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    Safety, infectivity and immunogenicity of a Chick for genetically attenuated blood-stage malaria updates vaccine
    Webster, R ; Sekuloski, S ; Odedra, A ; Woolley, S ; Jennings, H ; Amante, F ; Trenholme, KR ; Healer, J ; Cowman, AF ; Eriksson, EM ; Sathe, P ; Penington, J ; Blanch, AJ ; Dixon, MWA ; Tilley, L ; Duffy, MF ; Craig, A ; Storm, J ; Chan, J-A ; Evans, K ; Papenfuss, AT ; Schofield, L ; Griffin, P ; Barber, BE ; Andrew, D ; Boyle, MJ ; Rivera, FDL ; Engwerda, C ; McCarthy, JS (BMC, 2021-11-22)
    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).
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    Dual Plasmepsin-Targeting Antimalarial Agents Disrupt Multiple Stages of the Malaria Parasite Life Cycle
    Favuzza, P ; Ruiz, MDL ; Thompson, JK ; Triglia, T ; Ngo, A ; Steel, RWJ ; Vavrek, M ; Christensen, J ; Healer, J ; Boyce, C ; Guo, Z ; Hu, M ; Khan, T ; Murgolo, N ; Zhao, L ; Penington, JS ; Reaksudsan, K ; Jarman, K ; Dietrich, MH ; Richardson, L ; Guo, K-Y ; Lopaticki, S ; Tham, W-H ; Rottmann, M ; Papenfuss, T ; Robbins, JA ; Boddey, JA ; Sleebs, BE ; Sabroux, HJ ; McCauley, JA ; Olsen, DB ; Cowman, AF (CELL PRESS, 2020-04-08)
    Artemisin combination therapy (ACT) is the main treatment option for malaria, which is caused by the intracellular parasite Plasmodium. However, increased resistance to ACT highlights the importance of finding new drugs. Recently, the aspartic proteases Plasmepsin IX and X (PMIX and PMX) were identified as promising drug targets. In this study, we describe dual inhibitors of PMIX and PMX, including WM382, that block multiple stages of the Plasmodium life cycle. We demonstrate that PMX is a master modulator of merozoite invasion and direct maturation of proteins required for invasion, parasite development, and egress. Oral administration of WM382 cured mice of P. berghei and prevented blood infection from the liver. In addition, WM382 was efficacious against P. falciparum asexual infection in humanized mice and prevented transmission to mosquitoes. Selection of resistant P. falciparum in vitro was not achievable. Together, these show that dual PMIX and PMX inhibitors are promising candidates for malaria treatment and prevention.
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    A newly discovered protein export machine in malaria parasites
    de Koning-Ward, TF ; Gilson, PR ; Boddey, JA ; Rug, M ; Smith, BJ ; Papenfuss, AT ; Sanders, PR ; Lundie, RJ ; Maier, AG ; Cowman, AF ; Crabb, BS (NATURE PORTFOLIO, 2009-06-18)
    Several hundred malaria parasite proteins are exported beyond an encasing vacuole and into the cytosol of the host erythrocyte, a process that is central to the virulence and viability of the causative Plasmodium species. The trafficking machinery responsible for this export is unknown. Here we identify in Plasmodium falciparum a translocon of exported proteins (PTEX), which is located in the vacuole membrane. The PTEX complex is ATP-powered, and comprises heat shock protein 101 (HSP101; a ClpA/B-like ATPase from the AAA+ superfamily, of a type commonly associated with protein translocons), a novel protein termed PTEX150 and a known parasite protein, exported protein 2 (EXP2). EXP2 is the potential channel, as it is the membrane-associated component of the core PTEX complex. Two other proteins, a new protein PTEX88 and thioredoxin 2 (TRX2), were also identified as PTEX components. As a common portal for numerous crucial processes, this translocon offers a new avenue for therapeutic intervention.
