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Title:
Mechanistic insights into dual-active liver and blood-stage antiplasmodials
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Author(s):
Rawat, M (Rawat, Mukul); Boonyalai, N (Boonyalai, Nonlawat); Smidt, C (Smidt, Cindy); Luth, MR (Luth, Madeline R.); Chen, D (Chen, Daisy); Plater, A (Plater, Andrew); Post, J (Post, John); De Lin (De Lin); McMillan, J (McMillan, Joel); Eadsforth, T (Eadsforth, Thomas); Moliner-Cubel, S (Moliner-Cubel, Sonia); Billker, O (Billker, Oliver); Rayner, JC (Rayner, Julian C.); Gamo, FJ (Gamo, Fransisco-Javier); Baragaña, B (Baragana, Beatriz); Winzeler, EA (Winzeler, Elizabeth A.); Lee, MCS (Lee, Marcus C. S.) |
| Source:
MBIO Volume:
17 Issue:
1 DOI:
10.1128/mbio.02423-25 Early Access Date:
NOV 2025 Published Date:
2026 JAN 14 |
|
Abstract:
The identification of novel antimalarials with activity against both the liver and blood stages of the parasite lifecycle would have the dual benefit of prophylactic and curative potential. However, one challenge of leveraging chemical hits from phenotypic screens is subsequent target identification. Here, we use in vitro evolution of resistance to investigate nine compounds from the Tres Cantos Antimalarial Set (TCAMS) with dual liver and asexual blood stage activity. We succeeded in eliciting resistance to four compounds, yielding mutations in acetyl CoA synthetase (AcAS), cytoplasmic isoleucine tRNA synthetase (cIRS), and protein kinase G (PKG), respectively. Using a combination of CRISPR editing and in vitro activity assays with recombinant proteins, we validate these as targets for TCMDC-125075 (AcAS), TCMDC-124602 (cIRS), TCMDC-141334, and TCDMC-140674 (PKG). Notably, for the latter two compounds, we obtained a T618I mutation in the gatekeeper residue of PKG, consistent with direct interaction with the active site, which we modeled with molecular docking. Finally, we performed cross-resistance evaluation of the remaining five resistance-refractory compounds using the Antimalarial Resistome Barcode sequencing assay (AReBar), which examined a pool of 52 barcoded lines with mutations covering >30 common modes of action. None of the five compounds where in vitro evolution of resistance was not successful yielded validated hits using AReBar, indicating they likely act via novel mechanisms and may be candidates for further exploration. |
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Title:
Extracellular ATP is an environmental cue in bacteria
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Author(s):
Tronnet, S (Tronnet, Sophie); Pandey, V (Pandey, Vikash); Lloret-Berrocal, M (Lloret-Berrocal, Miriam); Pérez-del-Pozo, M (Perez-del-Pozo, Mario); Hernández-Ortego, C (Hernandez-Ortego, Carlos); Söderholm, N (Soderholm, Niklas); Billker, O (Billker, Oliver); Nordström, A (Nordstrom, Anders); Puhar, A (Puhar, Andrea) |
| Source:
CELL REPORTS Volume:
44 Issue:
10 Article Number:
116356 DOI:
10.1016/j.celrep.2025.116356 Early Access Date:
OCT 2025 Published Date:
2025 OCT 28 |
|
Abstract:
In animals and plants, extracellular ATP (eATP) functions as a signal and regulates the immune response. During inflammation, intestinal bacteria are exposed to elevated eATP originating from the mucosa. However, whether bacteria respond to eATP is unclear. Here, we show that non-pathogenic Escherichia coli responds to eATP by modifying its transcriptional and metabolic landscapes. A genome-scale promoter library showed that the response is dependent on time, concentration, and medium and ATP specific. Second messengers and genes related to metabolism, biofilm formation, and envelope stress were regulated downstream of eATP. Metabolomics confirmed that eATP triggers enrichment of compounds with bioactive properties in the host or bacteria. Combined genome-scale modeling revealed modifications to global metabolic and biomass building blocks. Consequently, eATP altered the sensitivity to antibiotics and antimicrobial peptides. Finally, in pathogens, eATP controlled virulence factor expression. Our results indicate that eATP is an environmental cue in prokaryotes, which broadly regulates physiology, antimicrobial resistance, and virulence. |
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Title:
Cellular Hallmarks From Volume Electron Microscopy Reveal Developmental Progression of Plasmodium Ookinetes
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Author(s):
Darif, N (Darif, Nedal); Rheinnecker, M (Rheinnecker, Marco); Hildenbrand, K (Hildenbrand, Kolja); Chookajorn, T (Chookajorn, Thanat); Dorner, LP (Dorner, Lilian P.); Heriche, J (Heriche, Jean-Karim); Henriksson, S (Henriksson, Sara); Funaya, C (Funaya, Charlotta); Hentzschel, F (Hentzschel, Franziska); Sandblad, L (Sandblad, Linda); Billker, O (Billker, Oliver); Schwab, Y (Schwab, Yannick); Frischknecht, F (Frischknecht, Friedrich) |
| Source:
ADVANCED SCIENCE Volume:
13 Issue:
4 DOI:
10.1002/advs.202508250 Early Access Date:
SEP 2025 Published Date:
2026 JAN |
|
Abstract:
Unicellular organisms or cells of metazoans often change their morphology during development or life cycle progression to adapt to environmental changes. Malaria parasites undergo a striking range of morphological transformations as they navigate through the different environments of mammalian hosts and mosquito vectors. These developmental transitions are accompanied by changes in the subcellular organelles. Here, this work introduces an unbiased approach using volume electron microscopy (vEM) to facilitate cluster analyses of morphometric parameters during developmental transformation. Investigating the transformation of fertilized Plasmodium zygotes into the motile ookinetes with three complementary vEM techniques revealed intimate mitochondrion-nucleus interactions, different microtubule arrangements, elongated shapes of micronemes and their close interaction with the apicoplast. The presented data and approach provide an open-access subcellular atlas for ookinete development to aid mechanistic molecular insights from reverse genetic studies and a framework for the ultrastructural study of other parasite stages and developmental transitions in general. |
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Title:
A CRISPR homing screen finds a chloroquine resistance transporter-like protein of the Plasmodium oocyst essential for mosquito transmission of malaria
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Author(s):
Balakrishnan, A (Balakrishnan, Arjun); Hunziker, M (Hunziker, Mirjam); Tiwary, P (Tiwary, Puja); Pandey, V (Pandey, Vikash); Drew, D (Drew, David); Billker, O (Billker, Oliver) |
| Source:
NATURE COMMUNICATIONS Volume:
16 Issue:
1 Article Number:
3895 DOI:
10.1038/s41467-025-59099-1 Published Date:
2025 APR 24 |
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Abstract:
Genetic screens with barcoded PlasmoGEM vectors have identified thousands of Plasmodium berghei gene functions in haploid blood stages, gametocytes and liver stages. However, the formation of diploid cells by fertilisation has hindered similar research on the parasites’ mosquito stages. In this study, we develop a scalable genetic system that uses barcoded gene targeting vectors equipped with a CRISPR-mediated homing mechanism to generate homozygous loss-of-function mutants after one parent introduces a modified allele into the zygote. To achieve this, we use vectors additionally expressing a target gene specific gRNA. When integrated into one of the parental alleles it directs Cas9 to the intact allele after fertilisation, leading to its disruption. This homing strategy is 90% effective at generating homozygous gene editing of a fluorescence-tagged reporter locus in the oocyst. A pilot screen identifies PBANKA_0916000 as a chloroquine resistance transporter-like protein (CRTL) essential for oocyst growth and sporogony, pointing to an unexpected importance for malaria transmission of the poorly understood digestive vacuole of the oocyst that contains hemozoin granules. Homing screens provide a method for the systematic discovery of malaria transmission genes whose first essential functions are after fertilisation in the bloodmeal, enabling their potential as targets for transmission-blocking interventions to be assessed. |
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Title:
Systematic screens for fertility genes essential for malaria parasite transmission reveal conserved aspects of sex in a divergent eukaryote
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Author(s):
Sayers, C (Sayers, Claire); Pandey, V (Pandey, Vikash); Balakrishnan, A (Balakrishnan, Arjun); Michie, K (Michie, Katharine); Svedberg, D (Svedberg, Dennis); Hunziker, M (Hunziker, Mirjam); Pardo, M (Pardo, Mercedes); Choudhary, J (Choudhary, Jyoti); Berntsson, R (Berntsson, Ronnie); Billker, O (Billker, Oliver) |
| Source:
CELL SYSTEMS Volume:
15 Issue:
11 DOI:
10.1016/j.cels.2024.10.008 Early Access Date:
NOV 2024 Published Date:
2024 NOV 20 |
|
Abstract:
Sexual reproduction in malaria parasites is essential for their transmission to mosquitoes and offers a divergent eukaryote model to understand the evolution of sex. Through a panel of genetic screens in Plasmodium berghei, we identify 348 sex and transmission-related genes and define roles for unstudied genes as putative targets for transmission-blocking interventions. The functional data provide a deeper understanding of female metabolic reprogramming, meiosis, and the axoneme. We identify a complex of a SUN domain protein (SUN1) and a putative allantoicase (ALLC1) that is essential for male fertility by linking the microtubule organizing center to the nuclear envelope and enabling mitotic spindle formation during male gametogenesis. Both proteins have orthologs in mouse testis, and the data raise the possibility of an ancient role for atypical SUN domain proteins in coupling the nucleus and axoneme. Altogether, our data provide an unbiased picture of the molecular processes that underpin malaria parasite transmission. A record of this paper’s transparent peer review process is included in the supplemental information. |
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Title:
The global transcriptome of Plasmodium falciparum mid-stage gametocytes (stages II-IV) appears largely conserved and gametocyte-specific gene expression patterns vary in clinical isolates
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Author(s):
Kengne-Ouafo, JA (Kengne-Ouafo, Jonas A.); Bah, SY (Bah, Saikou Y.); Kemp, A (Kemp, Alison); Stewart, L (Stewart, Lindsay); Amenga-Etego, L (Amenga-Etego, Lucas); Deitsch, KW (Deitsch, Kirk W.); Rayner, JC (Rayner, Julian C.); Billker, O (Billker, Oliver); Binka, FN (Binka, Fred N.); Sutherland, CJ (Sutherland, Colin J.); Awandare, GA (Awandare, Gordon A.); Urban, BC (Urban, Britta C.); Dinko, B (Dinko, Bismarck) |
| Source:
MICROBIOLOGY SPECTRUM Volume:
11 Issue:
5 Article Number:
e03820-22 DOI:
10.1128/spectrum.03820-22 Published Date:
2023 OCT |
Abstract:
Our overall understanding of the developmental biology of malaria parasites has been greatly enhanced by recent advances in transcriptomic analysis. However, most of these investigations rely on laboratory strains (LS) that were adapted into in vitro culture many years ago, and the transcriptomes of clinical isolates (CI) circulating in human populations have not been assessed. In this study, RNA-seq was used to compare the global transcriptome of mid-stage gametocytes derived from three short-term cultured CI, with gametocytes derived from the NF54 reference laboratory strain. The core transcriptome appeared to be consistent between CI- and LS-derived gametocyte preparations, but some important differences were also observed. A majority of gametocyte-specific genes (43/53) appear to have relatively higher expression in CI-derived gametocytes than in LS-derived gametocytes, but a K-means clustering analysis showed that genes involved in flagellum- and microtubule-based processes (movement/motility) were more abundant in both groups, albeit with some differences between them. In addition, gametocytes from one CI described as CI group II gametocytes (CI:GGII) showed gene expression variation in the form of reduced gametocyte-specific gene expression compared to the other two CI-derived gametocytes (CI gametocyte group I, CI:GGI), although the mixed developmental stages used in our study is a potential confounder, only partially mitigated by the inclusion of multiple replicates for each CI. Overall, our study suggests that there may be subtle differences in the gene expression profiles of mid-stage gametocytes from CI relative to the NF54 reference strain of Plasmodium falciparum. Thus, it is necessary to deploy gametocyte-producing clinical parasite isolates to fully understand the diversity of gene expression strategies that may occur during the sequestered development of parasite sexual stages.
IMPORTANCE Maturing gametocytes of Plasmodium falciparum are known to sequester away from peripheral circulation into the bone marrow until they are mature. Blocking gametocyte sequestration can prevent malaria transmission from humans to mosquitoes, but most studies aim to understand gametocyte development utilizing long-term adapted laboratory lines instead of clinical isolates. This is a particular issue for our understanding of the sexual stages, which are known to decrease rapidly during adaptation to long-term culture, meaning that many LS are unable to produce transmissible gametocytes. Using RNA-seq, we investigated the global transcriptome of mid-stage gametocytes derived from three clinical isolates and a reference strain (NF54). This identified important differences in gene expression profiles between immature gametocytes of CI and the NF54 reference strain of P. falciparum, suggesting increased investment in gametocytogenesis in clinical isolates. Our transcriptomic data highlight the use of clinical isolates in studying the morphological, cellular features and molecular biology of gametocytes. |
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Title:
Identification of genes required for Plasmodium gametocyte-to-sporozoite development in the mosquito vector
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Author(s):
Ukegbu, CV (Ukegbu, Chiamaka Valerie); Gomes, AR (Gomes, Ana Rita); Giorgalli, M (Giorgalli, Maria); Campos, M (Campos, Melina); Bailey, AJ (Bailey, Alexander J.); Besson, TRB (Besson, Tanguy Rene Balthazar); Billker, O (Billker, Oliver); Vlachou, D (Vlachou, Dina); Christophides, GK (Christophides, George K.) |
| Source:
CELL HOST & MICROBE Volume:
31 Issue:
9 Pages:
1539-+ DOI:
10.1016/j.chom.2023.08.010 Early Access Date:
SEP 2023 Published Date:
2023 SEP 13 |
|
Abstract:
Malaria remains one of the most devastating infectious diseases. Reverse genetic screens offer a powerful approach to identify genes and molecular processes governing malaria parasite biology. However, the com-plex regulation of gene expression and genotype-phenotype associations in the mosquito vector, along with sexual reproduction, have hindered the development of screens in this critical part of the parasite life cycle. To address this, we developed a genetic approach in the rodent parasite Plasmodium berghei that, in com-bination with barcode sequencing, circumvents the fertilization roadblock and enables screening for game-tocyte-expressed genes required for parasite infection of the mosquito Anopheles coluzzii. Our results confirm previous findings, validating our approach for scaling up, and identify genes necessary for mosquito midgut infection, oocyst development, and salivary gland infection. These findings can aid efforts to study malaria transmission biology and to develop interventions for controlling disease transmission. |
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Title:
Sideways: road to gene-by-gene functional screening in malaria parasites
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Author(s):
Chookajorn, T (Chookajorn, Thanat); Billker, O (Billker, Oliver) |
| Source:
TRENDS IN PARASITOLOGY Volume:
39 Issue:
5 Pages:
317-318 DOI:
10.1016/j.pt.2023.03.007 Early Access Date:
APR 2023 Published Date:
2023 MAY |
|
Title:
A non-canonical sensing pathway mediates Plasmodium adaptation to amino acid deficiency
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Author(s):
Marreiros, IM (Marreiros, Ines M.); Marques, S (Marques, Sofia); Parreira, A (Parreira, Ana); Mastrodomenico, V (Mastrodomenico, Vincent); Mounce, BC (Mounce, Bryan C.); Harris, CT (Harris, Chantal T.); Kafsack, BF (Kafsack, Bjoern F.); Billker, O (Billker, Oliver); Zuzarte-Luís, V (Zuzarte-Luis, Vanessa); Mota, MM (Mota, Maria M.) |
| Source:
COMMUNICATIONS BIOLOGY Volume:
6 Issue:
1 Article Number:
205 DOI:
10.1038/s42003-023-04566-y Published Date:
2023 FEB 21 |
Abstract:
Eukaryotes have canonical pathways for responding to amino acid (AA) availability. Under AA-limiting conditions, the TOR complex is repressed, whereas the sensor kinase GCN2 is activated. While these pathways have been highly conserved throughout evolution, malaria parasites are a rare exception. Despite auxotrophic for most AA, Plasmodium does not have either a TOR complex nor the GCN2-downstream transcription factors. While Ile starvation has been shown to trigger eIF2 alpha phosphorylation and a hibernation-like response, the overall mechanisms mediating detection and response to AA fluctuation in the absence of such pathways has remained elusive. Here we show that Plasmodium parasites rely on an efficient sensing pathway to respond to AA fluctuations. A phenotypic screen of kinase knockout mutant parasites identified nek4, eIK1 and eIK2-the last two clustering with the eukaryotic eIF2 alpha kinases-as critical for Plasmodium to sense and respond to distinct AA-limiting conditions. Such AA-sensing pathway is temporally regulated at distinct life cycle stages, allowing parasites to actively fine-tune replication and development in response to AA availability. Collectively, our data disclose a set of heterogeneous responses to AA depletion in malaria parasites, mediated by a complex mechanism that is critical for modulating parasite growth and survival.
