Cellular Microbiology

Immediately after invading their chosen host cell, the mature human erythrocyte, malaria parasites begin to export an array of proteins to this compartment, where they initiate processes that are prerequisite for parasite survival and propagation, including nutrient import and immune evasion. One consequence of these activities is the emergence of novel adhesive phenotypes that can lead directly to pathology in the human host. To identify parasite proteins involved in this process we used modern genetic tools to target genes encoding 15 exported parasite proteins, selected by an in-silico workflow. This resulted in 4 genetically modified parasite lines that were then characterised in detail. Of these lines, 3 could be shown to have aberrations in adhesion, and of these 1 appears to have a block in the transport and/or correct folding of the major surface adhesin PfEMP1 (Plasmodium falciparum erythrocyte membrane protein 1). Our data expand the known factors involved in this important process, and once again highlight the complexity of this phenomenon.

Plasmodium falciparum, which causes the most severe malaria, remodels infected erythrocytes by exporting several hundred effector proteins. Parasites express the aspartyl protease plasmepsin V that processes proteins containing a PEXEL motif and the PTEX translocon to successfully export proteins. During liver-stage infection, PTEX is required for P. falciparum development, but which proteins are exported remain unknown; these proteins may serve important biological functions and be presented by MHC-I molecules, thereby representing potential vaccine candidates. Here, we investigated liver stage antigen 3 (LSA3), an immunogenic protein of the Laverania subgenus of Plasmodium. We show that LSA3 possesses a PEXEL motif processed by plasmepsin V and is targeted to one or more membranes surrounding the blood-stage parasite, suggestive of the parasitophorous vacuole membrane (PVM). A subset of LSA3 also localizes in the erythrocyte, where it forms punctate structures that are not Maurers clefts but are soluble in biochemical fractionation assays reminiscent of J-dot proteins. During infection of human hepatocytes, antibodies to LSA3 co-localize with EXP1 and EXP2 at the PVM, yet these antibodies were rarely detected beyond this membrane. Finally, genetic disruption of LSA3 in P. falciparum NF54 attenuated fitness at the liver stage, manifest as a 40% reduction in parasite liver load by day 5 postinfection of humanized mice. The identification of LSA3 as a previously unrecognized member of the P. falciparum exportome, essential for normal liver-stage development and capable of eliciting protective pre-erythrocytic immunity, confirms the hypothesized potential of exported proteins as promising malaria vaccine candidates, underscoring the need for continued investigation into their discovery and biological characterization.

Malaria is an important infectious disease that continues to claim hundreds of thousands of lives annually. The disease is caused by infection of host erythrocytes by apicomplexan parasites of the genus Plasmodium. The parasite contains three different apical organelles - micronemes, rhoptries and dense granules - whose contents are secreted to mediate binding to and invasion of the host cell and the extensive remodelling of the host cell that occurs following invasion. Whereas the roles of micronemes and rhoptries in binding and invasion of the host erythrocyte have been studied in detail, the role of dense granules (DGs) in Plasmodium parasites are poorly understood. They have been proposed to control host cell remodelling through regulated protein secretion after invasion, but many basic aspects of the biology of DGs remain unknown. Here we describe DG biogenesis timing for the first time, using RESA localisation as a proxy for DG formation timing. We show that DG formation commences approximately 37 minutes prior to schizont egress, as measured by the recruitment of the DG marker RESA. Furthermore, using a bioinformatics approach, we aimed to predict additional cargo of the DGs and identified the J-dot protein HSP40 as a DG protein, further supporting the very early role of these organelles in the interaction of the parasite with the host cell.

Apicomplexan parasites such as Plasmodium spp. and Toxoplasma gondii possess unique tubulin-based structures, including subpellicular microtubules and apical polar rings, which are essential for parasite motility, host cell invasion, and replication. How the stability of these structures is maintained is poorly understood, but it may involve Apicortin, a microtubule-associated protein, so-far found only in apicomplexans and the placozoan Trichoplax adhaerens. Apicortin contains a doublecortin (DC) domain and a partial tubulin polymerisation-promoting protein (TPPP) domain, both implicated in microtubule binding and stabilization. In this study, we investigated the location and function of Apicortin in Plasmodium berghei. Live cell imaging of a transgenic parasite line expressing GFP-tagged Apicortin showed that it was present at the apical end of invasive parasites only during development of transmission stages within the mosquito vector. High-resolution imaging using super-resolution and expansion microscopy, revealed that Apicortin forms a distinct ring-like structure in the apical complex region at the apical end of ookinetes and sporozoites. However, deletion of the Apicortin gene had no effect on parasite development and transmission through the mosquito, indicating that this protein is not essential. This suggests that there may be redundancy or compensatory functions in the mechanisms that stabilise the apical complex.