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    Molecular genetics and comparative genomics reveal RNAi is not functional in malaria parasites
    Baum, J ; Papenfuss, AT ; Mair, GR ; Janse, CJ ; Vlachou, D ; Waters, AP ; Cowman, AF ; Crabb, BS ; de Koning-Ward, TF (OXFORD UNIV PRESS, 2009-06)
    Techniques for targeted genetic disruption in Plasmodium, the causative agent of malaria, are currently intractable for those genes that are essential for blood stage development. The ability to use RNA interference (RNAi) to silence gene expression would provide a powerful means to gain valuable insight into the pathogenic blood stages but its functionality in Plasmodium remains controversial. Here we have used various RNA-based gene silencing approaches to test the utility of RNAi in malaria parasites and have undertaken an extensive comparative genomics search using profile hidden Markov models to clarify whether RNAi machinery exists in malaria. These investigative approaches revealed that Plasmodium lacks the enzymology required for RNAi-based ablation of gene expression and indeed no experimental evidence for RNAi was observed. In its absence, the most likely explanations for previously reported RNAi-mediated knockdown are either the general toxicity of introduced RNA (with global down-regulation of gene expression) or a specific antisense effect mechanistically distinct from RNAi, which will need systematic analysis if it is to be of use as a molecular genetic tool for malaria parasites.
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    A Novel Family of Apicomplexan Glideosome-associated Proteins with an Inner Membrane-anchoring Role
    Bullen, HE ; Tonkin, CJ ; O'Donnell, RA ; Tham, W-H ; Papenfuss, AT ; Gould, S ; Cowman, AF ; Crabb, BS ; Gilson, PR (AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC, 2009-09-11)
    The phylum Apicomplexa are a group of obligate intracellular parasites responsible for a wide range of important diseases. Central to the lifecycle of these unicellular parasites is their ability to migrate through animal tissue and invade target host cells. Apicomplexan movement is generated by a unique system of gliding motility in which substrate adhesins and invasion-related proteins are pulled across the plasma membrane by an underlying actin-myosin motor. The myosins of this motor are inserted into a dual membrane layer called the inner membrane complex (IMC) that is sandwiched between the plasma membrane and an underlying cytoskeletal basket. Central to our understanding of gliding motility is the characterization of proteins residing within the IMC, but to date only a few proteins are known. We report here a novel family of six-pass transmembrane proteins, termed the GAPM family, which are highly conserved and specific to Apicomplexa. In Plasmodium falciparum and Toxoplasma gondii the GAPMs localize to the IMC where they form highly SDS-resistant oligomeric complexes. The GAPMs co-purify with the cytoskeletal alveolin proteins and also to some degree with the actin-myosin motor itself. Hence, these proteins are strong candidates for an IMC-anchoring role, either directly or indirectly tethering the motor to the cytoskeleton.
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    A 4-cyano-3-methylisoquinoline inhibitor of Plasmodium falciparum growth targets the sodium efflux pump PfATP4
    Gilson, PR ; Kumarasingha, R ; Thompson, J ; Zhang, X ; Sietsma Penington, J ; Kalhor, R ; Bullen, HE ; Lehane, AM ; Dans, MG ; de Koning-Ward, TF ; Holien, JK ; Soares da Costa, TP ; Hulett, MD ; Buskes, MJ ; Crabb, BS ; Kirk, K ; Papenfuss, AT ; Cowman, AF ; Abbott, BM (NATURE PORTFOLIO, 2019-07-16)
    We developed a novel series of antimalarial compounds based on a 4-cyano-3-methylisoquinoline. Our lead compound MB14 achieved modest inhibition of the growth in vitro of the human malaria parasite, Plasmodium falciparum. To identify its biological target we selected for parasites resistant to MB14. Genome sequencing revealed that all resistant parasites bore a single point S374R mutation in the sodium (Na+) efflux transporter PfATP4. There are many compounds known to inhibit PfATP4 and some are under preclinical development. MB14 was shown to inhibit Na+ dependent ATPase activity in parasite membranes, consistent with the compound targeting PfATP4 directly. PfATP4 inhibitors cause swelling and lysis of infected erythrocytes, attributed to the accumulation of Na+ inside the intracellular parasites and the resultant parasite swelling. We show here that inhibitor-induced lysis of infected erythrocytes is dependent upon the parasite protein RhopH2, a component of the new permeability pathways that are induced by the parasite in the erythrocyte membrane. These pathways mediate the influx of Na+ into the infected erythrocyte and their suppression via RhopH2 knockdown limits the accumulation of Na+ within the parasite hence protecting the infected erythrocyte from lysis. This study reveals a role for the parasite-induced new permeability pathways in the mechanism of action of PfATP4 inhibitors.