Safety, infectivity and immunogenicity of a genetically attenuated blood-stage malaria vaccine.

BACKGROUND: There is a clear need for novel approaches to malaria vaccine development. We aimed to develop a genetically attenuated blood-stage vaccine and test its safety, infectivity, and immunogenicity in healthy volunteers. Our approach was to target the gene encoding the knob-associated histidine-rich protein (KAHRP), which is responsible for the assembly of knob structures at the infected erythrocyte surface. Knobs are required for correct display of the polymorphic adhesion ligand P. falciparum erythrocyte membrane protein 1 (PfEMP1), a key virulence determinant encoded by a repertoire of var genes. METHODS: The gene encoding KAHRP was deleted from P. falciparum 3D7 and a master cell bank was produced in accordance with Good Manufacturing Practice. Eight malaria naïve males were intravenously inoculated (day 0) with 1800 (2 subjects), 1.8 × 105 (2 subjects), or 3 × 106 viable parasites (4 subjects). Parasitemia was measured using qPCR; immunogenicity was determined using standard assays. Parasites were rescued into culture for in vitro analyses (genome sequencing, cytoadhesion assays, scanning electron microscopy, var gene expression). RESULTS: None of the subjects who were administered with 1800 or 1.8 × 105 parasites developed parasitemia; 3/4 subjects administered 3× 106 parasites developed significant parasitemia, first detected on days 13, 18, and 22. One of these three subjects developed symptoms of malaria simultaneously with influenza B (day 17; 14,022 parasites/mL); one subject developed mild symptoms on day 28 (19,956 parasites/mL); and one subject remained asymptomatic up to day 35 (5046 parasites/mL). Parasitemia rapidly cleared with artemether/lumefantrine. Parasitemia induced a parasite-specific antibody and cell-mediated immune response. Parasites cultured ex vivo exhibited genotypic and phenotypic properties similar to inoculated parasites, although the var gene expression profile changed during growth in vivo. CONCLUSIONS: This study represents the first clinical investigation of a genetically attenuated blood-stage human malaria vaccine. A P. falciparum 3D7 kahrp- strain was tested in vivo and found to be immunogenic but can lead to patent parasitemia at high doses. TRIAL REGISTRATION: Australian New Zealand Clinical Trials Registry (number: ACTRN12617000824369 ; date: 06 June 2017).

Development and evaluation of a new Plasmodium falciparum 3D7 blood stage malaria cell bank for use in malaria volunteer infection studies.

BACKGROUND: New anti-malarial therapeutics are required to counter the threat of increasing drug resistance. Malaria volunteer infection studies (VIS), particularly the induced blood stage malaria (IBSM) model, play a key role in accelerating anti-malarial drug development. Supply of the reference 3D7-V2 Plasmodium falciparum malaria cell bank (MCB) is limited. This study aimed to develop a new MCB, and compare the safety and infectivity of this MCB with the existing 3D7-V2 MCB, in a VIS. A second bank (3D7-V1) developed in 1995 was also evaluated. METHODS: The 3D7-V2 MCB was expanded in vitro using a bioreactor to produce a new MCB designated 3D7-MBE-008. This bank and 3D7-V1 were then evaluated using the IBSM model, where healthy participants were intravenously inoculated with blood-stage parasites. Participants were treated with artemether-lumefantrine when parasitaemia or clinical thresholds were reached. Safety, infectivity and parasite growth and clearance were evaluated. RESULTS: The in vitro expansion of 3D7-V2 produced 200 vials of the 3D7-MBE-008 MCB, with a parasitaemia of 4.3%. This compares to 0.1% in the existing 3D7-V2 MCB, and < 0.01% in the 3D7-V1 MCB. All four participants (two per MCB) developed detectable P. falciparum infection after inoculation with approximately 2800 parasites. For the 3D7-MBE-008 MCB, the parasite multiplication rate of 48 h (PMR48) using non-linear mixed effects modelling was 34.6 (95% CI 18.5-64.6), similar to the parental 3D7-V2 line; parasitaemia in both participants exceeded 10,000/mL by day 8. Growth of the 3D7-V1 was slower (PMR48 of 11.5 [95% CI 8.5-15.6]), with parasitaemia exceeding 10,000 parasites/mL on days 10 and 8.5. Rapid parasite clearance followed artemether-lumefantrine treatment in all four participants, with clearance half-lives of 4.01 and 4.06 (weighted mean 4.04 [95% CI 3.61-4.57]) hours for 3D7-MBE-008 and 4.11 and 4.52 (weighted mean 4.31 [95% CI 4.16-4.47]) hours for 3D7-V1. A total of 59 adverse events occurred; most were of mild severity with three being severe in the 3D7-MBE-008 study. CONCLUSION: The safety, growth and clearance profiles of the expanded 3D7-MBE-008 MCB closely resemble that of its parent, indicating its suitability for future studies. TRIAL REGISTRATION: Australian New Zealand Clinical Trials registry numbers: P3487 (3D7-V1): ACTRN12619001085167. P3491 (3D7-MBE-008): ACTRN12619001079134.