The nek4, eIK1, and eIK2 sensor kinases play a key role in Plasmodium amino acid sensing, enabling these parasites to fine-tune replication and development in response to amino acid availability. |
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Title:
Regulators of male and female sexual development are critical for the transmission of a malaria parasite
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Author(s):
Russell, AJC (Russell, Andrew J. C.); Sanderson, T (Sanderson, Theo); Bushell, E (Bushell, Ellen); Talman, AM (Talman, Arthur M.); Anar, B (Anar, Burcu); Girling, G (Girling, Gareth); Hunziker, M (Hunziker, Mirjam); Kent, RS (Kent, Robyn S.); Martin, JS (Martin, Julie S.); Metcalf, T (Metcalf, Tom); Montandon, R (Montandon, Ruddy); Pandey, V (Pandey, Vikash); Pardo, M (Pardo, Mercedes); Roberts, AB (Roberts, A. Brett); Sayers, C (Sayers, Claire); Schwach, F (Schwach, Frank); Choudhary, JS (Choudhary, Jyoti S.); Rayner, JC (Rayner, Julian C.); Voet, T (Voet, Thierry); Modrzynska, KK (Modrzynska, Katarzyna K.); Waters, AP (Waters, Andrew P.); Lawniczak, MKN (Lawniczak, Mara K. N.); Billker, O (Billker, Oliver) |
| Source:
CELL HOST & MICROBE Volume:
31 Issue:
2 Pages:
305-+ DOI:
10.1016/j.chom.2022.12.011 Early Access Date:
FEB 2023 Published Date:
2023 FEB 8 |
|
Abstract:
Malaria transmission to mosquitoes requires a developmental switch in asexually dividing blood-stage par-asites to sexual reproduction. In Plasmodium berghei, the transcription factor AP2-G is required and suffi-cient for this switch, but how a particular sex is determined in a haploid parasite remains unknown. Using a global screen of barcoded mutants, we here identify genes essential for the formation of either male or fe-male sexual forms and validate their importance for transmission. High-resolution single-cell transcriptomics of ten mutant parasites portrays the developmental bifurcation and reveals a regulatory cascade of putative gene functions in the determination and subsequent differentiation of each sex. A male-determining gene with a LOTUS/OST-HTH domain as well as the protein interactors of a female-determining zinc-finger protein indicate that germ-granule-like ribonucleoprotein complexes complement transcriptional processes in the regulation of both male and female development of a malaria parasite. |
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Title:
Genome reconstructions of metabolism of Plasmodium RBC and liver stages
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Author(s):
Chiappino-Pepe, A (Chiappino-Pepe, Anush); Pandey, V (Pandey, Vikash); Billker, O (Billker, Oliver) |
| Source:
CURRENT OPINION IN MICROBIOLOGY Volume:
63 Pages:
259-266 DOI:
10.1016/j.mib.2021.08.006 Published Date:
2021 OCT |
|
Abstract:
Genome scale metabolic models (GEMs) offer a powerful means of integrating genome and biochemical information on an organism to make testable predictions of metabolic functions at different conditions and to systematically predict essential genes that may be targeted by drugs. This review describes how Plasmodium GEMs have become increasingly more accurate through the integration of omics and experimental genetic data. We also discuss how GEMs contribute to our increasing understanding of how Plasmodium metabolism is reprogrammed between life cycle stages. |
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Title:
A 39-Amino-Acid C-Terminal Truncation of GDV1 Disrupts Sexual Commitment in Plasmodium falciparum
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Author(s):
Tibúrcio, M (Tiburcio, Marta); Hitz, E (Hitz, Eva); Niederwieser, I (Niederwieser, Igor); Kelly, G (Kelly, Gavin); Davies, H (Davies, Heledd); Doerig, C (Doerig, Christian); Billker, O (Billker, Oliver); Voss, TS (Voss, Till S.); Treeck, M (Treeck, Moritz) |
| Source:
MSPHERE Volume:
6 Issue:
3 Article Number:
e01093-20 DOI:
10.1128/mSphere.01093-20 Published Date:
2021 MAY-JUN |
Abstract:
Malaria is a mosquito-borne disease caused by apicomplexan parasites of the genus Plasmodium. Completion of the parasite’s life cycle depends on the transmission of sexual stages, the gametocytes, from an infected human host to the mosquito vector. Sexual commitment occurs in only a small fraction of asexual blood-stage parasites and is initiated by external cues. The gametocyte development protein 1 (GDV1) has been described as a key facilitator to trigger sexual commitment. GDV1 interacts with the silencing factor heterochromatin protein 1 (HP1), leading to its dissociation from heterochromatic DNA at the genomic locus encoding AP2-G, the master transcription factor of gametocytogenesis. How this process is regulated is not known. In this study, we have addressed the role of protein kinases implicated in gametocyte development. From a pool of available protein kinase knockout (KO) lines, we identified two kinase knockout lines which fail to produce gametocytes. However, independent genetic verification revealed that both kinases are not required for gametocytogenesis but that both lines harbor the same mutation that leads to a truncation in the extreme C terminus of GDV1. Introduction of the identified nonsense mutation into the genome of wild-type parasite lines replicates the observed phenotype. Using a GDV1 over expression line, we show that the truncation in the GDV1 C terminus does not interfere with the nuclear import of GDV1 or its interaction with HP1 in vitro but appears to be important to sustain GDV1 protein levels and thereby sexual commitment.
IMPORTANCE Transmission of malaria-causing Plasmodium species by mosquitos requires the parasite to change from a continuously growing asexual parasite form growing in the blood to a sexually differentiated form, the gametocyte. Only a small subset of asexual parasites differentiates into gametocytes that are taken up by the mosquito. Transmission represents a bottleneck in the life cycle of the parasite, so a molecular understanding of the events that lead to stage conversion may identify novel intervention points. Here, we screened a subset of kinases we hypothesized to play a role in this process. While we did not identify kinases required for sexual conversion, we identified a mutation in the C terminus of the gametocyte development 1 protein (GDV1), which abrogates sexual development. The mutation destabilizes the protein but not its interaction with its cognate binding partner HP1. This suggests an important role for the GDV1 C terminus beyond trafficking and protein stability. |
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Title:
The antimalarial efficacy and mechanism of resistance of the novel chemotype DDD01034957
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Author(s):
Miguel-Blanco, C (Miguel-Blanco, Celia); Murithi, JM (Murithi, James M.); Benavente, ED (Benavente, Ernest Diez); Angrisano, F (Angrisano, Fiona); Sala, KA (Sala, Katarzyna A.); van Schalkwyk, DA (van Schalkwyk, Donelly A.); Vanaerschot, M (Vanaerschot, Manu); Schwach, F (Schwach, Frank); Fuchter, MJ (Fuchter, Matthew J.); Billker, O (Billker, Oliver); Sutherland, CJ (Sutherland, Colin J.); Campino, SG (Campino, Susana G.); Clark, TG (Clark, Taane G.); Blagborough, AM (Blagborough, Andrew M.); Fidock, DA (Fidock, David A.); Herreros, E (Herreros, Esperanza); Gamo, FJ (Gamo, Francisco Javier); Baum, J (Baum, Jake); Delves, MJ (Delves, Michael J.) |
| Source:
SCIENTIFIC REPORTS Volume:
11 Issue:
1 Article Number:
1888 DOI:
10.1038/s41598-021-81343-z Published Date:
2021 JAN 21 |
|
Abstract:
New antimalarial therapeutics are needed to ensure that malaria cases continue to be driven down, as both emerging parasite resistance to frontline chemotherapies and mosquito resistance to current insecticides threaten control programmes. Plasmodium, the apicomplexan parasite responsible for malaria, causes disease pathology through repeated cycles of invasion and replication within host erythrocytes (the asexual cycle). Antimalarial drugs primarily target this cycle, seeking to reduce parasite burden within the host as fast as possible and to supress recrudescence for as long as possible. Intense phenotypic drug screening efforts have identified a number of promising new antimalarial molecules. Particularly important is the identification of compounds with new modes of action within the parasite to combat existing drug resistance and suitable for formulation of efficacious combination therapies. Here we detail the antimalarial properties of DDD01034957-a novel antimalarial molecule which is fast-acting and potent against drug resistant strains in vitro, shows activity in vivo, and possesses a resistance mechanism linked to the membrane transporter PfABCI3. These data support further medicinal chemistry lead-optimization of DDD01034957 as a novel antimalarial chemical class and provide new insights to further reduce in vivo metabolic clearance. |
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Title:
Systematic Identification of Plasmodium Falciparum Sporozoite Membrane Protein Interactions Reveals an Essential Role for the p24 Complex in Host Infection
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Author(s):
Knöckel, J (Knockel, Julia); Dundas, K (Dundas, Kirsten); Yang, ASP (Yang, Annie S. P.); Galaway, F (Galaway, Francis); Metcalf, T (Metcalf, Tom); Van Gemert, GJ (Van Gemert, Geert-Jan); Sauerwein, RW (Sauerwein, Robert W.); Rayner, JC (Rayner, Julian C.); Billker, O (Billker, Oliver); Wright, GJ (Wright, Gavin J.) |
| Source:
MOLECULAR & CELLULAR PROTEOMICS Volume:
20 Article Number:
100038 DOI:
10.1074/mcp.RA120.002432 Published Date:
2021 |
|
Abstract:
Sporozoites are a motile form of malaria-causing Plasmodium falciparum parasites that migrate from the site of transmission in the dermis through the bloodstream to invade hepatocytes. Sporozoites interact with many cells within the host, but the molecular identity of these interactions and their role in the pathology of malaria is poorly understood. Parasite proteins that are secreted and embedded within membranes are known to be important for these interactions, but our understanding of how they interact with each other to form functional complexes is largely unknown. Here, we compile a library of recombinant proteins representing the repertoire of cell surface and secreted proteins from the P. falciparum sporozoite and use an assay designed to detect extracellular interactions to systematically identify complexes. We identify three protein complexes including an interaction between two components of the p24 complex that is involved in the trafficking of glycosylphosphatidylinositolanchored proteins through the secretory pathway. Plasmodium parasites lacking either gene are strongly inhibited in the establishment of liver-stage infections. These findings reveal an important role for the p24 complex in malaria pathogenesis and show that the library of recombinant proteins represents a valuable resource to investigate P. falciparum sporozoite biology. |
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Title:
Multiple blood feeding in mosquitoes shortens the Plasmodium falciparum incubation period and increases malaria transmission potential
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Author(s):
Shaw, WR (Shaw, W. Robert); Holmdahl, IE (Holmdahl, Inga E.); Itoe, MA (Itoe, Maurice A.); Werling, K (Werling, Kristine); Marquette, M (Marquette, Meghan); Paton, DG (Paton, Douglas G.); Singh, N (Singh, Naresh); Buckee, CO (Buckee, Caroline O.); Childs, LM (Childs, Lauren M.); Catteruccia, F (Catteruccia, Flaminia); Kim, K (Kim, Kami); Billker, O (Billker, Oliver) |
| Source:
PLOS PATHOGENS Volume:
16 Issue:
12 Article Number:
e1009131 DOI:
10.1371/journal.ppat.1009131 Published Date:
2020 DEC |
Abstract:
Author summary
In natural settings the female Anopheles gambiae mosquito, the major malaria vector, blood feeds multiple times in her lifespan. Here we demonstrate that an additional blood feed accelerates the growth of Plasmodium falciparum malaria parasites in this mosquito. Incorporating these data into a mathematical model across sub-Saharan Africa reveals that malaria transmission potential is likely to be higher than previously thought, making disease elimination more difficult. Additionally, we show that control strategies that manipulate mosquito reproduction with the aim of suppressing Anopheles populations may inadvertently favor malaria transmission. Our data also suggest that parasites can be transmitted by younger mosquitoes, which are less susceptible to insecticide killing, with negative implications for the success of insecticide-based strategies.
