was delivered by three (n = 6) or five (n = 8) immunizations with ~200 PfGAP3KO-infected mosquito bites per immunization. PfGAP3KO was safe and well tolerated with no breakthrough P. falciparum blood stage infections. Vaccine-related adverse events were predominately localized urticaria related to the numerous mosquito bites administered per vaccination. CHMI via bites with mosquitoes carrying fully infectious Pf NF54 parasites was carried out 1 month after the last immunization. Half of the study participants who received either three or five PfGAP3KO immunizations remained P. falciparum blood stage negative, as shown by a lack of detection of Plasmodium 18S rRNA in the blood for 28 days after CHMI. Six protected study participants received a second CHMI 6 months later, and one remained completely protected. Thus, the PfGAP3KO vaccine was safe and immunogenic and was capable of inducing protection against sporozoite infection. These results warrant further evaluation of PfGAP3KO vaccine efficacy in dose-range finding trials with an injectable formulation.

A combined transcriptome and proteome survey of malaria parasite liver stages.

For 50 years since their discovery, the malaria parasite liver stages (LS) have been difficult to analyze, impeding their utilization as a critical target for antiinfection vaccines and drugs. We have undertaken a comprehensive transcriptome analysis in combination with a proteomic survey of LS. Green fluorescent protein-tagged Plasmodium yoelii (PyGFP) was used to efficiently isolate LS-infected hepatocytes from the rodent host. Genome-wide LS gene expression was profiled and compared with other parasite life cycle stages. The analysis revealed approximately 2,000 genes active during LS development, and proteomic analysis identified 816 proteins. A subset of proteins appeared to be expressed in LS only. The data revealed exported parasite proteins and LS metabolic pathways including expression of FASII pathway enzymes. The FASII inhibitor hexachlorophene and the antibiotics, tetracycline and rifampicin, that target the apicoplast inhibited LS development, identifying FASII and other pathways localized in the apicoplast as potential drug targets to prevent malaria infection.

Platelet derived growth factor receptor ß (PDGFRß) is a host receptor for the human malaria parasite adhesin TRAP.

Following their inoculation by the bite of an infected Anopheles mosquito, the malaria parasite sporozoite forms travel from the bite site in the skin into the bloodstream, which transports them to the liver. The thrombospondin-related anonymous protein (TRAP) is a type 1 transmembrane protein that is released from secretory organelles and relocalized on the sporozoite plasma membrane. TRAP is required for sporozoite motility and host infection, and its extracellular portion contains adhesive domains that are predicted to engage host receptors. Here, we identified the human platelet-derived growth factor receptor ß (hPDGFRß) as one such protein receptor. Deletion constructs showed that the von Willebrand factor type A and thrombospondin repeat domains of TRAP are both required for optimal binding to hPDGFRß-expressing cells. We also demonstrate that this interaction is conserved in the human-infective parasite Plasmodium vivax, but not the rodent-infective parasite Plasmodium yoelii. We observed expression of hPDGFRß mainly in cells associated with the vasculature suggesting that TRAP:hPDGFRß interaction may play a role in the recognition of blood vessels by invading sporozoites.

Transformation of sporozoites into early exoerythrocytic malaria parasites does not require host cells.

Malaria parasite species that infect mammals, including humans, must first take up residence in hepatic host cells as exoerythrocytic forms (EEF) before initiating infection of red blood cells that leads to malaria disease. Despite the importance of hepatic stages for immunity against malaria, little is known about their biology and antigenic composition. Here, we show that sporozoites, the parasites' transmission stage that resides in the mosquito vector salivary glands, can transform into early EEF without intracellular residence in host hepatocytes. The morphological sequence of transformation and the expression of proteins in the EEF appear indistinguishable from parasites that develop within host cells. Transformation depends on temperature elevation to 37 degrees C and serum. Our findings demonstrate that residence in a host hepatocyte or specific host cell-derived factors are not necessary to bring about the profound morphological and biochemical changes of the parasite that occur after its transmission from vector to mammalian host.

Type II fatty acid synthesis is essential only for malaria parasite late liver stage development.

Intracellular malaria parasites require lipids for growth and replication. They possess a prokaryotic type II fatty acid synthesis (FAS II) pathway that localizes to the apicoplast plastid organelle and is assumed to be necessary for pathogenic blood stage replication. However, the importance of FAS II throughout the complex parasite life cycle remains unknown. We show in a rodent malaria model that FAS II enzymes localize to the sporozoite and liver stage apicoplast. Targeted deletion of FabB/F, a critical enzyme in fatty acid synthesis, did not affect parasite blood stage replication, mosquito stage development and initial infection in the liver. This was confirmed by knockout of FabZ, another critical FAS II enzyme. However, FAS II-deficient Plasmodium yoelii liver stages failed to form exo-erythrocytic merozoites, the invasive stage that first initiates blood stage infection. Furthermore, deletion of FabI in the human malaria parasite Plasmodium falciparum did not show a reduction in asexual blood stage replication in vitro. Malaria parasites therefore depend on the intrinsic FAS II pathway only at one specific life cycle transition point, from liver to blood.