Anti-Giardia Drug Discovery: Current Status and Gut Feelings.

Giardia parasites are ubiquitous protozoans of global importance that impact a wide range of animals including humans. They are the most common enteric pathogen of cats and dogs in developed countries and infect ∼1 billion people worldwide. While Giardia infections can be asymptomatic, they often result in severe and chronic diseases. There is also mounting evidence that they are linked to postinfection disorders. Despite growing evidence of the widespread morbidity associated with Giardia infections, current treatment options are limited to compound classes with broad antimicrobial activity. Frontline anti-Giardia drugs are also associated with increasing drug resistance and treatment failures. To improve the health and well-being of millions, new selective anti-Giardia drugs are needed alongside improved health education initiatives. Here we discuss current treatment options together with recent advances and gaps in drug discovery. We also propose criteria to guide the discovery of new anti-Giardia compounds.

Identification of Antimalarial Inhibitors Using Late-Stage Gametocytes in a Phenotypic Live/Dead Assay.

Malaria remains a major cause of morbidity and mortality worldwide with ~3.3 billion people at risk of contracting malaria and an estimated 450,000 deaths each year. While tools to reduce the infection prevalence to low levels are currently under development, additional efforts will be required to interrupt transmission. Transmission between human host and vector by the malaria parasite involves gametogenesis in the host and uptake of gametocytes by the mosquito vector. This stage is a bottleneck for reproduction of the parasite, making it a target for small-molecule drug discovery. Targeting this stage, we used whole Plasmodium falciparum gametocytes from in vitro culture and implemented them into 1536-well plates to create a live/dead phenotypic antigametocyte assay. Using specialized equipment and upon further validation, we screened ~150,000 compounds from the NIH repository currently housed at Scripps Florida. We identified 100 primary screening hits that were tested for concentration response. Additional follow-up studies to determine specificity, potency, and increased efficacy of the antigametocyte candidate compounds resulted in a starting point for initial medicinal chemistry intervention. From this, 13 chemical analogs were subsequently tested as de novo powders, which confirmed original activity from the initial analysis and now provide a point of future engagement.

A bioreactor system for the manufacture of a genetically modified Plasmodium falciparum blood stage malaria cell bank for use in a clinical trial.

BACKGROUND: Although the use of induced blood stage malaria infection has proven to be a valuable tool for testing the efficacy of vaccines and drugs against Plasmodium falciparum, a limiting factor has been the availability of Good Manufacturing Practice (GMP)-compliant defined P. falciparum strains for in vivo use. The aim of this study was to develop a cost-effective method for the large-scale production of P. falciparum cell banks suitable for use in clinical trials. METHODS: Genetically-attenuated parasites (GAP) were produced by targeted deletion of the gene encoding the knob associated histidine rich protein (kahrp) from P. falciparum strain 3D7. A GAP master cell bank (MCB) was manufactured by culturing parasites in an FDA approved single use, closed system sterile plastic bioreactor. All components used to manufacture the MCB were screened to comply with standards appropriate for in vivo use. The cryopreserved MCB was subjected to extensive testing to ensure GMP compliance for a phase 1 investigational product. RESULTS: Two hundred vials of the GAP MCB were successfully manufactured. At harvest, the GAP MCB had a parasitaemia of 6.3%, with 96% of parasites at ring stage. Testing confirmed that all release criteria were met (sterility, absence of viral contaminants and endotoxins, parasite viability following cryopreservation, identity and anti-malarial drug sensitivity of parasites). CONCLUSION: Large-scale in vitro culture of P. falciparum parasites using a wave bioreactor can be achieved under GMP-compliant conditions. This provides a cost-effective methodology for the production of malaria parasites suitable for administration in clinical trials.