Many mosquito species, including the major malaria vector Anopheles gambiae, naturally undergo multiple reproductive cycles of blood feeding, egg development and egg laying in their lifespan. Such complex mosquito behavior is regularly overlooked when mosquitoes are experimentally infected with malaria parasites, limiting our ability to accurately describe potential effects on transmission. Here, we examine how Plasmodium falciparum development and transmission potential is impacted when infected mosquitoes feed an additional time. We measured P. falciparum oocyst size and performed sporozoite time course analyses to determine the parasite’s extrinsic incubation period (EIP), i.e. the time required by parasites to reach infectious sporozoite stages, in An. gambiae females blood fed either once or twice. An additional blood feed at 3 days post infection drastically accelerates oocyst growth rates, causing earlier sporozoite accumulation in the salivary glands, thereby shortening the EIP (reduction of 2.3 +/- 0.4 days). Moreover, parasite growth is further accelerated in transgenic mosquitoes with reduced reproductive capacity, which mimic genetic modifications currently proposed in population suppression gene drives. We incorporate our shortened EIP values into a measure of transmission potential, the basic reproduction number R-0, and find the average R-0 is higher (range: 10.1%-12.1% increase) across sub-Saharan Africa than when using traditional EIP measurements. These data suggest that malaria elimination may be substantially more challenging and that younger mosquitoes or those with reduced reproductive ability may provide a larger contribution to infection than currently believed. Our findings have profound implications for current and future mosquito control interventions. |
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Title:
Mosquito cellular immunity at single-cell resolution
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Author(s):
Raddi, G (Raddi, Gianmarco); Barletta, ABF (Barletta, Ana Beatriz F.); Efremova, M (Efremova, Mirjana); Ramirez, JL (Ramirez, Jose Luis); Cantera, R (Cantera, Rafael); Teichmann, SA (Teichmann, Sarah A.); Barillas-Mury, C (Barillas-Mury, Carolina); Billker, O (Billker, Oliver) |
| Source:
SCIENCE Volume:
369 Issue:
6507 Pages:
1128-+ DOI:
10.1126/science.abc0322 Published Date:
2020 AUG 28 |
|
Abstract:
Hemocytes limit the capacity of mosquitoes to transmit human pathogens. Here we profile the transcriptomes of 8506 hemocytes of Anopheles gambiae and Aedes aegypti mosquito vectors. Our data reveal the functional diversity of hemocytes, with different subtypes of granulocytes expressing distinct and evolutionarily conserved subsets of effector genes. A previously unidentified cell type in An. gambiae, which we term “megacyte,” is defined by a specific transmembrane protein marker (TM7318) and high expression of lipopolysaccharide-induced tumor necrosis factor-a transcription factor 3 (LL3). Knockdown experiments indicate that LL3 mediates hemocyte differentiation during immune priming. We identify and validate two main hemocyte lineages and find evidence of proliferating granulocyte populations. This atlas of medically relevant invertebrate immune cells at single-cell resolution identifies cellular events that underpin mosquito immunity to malaria infection. |
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Title:
Testing the impact of a single nucleotide polymorphism in a Plasmodium berghei ApiAP2 transcription factor on experimental cerebral malaria in mice
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Author(s):
Akkaya, M (Akkaya, Munir); Bansal, A (Bansal, Abhisheka); Sheehan, PW (Sheehan, Patrick W.); Pena, M (Pena, Mirna); Cimperman, CK (Cimperman, Clare K.); Qi, CF (Qi, Chen Feng); Yazew, T (Yazew, Takele); Otto, TD (Otto, Thomas D.); Billker, O (Billker, Oliver); Miller, LH (Miller, Louis H.); Pierce, SK (Pierce, Susan K.) |
| Source:
SCIENTIFIC REPORTS Volume:
10 Issue:
1 Article Number:
13630 DOI:
10.1038/s41598-020-70617-7 Published Date:
2020 AUG 12 |
|
Abstract:
Cerebral malaria (CM) is the deadliest form of severe Plasmodium infections. Currently, we have limited understanding of the mechanisms by which Plasmodium parasites induce CM. The mouse model of CM, experimental CM (ECM), induced by infection with the rodent parasite, Plasmodium berghei ANKA (PbANKA) has been extensively used to study the pathophysiology of CM. Recent genomic analyses revealed that the coding regions of PbANKA and the closely related Plasmodium berghei NK65 (PbNK65), that does not cause ECM, differ in only 21 single nucleotide polymorphysims (SNPs). Thus, the SNP-containing genes might contribute to the pathogenesis of ECM. Although the majority of these SNPs are located in genes of unknown function, one SNP is located in the DNA binding site of a member of the Plasmodium ApiAP2 transcription factor family, that we recently showed functions as a virulence factor alternating the host’s immune response to the parasite. Here, we investigated the impact of this SNP on the development of ECM. Our results using CRISPR-Cas9 engineered parasites indicate that despite its immune modulatory function, the SNP is neither necessary nor sufficient to induce ECM and thus cannot account for parasite strain-specific differences in ECM phenotypes. |
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Title:
Analysis of erythrocyte signalling pathways during Plasmodium falciparum infection identifies targets for host-directed antimalarial intervention
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Author(s):
Adderley, JD (Adderley, Jack D.); von Freyend, SJ (von Freyend, Simona John); Jackson, SA (Jackson, Sarah A.); Bird, MJ (Bird, Megan J.); Burns, AL (Burns, Amy L.); Anar, B (Anar, Burcu); Metcalf, T (Metcalf, Tom); Semblat, JP (Semblat, Jean-Philippe); Billker, O (Billker, Oliver); Wilson, DW (Wilson, Danny W.); Doerig, C (Doerig, Christian) |
| Source:
NATURE COMMUNICATIONS Volume:
11 Issue:
1 Article Number:
4015 DOI:
10.1038/s41467-020-17829-7 Published Date:
2020 AUG 11 |
|
Abstract:
Intracellular pathogens mobilize host signaling pathways of their host cell to promote their own survival. Evidence is emerging that signal transduction elements are activated in a-nucleated erythrocytes in response to infection with malaria parasites, but the extent of this phenomenon remains unknown. Here, we fill this knowledge gap through a comprehensive and dynamic assessment of host erythrocyte signaling during infection with Plasmodium falciparum. We used arrays of 878 antibodies directed against human signaling proteins to interrogate the activation status of host erythrocyte phospho-signaling pathways at three blood stages of parasite asexual development. This analysis reveals a dynamic modulation of many host signalling proteins across parasite development. Here we focus on the hepatocyte growth factor receptor (c-MET) and the MAP kinase pathway component B-Raf, providing a proof of concept that human signaling kinases identified as activated by malaria infection represent attractive targets for antimalarial intervention.Plasmodium infection activates signaling pathways in a-nucleated erythrocytes. Here, Adderley et al. use a comprehensive antibody microarray to show that infection extensively modulates host cell signalling and that the host receptor tyrosine kinase c-MET supports Plasmodium falciparum proliferation. |
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Title:
A Novel Chemically Differentiated Mouse Embryonic Stem Cell-Based Model to Study Liver Stages of Plasmodium berghei
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Author(s):
Tripathi, J (Tripathi, Jaishree); Segeritz, CP (Segeritz, Charis-Patricia); Griffiths, G (Griffiths, Gareth); Bushell, W (Bushell, Wendy); Vallier, L (Vallier, Ludovic); Skarnes, WC (Skarnes, William C.); Mota, MM (Mota, Maria M.); Billker, O (Billker, Oliver) |
| Source:
STEM CELL REPORTS Volume:
14 Issue:
6 Pages:
1123-1134 DOI:
10.1016/j.stemcr.2020.04.010 Published Date:
2020 JUN 9 |
|
Abstract:
Asymptomatic and obligatory liver stage (LS) infection of Plasmodium parasites presents an attractive target for antimalarial vaccine and drug development. Lack of robust cellular models to study LS infection has hindered the discovery and validation of host genes essential for intrahepatic parasite development. Here, we present a chemically differentiated mouse embryonic stem cell (ESC)-based LS model, which supports complete development of Plasmodium berghei exoerythrocytic forms (EEFs) and can be used to define new host-parasite interactions. Using our model, we established that host Pnpla2, coding for adipose triglyceride lipase, is dispensable for P. berghei EEF development. In addition, we also evaluated in-vitro-differentiated human hepatocyte-like cells (iHLCs) to study LS of P. berghei and found it to be a sub-optimal infection model. Overall, our results present a new mouse ESC-based P. berghei LS infection model that can be utilized to study the impact of host genetic variation on parasite development. |
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Title:
A single-nucleotide polymorphism in a Plasmodium berghei ApiAP2 transcription factor alters the development of host immunity
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Author(s):
Akkaya, M (Akkaya, Munir); Bansal, A (Bansal, Abhisheka); Sheehan, PW (Sheehan, Patrick W.); Pena, M (Pena, Mirna); Molina-Cruz, A (Molina-Cruz, Alvaro); Orchard, LM (Orchard, Lindsey M.); Cimperman, CK (Cimperman, Clare K.); Qi, CF (Qi, Chen-Feng); Ross, P (Ross, Philipp); Yazew, T (Yazew, Takele); Sturdevant, D (Sturdevant, Daniel); Anzick, SL (Anzick, Sarah L.); Thiruvengadam, G (Thiruvengadam, Girija); Otto, TD (Otto, Thomas Dan); Billker, O (Billker, Oliver); Llinás, M (Llinas, Manuel); Miller, LH (Miller, Louis H.); Pierce, SK (Pierce, Susan K.) |
| Source:
SCIENCE ADVANCES Volume:
6 Issue:
6 Article Number:
eaaw6957 DOI:
10.1126/sciadv.aaw6957 Published Date:
2020 FEB |
|
Abstract:
The acquisition of malaria immunity is both remarkably slow and unpredictable. At present, we know little about the malaria parasite genes that influence the host’s ability to mount a protective immune response. Here, we show that a single-nucleotide polymorphism (SNP) resulting in a single amino acid change (S to F) in an ApiAP2 transcription factor in the rodent malaria parasite Plasmodium berghei (Pb) NK65 allowed infected mice to mount a T helper cell 1 (T(H)1)-type immune response that controlled subsequent infections. As compared to PbNK65(S), PbNK65(F) parasites differentially expressed 46 genes, most of which are predicted to play roles in immune evasion. PbNK65(F) infections resulted in an early interferon-gamma response and a later expansion of germinal centers, resulting in high levels of infected red blood cell-specific T(H)1-type immunoglobulin G2b (IgG2b) and IgG2c antibodies. Thus, the Pb ApiAP2 transcription factor functions as a critical parasite virulence factor in malaria infections. |
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Title:
Calcium and cyclic nucleotide signaling networks in Toxoplasma gondii
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Author(s):
Brown, KM (Brown, Kevin M.); Tonkin, CJ (Tonkin, Christopher J.); Billker, O (Billker, Oliver); Sibley, LD (Sibley, L. David) |
|
Edited by:
Weiss LM; Kim K |
| Source:
TOXOPLASMA GONDII: THE MODEL APICOMPLEXAN-PERSPECTIVES AND METHODS, 3RD EDITION Pages:
577-605 DOI:
10.1016/B978-0-12-815041-2.00013-X Published Date:
2020 |
|
Book DOI:
10.1016/C2011-0-07157-0
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|
Title:
Genome-Scale Identification of Essential Metabolic Processes for Targeting the Plasmodium Liver Stage
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Author(s):
Stanway, RR (Stanway, Rebecca R.); Bushell, E (Bushell, Ellen); Chiappino-Pepe, A (Chiappino-Pepe, Anush); Roques, M (Roques, Magali); Sanderson, T (Sanderson, Theo); Franke-Fayard, B (Franke-Fayard, Blandine); Caldelari, R (Caldelari, Reto); Golomingi, M (Golomingi, Murielle); Nyonda, M (Nyonda, Mary); Pandey, V (Pandey, Vikash); Schwach, F (Schwach, Frank); Chevalley, S (Chevalley, Severine); Ramesar, J (Ramesar, Jai); Metcalf, T (Metcalf, Tom); Herd, C (Herd, Colin); Burda, PC (Burda, Paul-Christian); Rayner, JC (Rayner, Julian C.); Soldati-Favre, D (Soldati-Favre, Dominique); Janse, CJ (Janse, Chris J.); Hatzimanikatis, V (Hatzimanikatis, Vassily); Billker, O (Billker, Oliver); Heussler, VT (Heussler, Volker T.) |
| Source:
CELL Volume:
179 Issue:
5 Pages:
1112-+ DOI:
10.1016/j.cell.2019.10.030 Published Date:
2019 NOV 14 |
|
Abstract:
Plasmodium gene functions in mosquito and liver stages remain poorly characterized due to limitations in the throughput of phenotyping at these stages. To fill this gap, we followed more than 1,300 barcoded P. berghei mutants through the life cycle. We discover 461 genes required for efficient parasite transmission to mosquitoes through the liver stage and back into the bloodstream of mice. We analyze the screen in the context of genomic, transcriptomic, and metabolomic data by building a thermodynamic model of P. berghei liver-stage metabolism, which shows a major reprogramming of parasite metabolism to achieve rapid growth in the liver. We identify seven metabolic subsystems that become essential at the liver stages compared with asexual blood stages: type II fatly acid synthesis and elongation (FAE), tricarboxylic acid, amino sugar, heme, lipoate, and shikimate metabolism. Selected predictions from the model are individually validated in single mutants to provide future targets for drug development. |
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Title:
The Malaria Cell Atlas: Single parasite transcriptomes across the complete Plasmodium life cycle
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|
Author(s):
Howick, VM (Howick, Virginia M.); Russell, AJC (Russell, Andrew J. C.); Andrews, T (Andrews, Tallulah); Heaton, H (Heaton, Haynes); Reid, AJ (Reid, Adam J.); Natarajan, K (Natarajan, Kedar); Butungi, H (Butungi, Hellen); Metcalf, T (Metcalf, Tom); Verzier, LH (Verzier, Lisa H.); Rayner, JC (Rayner, Julian C.); Berriman, M (Berriman, Matthew); Herren, JK (Herren, Jeremy K.); Billker, O (Billker, Oliver); Hemberg, M (Hemberg, Martin); Talman, AM (Talman, Arthur M.); Lawniczak, MKN (Lawniczak, Mara K. N.) |
| Source:
SCIENCE Volume:
365 Issue:
6455 Special Issue:
SI Pages:
774-+ Article Number:
eaaw2619 DOI:
10.1126/science.aaw2619 Published Date:
2019 AUG 23 |
|
Abstract:
Malaria parasites adopt a remarkable variety of morphological life stages as they transition through multiple mammalian host and mosquito vector environments. We profiled the single-cell transcriptomes of thousands of individual parasites, deriving the first high-resolution transcriptional atlas of the entire Plasmodium berghei life cycle. We then used our atlas to precisely define developmental stages of single cells from three different human malaria parasite species, including parasites isolated directly from infected individuals. The Malaria Cell Atlas provides both a comprehensive view of gene usage in a eukaryotic parasite and an open-access reference dataset for the study of malaria parasites. |
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Title:
Landscape of the Plasmodium Interactome Reveals Both Conserved and Species-Specific Functionality
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Author(s):
Hillier, C (Hillier, Charles); Pardo, M (Pardo, Mercedes); Yu, L (Yu, Lu); Bushell, E (Bushell, Ellen); Sanderson, T (Sanderson, Theo); Metcalf, T (Metcalf, Tom); Herd, C (Herd, Colin); Anar, B (Anar, Burcu); Rayner, JC (Rayner, Julian C.); Billker, O (Billker, Oliver); Choudhary, JS (Choudhary, Jyoti S.) |
| Source:
CELL REPORTS Volume:
28 Issue:
6 Pages:
1635-+ DOI:
10.1016/j.celrep.2019.07.019 Published Date:
2019 AUG 6 |
|
Abstract:
Malaria represents a major global health issue, and the identification of new intervention targets remains an urgent priority. This search is hampered by more than one-third of the genes of malaria-causing Plasmodium parasites being uncharacterized. We report a large-scale protein interaction network in Plasmodium schizonts, generated by combining blue native-polyacrylamide electrophoresis with quantitative mass spectrometry and machine learning. This integrative approach, spanning 3 species, identifies > 20,000 putative protein interactions, organized into 600 protein clusters. We validate selected interactions, assigning functions in chromatin regulation to previously unannotated proteins and suggesting a role for an EELM2 domain-containing protein and a putative microrchidia protein as mechanistic links between AP2-domain transcription factors and epigenetic regulation. Our interactome represents a high-confidence map of the native organization of core cellular processes in Plasmodium parasites. The network reveals putative functions for uncharacterized proteins, provides mechanistic and structural insight, and uncovers potential alternative therapeutic targets. |
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Title:
CRISPRing the Elephant in the Room
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|
Author(s):
Billker, O (Billker, Oliver) |
| Source:
CELL HOST & MICROBE Volume:
24 Issue:
6 Pages:
754-755 DOI:
10.1016/j.chom.2018.11.015 Published Date:
2018 DEC 12 |
|
Abstract:
The importance of guanylyl-cyclases (GCs) in apicomplexa has remained elusive due to the large size of the genes. Two recent studies, including Brown and Sibley, 2018 in this issue of Cell Host & Microbe, make elegant use of genome editing with CRISPR-Cas9 to characterize roles of GCs in Toxoplasma and Plasmodium. |
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Title:
Inducible developmental reprogramming redefines commitment to sexual development in the malaria parasite Plasmodium berghei
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Author(s):
Kent, RS (Kent, Robyn S.); Modrzynska, KK (Modrzynska, Katarzyna K.); Cameron, R (Cameron, Rachael); Philip, N (Philip, Nisha); Billker, O (Billker, Oliver); Waters, AP (Waters, Andrew P.) |
| Source:
NATURE MICROBIOLOGY Volume:
3 Issue:
11 Pages:
1206-1213 DOI:
10.1038/s41564-018-0223-6 Published Date:
2018 NOV |
|
Abstract:
During malaria infection, Plasmodium spp. parasites cyclically invade red blood cells and can follow two different developmental pathways. They can either replicate asexually to sustain the infection, or differentiate into gametocytes, the sexual stage that can be taken up by mosquitoes, ultimately leading to disease transmission. Despite its importance for malaria control, the process of gametocytogenesis remains poorly understood, partially due to the difficulty of generating high numbers of sexually committed parasites in laboratory conditions(1). Recently, an apicomplexa-specific transcription factor (AP2-G) was identified as necessary for gametocyte production in multiple Plasmodium species(2,3), and suggested to be an epigenetically regulated master switch that initiates gametocytogenesis(4,5). Here we show that in a rodent malaria parasite, Plasmodium berghei, conditional overexpression of AP2-G can be used to synchronously convert the great majority of the population into fertile gametocytes. This discovery allowed us to redefine the time frame of sexual commitment, identify a number of putative AP2-G targets and chart the sequence of transcriptional changes through gametocyte development, including the observation that gender-specific transcription occurred within 6 h of induction. These data provide entry points for further detailed characterization of the key process required for malaria transmission. |
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Title:
Epistasis studies reveal redundancy among calcium-dependent protein kinases in motility and invasion of malaria parasites
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|
Author(s):
Fang, HW (Fang, Hanwei); Gomes, AR (Gomes, Ana Rita); Klages, N (Klages, Natacha); Pino, P (Pino, Paco); Maco, B (Maco, Bohumil); Walker, EM (Walker, Eloise M.); Zenonos, ZA (Zenonos, Zenon A.); Angrisano, F (Angrisano, Fiona); Baum, J (Baum, Jake); Doerig, C (Doerig, Christian); Baker, DA (Baker, David A.); Billker, O (Billker, Oliver); Brochet, M (Brochet, Mathieu) |
| Source:
NATURE COMMUNICATIONS Volume:
9 Article Number:
4248 DOI:
10.1038/s41467-018-06733-w Published Date:
2018 OCT 12 |
|
Abstract:
In malaria parasites, evolution of parasitism has been linked to functional optimisation. Despite this optimisation, most members of a calcium-dependent protein kinase (CDPK) family show genetic redundancy during erythrocytic proliferation. To identify relationships between phospho-signalling pathways, we here screen 294 genetic interactions among protein kinases in Plasmodium berghei. This reveals a synthetic negative interaction between a hypomorphic allele of the protein kinase G (PKG) and CDPK4 to control erythrocyte invasion which is conserved in P. falciparum. CDPK4 becomes critical when PKG-dependent calcium signals are attenuated to phosphorylate proteins important for the stability of the inner membrane complex, which serves as an anchor for the acto-myosin motor required for motility and invasion. Finally, we show that multiple kinases functionally complement CDPK4 during erythrocytic proliferation and transmission to the mosquito. This study reveals how CDPKs are wired within a stage-transcending signalling network to control motility and host cell invasion in malaria parasites. |
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Title:
Proteomic profiling of the brain of mice with experimental cerebral malaria
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|
Author(s):
Moussa, E (Moussa, Ehab); Huang, HL (Huang, Honglei); Ahras, M (Ahras, Malika); Lall, A (Lall, Amar); Thezenas, ML (Thezenas, Marie L.); Fischer, R (Fischer, Roman); Kessler, BM (Kessler, Benedikt M.); Pain, A (Pain, Arnab); Billker, O (Billker, Oliver); Casals-Pascual, C (Casals-Pascual, Climent) |
| Source:
JOURNAL OF PROTEOMICS Volume:
180 Special Issue:
SI Pages:
61-69 DOI:
10.1016/j.jprot.2017.06.002 Published Date:
2018 MAY 30 |
Abstract:
Cerebral malaria (CM) is a severe neurological complication of malaria infection in both adults and children. In pursuit of effective treatment of CM, clinical studies, postmortem analysis and animal models have been employed to understand the pathology and identify effective interventions. In this study, a shotgun proteomics analysis was conducted to profile the proteomic signature of the brain tissue of mice with experimental cerebral malaria (ECM) in order to further understand the underlying pathology. To identify CM-associated response, proteomic signatures of the brains of C57/B16N mice infected with P. berghei ANKA that developed neurological syndrome were compared to those of mice infected with P. berghei NK65 that developed equally high parasite burdens without neurological signs, and to those of non-infected mice. The results show that the CM-associated response in mice that developed neurological signs comprise mainly acute-phase reaction and coagulation cascade activation, and indicate the leakage of plasma proteins into the brain parenchyma.