An essential process in transmission of the malaria parasite to the Anopheles vector is the conversion of mature gametocytes into gametes within the mosquito gut, where they egress from the red blood cell (RBC). During egress, male gametocytes undergo exflagellation, leading to the formation of eight haploid motile microgametes, while female gametes retain their spherical shape. Gametocyte egress depends on sequential disruption of the parasitophorous vacuole membrane and the host cell membrane. In other life cycle stages of the malaria parasite, phospholipases have been implicated in membrane disruption processes during egress, however their importance for gametocyte egress is relatively unknown. Here, we performed comprehensive functional analyses of six putative phospholipases for their role during development and egress of Plasmodium falciparum gametocytes. We localize two of them, the prodrug activation and resistance esterase (PF3D7_0709700) and the lysophospholipase 1 (PF3D7_1476700), to the parasite plasma membrane. Subsequently, we show that disruption of most of the studied phospholipase genes does neither affect gametocyte development nor egress. The exception is the putative patatin-like phospholipase PF3D7_0924000, whose gene deletion leads to a delay in male gametocyte exflagellation, indicating an important, albeit not essential, role of this enzyme in male gametogenesis.

Malaria parasites alter multiple properties of the host erythrocyte by exporting proteins into the host cell. Many exported proteins contain a five-amino acid motif called the Plasmodium Export Element (PEXEL) that is cleaved by the parasite protease Plasmepsin V (PM V). The presence of a PEXEL is considered a signature of protein export and has been used to identify a large number of exported proteins. The export of proteins becomes essential midway through the intraerythrocytic cycle - preventing protein export blocks parasite development 18-24 h after invasion. However, a genetic investigation revealed that the absence of the PEXEL protein PFA0210c causes parasite development to arrest immediately after invasion. We now show that this protein (renamed PV6) is cleaved by PM V but not exported into the host erythrocyte and instead functions in the parasitophorous vacuole. Furthermore, we show that the lysine residue that becomes the N terminus of PV6 after processing by PM V prevents export. This is the first example of a native Plasmodium falciparum PM V substrate that remains in the parasitophorous vacuole. We also provide evidence that the parasite produces at least one additional essential, non-exported PM V substrate. These results reveal that the presence of a PEXEL, and hence processing of a protein by PM V, does not always target a protein for export and that PM V has a broader function in parasite growth beyond processing exported proteins. Furthermore, we utilized this finding to investigate possible requirements for protein export further.

The centriole/basal body (CBB) is an evolutionarily conserved organelle acting as a microtubule organising centre (MTOC) to nucleate cilia, flagella and the centrosome. SAS4/CPAP is a conserved component associated with BB biogenesis in many model flagellated cells. Plasmodium, a divergent unicellular eukaryote and causative agent of malaria, displays an atypical closed mitosis with an MTOC, reminiscent of the acentriolar MTOC, embedded in the nuclear membrane at most proliferative stages. Mitosis during male gamete formation is accompanied by flagellum formation: within 15 minutes, genome replication (from 1N to 8N) and three successive rounds of mitosis without nuclear division occur, with coordinated axoneme biogenesis in the cytoplasm resulting in eight flagellated gametes. There are two MTOCs in male gametocytes. An acentriolar MTOC located with the nuclear envelope and a centriolar MTOC (basal body) located within the cytoplasm that are required for flagellum assembly. To study the location and function of SAS4 during this rapid process, we examined the spatial profile of SAS4 in real time by live cell imaging and its function by gene deletion. We show its absence during asexual proliferation but its presence and coordinated association and assembly of SAS4 with another basal body component, kinesin8B, which is involved in axoneme biogenesis. In contrast its separation from the nuclear kinetochore marker NDC80 suggests that SAS4 is part of the basal body and outer centriolar MTOC residing in the cytoplasm. However, deletion of the SAS4 gene produced no phenotype, indicating that it is not essential for male gamete formation or parasite transmission through the mosquito.