The development of sexual stage malaria gametocytes in a Wave Bioreactor.

BACKGROUND: Blocking malaria gametocyte development in RBCs or their fertilization in the mosquito gut can prevent infection of the mosquito vector and passage of disease to the human host. A 'transmission blocking' strategy is a component of future malaria control. However, the lack of robust culture systems for producing large amounts of Plasmodium falciparum gametocytes has limited our understanding of sexual-stage malaria biology and made vaccine or chemotherapeutic discoveries more difficult. METHODS: The Wave BioreactorTM 20/50 EHT culture system was used to develop a convenient and low-maintenance protocol for inducing commitment of P. falciparum parasites to gametocytogenesis. Culture conditions were optimised to obtain mature stage V gametocytes within 2 weeks in a large-scale culture of up to a 1 l. RESULTS: We report a simple method for the induction of gametocytogenesis with N-acetylglucosamine (10 mM) within a Wave Bioreactor. By maintaining the culture for 14-16 days as many as 100 million gametocytes (stage V) were produced in a 1 l culture. Gametocytes isolated using magnetic activated cell sorting (MACS) columns were frozen in aliquots for storage. These were revitalised by thawing and shown to retain their ability to exflagellate and infect mosquitoes (Anopheles stephansi). CONCLUSIONS: The production of gametocytes in the Wave Bioreactor under GMP-compliant conditions will not only facilitate cellular, developmental and molecular studies of gametocytes, but also the high-throughput screening for new anti-malarial drugs and, possibly, the development of whole-cell gametocyte or sporozoite-based vaccines.

Infectivity of Plasmodium falciparum in Malaria-Naive Individuals Is Related to Knob Expression and Cytoadherence of the Parasite.

Plasmodium falciparum is the most virulent human malaria parasite because of its ability to cytoadhere in the microvasculature. Nonhuman primate studies demonstrated relationships among knob expression, cytoadherence, and infectivity. This has not been examined in humans. Cultured clinical-grade P. falciparum parasites (NF54, 7G8, and 3D7B) and ex vivo-derived cell banks were characterized. Knob and knob-associated histidine-rich protein expression, CD36 adhesion, and antibody recognition of parasitized erythrocytes (PEs) were evaluated. Parasites from the cell banks were administered to malaria-naive human volunteers to explore infectivity. For the NF54 and 3D7B cell banks, blood was collected from the study participants for in vitro characterization. All parasites were infective in vivo However, infectivity of NF54 was dramatically reduced. In vitro characterization revealed that unlike other cell bank parasites, NF54 PEs lacked knobs and did not cytoadhere. Recognition of NF54 PEs by immune sera was observed, suggesting P. falciparum erythrocyte membrane protein 1 expression. Subsequent recovery of knob expression and CD36-mediated adhesion were observed in PEs derived from participants infected with NF54. Knobless cell bank parasites have a dramatic reduction in infectivity and the ability to adhere to CD36. Subsequent infection of malaria-naive volunteers restored knob expression and CD36-mediated cytoadherence, thereby showing that the human environment can modulate virulence.

A Plasmodium falciparum S33 proline aminopeptidase is associated with changes in erythrocyte deformability.