Significance: Cerebral malaria (CM) remains a major cause of death in children. The majority of these deaths occur in sub-Saharan Africa. Even with adequate access to treatment, mortality remains high and neurological sequelae can be found in up to 20% of survivors. No adjuvant treatment to date has been shown to reduce mortality and the pathophysiology of CM is largely unknown. Experimental cerebral malaria (ECM) is a well-established model that may contribute to identify and test druggable targets. In this study we have identified the disruption of the blood-brain barrier following inflammatory and vascular injury as a mechanism of disease. In this study we report a number of proteins that could be validated as potential biomarkers of ECM. Further studies, will be required to validate the clinical relevance of these biomarkers in human CM. (c) 2017 Elsevier B.V. All rights reserved. |
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Title:
Alpha-v-containing integrins are host receptors for the Plasmodium falciparum sporozoite surface protein, TRAP
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Author(s):
Dundas, K (Dundas, Kirsten); Shears, MJ (Shears, Melanie J.); Sun, Y (Sun, Yi); Hopp, CS (Hopp, Christine S.); Crosnier, C (Crosnier, Cecile); Metcalf, T (Metcalf, Tom); Girling, G (Girling, Gareth); Sinnis, P (Sinnis, Photini); Billker, O (Billker, Oliver); Wright, GJ (Wright, Gavin J.) |
| Source:
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Volume:
115 Issue:
17 Pages:
4477-4482 DOI:
10.1073/pnas.1719660115 Published Date:
2018 APR 24 |
|
Abstract:
Malaria-causing Plasmodium sporozoites are deposited in the dermis by the bite of an infected mosquito and move by gliding motility to the liver where they invade and develop within host hepatocytes. Although extracellular interactions between Plasmodium sporozoite ligands and host receptors provide important guidance cues for productive infection and are good vaccine targets, these interactions remain largely uncharacterized. Thrombo-spondin-related anonymous protein (TRAP) is a parasite cell surface ligand that is essential for both gliding motility and invasion because it couples the extracellular binding of host receptors to the parasite cytoplasmic actinomyosin motor; however, the molecular nature of the host TRAP receptors is poorly defined. Here, we use a systematic extracellular protein interaction screening approach to identify the integrin alpha v beta 3 as a directly interacting host receptor for Plasmodium falciparum TRAP. Biochemical characterization of the interaction suggests a two-site binding model, requiring contributions from both the von Willebrand factor A domain and the RGD motif of TRAP for integrin binding. We show that TRAP binding to cells is promoted in the presence of integrin-activating proadhesive Mn2+ ions, and that cells genetically targeted so that they lack cell surface expression of the integrin alpha v-subunit are no longer able to bind TRAP. P. falciparum sporozoites moved with greater speed in the dermis of Itgb3-deficient mice, suggesting that the interaction has a role in sporozoite migration. The identification of the integrin alpha v beta 3 as the host receptor for TRAP provides an important demonstration of a sporozoite surface ligand that directly interacts with host receptors. |
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Title:
Complete avian malaria parasite genomes reveal features associated with lineage-specific evolution in birds and mammals
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Author(s):
Böhme, U (Bohme, Ulrike); Otto, TD (Otto, Thomas D.); Cotton, JA (Cotton, James A.); Steinbiss, S (Steinbiss, Sascha); Sanders, M (Sanders, Mandy); Oyola, SO (Oyola, Samuel O.); Nicot, A (Nicot, Antoine); Gandon, S (Gandon, Sylvain); Patra, KP (Patra, Kailash P.); Herd, C (Herd, Colin); Bushell, E (Bushell, Ellen); Modrzynska, KK (Modrzynska, Katarzyna K.); Billker, O (Billker, Oliver); Vinetz, JM (Vinetz, Joseph M.); Rivero, A (Rivero, Ana); Newbold, CI (Newbold, Chris I.); Berriman, M (Berriman, Matthew) |
| Source:
GENOME RESEARCH Volume:
28 Issue:
4 Pages:
547-560 DOI:
10.1101/gr.218123.116 Published Date:
2018 APR |
|
Abstract:
Avian malaria parasites are prevalent around the world and infect a wide diversity of bird species. Here, we report the sequencing and analysis of high-quality draft genome sequences for two avian malaria species, Plasmodium relictum and Plasmodium gallinaceum. We identify 50 genes that are specific to avian malaria, located in an otherwise conserved core of the genome that shares gene synteny with all other sequenced malaria genomes. Phylogenetic analysis suggests that the avian malaria species form an outgroup to the mammalian Plasmodium species, and using amino acid divergence between species, we estimate the avian- and mammalian-infective lineages diverged in the order of 10 million years ago. Consistent with their phylogenetic position, we identify orthologs of genes that had previously appeared to be restricted to the clades of parasites containing Plasmodium falciparum and Plasmodium vivax, the species with the greatest impact on human health. From these orthologs, we explore differential diversifying selection across the genus and show that the avian lineage is remarkable in the extent to which invasion-related genes are evolving. The subtelomeres of the P. relictum and P. gallinaceum genomes contain several novel gene families, including an expanded surf multigene family. We also identify an expansion of reticulocyte binding protein homologs in P. relictum, and within these proteins, we detect distinct regions that are specific to nonhuman primate, humans, rodent, and avian hosts. For the first time in the Plasmodium lineage, we find evidence of transposable elements, including several hundred fragments of LTR-retrotransposons in both species and an apparently complete LTR-retrotransposon in the genome of P. gallinaceum. |
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Title:
Single-cell RNA-seq reveals hidden transcriptional variation in malaria parasites
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Author(s):
Reid, AJ (Reid, Adam J.); Talman, AM (Talman, Arthur M.); Bennett, HM (Bennett, Hayley M.); Gomes, AR (Gomes, Ana R.); Sanders, MJ (Sanders, Mandy J.); Illingwoth, CJR (Illingwoth, Christopher J. R.); Billker, O (Billker, Oliver); Berriman, M (Berriman, Matthew); Lawniczak, MKN (Lawniczak, Mara K. N.) |
| Source:
ELIFE Volume:
7 Article Number:
e33105 DOI:
10.7554/eLife.33105 Published Date:
2018 MAR 27 |
|
Abstract:
Single-cell RNA-sequencing is revolutionising our understanding of seemingly homogeneous cell populations but has not yet been widely applied to single-celled organisms. Transcriptional variation in unicellular malaria parasites from the Plasmodium genus is associated with critical phenotypes including red blood cell invasion and immune evasion, yet transcriptional variation at an individual parasite level has not been examined in depth. Here, we describe the adaptation of a single-cell RNA-sequencing (scRNA-seq) protocol to deconvolute transcriptional variation for more than 500 individual parasites of both rodent and human malaria comprising asexual and sexual life-cycle stages. We uncover previously hidden discrete transcriptional signatures during the pathogenic part of the life cycle, suggesting that expression over development is not as continuous as commonly thought. In transmission stages, we find novel, sex-specific roles for differential expression of contingency gene families that are usually associated with immune evasion and pathogenesis. |
|
Title:
IMMUNITY AND MEMORY AGAINST MALARIA: AN ATLAS OF THE MOSQUITO IMMUNE SYSTEM AT SINGLE CELL RESOLUTION
|
|
Author(s):
Raddi, G (Raddi, Gianmarco); Barillas-Mury, C (Barillas-Mury, Carolina); Billker, O (Billker, Oliver) |
| Source:
AMERICAN JOURNAL OF TROPICAL MEDICINE AND HYGIENE Meeting Abstract:
1453 Volume:
99 Issue:
4 Pages:
458-458 Published Date:
2018 Supplement:
S |
|
Title:
Sub-minute Phosphoregulation of Cell Cycle Systems during Plasmodium Gamete Formation
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Author(s):
Invergo, BM (Invergo, Brandon M.); Brochet, M (Brochet, Mathieu); Yu, L (Yu, Lu); Choudhary, J (Choudhary, Jyoti); Beltrao, P (Beltrao, Pedro); Billker, O (Billker, Oliver) |
| Source:
CELL REPORTS Volume:
21 Issue:
7 Pages:
2017-2029 DOI:
10.1016/j.celrep.2017.10.071 Published Date:
2017 NOV 14 |
|
Abstract:
The transmission of malaria parasites to mosquitoes relies on the rapid induction of sexual reproduction upon their ingestion into a blood meal. Haploid female and male gametocytes become activated and emerge from their host cells, and the males enter the cell cycle to produce eight microgametes. The synchronized nature of gametogenesis allowed us to investigate phosphorylation signaling during its first minute in Plasmodium berghei via a high-resolution time course of the phosphoproteome. This revealed an unexpectedly broad response, with proteins related to distinct cell cycle events undergoing simultaneous phosphoregulation. We implicate several protein kinases in the process, and we validate our analyses on the plant-like calcium-dependent protein kinase 4 (CDPK4) and a homolog of serine/arginine-rich protein kinases (SRPK1). Mutants in these kinases displayed distinct phosphoproteomic disruptions, consistent with differences in their phenotypes. The results reveal the central role of protein phosphorylation in the atypical cell cycle regulation of a divergent eukaryote. |
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Title:
malERA: An updated research agenda for basic science and enabling technologies in malaria elimination and eradication
|
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Author(s):
Wirth, DF (Wirth, Dyann F.); Winzeler, EA (Winzeler, Elizabeth A.); Fenton, B (Fenton, B.); Adams, JH (Adams, John H.); Ariey, F (Ariey, Frederic); Barillas-Mury, CV (Barillas-Mury, Carolina V.); Baum, J (Baum, Jake); Bhatia, SN (Bhatia, Sangeeta N.); Billker, O (Billker, Oliver); Bopp, SE (Bopp, Selina E.); Catteruccia, F (Catteruccia, Flaminia); Cowman, AF (Cowman, Alan F.); Chitnis, CE (Chitnis, Chetan E.); Crabb, BS (Crabb, Brendan S.); Deitsch, KW (Deitsch, Kirk W.); Del Portillo, HA (Del Portillo, Hernando A.); Djimde, AA (Djimde, Abdoulaye A.); Dobaño, C (Dobano, Carlota); Duffy, PE (Duffy, Patrick E.); Duraisingh, MT (Duraisingh, Manoj T.); Happi, C (Happi, Christian); James, AA (James, Anthony A.); LaMonte, G (LaMonte, Gregory); Lukens, AK (Lukens, Amanda K.); Llinas, M (Llinas, Manuel); Mali, P (Mali, Prashant); Marti, M (Marti, Matthias); Martinez-Barnetche, J (Martinez-Barnetche, Jesus); McGovern, V (McGovern, Victoria); Mota, MM (Mota, Maria M.); Mueller, I (Mueller, Ivo); Ndiaye, D (Ndiaye, Daouda); Neafsey, DE (Neafsey, Daniel E.); Ntoumi, F (Ntoumi, Francine); Prachumsri, J (Prachumsri, Jetsumon); Sharma, P (Sharma, Pushkar); Shaw, WR (Shaw, W. Robert); Sinnis, P (Sinnis, Photini); Tolia, NH (Tolia, Niraj H.); Volkman, SK (Volkman, Sarah K.); Wenger, EA (Wenger, Edward A.); Williamson, K (Williamson, Kim) |
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Group Author(s):
The Malera Refresh Consultative Pa |
| Source:
PLOS MEDICINE Volume:
14 Issue:
11 Article Number:
e1002451 DOI:
10.1371/journal.pmed.1002451 Published Date:
2017 NOV |
|
Abstract:
Basic science holds enormous power for revealing the biological mechanisms of disease and, in turn, paving the way toward new, effective interventions. Recognizing this power, the 2011 Research Agenda for Malaria Eradication included key priorities in fundamental research that, if attained, could help accelerate progress toward disease elimination and eradication. The Malaria Eradication Research Agenda (malERA) Consultative Panel on Basic Science and Enabling Technologies reviewed the progress, continuing challenges, and major opportunities for future research. The recommendations come from a literature of published and unpublished materials and the deliberations of the malERA Refresh Consultative Panel. These areas span multiple aspects of the Plasmodium life cycle in both the human host and the Anopheles vector and include critical, unanswered questions about parasite transmission, human infection in the liver, asexual-stage biology, and malaria persistence. We believe an integrated approach encompassing human immunology, parasitology, and entomology, and harnessing new and emerging biomedical technologies offers the best path toward addressing these questions and, ultimately, lowering the worldwide burden of malaria. |
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Title:
Functional Profiling of a Plasmodium Genome Reveals an Abundance of Essential Genes
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Author(s):
Bushell, E (Bushell, Ellen); Gomes, AR (Gomes, Ana Rita); Sanderson, T (Sanderson, Theo); Anar, B (Anar, Burcu); Girling, G (Girling, Gareth); Herd, C (Herd, Colin); Metcalf, T (Metcalf, Tom); Modrzynska, K (Modrzynska, Katarzyna); Schwach, F (Schwach, Frank); Martin, RE (Martin, Rowena E.); Mather, MW (Mather, Michael W.); McFadden, GI (McFadden, Geoffrey I.); Parts, L (Parts, Leopold); Rutledge, GG (Rutledge, Gavin G.); Vaidya, AB (Vaidya, Akhil B.); Wengelnik, K (Wengelnik, Kai); Rayner, JC (Rayner, Julian C.); Billker, O (Billker, Oliver) |
| Source:
CELL Volume:
170 Issue:
2 Pages:
260-+ DOI:
10.1016/j.cell.2017.06.030 Published Date:
2017 JUL 13 |
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Abstract:
The genomes of malaria parasites contain many genes of unknown function. To assist drug development through the identification of essential genes and pathways, we have measured competitive growth rates in mice of 2,578 barcoded Plasmodium berghei knockout mutants, representing >50% of the genome, and created a phenotype database. At a single stage of its complex life cycle, P. berghei requires two-thirds of genes for optimal growth, the highest proportion reported from any organism and a probable consequence of functional optimization necessitated by genomic reductions during the evolution of parasitism. In contrast, extreme functional redundancy has evolved among expanded gene families operating at the parasite-host interface. The level of genetic redundancy in a single-celled organism may thus reflect the degree of environmental variation it experiences. In the case of Plasmodium parasites, this helps rationalize both the relative successes of drugs and the greater difficulty of making an effective vaccine. |
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Title:
Nutrient sensing modulates malaria parasite virulence
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Author(s):
Mancio-Silva, L (Mancio-Silva, Liliana); Slavic, K (Slavic, Ksenija); Ruivo, MTG (Ruivo, Margarida T. Grilo); Grosso, AR (Grosso, Ana Rita); Modrzynska, KK (Modrzynska, Katarzyna K.); Vera, IM (Vera, Iset Medina); Sales-Dias, J (Sales-Dias, Joana); Gomes, AR (Gomes, Ana Rita); MacPherson, CR (MacPherson, Cameron Ross); Crozet, P (Crozet, Pierre); Adamo, M (Adamo, Mattia); Baena-Gonzalez, E (Baena-Gonzalez, Elena); Tewari, R (Tewari, Rita); Llinás, M (Llinas, Manuel); Billker, O (Billker, Oliver); Mota, MM (Mota, Maria M.) |
| Source:
NATURE Volume:
547 Issue:
7662 Pages:
213-+ DOI:
10.1038/nature23009 Published Date:
2017 JUL 13 |
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Abstract:
The lifestyle of intracellular pathogens, such as malaria parasites, is intimately connected to that of their host, primarily for nutrient supply. Nutrients act not only as primary sources of energy but also as regulators of gene expression, metabolism and growth, through various signalling networks that enable cells to sense and adapt to varying environmental conditions(1,2). Canonical nutrient-sensing pathways are presumed to be absent from the causative agent of malaria, Plasmodium(3-5), thus raising the question of whether these parasites can sense and cope with fluctuations in host nutrient levels. Here we show that Plasmodium blood-stage parasites actively respond to host dietary calorie alterations through rearrangement of their transcriptome accompanied by substantial adjustment of their multiplication rate. A kinome analysis combined with chemical and genetic approaches identified KIN as a critical regulator that mediates sensing of nutrients and controls a transcriptional response to the host nutritional status. KIN shares homology with SNF1/AMPK alpha, and yeast complementation studies suggest that it is part of a functionally conserved cellular energy-sensing pathway. Overall, these findings reveal a key parasite nutrient-sensing mechanism that is critical for modulating parasite replication and virulence. |
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Title:
Malaria: Cracking Ali Baba’s code
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Author(s):
Billker, O (Billker, Oliver) |
| Source:
ELIFE Volume:
6 Article Number:
e28600 DOI:
10.7554/eLife.28600 Published Date:
2017 JUN 14 |
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Title:
A single nucleotide polymorphism in an AP2 transcription factor encoded in the malaria-causing Plasmodium berghei alters the development of host immunity
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Author(s):
Sheehan, PW (Sheehan, Patrick William); Akkaya, M (Akkaya, Munir); Bansal, A (Bansal, Abhisheka); Arora, G (Arora, Gunjan); Otto, TD (Otto, Thomas Dan); Qi, CF (Qi, Cheng-Feng); Pena, M (Pena, Mirna); Yazew, T (Yazew, Takele); Billker, O (Billker, Oliver); Miller, L (Miller, Louis); Pierce, SK (Pierce, Susan K.) |
| Source:
JOURNAL OF IMMUNOLOGY Meeting Abstract:
77.5 Volume:
198 Issue:
1 Published Date:
2017 MAY 1 Supplement:
S |
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Title:
Antibody-independent mechanisms regulate the establishment of chronic Plasmodium infection
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Author(s):
Brugat, T (Brugat, Thibaut); Reid, AJ (Reid, Adam James); Lin, JW (Lin, Jing-wen); Cunningham, D (Cunningham, Deirdre); Tumwine, I (Tumwine, Irene); Kushinga, G (Kushinga, Garikai); McLaughlin, S (McLaughlin, Sarah); Spence, P (Spence, Philip); Boehme, U (Boehme, Ulrike); Sanders, M (Sanders, Mandy); Conteh, S (Conteh, Solomon); Bushell, E (Bushell, Ellen); Metcalf, T (Metcalf, Tom); Billker, O (Billker, Oliver); Duffy, PE (Duffy, Patrick E.); Newbold, C (Newbold, Chris); Berriman, M (Berriman, Matthew); Langhorne, J (Langhorne, Jean) |
| Source:
NATURE MICROBIOLOGY Volume:
2 Issue:
4 Article Number:
16276 DOI:
10.1038/nmicrobiol.2016.276 Published Date:
2017 APR |
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Abstract:
Malaria is caused by parasites of the genus Plasmodium. All human-infecting Plasmodium species can establish longlasting chronic infections(1-5), creating an infectious reservoir to sustain transmission(1),(6). It is widely accepted that the maintenance of chronic infection involves evasion of adaptive immunity by antigenic variation(7). However, genes involved in this process have been identified in only two of five human-infecting species: Plasmodium falciparum and Plasmodium knowlesi. Furthermore, little is understood about the early events in the establishment of chronic infection in these species. Using a rodent model we demonstrate that from the infecting population, only a minority of parasites, expressing one of several clusters of virulence- associated pir genes, establishes a chronic infection. This process occurs in different species of parasites and in different hosts. Establishment of chronicity is independent of adaptive immunity and therefore different from the mechanism proposed for maintenance of chronic P. falciparum infections(7)- . Furthermore, we show that the proportions of parasites expressing different types of pir genes regulate the time taken to establish a chronic infection. Because pir genes are common to most, if not all, species of Plasmodium(10,) this process may be a common way of regulating the establishment of chronic infections. |
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Title:
Single-cell RNA-seq and computational analysis using temporal mixture modeling resolves T<sub>H</sub>1/T<sub>FH</sub> fate bifurcation in malaria
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Author(s):
Lönnberg, T (Lonnberg, Tapio); Svensson, V (Svensson, Valentine); James, KR (James, Kylie R.); Fernandez-Ruiz, D (Fernandez-Ruiz, Daniel); Sebina, I (Sebina, Ismail); Montandon, R (Montandon, Ruddy); Soon, MSF (Soon, Megan S. F.); Fogg, LG (Fogg, Lily G.); Nair, AS (Nair, Arya Sheela); Liligeto, UN (Liligeto, Urijah N.); Stubbington, MJT (Stubbington, Michael J. T.); Ly, LH (Ly, Lam-Ha); Bagger, FO (Bagger, Frederik Otzen); Zwiessele, M (Zwiessele, Max); Lawrence, ND (Lawrence, Neil D.); Souza-Fonseca-Guimaraes, F (Souza-Fonseca-Guimaraes, Fernando); Bunn, PT (Bunn, Patrick T.); Engwerda, CR (Engwerda, Christian R.); Heath, WR (Heath, William R.); Billker, O (Billker, Oliver); Stegle, O (Stegle, Oliver); Haque, A (Haque, Ashraful); Teichmann, SA (Teichmann, Sarah A.) |
| Source:
SCIENCE IMMUNOLOGY Volume:
2 Issue:
9 Article Number:
eaal2192 DOI:
10.1126/sciimmunol.aal2192 Published Date:
2017 MAR |
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Abstract:
Differentiation of naive CD4(+) T cells into functionally distinct T helper (T-H) subsets is crucial for the orchestration of immune responses. Because of extensive heterogeneity and multiple overlapping transcriptional programs in differentiating T cell populations, this process has remained a challenge for systematic dissection in vivo. By using single-cell transcriptomics and computational analysis with a temporal mixtures of Gaussian processes model, termed GPfates, we reconstructed the developmental trajectories of T(H)1 and T-FH (T follicular helper) cells during blood-stage Plasmodium infection in mice. By tracking clonality using endogenous T cell receptor sequences, we first demonstrated that T(H)1/T-FH bifurcation had occurred at both population and single-clone levels. Next, we identified genes whose expression was associated with T(H)1 or T-FH fates and demonstrated a T cell-intrinsic role for Galectin-1 in supporting T(H)1 differentiation. We also revealed the close molecular relationship between T(H)1 and interleukin-10-producing Tr1 cells in this infection. T(H)1 and T-FH fates emerged from a highly proliferative precursor that up-regulated aerobic glycolysis and accelerated cell cycling as cytokine expression began. Dynamic gene expression of chemokine receptors around bifurcation predicted roles for cell-cell interaction in driving T(H)1/T-FH fates. In particular, we found that precursor T-H cells were coached toward a T(H)1 but not a T-FH fate by inflammatory monocytes. Thus, by integrating genomic and computational approaches, our study has provided two unique resources: a database, www.PlasmoTH.org, which facilitates discovery of novel factors controlling T(H)1/T-FH fate commitment, and, more generally, GPfates, a modeling framework for characterizing cell differentiation toward multiple fates. |
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Title:
A Knockout Screen of ApiAP2 Genes Reveals Networks of Interacting Transcriptional Regulators Controlling the Plasmodium Life Cycle
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Author(s):
Modrzynska, K (Modrzynska, Katarzyna); Pfander, C (Pfander, Claudia); Chappell, L (Chappell, Lia); Yu, L (Yu, Lu); Suarez, C (Suarez, Catherine); Dundas, K (Dundas, Kirsten); Gomes, AR (Gomes, Ana Rita); Goulding, D (Goulding, David); Rayner, JC (Rayner, Julian C.); Choudhary, J (Choudhary, Jyoti); Billker, O (Billker, Oliver) |
| Source:
CELL HOST & MICROBE Volume:
21 Issue:
1 Pages:
11-22 DOI:
10.1016/j.chom.2016.12.003 Published Date:
2017 JAN 11 |
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Abstract:
A family of apicomplexa-specific proteins containing AP2 DNA-binding domains (ApiAP2s) was identified in malaria parasites. This family includes sequencespecific transcription factors that are key regulators of development. However, functions for the majority of ApiAP2 genes remain unknown. Here, a systematic knockout screen in Plasmodium berghei identified ten ApiAP2 genes that were essential for mosquito transmission: four were critical for the formation of infectious ookinetes, and three were required for sporogony. We describe non-essential functions for AP2-O and AP2-SP proteins in blood stages, and identify AP2-G2 as a repressor active in both asexual and sexual stages. Comparative transcriptomics across mutants and developmental stages revealed clusters of co-regulated genes with shared cis promoter elements, whose expression can be controlled positively or negatively by different ApiAP2 factors. We propose that stage-specific interactions between ApiAP2 proteins on partly overlapping sets of target genes generate the complex transcriptional network that controls the Plasmodium life cycle. |
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Title:
MULTIPLEX COMPETITIVE GROWTH ASSAYS FOR MEASURING THE BIOLOGICAL IMPACT OF FITNESS IN DRUG-RESISTANT PLASMODIUM FALCIPARUM
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Author(s):
Carrasquilla, M (Carrasquilla, Manuela); Billker, O (Billker, Oliver); Rayner, J (Rayner, Julian); Lee, M (Lee, Marcus) |
| Source:
AMERICAN JOURNAL OF TROPICAL MEDICINE AND HYGIENE Meeting Abstract:
1296 Volume:
97 Issue:
5 Pages:
403-403 Published Date:
2017 Supplement:
S |
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Title:
Decreased Rate of Plasma Arginine Appearance in Murine Malaria May Explain Hypoargininemia in Children With Cerebral Malaria
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Author(s):
Alkaitis, MS (Alkaitis, Matthew S.); Wang, HH (Wang, Honghui); Ikeda, AK (Ikeda, Allison K.); Rowley, CA (Rowley, Carol A.); MacCormick, IJC (MacCormick, Ian J. C.); Chertow, JH (Chertow, Jessica H.); Billker, O (Billker, Oliver); Suffredini, AF (Suffredini, Anthony F.); Roberts, DJ (Roberts, David J.); Taylor, TE (Taylor, Terrie E.); Seydel, KB (Seydel, Karl B.); Ackerman, HC (Ackerman, Hans C.) |
| Source:
JOURNAL OF INFECTIOUS DISEASES Volume:
214 Issue:
12 Pages:
1840-1849 DOI:
10.1093/infdis/jiw452 Published Date:
2016 DEC 15 |
Abstract:
Background. Plasmodium infection depletes arginine, the substrate for nitric oxide synthesis, and impairs endothelium-dependent vasodilation. Increased conversion of arginine to ornithine by parasites or host arginase is a proposed mechanism of arginine depletion.
Methods. We used high-performance liquid chromatography to measure plasma arginine, ornithine, and citrulline levels in Malawian children with cerebral malaria and in mice infected with Plasmodium berghei ANKA with or without the arginase gene. Heavy isotope-labeled tracers measured by quadrupole time-of-flight liquid chromatography-mass spectrometry were used to quantify the in vivo rate of appearance and interconversion of plasma arginine, ornithine, and citrulline in infected mice.
Results. Children with cerebral malaria and P. berghei-infected mice demonstrated depletion of plasma arginine, ornithine, and citrulline. Knock out of Plasmodium arginase did not alter arginine depletion in infected mice. Metabolic tracer analysis demonstrated that plasma arginase flux was unchanged by P. berghei infection. Instead, infected mice exhibited decreased rates of plasma arginine, ornithine, and citrulline appearance and decreased conversion of plasma citrulline to arginine. Notably, plasma arginine use by nitric oxide synthase was decreased in infected mice.