Throughout its complex life cycle, the uniflagellate parasitic protist, Trypanosoma cruzi, adapts to different host environments by transitioning between elongated motile extracellular forms and non-motile intracellular amastigote forms that replicate in the cytoplasm of mammalian host cells. Despite their name, intracellular T. cruzi amastigotes retain a short flagellum that extends beyond the opening of the flagellar pocket with access to the extracellular milieu. Contrary to the long-held view that the T. cruzi amastigote flagellum is inert, we now report that this organelle is motile and displays quasiperiodic beating inside mammalian host cells. Kymograph analysis determined an average flagellar beat frequency of ~0.7 Hz for intracellular amastigotes. Similar beat frequencies were measured in extracellular amastigotes following their isolation from host cells. Inhibitor studies reveal roles for parasite mitochondrial respiration and intracellular calcium availability in modulating flagellar beat in T. cruzi amastigotes. Together, these findings demonstrate that flagellar motility is an intrinsic property of T. cruzi amastigotes and suggest that this organelle may play an active role in the parasite infection process. To our knowledge, this is the first record of an intracellular eukaryotic flagellum beating within another eukaryotic cell.

The intraerythrocytic malaria parasite Plasmodium falciparum exports hundreds of proteins into the host red blood cell (RBC). Most are targeted to the ER by a stretch of hydrophobic amino acids and cleaved further downstream at a conserved motif called the Protein Export Element (PEXEL) by the ER protease plasmepsin V (PM V). The mature effectors then travel through the secretory pathway to the parasitophorous vacuole (PV) that surrounds the parasite. There, PEXEL proteins are somehow recognized as export-destined proteins, as opposed to PV- resident proteins, and are selectively translocated out into the RBC. The mature N terminus appears to be important for export. There is conflicting data on whether PM V cleavage is needed for proper export, or whether any means of generating the mature N terminus would suffice. We replaced the PEXEL-containing N-terminal sequence of an exported GFP reporter with a signal peptide sequence and showed that precise cleavage by signal peptidase, generating the proper mature N terminus, yields export competence. Expressing a construct with only the native ER targeting signal without the PM V cleavage site dramatically decreased the amount of a mature PEXEL reporter, indicating that the hydrophobic stretch lacks an efficient cleavage signal. Therefore, the PEXEL motif functions as a specialized signal cleavage site when appropriately located after an ER targeting sequence. Our data suggest that PM V cleavage and RBC export are two independent events for PEXEL proteins. We also tested and rejected the hypothesis that an alpha-helical mature N terminus is necessary for export. ImportanceMalaria parasites export hundreds of proteins to the cytoplasm of the host red blood cells for their survival. A five amino acid sequence, called the PEXEL motif, is conserved among many exported proteins and is thought to be a signal for export. However, the motif is cleaved inside the endoplasmic reticulum of the parasite and mature proteins starting from the fourth PEXEL residue travel to the parasite periphery for export. We showed that the PEXEL motif is dispensable for export as long as identical mature proteins can be efficiently produced via alternative means in the ER. We also showed that the exported and non-exported proteins are differentiated at the parasite periphery based on their mature N termini, however, any discernible export signal within that region remained cryptic. Our study resolves a longstanding paradox in PEXEL protein trafficking.

The single flagellum of African trypanosomes is essential in multiple aspects of the parasite development. The FLAgellar Member 8 protein (FLAM8), localised to the tip of the flagellum in cultured insect forms, was identified as a marker of the locking event that controls flagellum length. Here, we investigated whether FLAM8 could also reflect the flagellum maturation state in other stages. We observed that FLAM8 distribution extended along the entire flagellar cytoskeleton in mammalian infective forms. Then, a rapid FLAM8 concentration to the distal tip occurs during differentiation into early insect forms, illustrating for the first time the remodeling of an existing flagellum in trypanosomes. In the tsetse cardia, FLAM8 further localizes to the entire length of the new flagellum during an asymmetric division. Strikingly, in parasites dividing in the tsetse midgut and in the salivary glands, the amount and distribution of FLAM8 in the new flagellum was seen to predict the daughter cell fate. We propose and discuss how FLAM8 could be considered as a meta-marker of the flagellum stage and maturation state in trypanosomes. Summary statementThe trypanosome protein FLAM8 displays a dynamic and stage-specific distribution during the entire parasite cycle, representing a novel marker of the flagellum stage and maturation state.