Infection with the apicomplexan parasite Plasmodium falciparum is a major cause of morbidity and mortality worldwide. One of the striking features of this parasite is its ability to remodel and decrease the deformability of host red blood cells, a process that contributes to disease. To further understand the virulence of Pf we investigated the biochemistry and function of a putative Pf S33 proline aminopeptidase (PfPAP). Unlike other P. falciparum aminopeptidases, PfPAP contains a predicted protein export element that is non-syntenic with other human infecting Plasmodium species. Characterization of PfPAP demonstrated that it is exported into the host red blood cell and that it is a prolyl aminopeptidase with a preference for N-terminal proline substrates. In addition genetic deletion of this exopeptidase was shown to lead to an increase in the deformability of parasite-infected red cells and in reduced adherence to the endothelial cell receptor CD36 under flow conditions. Our studies suggest that PfPAP plays a role in the rigidification and adhesion of infected red blood cells to endothelial surface receptors, a role that may make this protein a novel target for anti-disease interventions strategies.

Linking Murine and Human Plasmodium falciparum Challenge Models in a Translational Path for Antimalarial Drug Development.

Effective progression of candidate antimalarials is dependent on optimal dosing in clinical studies, which is determined by a sound understanding of pharmacokinetics and pharmacodynamics (PK/PD). Recently, two important translational models for antimalarials have been developed: the NOD/SCID/IL2Rγ(-/-) (NSG) model, whereby mice are engrafted with noninfected and Plasmodium falciparum-infected human erythrocytes, and the induced blood-stage malaria (IBSM) model in human volunteers. The antimalarial mefloquine was used to directly measure the PK/PD in both models, which were compared to previously published trial data for malaria patients. The clinical part was a single-center, controlled study using a blood-stage Plasmodium falciparum challenge inoculum in volunteers to characterize the effectiveness of mefloquine against early malaria. The study was conducted in three cohorts (n = 8 each) using different doses of mefloquine. The characteristic delay in onset of action of about 24 h was seen in both NSG and IBSM systems. In vivo 50% inhibitory concentrations (IC50s) were estimated at 2.0 µg/ml and 1.8 µg/ml in the NSG and IBSM models, respectively, aligning with 1.8 µg/ml reported previously for patients. In the IBSM model, the parasite reduction ratios were 157 and 195 for the 10- and 15-mg/kg doses, within the range of previously reported clinical data for patients but significantly lower than observed in the mouse model. Linking mouse and human challenge models to clinical trial data can accelerate the accrual of critical data on antimalarial drug activity. Such data can guide large clinical trials required for development of urgently needed novel antimalarial combinations. (This trial was registered at the Australian New Zealand Clinical Trials Registry [http://anzctr.org.au] under registration number ACTRN12612000323820.).

A repeat sequence domain of the ring-exported protein-1 of Plasmodium falciparum controls export machinery architecture and virulence protein trafficking.

The malaria parasite Plasmodium falciparum dramatically remodels its host red blood cell to enhance its own survival, using a secretory membrane system that it establishes outside its own cell. Cisternal organelles, called Maurer's clefts, act as a staging point for the forward trafficking of virulence proteins to the red blood cell (RBC) membrane. The Ring-EXported Protein-1 (REX1) is a Maurer's cleft resident protein. We show that inducible knockdown of REX1 causes stacking of Maurer's cleft cisternae without disrupting the organization of the knob-associated histidine-rich protein at the RBC membrane. Genetic dissection of the REX1 sequence shows that loss of a repeat sequence domain results in the formation of giant Maurer's cleft stacks. The stacked Maurer's clefts are decorated with tether-like structures and retain the ability to dock onto the RBC membrane skeleton. The REX1 mutant parasites show deficient export of the major virulence protein, PfEMP1, to the red blood cell surface and markedly reduced binding to the endothelial cell receptor, CD36. REX1 is predicted to form a largely α-helical structure, with a repetitive charge pattern in the repeat sequence domain, providing potential insights into the role of REX1 in Maurer's cleft sculpting.

Development of cultured Plasmodium falciparum blood-stage malaria cell banks for early phase in vivo clinical trial assessment of anti-malaria drugs and vaccines.