Conclusions. Simultaneous arginine and ornithine depletion in malaria parasite-infected children cannot be fully explained by plasma arginase activity. Our mouse model studies suggest that plasma arginine depletion is driven primarily by a decreased rate of appearance. |
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Title:
Palmitoyl transferases have critical roles in the development of mosquito and liver stages of Plasmodium
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Author(s):
Hopp, CS (Hopp, Christine S.); Balaban, AE (Balaban, Amanda E.); Bushell, ESC (Bushell, Ellen S. C.); Billker, O (Billker, Oliver); Rayner, JC (Rayner, Julian C.); Sinnis, P (Sinnis, Photini) |
| Source:
CELLULAR MICROBIOLOGY Volume:
18 Issue:
11 Pages:
1625-1641 DOI:
10.1111/cmi.12601 Published Date:
2016 NOV |
|
Abstract:
As the Plasmodium parasite transitions between mammalian and mosquito host, it has to adjust quickly to new environments. Palmitoylation, a reversible and dynamic lipid post-translational modification, plays a central role in regulating this process and has been implicated with functions for parasite morphology, motility and host cell invasion. While proteins associated with the gliding motility machinery have been described to be palmitoylated, no palmitoyl transferase responsible for regulating gliding motility has previously been identified. Here, we characterize two palmityol transferases with gene tagging and gene deletion approaches. We identify DHHC3, a palmitoyl transferase, as a mediator of ookinete development, with a crucial role for gliding motility in ookinetes and sporozoites, and we co-localize the protein with a marker for the inner membrane complex in the ookinete stage. Ookinetes and sporozoites lacking DHHC3 are impaired in gliding motility and exhibit a strong phenotype in vivo; with ookinetes being significantly less infectious to their mosquito host and sporozoites being non-infectious to mice. Importantly, genetic complementation of the DHHC3-ko parasite completely restored virulence. We generated parasites lacking both DHHC3, as well as the palmitoyl transferase DHHC9, and found an enhanced phenotype for these double knockout parasites, allowing insights into the functional overlap and compensational nature of the large family of PbDHHCs. These findings contribute to our understanding of the organization and mechanism of the gliding motility machinery, which as is becoming increasingly clear, is mediated by palmitoylation. |
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Title:
A Stem Cell Strategy Identifies Glycophorin C as a Major Erythrocyte Receptor for the Rodent Malaria Parasite Plasmodium berghei
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Author(s):
Yiangou, L (Yiangou, Loukia); Montandon, R (Montandon, Ruddy); Modrzynska, K (Modrzynska, Katarzyna); Rosen, B (Rosen, Barry); Bushell, W (Bushell, Wendy); Hale, C (Hale, Christine); Billker, O (Billker, Oliver); Rayner, JC (Rayner, Julian C.); Pance, A (Pance, Alena) |
| Source:
PLOS ONE Volume:
11 Issue:
6 Article Number:
e0158238 DOI:
10.1371/journal.pone.0158238 Published Date:
2016 JUN 30 |
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Abstract:
The clinical complications of malaria are caused by the parasite expansion in the blood. Invasion of erythrocytes is a complex process that depends on multiple receptor-ligand interactions. Identification of host receptors is paramount for fighting the disease as it could reveal new intervention targets, but the enucleated nature of erythrocytes makes genetic approaches impossible and many receptors remain unknown. Host-parasite interactions evolve rapidly and are therefore likely to be species-specific. As a results, understanding of invasion receptors outside the major human pathogen Plasmodium falciparum is very limited. Here we use mouse embryonic stem cells (mESCs) that can be genetically engineered and differentiated into erythrocytes to identify receptors for the rodent malaria parasite Plasmodium berghei. Two proteins previously implicated in human malaria infection: glycophorin C (GYPC) and Band-3 (Slc4a1) were deleted in mESCs to generate stable cell lines, which were differentiated towards erythropoiesis. In vitro infection assays revealed that while deletion of Band-3 has no effect, absence of GYPC results in a dramatic decrease in invasion, demonstrating the crucial role of this protein for P. berghei infection. This stem cell approach offers the possibility of targeting genes that may be essential and therefore difficult to disrupt in whole organisms and has the potential to be applied to a variety of parasites in diverse host cell types. |
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Title:
Single cell analysis of CD4+ T cell differentiation reveals three major cell states and progressive acceleration of proliferation (vol 17, 103, 2016)
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Author(s):
Proserpio, V (Proserpio, Valentina); Piccolo, A (Piccolo, Andrea); Haim-Vilmovsky, L (Haim-Vilmovsky, Liora); Kar, G (Kar, Gozde); Lönnberg, T (Lonnberg, Tapio); Svensson, V (Svensson, Valentine); Pramanik, J (Pramanik, Jhuma); Natarajan, KN (Natarajan, Kedar Nath); Zhai, WC (Zhai, Weichao); Zhang, XW (Zhang, Xiuwei); Donati, G (Donati, Giacomo); Kayikci, M (Kayikci, Melis); Kotar, J (Kotar, Jurij); McKenzie, ANJ (McKenzie, Andrew N. J.); Montandon, R (Montandon, Ruddy); James, KR (James, Kylie R.); Fernandez-Ruiz, D (Fernandez-Ruiz, Daniel); Heath, WR (Heath, William R.); Haque, A (Haque, Ashraful); Billker, O (Billker, Oliver); Woodhouse, S (Woodhouse, Steven); Cicuta, P (Cicuta, Pietro); Nicodemi, M (Nicodemi, Mario); Teichmann, SA (Teichmann, Sarah A.) |
| Source:
GENOME BIOLOGY Volume:
17 Article Number:
133 DOI:
10.1186/s13059-016-0998-9 Published Date:
2016 JUN 22 |
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Title:
Single-cell analysis of CD4+T-cell differentiation reveals three major cell states and progressive acceleration of proliferation
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Author(s):
Proserpio, V (Proserpio, Valentina); Piccolo, A (Piccolo, Andrea); Haim-Vilmovsky, L (Haim-Vilmovsky, Liora); Kar, G (Kar, Gozde); Lönnberg, T (Lonnberg, Tapio); Svensson, V (Svensson, Valentine); Pramanik, J (Pramanik, Jhuma); Natarajan, KN (Natarajan, Kedar Nath); Zhai, WC (Zhai, Weichao); Zhang, XW (Zhang, Xiuwei); Donati, G (Donati, Giacomo); Kayikci, M (Kayikci, Melis); Kotar, J (Kotar, Jurij); McKenzie, ANJ (McKenzie, Andrew N. J.); Montandon, R (Montandon, Ruddy); Billker, O (Billker, Oliver); Woodhouse, S (Woodhouse, Steven); Cicuta, P (Cicuta, Pietro); Nicodemi, M (Nicodemi, Mario); Teichmann, SA (Teichmann, Sarah A.) |
| Source:
GENOME BIOLOGY Volume:
17 Article Number:
103 DOI:
10.1186/s13059-016-0957-5 Published Date:
2016 MAY 12 |
Abstract:
Background: Differentiation of lymphocytes is frequently accompanied by cell cycle changes, interplay that is of central importance for immunity but is still incompletely understood. Here, we interrogate and quantitatively model how proliferation is linked to differentiation in CD4+ T cells.
Results: We perform ex vivo single-cell RNA-sequencing of CD4+ T cells during a mouse model of infection that elicits a type 2 immune response and infer that the differentiated, cytokine-producing cells cycle faster than early activated precursor cells. To dissect this phenomenon quantitatively, we determine expression profiles across consecutive generations of differentiated and undifferentiated cells during Th2 polarization in vitro. We predict three discrete cell states, which we verify by single-cell quantitative PCR. Based on these three states, we extract rates of death, division and differentiation with a branching state Markov model to describe the cell population dynamics. From this multi-scale modelling, we infer a significant acceleration in proliferation from the intermediate activated cell state to the mature cytokine-secreting effector state. We confirm this acceleration both by live imaging of single Th2 cells and in an ex vivo Th1 malaria model by single-cell RNA-sequencing.
Conclusion: The link between cytokine secretion and proliferation rate holds both in Th1 and Th2 cells in vivo and in vitro, indicating that this is likely a general phenomenon in adaptive immunity. |
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Title:
Calcium signalling in malaria parasites
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Author(s):
Brochet, M (Brochet, Mathieu); Billker, O (Billker, Oliver) |
| Source:
MOLECULAR MICROBIOLOGY Volume:
100 Issue:
3 Pages:
397-408 DOI:
10.1111/mmi.13324 Published Date:
2016 MAY |
|
Abstract:
Ca2+ is a ubiquitous intracellular messenger in malaria parasites with important functions in asexual blood stages responsible for malaria symptoms, the preceding liver-stage infection and transmission through the mosquito. Intracellular messengers amplify signals by binding to effector molecules that trigger physiological changes. The characterisation of some Ca2+ effector proteins has begun to provide insights into the vast range of biological processes controlled by Ca2+ signalling in malaria parasites, including host cell egress and invasion, protein secretion, motility and cell cycle regulation. Despite the importance of Ca2+ signalling during the life cycle of malaria parasites, little is known about Ca2+ homeostasis. Recent findings highlighted that upstream of stage-specific Ca2+ effectors is a conserved interplay between second messengers to control critical intracellular Ca2+ signals throughout the life cycle. The identification of the molecular mechanisms integrating stage-transcending mechanisms of Ca2+ homeostasis in a network of stage-specific regulator and effector pathways now represents a major challenge for a meaningful understanding of Ca2+ signalling in malaria parasites. |
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Title:
Resolving T helper cell fate decisions using single-cell RNA-sequencing
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Author(s):
Lönnberg, T (Loennberg, Tapio); James, K (James, Kylie); Svensson, V (Svensson, Valentine); Fernandez-Ruiz, D (Fernandez-Ruiz, Daniel); Sebina, I (Sebina, Ismail); Montandon, R (Montandon, Ruddy); Soon, M (Soon, Megan); Stubbington, M (Stubbington, Michael); Souza-Fonseca-Guimaraes, F (Souza-Fonseca-Guimaraes, Fernando); Heath, W (Heath, William); Billker, O (Billker, Oliver); Haque, A (Haque, Ashraful); Teichmann, S (Teichmann, Sarah) |
| Source:
SCANDINAVIAN JOURNAL OF IMMUNOLOGY Meeting Abstract:
O702 Volume:
83 Issue:
5 Pages:
377-377 Published Date:
2016 MAY |
|
Title:
Enhanced Methylation Analysis by Recovery of Unsequenceable Fragments
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Author(s):
McInroy, GR (McInroy, Gordon R.); Beraldi, D (Beraldi, Dario); Raiber, EA (Raiber, Eun-Ang); Modrzynska, K (Modrzynska, Katarzyna); van Delft, P (van Delft, Pieter); Billker, O (Billker, Oliver); Balasubramanian, S (Balasubramanian, Shankar) |
| Source:
PLOS ONE Volume:
11 Issue:
3 Article Number:
e0152322 DOI:
10.1371/journal.pone.0152322 Published Date:
2016 MAR 31 |
|
Abstract:
Bisulfite sequencing is a valuable tool for mapping the position of 5-methylcytosine in the genome at single base resolution. However, the associated chemical treatment causes strand scission, which depletes the number of sequenceable DNA fragments in a library and thus necessitates PCR amplification. The AT-rich nature of the library generated from bisulfite treatment adversely affects this amplification, resulting in the introduction of major biases that can confound methylation analysis. Here, we report a method that enables more accurate methylation analysis, by rebuilding bisulfite-damaged components of a DNA library. This recovery after bisulfite treatment (ReBuilT) approach enables PCR-free bisulfite sequencing from low nanogram quantities of genomic DNA. We apply the ReBuilT method for the first whole methylome analysis of the highly AT-rich genome of Plasmodium berghei. Side-by-side comparison to a commercial protocol involving amplification demonstrates a substantial improvement in uniformity of coverage and reduction of sequence context bias. Our method will be widely applicable for quantitative methylation analysis, even for technically challenging genomes, and where limited sample DNA is available. |
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Title:
Plasmodium Infection Is Associated with Impaired Hepatic Dimethylarginine Dimethylaminohydrolase Activity and Disruption of Nitric Oxide Synthase Inhibitor/Substrate Homeostasis
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Author(s):
Chertow, JH (Chertow, Jessica H.); Alkaitis, MS (Alkaitis, Matthew S.); Nardone, G (Nardone, Glenn); Ikeda, AK (Ikeda, Allison K.); Cunnington, AJ (Cunnington, Aubrey J.); Okebe, J (Okebe, Joseph); Ebonyi, AO (Ebonyi, Augustine O.); Njie, M (Njie, Madi); Correa, S (Correa, Simon); Jayasooriya, S (Jayasooriya, Shamanthi); Casals-Pascual, C (Casals-Pascual, Climent); Billker, O (Billker, Oliver); Conway, DJ (Conway, David J.); Walther, M (Walther, Michael); Ackerman, H (Ackerman, Hans) |
| Source:
PLOS PATHOGENS Volume:
11 Issue:
9 Article Number:
e1005119 DOI:
10.1371/journal.ppat.1005119 Published Date:
2015 SEP |
|
Abstract:
Inhibition of nitric oxide (NO) signaling may contribute to pathological activation of the vascular endothelium during severe malaria infection. Dimethylarginine dimethylaminohydrolase (DDAH) regulates endothelial NO synthesis by maintaining homeostasis between asymmetric dimethylarginine (ADMA), an endogenous NO synthase (NOS) inhibitor, and arginine, the NOS substrate. We carried out a community-based case-control study of Gambian children to determine whether ADMA and arginine homeostasis is disrupted during severe or uncomplicated malaria infections. Circulating plasma levels of ADMA and arginine were determined at initial presentation and 28 days later. Plasma ADMA/arginine ratios were elevated in children with acute severe malaria compared to 28-day follow-up values and compared to children with uncomplicated malaria or healthy children (p<0.0001 for each comparison). To test the hypothesis that DDAH1 is inactivated during Plasmodium infection, we examined DDAH1 in a mouse model of severe malaria. Plasmodium berghei ANKA infection inactivated hepatic DDAH1 via a post-transcriptional mechanism as evidenced by stable mRNA transcript number, decreased DDAH1 protein concentration, decreased enzyme activity, elevated tissue ADMA, elevated ADMA/arginine ratio in plasma, and decreased whole blood nitrite concentration. Loss of hepatic DDAH1 activity and disruption of ADMA/arginine homeostasis may contribute to severe malaria pathogenesis by inhibiting NO synthesis. |
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Title:
Calcium Builds Strong Host-Parasite Interactions
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Author(s):
Billker, O (Billker, Oliver); Rayner, JC (Rayner, Julian C.) |
| Source:
CELL HOST & MICROBE Volume:
18 Issue:
1 Pages:
9-10 DOI:
10.1016/j.chom.2015.06.012 Published Date:
2015 JUL 8 |
|
Abstract:
Apicomplexan parasite invasion of host cells is a multistep process, requiring coordinated events. In this issue of Cell Host & Microbe, Paul et al. (2015) and Philip and Waters (2015) leverage experimental genetics to show that the calcium-regulated protein phosphatase, calcinuerin, regulates invasion in multiple parasite species. |
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Title:
A Genome-Scale Vector Resource Enables High-Throughput Reverse Genetic Screening in a Malaria Parasite
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Author(s):
Gomes, AR (Gomes, Ana Rita); Bushell, E (Bushell, Ellen); Schwach, F (Schwach, Frank); Girling, G (Girling, Gareth); Anar, B (Anar, Burcu); Quail, MA (Quail, Michael A.); Herd, C (Herd, Colin); Pfander, C (Pfander, Claudia); Modrzynska, K (Modrzynska, Katarzyna); Rayner, JC (Rayner, Julian C.); Billker, O (Billker, Oliver) |
| Source:
CELL HOST & MICROBE Volume:
17 Issue:
3 Pages:
404-413 DOI:
10.1016/j.chom.2015.01.014 Published Date:
2015 MAR 11 |
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Abstract:
The genome-wide identification of gene functions in malaria parasites is hampered by a lack of reverse genetic screening methods. We present a large-scale resource of barcoded vectors with long homology arms for effective modification of the Plasmodium berghei genome. Cotransfecting dozens of vectors into the haploid blood stages creates complex pools of barcoded mutants, whose competitive fitness can be measured during infection of a single mouse using barcode sequencing (barseq). To validate the utility of this resource, we rescreen the P. berghei kinome, using published kinome screens for comparison. We find that several protein kinases function redundantly in asexual blood stages and confirm the targetability of kinases cdpk1, gsk3, tkl3, and PBANKA_082960 by genotyping cloned mutants. Thus, parallel phenotyping of barcoded mutants unlocks the power of reverse genetic screening for a malaria parasite and will enable the systematic identification of genes essential for in vivo parasite growth and transmission. |
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Title:
PlasmoGEM, a database supporting a community resource for large-scale experimental genetics in malaria parasites
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Author(s):
Schwach, F (Schwach, Frank); Bushell, E (Bushell, Ellen); Gomes, AR (Gomes, Ana Rita); Anar, B (Anar, Burcu); Girling, G (Girling, Gareth); Herd, C (Herd, Colin); Rayner, JC (Rayner, Julian C.); Billker, O (Billker, Oliver) |
| Source:
NUCLEIC ACIDS RESEARCH Volume:
43 Issue:
D1 Pages:
D1176-D1182 DOI:
10.1093/nar/gku1143 Published Date:
2015 JAN 28 |
|
Abstract:
The Plasmodium Genetic Modification (PlasmoGEM) database (http://plasmogem.sanger.ac.uk) provides access to a resource of modular, versatile and adaptable vectors for genome modification of Plasmodium spp. parasites. PlasmoGEM currently consists of > 2000 plasmids designed to modify the genome of Plasmodium berghei, a malaria parasite of rodents, which can be requested by non-profit research organisations free of charge. PlasmoGEM vectors are designed with long homology arms for efficient genome integration and carry gene specific barcodes to identify individual mutants. They can be used for a wide array of applications, including protein localisation, gene interaction studies and high-throughput genetic screens. The vector production pipeline is supported by a custom software suite that automates both the vector design process and quality control by full-length sequencing of the finished vectors. The PlasmoGEM web interface allows users to search a database of finished knock-out and gene tagging vectors, view details of their designs, download vector sequence in different formats and view available quality control data as well as suggested genotyping strategies. We also make gDNA library clones and intermediate vectors available for researchers to produce vectors for themselves. |
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Title:
A comprehensive evaluation of rodent malaria parasite genomes and gene expression
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Author(s):
Otto, TD (Otto, Thomas D.); Böhme, U (Boehme, Ulrike); Jackson, AP (Jackson, Andrew P.); Hunt, M (Hunt, Martin); Franke-Fayard, B (Franke-Fayard, Blandine); Hoeijmakers, WAM (Hoeijmakers, Wieteke A. M.); Religa, AA (Religa, Agnieszka A.); Robertson, L (Robertson, Lauren); Sanders, M (Sanders, Mandy); Ogun, SA (Ogun, Solabomi A.); Cunningham, D (Cunningham, Deirdre); Erhart, A (Erhart, Annette); Billker, O (Billker, Oliver); Khan, SM (Khan, Shahid M.); Stunnenberg, HG (Stunnenberg, Hendrik G.); Langhorne, J (Langhorne, Jean); Holder, AA (Holder, Anthony A.); Waters, AP (Waters, Andrew P.); Newbold, CI (Newbold, Chris I.); Pain, A (Pain, Arnab); Berriman, M (Berriman, Matthew); Janse, CJ (Janse, Chris J.) |
| Source:
BMC BIOLOGY Volume:
12 Article Number:
86 DOI:
10.1186/s12915-014-0086-0 Published Date:
2014 OCT 30 |
Abstract:
Background: Rodent malaria parasites (RMP) are used extensively as models of human malaria. Draft RMP genomes have been published for Plasmodium yoelii, P. berghei ANKA (PbA) and P. chabaudi AS (PcAS). Although availability of these genomes made a significant impact on recent malaria research, these genomes were highly fragmented and were annotated with little manual curation. The fragmented nature of the genomes has hampered genome wide analysis of Plasmodium gene regulation and function.