Plasmodium sporozoites are the highly polarized and motile forms of the malaria parasite that are transmitted by mosquitoes to the vertebrate hosts. Sporozoites use myosin molecular motors to generate retrograde flow of actin filaments. These are linked to plasma-membrane spanning adhesins, which in turn bind to the extracellular environment, resulting in forward directed gliding motility. The gliding motility machine of sporozoites leads to high speeds in the range of micrometer per second, which are essential for efficient migration in the skin. Yet, it is not clear how the individual parts of the machinery work together to generate force during migration. Sporozoites are elongated and curved cells and move on circular tracks in vitro. Sporozoites lacking the adhesin thrombospondin related anonymous protein (TRAP) like protein, TLP, can still migrate in the skin, but at a lower level. TLP lacking sporozoites generate a lower force on the dorsal (non-substrate facing) surface as measured by laser tweezers. Here we use traction force microscopy to investigate motile sporozoites and the forces they produce during migration on their ventral surface. Both wild type and tlp(-) sporozoites show distinct foci of force generation, but tlp(-) sporozoites generating overall lower forces. Our findings demonstrate that TLP is an important element of the force-generating machinery during sporozoite gliding motility.

The intracellular lifestyle represents a challenge for the rapidly proliferating liver stage Plasmodium parasite. In order to scavenge host resources, Plasmodium has evolved the ability to target and manipulate host cell organelles. Using dynamic fluorescence-based imaging, we show a direct interplay between the pre-erythrocytic stages of Plasmodium berghei and the host cell Golgi during the entire liver stage development. Liver stage schizonts fragment the host cell Golgi into miniaturized stacks, which increases surface interactions with the parasites parasitophorous vacuole membrane. Interference with the host cell Golgi-linked vesicular machinery using specific dominant-negative Arf and Rab GTPases results in developmental arrest and diminished survival of liver stage parasites. Moreover, functional Rab11a is critical for the parasites ability to induce Golgi fragmentation. Altogether, we demonstrate that the structural and functional integrity of the host cell Golgi is necessary for optimal pre-erythrocytic development of P. berghei. The parasite hijacks the hepatocytes Golgi structure to optimize its own intracellular development.

Micronutrient sequestration is a powerful host defence mechanism against intracellular pathogens. Central to this is the iron transporter Nramp1, which actively effluxes iron from phagolysosomes, depriving the engulfed pathogens of this essential element. Leishmania have evolved a clever strategy to counteract this by triggering hepcidin-mediated proteasomal degradation of Nramp1. Interestingly, treatment with Leishmania major conditioned media induced hepcidin upregulation and Nramp1 degradation even in uninfected macrophages, with simultaneous increase in endo/lysosomal iron content. This suggested the involvement of a parasite-derived secretory factor, ultimately leading to the identification of the metalloprotease GP63 as the effector responsible for Nramp1 degradation. Conditioned medium from the GP63 knockout strain (LmGP63-/-) failed to upregulate hepcidin expression or promote Nramp1 degradation. Further, we show that GP63 depletes DICER1 in macrophages, blocking maturation of miR-122, a known negative regulator of hepcidin. These in vitro findings were validated in a mouse model, where the wild type L. major, but not the LmGP63-/- strain, could deplete DICER1, induce hepcidin expression and reduce Nramp1 levels in the infected tissue. Collectively, we uncover a novel role for Leishmania GP63 in targeting the DICER1/miR-122 axis in host macrophages to trigger hepcidin expression and Nramp1 degradation, thereby facilitating iron acquisition by the parasite.