BACKGROUND: The ability to undertake controlled human malaria infection (CHMI) studies for preliminary evaluation of malaria vaccine candidates and anti-malaria drug efficacy has been limited by the need for access to sporozoite infected mosquitoes, aseptic, purified, cryopreserved sporozoites or blood-stage malaria parasites derived ex vivo from malaria infected individuals. Three different strategies are described for the manufacture of clinical grade cultured malaria cell banks suitable for use in CHMI studies. METHODS: Good Manufacturing Practices (GMP)-grade Plasmodium falciparum NF54, clinically isolated 3D7, and research-grade P. falciparum 7G8 blood-stage malaria parasites were cultured separately in GMP-compliant facilities using screened blood components and then cryopreserved to produce three P. falciparum blood-stage malaria cell banks. These cell banks were evaluated according to specific criteria (parasitaemia, identity, viability, sterility, presence of endotoxin, presence of mycoplasma or other viral agents and in vitro anti-malarial drug sensitivity of the cell bank malaria parasites) to ensure they met the criteria to permit product release according to GMP requirements. RESULTS: The P. falciparum NF54, 3D7 and 7G8 cell banks consisted of >78% ring stage parasites with a ring stage parasitaemia of >1.4%. Parasites were viable in vitro following thawing. The cell banks were free from contamination with bacteria, mycoplasma and a broad panel of viruses. The P. falciparum NF54, 3D7 and 7G8 parasites exhibited differential anti-malarial drug susceptibilities. The P. falciparum NF54 and 3D7 parasites were susceptible to all anti-malaria compounds tested, whereas the P. falciparum 7G8 parasites were resistant/had decreased susceptibility to four compounds. Following testing, all defined release criteria were met and the P. falciparum cell banks were deemed suitable for release. Ethical approval has been obtained for administration to human volunteers. CONCLUSIONS: The production of cultured P. falciparum blood-stage malaria cell banks represents a suitable approach for the generation of material suitable for CHMI studies. A key feature of this culture-based approach is the ability to take research-grade material through to a product suitable for administration in clinical trials.

Lysine acetylation in sexual stage malaria parasites is a target for antimalarial small molecules.

Therapies to prevent transmission of malaria parasites to the mosquito vector are a vital part of the global malaria elimination agenda. Primaquine is currently the only drug with such activity; however, its use is limited by side effects. The development of transmission-blocking strategies requires an understanding of sexual stage malaria parasite (gametocyte) biology and the identification of new drug leads. Lysine acetylation is an important posttranslational modification involved in regulating eukaryotic gene expression and other essential processes. Interfering with this process with histone deacetylase (HDAC) inhibitors is a validated strategy for cancer and other diseases, including asexual stage malaria parasites. Here we confirm the expression of at least one HDAC protein in Plasmodium falciparum gametocytes and show that histone and nonhistone protein acetylation occurs in this life cycle stage. The activity of the canonical HDAC inhibitors trichostatin A (TSA) and suberoylanilide hydroxamic acid (SAHA; Vorinostat) and a panel of novel HDAC inhibitors on early/late-stage gametocytes and on gamete formation was examined. Several compounds displayed early/late-stage gametocytocidal activity, with TSA being the most potent (50% inhibitory concentration, 70 to 90 nM). In contrast, no inhibitory activity was observed in P. falciparum gametocyte exflagellation experiments. Gametocytocidal HDAC inhibitors caused hyperacetylation of gametocyte histones, consistent with a mode of action targeting HDAC activity. Our data identify HDAC inhibitors as being among a limited number of compounds that target both asexual and sexual stage malaria parasites, making them a potential new starting point for gametocytocidal drug leads and valuable tools for dissecting gametocyte biology.

Identification of Potent and Selective Inhibitors of the Plasmodium falciparum M18 Aspartyl Aminopeptidase (PfM18AAP) of Human Malaria via High-Throughput Screening.