Results: We have greatly improved the genome assemblies of PbA and PcAS, newly sequenced the virulent parasite P. yoelii YM genome, sequenced additional RMP isolates/lines and have characterized genotypic diversity within RMP species. We have produced RNA-seq data and utilised it to improve gene-model prediction and to provide quantitative, genome-wide, data on gene expression. Comparison of the RMP genomes with the genome of the human malaria parasite P. falciparum and RNA-seq mapping permitted gene annotation at base-pair resolution. Full-length chromosomal annotation permitted a comprehensive classification of all subtelomeric multigene families including the ‘Plasmodium interspersed repeat genes’ (pir). Phylogenetic classification of the pir family, combined with pir expression patterns, indicates functional diversification within this family.
Conclusions: Complete RMP genomes, RNA-seq and genotypic diversity data are excellent and important resources for gene-function and post-genomic analyses and to better interrogate Plasmodium biology. Genotypic diversity between P. chabaudi isolates makes this species an excellent parasite to study genotype-phenotype relationships. The improved classification of multigene families will enhance studies on the role of (variant) exported proteins in virulence and immune evasion/modulation. |
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Title:
BCKDH: The Missing Link in Apicomplexan Mitochondrial Metabolism Is Required for Full Virulence of Toxoplasma gondii and Plasmodium berghei
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Author(s):
Oppenheim, RD (Oppenheim, Rebecca D.); Creek, DJ (Creek, Darren J.); Macrae, JI (Macrae, James I.); Modrzynska, KK (Modrzynska, Katarzyna K.); Pino, P (Pino, Paco); Limenitakis, J (Limenitakis, Julien); Polonais, V (Polonais, Valerie); Seeber, F (Seeber, Frank); Barrett, MP (Barrett, Michael P.); Billker, O (Billker, Oliver); McConville, MJ (McConville, Malcolm J.); Soldati-Favre, D (Soldati-Favre, Dominique) |
| Source:
PLOS PATHOGENS Volume:
10 Issue:
7 Article Number:
e1004263 DOI:
10.1371/journal.ppat.1004263 Published Date:
2014 JUL |
|
Abstract:
While the apicomplexan parasites Plasmodium falciparum and Toxoplasma gondii are thought to primarily depend on glycolysis for ATP synthesis, recent studies have shown that they can fully catabolize glucose in a canonical TCA cycle. However, these parasites lack a mitochondrial isoform of pyruvate dehydrogenase and the identity of the enzyme that catalyses the conversion of pyruvate to acetyl-CoA remains enigmatic. Here we demonstrate that the mitochondrial branched chain ketoacid dehydrogenase (BCKDH) complex is the missing link, functionally replacing mitochondrial PDH in both T. gondii and P. berghei. Deletion of the E1a subunit of T. gondii and P. berghei BCKDH significantly impacted on intracellular growth and virulence of both parasites. Interestingly, disruption of the P. berghei E1a restricted parasite development to reticulocytes only and completely prevented maturation of oocysts during mosquito transmission. Overall this study highlights the importance of the molecular adaptation of BCKDH in this important class of pathogens. |
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Title:
A cascade of DNA-binding proteins for sexual commitment and development in Plasmodium
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Author(s):
Sinha, A (Sinha, Abhinav); Hughes, KR (Hughes, Katie R.); Modrzynska, KK (Modrzynska, Katarzyna K.); Otto, TD (Otto, Thomas D.); Pfander, C (Pfander, Claudia); Dickens, NJ (Dickens, Nicholas J.); Religa, AA (Religa, Agnieszka A.); Bushell, E (Bushell, Ellen); Graham, AL (Graham, Anne L.); Cameron, R (Cameron, Rachael); Kafsack, BFC (Kafsack, Bjorn F. C.); Williams, AE (Williams, April E.); Llinás, M (Llinas, Manuel); Berriman, M (Berriman, Matthew); Billker, O (Billker, Oliver); Waters, AP (Waters, Andrew P.) |
| Source:
NATURE Volume:
507 Issue:
7491 Pages:
253-+ DOI:
10.1038/nature12970 Published Date:
2014 MAR 13 |
|
Abstract:
Commitment to and completion of sexual development are essential for malaria parasites (protists of the genus Plasmodium) to be transmitted through mosquitoes(1). The molecular mechanism(s) responsible for commitment have been hitherto unknown. Here we show that PbAP2-G, a conserved member of the apicomplexan AP2 (ApiAP2) family of DNA-binding proteins, is essential for the commitment of asexually replicating forms to sexual development in Plasmodium berghei, a malaria parasite of rodents. PbAP2-G was identified from mutations in its encoding gene, PBANKA_143750, which account for the loss of sexual development frequently observed in parasites transmitted artificially by blood passage. Systematic gene deletion of conserved ApiAP2 genes in Plasmodium confirmed the role of PbAP2-G and revealed a second ApiAP2 member (PBANKA_103430, here termed PbAP2-G2) that significantly modulates but does not abolish gametocytogenesis, indicating that a cascade of ApiAP2 proteins are involved in commitment to the production and maturation of gametocytes. The data suggest a mechanism of commitment to gametocytogenesis in Plasmodium consistent with a positive feedback loop involving PbAP2-G that could be exploited to prevent the transmission of this pernicious parasite. |
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Title:
Efficacy of a Plasmodium vivax Malaria Vaccine Using ChAd63 and Modified Vaccinia Ankara Expressing Thrombospondin-Related Anonymous Protein as Assessed with Transgenic Plasmodium berghei Parasites
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Author(s):
Bauza, K (Bauza, Karolis); Malinauskas, T (Malinauskas, Tomas); Pfander, C (Pfander, Claudia); Anar, B (Anar, Burcu); Jones, EY (Jones, E. Yvonne); Billker, O (Billker, Oliver); Hill, AVS (Hill, Adrian V. S.); Reyes-Sandoval, A (Reyes-Sandoval, Arturo) |
| Source:
INFECTION AND IMMUNITY Volume:
82 Issue:
3 Pages:
1277-1286 DOI:
10.1128/IAI.01187-13 Published Date:
2014 MAR |
|
Abstract:
Plasmodium vivax is the world’s most widely distributed malaria parasite and a potential cause of morbidity and mortality for approximately 2.85 billion people living mainly in Southeast Asia and Latin America. Despite this dramatic burden, very few vaccines have been assessed in humans. The clinically relevant vectors modified vaccinia virus Ankara (MVA) and the chimpanzee adenovirus ChAd63 are promising delivery systems for malaria vaccines due to their safety profiles and proven ability to induce protective immune responses against Plasmodium falciparum thrombospondin-related anonymous protein (TRAP) in clinical trials. Here, we describe the development of new recombinant ChAd63 and MVA vectors expressing P. vivax TRAP (PvTRAP) and show their ability to induce high antibody titers and T cell responses in mice. In addition, we report a novel way of assessing the efficacy of new candidate vaccines against P. vivax using a fully infectious transgenic Plasmodium berghei parasite expressing P. vivax TRAP to allow studies of vaccine efficacy and protective mechanisms in rodents. Using this model, we found that both CD8(+) T cells and antibodies mediated protection against malaria using virus-vectored vaccines. Our data indicate that ChAd63 and MVA expressing PvTRAP are good preerythrocytic-stage vaccine candidates with potential for future clinical application. |
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Title:
Phosphoinositide Metabolism Links cGMP- Dependent Protein Kinase G to Essential Ca <SUP>2+</SUP> Signals at Key Decision Points in the Life Cycle of Malaria Parasites
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Author(s):
Brochet, M (Brochet, Mathieu); Collins, MO (Collins, Mark O.); Smith, TK (Smith, Terry K.); Thompson, E (Thompson, Eloise); Sebastian, S (Sebastian, Sarah); Volkmann, K (Volkmann, Katrin); Schwach, F (Schwach, Frank); Chappell, L (Chappell, Lia); Gomes, AR (Gomes, Ana Rita); Berriman, M (Berriman, Matthew); Rayner, JC (Rayner, Julian C.); Baker, DA (Baker, David A.); Choudhary, J (Choudhary, Jyoti); Billker, O (Billker, Oliver) |
| Source:
PLOS BIOLOGY Volume:
12 Issue:
3 Article Number:
e1001806 DOI:
10.1371/journal.pbio.1001806 Published Date:
2014 MAR |
Abstract:
Many critical events in the Plasmodium life cycle rely on the controlled release of Ca2+ from intracellular stores to activate stage-specific Ca2+-dependent protein kinases. Using the motility of Plasmodium berghei ookinetes as a signalling paradigm, we show that the cyclic guanosine monophosphate (cGMP)-dependent protein kinase, PKG, maintains the elevated level of cytosolic Ca2+ required for gliding motility. We find that the same PKG-dependent pathway operates upstream of the Ca2+ signals that mediate activation of P. berghei gametocytes in the mosquito and egress of Plasmodium falciparum merozoites from infected human erythrocytes. Perturbations of PKG signalling in gliding ookinetes have a marked impact on the phosphoproteome, with a significant enrichment of in vivo regulated sites in multiple pathways including vesicular trafficking and phosphoinositide metabolism. A global analysis of cellular phospholipids demonstrates that in gliding ookinetes PKG controls phosphoinositide biosynthesis, possibly through the subcellular localisation or activity of lipid kinases. Similarly, phosphoinositide metabolism links PKG to egress of P. falciparum merozoites, where inhibition of PKG blocks hydrolysis of phosphatidylinostitol (4,5)-bisphosphate. In the face of an increasing complexity of signalling through multiple Ca2+ effectors, PKG emerges as a unifying factor to control multiple cellular Ca2+ signals essential for malaria parasite development and transmission.