Trypanosoma brucei, the causal agent of Human and Animal African trypanosomiasis proliferates in the extracellular milieu of mammals. It acquires host macromolecular nutrients, by receptor mediated endocytosis. The best characterised cell surface receptor is for transferrin (TfR) and it has been reported to be preferentially localised in the flagellar pocket domain of the plasma membrane, the sole site of endocytosis. In this location the TfR may be inaccessible to adaptive immune system effectors. The T. brucei genome encodes [~]15 TfR variants, and here we compared two, the first attached to the plasma membrane by a single glycosylphosphatidylinositol (GPI)-anchor and the other by two. Transferrin uptake kinetics were similar and rapid for both. Unexpectedly, initial binding of transferrin occurred over the whole cell surface suggesting the TfR was not localised solely in the flagellar pocket. This localisation was confirmed by immunofluorescence assays and was independent of the number of GPI-anchors. Two other GPI-anchored receptors were investigated to determine whether localisation to the whole cell surface was a general property of GPI-anchored receptors. Haptoglobin-haemoglobin uptake assays and immunofluorescence localisation of complement factor H receptor showed both were also whole cell surface localised. The mechanisms by which trypanosome receptors are protected from antibody-mediated attack are more complex than hiding in a pocket.

Cells use fatty acids (FAs) for membrane biosynthesis, energy storage and the generation of signaling molecules. 3-hydroxyacyl-CoA dehydratase - DEH - is a key component of very long chain FA (VLCFA) synthesis. Here, we further characterized in-depth the location and function of DEH, applying in silico analysis, live cell imaging, reverse genetics and ultrastructure analysis using the mouse malaria model Plasmodium berghei. DEH is evolutionarily conserved across eukaryotes, with a single DEH in Plasmodium spp. and up to three orthologs in the other eukaryotes studied. DEH-GFP live-cell imaging showed strong GFP fluorescence throughout the life-cycle, with areas of localized expression in the cytoplasm and a circular ring pattern around the nucleus that colocalized with ER markers. {Delta}deh mutants showed a small but significant reduction in oocyst size compared to WT controls from day 10 post-infection onwards and endomitotic cell division and sporogony were completely ablated, blocking parasite transmission from mosquito to vertebrate host. Ultrastructure analysis confirmed degeneration of {Delta}deh oocysts, and a complete lack of sporozoite budding. Overall, DEH is evolutionarily conserved, localizes to the ER and plays a crucial role in sporogony.

Plasmodium sporozoites are the infective forms of the malaria parasite in the vertebrate host. Gliding motility allows sporozoites to migrate and invade the salivary gland and hepatocytes. Invasion is powered by an actin-myosin motor complex linked to glideosome. However, the gliding complex and the role of several glideosome-associated proteins (GAPs) are poorly understood. In silico analysis of a novel protein, S14, which is uniquely upregulated in salivary gland sporozoites, suggested its association with glideosome-associated proteins. We confirmed S14 expression in sporozoites using real-time PCR. Further, the S14 gene was endogenously tagged with 3XHA-mCherry to study expression and localization. We found its expression and localization on the inner membrane of sporozoites. By targeted gene deletion, we demonstrate that S14 is essential for sporozoite gliding motility, salivary gland, and hepatocyte invasion. The gliding and invasion-deficient S14 KO sporozoites showed normal expression and organization of IMC and surface proteins. Using in silico and the yeast two-hybrid system, we showed the interaction of S14 with the glideosome-associated proteins GAP45 and MTIP. Together, our data show that S14 is a glideosome-associated protein and plays an essential role in sporozoite gliding motility, which is critical for the invasion of the salivary gland, hepatocyte, and malaria transmission.

MicroRNAs (miRNAs) are small non-coding RNAs that can play critical roles in regulating various cellular processes including during many parasitic infections. Here, we report a regulatory role for miR-34c-3p in cAMP-independent regulation of PKA activity in Theileria annulata infection of bovine leukocytes. We identified prkar2b (cAMP-dependent protein kinase A type II-beta regulatory subunit), as a novel miR-34c-3p target gene and demonstrated how infection-induced up-regulation of miR-34c-3p in leukocytes repressed PRKAR2B expression to increase PKA activity and promote the virulent disseminating tumour phenotype of T. annulata-transformed macrophages. Finally, we demonstrate that miR-34c-3p regulation of prkar2b expression is generalizable, by confirming that Plasmodium falciparum infection of red blood cells also raises intracellular levels of miR-34c-3p and show that this negatively regulates host prkar2b expression so increasing PKA activity. Infection-induced increase in miR-34c-3p levels, therefore, represents a novel cAMP-independent way of regulating host cell PKA activity in infections by Theileria and Plasmodium parasites. Abstract ImportanceTheileria and Plasmodium infections of leukocytes and erythrocytes; respectively, lead to an increase in host cell miR-34c-3p levels and we identified prkar2b (cAMP-dependent protein kinase A type II-beta regulatory subunit), as a specific miR-34c-3p target gene. We demonstrate how infection-induced up-regulation of miR-34c-3p repressed PRKAR2B expression to increase PKA activity independent of fluxes in cAMP. Thus, in two different host-parasite combinations infection-induced increase in miR-34c-3p represents a novel epigenetic way of increasing in host PKA activity that contributes to the pathology of disease.