The target of this study, the PfM18 aspartyl aminopeptidase (PfM18AAP), is the only AAP present in the genome of the malaria parasite Plasmodium falciparum. PfM18AAP is a metallo-exopeptidase that exclusively cleaves N-terminal acidic amino acids glutamate and aspartate. It is expressed in parasite cytoplasm and may function in concert with other aminopeptidases in protein degradation, of, for example, hemoglobin. Previous antisense knockdown experiments identified a lethal phenotype associated with PfM18AAP, suggesting that it is a valid target for new antimalaria therapies. To identify inhibitors of PfM18AAP function, a fluorescence enzymatic assay was developed using recombinant PfM18AAP enzyme and a fluorogenic peptide substrate (H-Glu-NHMec). This was screened against the Molecular Libraries Probe Production Centers Network collection of ~292,000 compounds (the Molecular Libraries Small Molecule Repository). A cathepsin L1 (CTSL1) enzyme-based assay was developed and used as a counter screen to identify compounds with nonspecific activity. Enzymology and phenotypic assays were used to determine mechanism of action and efficacy of selective and potent compounds identified from high-throughput screening. Two structurally related compounds, CID 6852389 and CID 23724194, yielded micromolar potency and were inactive in CTSL1 titration experiments (IC50>59.6 µM). As measured by the K(i) assay, both compounds demonstrated micromolar noncompetitive inhibition in the PfM18AAP enzyme assay. Both CID 6852389 and CID 23724194 demonstrated potency in malaria growth assays (IC504 µM and 1.3 µM, respectively).

Plasmodium gametocyte inhibition identified from a natural-product-based fragment library.

Fragment-based screening is commonly used to identify compounds with relatively weak but efficient localized binding to protein surfaces. We used mass spectrometry to study fragment-sized three-dimensional natural products. We identified seven securinine-related compounds binding to Plasmodium falciparum 2'-deoxyuridine 5'-triphosphate nucleotidohydrolase (PfdUTPase). Securinine bound allosterically to PfdUTPase, enhancing enzyme activity and inhibiting viability of both P. falciparum gametocyte (sexual) and blood (asexual) stage parasites. Our results provide a new insight into mechanisms that may be applicable to transmission-blocking agents.

Plasmodium falciparum gametocytes: with a view to a kill.

Drugs that kill or inhibit the sexual stages of Plasmodium in order to prevent transmission are important components of malaria control programmes. Reducing gametocyte carriage is central to the control of Plasmodium falciparum transmission as infection can result in extended periods of gametocytaemia. Unfortunately the number of drugs with activity against gametocytes is limited. Primaquine is currently the only licensed drug with activity against the sexual stages of malaria parasites and its use is hampered by safety concerns. This shortcoming is likely the result of the technical challenges associated with gametocyte studies together with the focus of previous drug discovery campaigns on asexual parasite stages. However recent emphasis on malaria eradication has resulted in an upsurge of interest in identifying compounds with activity against gametocytes. This review examines the gametocytocidal properties of currently available drugs as well as those in the development pipeline and examines the prospects for discovery of new anti-gametocyte compounds.

Experimentally induced blood-stage Plasmodium vivax infection in healthy volunteers.

BACKGROUND: Major impediments to development of vaccines and drugs for Plasmodium vivax malaria are the inability to culture this species and the extreme difficulty in undertaking clinical research by experimental infection. METHODS: A parasite bank was collected from a 49-year-old woman with P. vivax infection, characterized, and used in an experimental infection study. RESULTS: The donor made a full recovery from malaria after collection of a parasite bank, which tested negative for agents screened for in blood donations. DNA sequence analysis of the isolate indicated that it was clonal. Two subjects inoculated with the isolate became polymerase chain reaction positive on days 8 and 9, with onset of symptoms and positive blood smears on day 14, when they were treated with artemether-lumefantrine, with rapid clinical and parasitologic response. Transcripts of the parasite gene pvs25 that is expressed in gametocytes, the life cycle stage infectious to mosquitoes, were first detected on days 11 and 12. CONCLUSIONS: This experimental system results in in vivo parasite growth, probably infectious to mosquitoes. It offers the opportunity to undertake studies previously impossible in P. vivax that will facilitate a better understanding of the pathology of vivax malaria and development of antimalarial drugs and vaccines. Trial Registration. ANZCTR: 12612001096842.