Phosphoinositide metabolism defines the foundation of a major signaling pathway that is conserved throughout the eukaryotic kingdom. The 4-OH phosphorylated phosphoinositides such as phosphatidylinositol-4-phosphate (PtdIns4P) and phosphatidylinositol-4,5-bisphosphate are particularly important molecules as these execute intrinsically essential activities required for the viability of all eukaryotic cells studied thus far. Using intracellular tachyzoites of the apicomplexan parasite Toxoplasma gondii as model for assessing primordial roles for PtdIns4P signaling, we demonstrate the presence of PtdIns4P pools in Golgi/trans-Golgi (TGN) system and in post-TGN compartments of the parasite. Moreover, we show that deficits in PtdIns4P signaling result in structural perturbation of compartments that house dense granule cargo with accompanying deficits in dense granule exocytosis. Taken together, the data report a direct role for PtdIns4P in dense granule biogenesis and exocytosis. The data further indicate that the biogenic pathway for secretion-competent dense granule formation in T. gondii is more complex than simple budding of fully matured dense granules from the TGN.

Artemisinin (ART) is a quick-killing and effective antimalarial activated by the haem derived from haemoglobin digestion. Mutations in the parasites Kelch 13 (K13) protein compromise the efficacy of this drug. Recent studies indicate an undefined role for K13 in haemoglobin uptake. Here, we show that K13 is associated with the collar that constricts cytostomal invaginations required for the parasite to ingest host cytosol. Induced mislocalisation of K13 led to the formation of atypical invaginations lacking the cytostomal ring and constricted neck normally associated with cytostomes. Moreover, the levels of haemoglobin degradation products, haem and haemozoin, are decreased when K13 is inactivated. Our findings demonstrate that K13 is required for normal formation and/or stabilisation of the cytostome, and thereby the parasites uptake of haemoglobin. This is consistent with perturbation of K13 function leading to decreased activation of ART and consequently, reduced killing. Significance StatementArtemisinin-resistant parasites contain mutations in the gene encoding the Kelch 13 protein (K13). How K13 mutations result in artemisinin resistance is unclear. Here, we present evidence that normal K13 is required for the formation of the cytostome, a specialised parasite feeding apparatus used to endocytose host cell haemoglobin. Our results suggest that artemisinin resistance is due to a decrease in artemisinin activation brought about by a decrease in efficiency of haemoglobin uptake and consequently reduced production of haem.

Kinesin-5 motors play essential roles in spindle apparatus assembly during cell division, by generating forces to establish and maintain the spindle bipolarity essential for proper chromosome segregation. Kinesin-5 is largely conserved structurally and functionally in model eukaryotes, but its role is unknown in the Plasmodium parasite, an evolutionarily divergent organism with several atypical features of both mitotic and meiotic cell division. We have investigated the function and subcellular location of kinesin-5 during cell division throughout the Plasmodium berghei life cycle. Deletion of kinesin-5 had little visible effect at any proliferative stage except sporozoite production in oocysts, resulting in a significant decrease in the number of motile sporozoites in mosquito salivary glands, which were able to infect a new vertebrate host. Live-cell imaging showed kinesin-5-GFP located on the spindle and at spindle poles during both atypical mitosis and meiosis. Fixed-cell immunofluorescence assays revealed kinesin-5 co-localized with -tubulin and centrin-2 and a partial overlap with kinetochore marker NDC80 during early blood stage schizogony. Dual-colour live-cell imaging showed that kinesin-5 is closely associated with NDC80 during male gametogony, but not with kinesin-8B, a marker of the basal body and axonemes of the forming flagella. Treatment of gametocytes with microtubule-specific inhibitors confirmed kinesin-5 association with nuclear spindles and not cytoplasmic axonemal microtubules. Altogether, our results demonstrate that kinesin-5 is associated with the spindle apparatus, expressed in proliferating parasite stages, and important for efficient production of infectious sporozoites.