Enhanced gametocyte formation in erythrocyte progenitor cells: a site-specific adaptation by Plasmodium falciparum.

Gametocytogenesis by Plasmodium falciparum is essential for transmission of the parasite from human to mosquito, yet developing gametocytes lack expression of surface proteins required for cytoadherence. Therefore, elimination from the circulation should occur unless they are sequestered in regions of low blood flow such as the extracellular spaces of the bone marrow. Our data indicate that gametocytogenesis is enhanced in the presence of erythroid progenitors found within the bone marrow. Furthermore, atomic force microscopy indicates that developing gametocytes undergo remarkable shifts in their erythrocyte membrane elasticity, which may allow them to be retained within the bone marrow until maturation.

Temporal evaluation of commitment to sexual development in Plasmodium falciparum.

BACKGROUND: The production of gametocytes is essential for transmission of malaria parasites from the mammalian host to the mosquito vector. However the process by which the asexual blood-stage parasite undergoes commitment to sexual development is not well understood. This process is known to be sensitive to environmental stimuli and it has been suggested that a G protein dependent system may mediate the switch, but there is little evidence that the Plasmodium falciparum genome encodes heterotrimeric G proteins. Previous studies have indicated that the malaria parasite can interact with endogenous erythrocyte G proteins, and other components of the cyclic nucleotide pathway have been identified in P. falciparum. Also, the polypeptide cholera toxin, which induces commitment to gametocytogenesis is known to catalyze the ADP-ribosylation of the α(s) class of heterotrimeric G protein α subunits in mammalian systems has been reported to detect a number of G(α) subunits in P. falciparum-infected red cells. METHODS: Cholera toxin and Mas 7 (a structural analogue of Mastoparan) were used to assess the role played by putative G protein signalling in the commitment process, both are reported to interact with different components of classical Gas and Gai/o signalling pathways. Their ability to induce gametocyte production in the transgenic P. falciparum line Pfs16-GFP was determined and downstream effects on the secondary messenger cAMP measured. RESULTS: Treatment of parasite cultures with either cholera toxin or MAS 7 resulted in increased gametocyte production, but only treatment with MAS 7 resulted in a significant increase in cAMP levels. This indicates that MAS 7 acts either directly or indirectly on the P. falciparum adenylyl cyclase. CONCLUSION: The observation that cholera toxin treatment did not affect cAMP levels indicates that while addition of cholera toxin does increase gametocytogenesis the method by which it induces increased commitment is not immediately obvious, except that is unlikely to be via heterotrimeric G proteins.

X-ray crystal structure and specificity of the Plasmodium falciparum malaria aminopeptidase PfM18AAP.

The malarial aminopeptidases have emerged as promising new drug targets for the development of novel antimalarial drugs. The M18AAP of Plasmodium falciparum malaria is a metallo-aminopeptidase that we show demonstrates a highly restricted specificity for peptides with an N-terminal Glu or Asp residue. Thus, the enzyme may function alongside other aminopeptidases in effecting the complete degradation or turnover of proteins, such as host hemoglobin, which provides a free amino acid pool for the growing parasite. Inhibition of PfM18AAP's function using antisense RNA is detrimental to the intra-erythrocytic malaria parasite and, hence, it has been proposed as a potential novel drug target. We report the X-ray crystal structure of the PfM18AAP aminopeptidase and reveal its complex dodecameric assembly arranged via dimer and trimer units that interact to form a large tetrahedron shape that completely encloses the 12 active sites within a central cavity. The four entry points to the catalytic lumen are each guarded by 12 large flexible loops that could control substrate entry into the catalytic sites. PfM18AAP thus resembles a proteasomal-like machine with multiple active sites able to degrade peptide substrates that enter the central lumen. The Plasmodium enzyme shows significant structural differences around the active site when compared to recently determined structures of its mammalian and human homologs, which provides a platform from which a rational approach to inhibitor design of new malaria-specific drugs can begin.