Author Summary Malaria, caused by Plasmodium spp. parasites, is a profound human health problem. Plasmodium parasites progress through a complex life cycle as they move between infected humans and blood-feeding mosquitoes. We know that tight regulation of calcium ion levels within the cytosol of the parasite is critical to control multiple signalling events in their life cycle. However, how these calcium levels are controlled remains a mystery. Here, we show that a single protein kinase, the cGMP-dependent protein kinase G (PKG), controls the calcium signals that are critical at three different points of the life cycle: (1) for the exit of the merozoite form of the parasite from human erythrocytes (red blood cells), (2) for the cellular activation that happens when Plasmodium sexual transmission stages are ingested by a blood-feeding mosquito, and (3) for the productive gliding of the ookinete, which is the parasite stage that invades the mosquito midgut. We provide initial evidence that the universal role of PKG relies on the production of lipid precursors which then give rise to inositol (1,4,5)-trisphosphate (IP3), a messenger molecule that serves as a signal for the release of calcium from stores within the parasite. This signalling pathway provides a potential target to block both malaria development in the human host and transmission to the mosquito vector. |
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Title:
The Malarial Serine Protease SUB1 Plays an Essential Role in Parasite Liver Stage Development
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Author(s):
Suarez, C (Suarez, Catherine); Volkmann, K (Volkmann, Katrin); Gomes, AR (Gomes, Ana Rita); Billker, O (Billker, Oliver); Blackman, MJ (Blackman, Michael J.) |
| Source:
PLOS PATHOGENS Volume:
9 Issue:
12 Article Number:
e1003811 DOI:
10.1371/journal.ppat.1003811 Published Date:
2013 DEC |
|
Abstract:
Transmission of the malaria parasite to its vertebrate host involves an obligatory exoerythrocytic stage in which extensive asexual replication of the parasite takes place in infected hepatocytes. The resulting liver schizont undergoes segmentation to produce thousands of daughter merozoites. These are released to initiate the blood stage life cycle, which causes all the pathology associated with the disease. Whilst elements of liver stage merozoite biology are similar to those in the much better-studied blood stage merozoites, little is known of the molecular players involved in liver stage merozoite production. To facilitate the study of liver stage biology we developed a strategy for the rapid production of complex conditional alleles by recombinase mediated engineering in Escherichia coli, which we used in combination with existing Plasmodium berghei deleter lines expressing Flp recombinase to study subtilisin-like protease 1 (SUB1), a conserved Plasmodium serine protease previously implicated in blood stage merozoite maturation and egress. We demonstrate that SUB1 is not required for the early stages of intrahepatic growth, but is essential for complete development of the liver stage schizont and for production of hepatic merozoites. Our results indicate that inhibitors of SUB1 could be used in prophylactic approaches to control or block the clinically silent pre-erythrocytic stage of the malaria parasite life cycle. |
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Title:
Defining the Range of Pathogens Susceptible to Ifitm3 Restriction Using a Knockout Mouse Model
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Author(s):
Everitt, AR (Everitt, Aaron R.); Clare, S (Clare, Simon); McDonald, JU (McDonald, Jacqueline U.); Kane, L (Kane, Leanne); Harcourt, K (Harcourt, Katherine); Ahras, M (Ahras, Malika); Lall, A (Lall, Amar); Hale, C (Hale, Christine); Rodgers, A (Rodgers, Angela); Young, DB (Young, Douglas B.); Haque, A (Haque, Ashraful); Billker, O (Billker, Oliver); Tregoning, JS (Tregoning, John S.); Dougan, G (Dougan, Gordon); Kellam, P (Kellam, Paul) |
| Source:
PLOS ONE Volume:
8 Issue:
11 Article Number:
e80723 DOI:
10.1371/journal.pone.0080723 Published Date:
2013 NOV 21 |
|
Abstract:
The interferon-inducible transmembrane (IFITM) family of proteins has been shown to restrict a broad range of viruses in vitro and in vivo by halting progress through the late endosomal pathway. Further, single nucleotide polymorphisms (SNPs) in its sequence have been linked with risk of developing severe influenza virus infections in humans. The number of viruses restricted by this host protein has continued to grow since it was first demonstrated as playing an antiviral role; all of which enter cells via the endosomal pathway. We therefore sought to test the limits of antimicrobial restriction by Ifitm3 using a knockout mouse model. We showed that Ifitm3 does not impact on the restriction or pathogenesis of bacterial (Salmonella typhimurium, Citrobacter rodentium, Mycobacterium tuberculosis) or protozoan (Plasmodium berghei) pathogens, despite in vitro evidence. However, Ifitm3 is capable of restricting respiratory syncytial virus (RSV) in vivo either through directly restricting RSV cell infection, or by exerting a previously uncharacterised function controlling disease pathogenesis. This represents the first demonstration of a virus that enters directly through the plasma membrane, without the need for the endosomal pathway, being restricted by the IFITM family; therefore further defining the role of these antiviral proteins. |
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Title:
Global Analysis of Apicomplexan Protein S-Acyl Transferases Reveals an Enzyme Essential for Invasion
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Author(s):
Frénal, K (Frenal, Karine); Tay, CL (Tay, Chwen L.); Mueller, C (Mueller, Christina); Bushell, ES (Bushell, Ellen S.); Jia, YG (Jia, Yonggen); Graindorge, A (Graindorge, Arnault); Billker, O (Billker, Oliver); Rayner, JC (Rayner, Julian C.); Soldati-Favre, D (Soldati-Favre, Dominique) |
| Source:
TRAFFIC Volume:
14 Issue:
8 Pages:
895-911 DOI:
10.1111/tra.12081 Published Date:
2013 AUG |
|
Abstract:
The advent of techniques to study palmitoylation on a whole proteome scale has revealed that it is an important reversible modification that plays a role in regulating multiple biological processes. Palmitoylation can control the affinity of a protein for lipid membranes, which allows it to impact protein trafficking, stability, folding, signalling and interactions. The publication of the palmitome of the schizont stage of Plasmodium falciparum implicated a role for palmitoylation in host cell invasion, protein export and organelle biogenesis. However, nothing is known so far about the repertoire of protein S-acyl transferases (PATs) that catalyse this modification in Apicomplexa. We undertook a comprehensive analysis of the repertoire of Asp-His-His-Cys cysteine-rich domain (DHHC-CRD) PAT family in Toxoplasma gondii and Plasmodium berghei by assessing their localization and essentiality. Unlike functional redundancies reported in other eukaryotes, some apicomplexan-specific DHHCs are essential for parasite growth, and several are targeted to organelles unique to this phylum. Of particular interest is DHHC7, which localizes to rhoptry organelles in all parasites tested, including the major human pathogen P. falciparum. TgDHHC7 interferes with the localization of the rhoptry palmitoylated protein TgARO and affects the apical positioning of the rhoptry organelles. This PAT has a major impact on T. gondii host cell invasion, but not on the parasite’s ability to egress. |
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Title:
Comparative genomics in Chlamydomonas and Plasmodium identifies an ancient nuclear envelope protein family essential for sexual reproduction in protists, fungi, plants, and vertebrates
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Author(s):
Ning, J (Ning, Jue); Otto, TD (Otto, Thomas D.); Pfander, C (Pfander, Claudia); Schwach, F (Schwach, Frank); Brochet, M (Brochet, Mathieu); Bushell, E (Bushell, Ellen); Goulding, D (Goulding, David); Sanders, M (Sanders, Mandy); Lefebvre, PA (Lefebvre, Paul A.); Pei, JM (Pei, Jimin); Grishin, NV (Grishin, Nick V.); Vanderlaan, G (Vanderlaan, Gary); Billker, O (Billker, Oliver); Snell, WJ (Snell, William J.) |
| Source:
GENES & DEVELOPMENT Volume:
27 Issue:
10 Pages:
1198-1215 DOI:
10.1101/gad.212746.112 Published Date:
2013 MAY 15 |
|
Abstract:
Fertilization is a crucial yet poorly characterized event in eukaryotes. Our previous discovery that the broadly conserved protein HAP2 (GCS1) functioned in gamete membrane fusion in the unicellular green alga Chlamydomonas and the malaria pathogen Plasmodium led us to exploit the rare biological phenomenon of isogamy in Chlamydomonas in a comparative transcriptomics strategy to uncover additional conserved sexual reproduction genes. All previously identified Chlamydomonas fertilization-essential genes fell into related clusters based on their expression patterns. Out of several conserved genes in a minus gamete cluster, we focused on Cre06.g280600, an ortholog of the fertilization-related Arabidopsis GEX1. Gene disruption, cell biological, and immunolocalization studies show that CrGEX1 functions in nuclear fusion in Chlamydomonas. Moreover, CrGEX1 and its Plasmodium ortholog, PBANKA_113980, are essential for production of viable meiotic progeny in both organisms and thus for mosquito transmission of malaria. Remarkably, we discovered that the genes are members of a large, previously unrecognized family whose first-characterized member, KAR5, is essential for nuclear fusion during yeast sexual reproduction. Our comparative transcriptomics approach provides a new resource for studying sexual development and demonstrates that exploiting the data can lead to the discovery of novel biology that is conserved across distant taxa. |
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Title:
A Tetracycline-Repressible Transactivator System to Study Essential Genes in Malaria Parasites
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Author(s):
Pino, P (Pino, Paco); Sebastian, S (Sebastian, Sarah); Kim, EA (Kim, EunBin Arin); Bush, E (Bush, Erin); Brochet, M (Brochet, Mathieu); Volkmann, K (Volkmann, Katrin); Kozlowski, E (Kozlowski, Elyse); Llinás, M (Llinas, Manuel); Billker, O (Billker, Oliver); Soldati-Favre, D (Soldati-Favre, Dominique) |
| Source:
CELL HOST & MICROBE Volume:
12 Issue:
6 Pages:
824-834 DOI:
10.1016/j.chom.2012.10.016 Published Date:
2012 DEC 13 |
|
Abstract:
A major obstacle in analyzing gene function in apicomplexan parasites is the absence of a practical regulatable expression system. Here, we identified functional transcriptional activation domains within Apicomplexan AP2 (ApiAP2) family transcription factors. These ApiAP2 transactivation domains were validated in blood-, liver-, and mosquito-stage parasites and used to create a robust conditional expression system for stage-specific, tetracycline-dependent gene regulation in Toxoplasma gondii, Plasmodium berghei, and Plasmodium falciparum. To demonstrate the utility of this system, we created conditional knockdowns of two essential P. berghei genes: profilin (PRF), a protein implicated in parasite invasion, and N-myristoyltransferase (NMT), which catalyzes protein acylation. Tetracycline-induced repression of PRF and NMT expression resulted in a dramatic reduction in parasite viability. This efficient regulatable system will allow for the functional characterization of essential proteins that are found in these important parasites. |
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Title:
The Alveolin IMC1h Is Required for Normal Ookinete and Sporozoite Motility Behaviour and Host Colonisation in Plasmodium berghei
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Author(s):
Volkmann, K (Volkmann, Katrin); Pfander, C (Pfander, Claudia); Burstroem, C (Burstroem, Charlotte); Ahras, M (Ahras, Malika); Goulding, D (Goulding, David); Rayner, JC (Rayner, Julian C.); Frischknecht, F (Frischknecht, Friedrich); Billker, O (Billker, Oliver); Brochet, M (Brochet, Mathieu) |
| Source:
PLOS ONE Volume:
7 Issue:
7 Article Number:
e41409 DOI:
10.1371/journal.pone.0041409 Published Date:
2012 JUL 23 |
|
Abstract:
Alveolins, or inner membrane complex (IMC) proteins, are components of the subpellicular network that forms a structural part of the pellicle of malaria parasites. In Plasmodium berghei, deletions of three alveolins, IMC1a, b, and h, each resulted in reduced mechanical strength and gliding velocity of ookinetes or sporozoites. Using time lapse imaging, we show here that deletion of IMC1h (PBANKA_143660) also has an impact on the directionality and motility behaviour of both ookinetes and sporozoites. Despite their marked motility defects, sporozoites lacking IMC1h were able to invade mosquito salivary glands, allowing us to investigate the role of IMC1h in colonisation of the mammalian host. We show that IMC1h is essential for sporozoites to progress through the dermis in vivo but does not play a significant role in hepatoma cell transmigration and invasion in vitro. Colocalisation of IMC1h with the residual IMC in liver stages was detected up to 30 hours after infection and parasites lacking IMC1h showed developmental defects in vitro and a delayed onset of blood stage infection in vivo. Together, these results suggest that IMC1h is involved in maintaining the cellular architecture which supports normal motility behaviour, access of the sporozoites to the blood stream, and further colonisation of the mammalian host. |
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Title:
A Plasmodium Calcium-Dependent Protein Kinase Controls Zygote Development and Transmission by Translationally Activating Repressed mRNAs
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Author(s):
Sebastian, S (Sebastian, Sarah); Brochet, M (Brochet, Mathieu); Collins, MO (Collins, Mark O.); Schwach, F (Schwach, Frank); Jones, ML (Jones, Matthew L.); Goulding, D (Goulding, David); Rayner, JC (Rayner, Julian C.); Choudhary, JS (Choudhary, Jyoti S.); Billker, O (Billker, Oliver) |
| Source:
CELL HOST & MICROBE Volume:
12 Issue:
1 Pages:
9-19 DOI:
10.1016/j.chom.2012.05.014 Published Date:
2012 JUL 19 |
|
Abstract:
Calcium-dependent protein kinases (CDPKs) play key regulatory roles in the life cycle of the malaria parasite, but in many cases their precise molecular functions are unknown. Using the rodent malaria parasite Plasmodium berghei, we show that CDPK1, which is known to be essential in the asexual blood stage of the parasite, is expressed in all life stages and is indispensable during the sexual mosquito life-cycle stages. Knockdown of CDPK1 in sexual stages resulted in developmentally arrested parasites and prevented mosquito transmission, and these effects were independent of the previously proposed function for CDPK1 in regulating parasite motility. In-depth translational and transcriptional profiling of arrested parasites revealed that CDPK1 translationally activates mRNA species in the developing zygote that in macrogametes remain repressed via their 3′ and 5’UTRs. These findings indicate that CDPK1 is a multifunctional protein that translationally regulates mRNAs to ensure timely and stage-specific protein expression. |
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Title:
Transmission of malaria to mosquitoes blocked by bumped kinase inhibitors
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Author(s):
Ojo, KK (Ojo, Kayode K.); Pfander, C (Pfander, Claudia); Mueller, NR (Mueller, Natascha R.); Burstroem, C (Burstroem, Charlotte); Larson, ET (Larson, Eric T.); Bryan, CM (Bryan, Cassie M.); Fox, AMW (Fox, Anna M. W.); Reid, MC (Reid, Molly C.); Johnson, SM (Johnson, Steven M.); Murphy, RC (Murphy, Ryan C.); Kennedy, M (Kennedy, Mark); Mann, H (Mann, Henning); Leibly, DJ (Leibly, David J.); Hewitt, SN (Hewitt, Stephen N.); Verlinde, CLMJ (Verlinde, Christophe L. M. J.); Kappe, S (Kappe, Stefan); Merritt, EA (Merritt, Ethan A.); Maly, DJ (Maly, Dustin J.); Billker, O (Billker, Oliver); Van Voorhis, WC (Van Voorhis, Wesley C.) |
| Source:
JOURNAL OF CLINICAL INVESTIGATION Volume:
122 Issue:
6 Pages:
2301-2305 DOI:
10.1172/JCI61822 Published Date:
2012 JUN |
|
Abstract:
Effective control and eradication of malaria will require new tools to prevent transmission. Current antimalarial therapies targeting the asexual stage of Plasmodium do not prevent transmission of circulating gametocytes from infected humans to mosquitoes. Here, we describe a new class of transmission-blocking compounds, bumped kinase inhibitors (BKIs), which inhibit microgametocyte exflagellation. Oocyst formation and sporozoite production, necessary for transmission to mammals, were inhibited in mosquitoes fed on either BKI-1-treated human blood or mice treated with BKI-1. BKIs are hypothesized to act via inhibition of Plasmodium calcium-dependent protein kinase 4 and predicted to have little activity against mammalian kinases. Our data show that BKIs do not inhibit proliferation of mammalian cell lines and are well tolerated in mice. Used in combination with drugs active against asexual stages of Plasmodium, BKIs could prove an important tool for malaria control and eradication. |
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Title:
A scalable pipeline for highly effective genetic modification of a malaria parasite
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Author(s):
Pfander, C (Pfander, Claudia); Anar, B (Anar, Burcu); Schwach, F (Schwach, Frank); Otto, TD (Otto, Thomas D.); Brochet, M (Brochet, Mathieu); Volkmann, K (Volkmann, Katrin); Quail, MA (Quail, Michael A.); Pain, A (Pain, Arnab); Rosen, B (Rosen, Barry); Skarnes, W (Skarnes, William); Rayner, JC (Rayner, Julian C.); Billker, O (Billker, Oliver) |
| Source:
NATURE METHODS Volume:
8 Issue:
12 Pages:
1078-+ DOI:
10.1038/NMETH.1742 Published Date:
2011 DEC |
|
Abstract:
In malaria parasites, the systematic experimental validation of drug and vaccine targets by reverse genetics is constrained by the inefficiency of homologous recombination and by the difficulty of manipulating adenine and thymine (A+T)-rich DNA of most Plasmodium species in Escherichia coli. We overcame these roadblocks by creating a high-integrity library of Plasmodium berghei genomic DNA (>77% A+T content) in a bacteriophage N15-based vector that can be modified efficiently using the lambda Red method of recombineering. We built a pipeline for generating P. berghei genetic modification vectors at genome scale in serial liquid cultures on 96-well plates. Vectors have long homology arms, which increase recombination frequency up to tenfold over conventional designs. The feasibility of efficient genetic modification at scale will stimulate collaborative, genome-wide knockout and tagging programs for P. berghei. |
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Title:
Genetic and transcriptional analysis of phosphoinositide-specific phospholipase C in Plasmodium
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Author(s):
Raabe, A (Raabe, Andreas); Berry, L (Berry, Laurence); Sollelis, L (Sollelis, Lauriane); Cerdan, R (Cerdan, Rachel); Tawk, L (Tawk, Lina); Vial, HJ (Vial, Henri J.); Billker, O (Billker, Oliver); Wengelnik, K (Wengelnik, Kai) |
| Source:
EXPERIMENTAL PARASITOLOGY Volume:
129 Issue:
1 Pages:
75-80 DOI:
10.1016/j.exppara.2011.05.023 Published Date:
2011 SEP |
|
Abstract:
Phosphoinositide-specific phospholipase C (PI-PLC) is a major regulator of calcium-dependent signal transduction, which has been shown to be important in various processes of the malaria parasite Plasmodium. PI-PLC is generally implicated in calcium liberation from intracellular stores through the action of its product, inositol-(1,4,5)-trisphosphate, and is itself dependent on calcium for its activation. Here we describe the plc genes from Plasmodium species. The encoded proteins contain all domains typically found in PI-PLCs of the 8 class but are almost twice as long as their orthologues in mammals. Transcriptional analysis by qRT-PCR of plc during the erythrocytic cycle of P. falciparum revealed steady expression levels that increased at the late schizont stages. Genetic analysis in the P. berghei model revealed that the plc locus was targetable but that plc gene knock-outs could not be obtained, thereby strongly indicating that the gene is essential during blood stage development. Alternatively, we attempted to modify plc expression through a promoter exchange approach but found the gene to be refractory to over-expression indicating that plc expression levels might additionally be tightly controlled. (C) 2011 Elsevier Inc. All rights reserved. |
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Title:
Multiple roles for Plasmodium berghei phosphoinositide-specific phospholipase C in regulating gametocyte activation and differentiation
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Author(s):
Raabe, AC (Raabe, Andreas C.); Wengelnik, K (Wengelnik, Kai); Billker, O (Billker, Oliver); Vial, HJ (Vial, Henri J.) |
| Source:
CELLULAR MICROBIOLOGY Volume:
13 Issue:
7 Pages:
955-966 DOI:
10.1111/j.1462-5822.2011.01591.x Published Date:
2011 JUL |
|
Abstract:
Critical events in the life cycle of malaria parasites are controlled by calcium-dependent signalling cascades, yet the molecular mechanisms of calcium release remain poorly understood. The synchronized development of Plasmodium berghei gametocytes relies on rapid calcium release from internal stores within 10 s of gametocytes being exposed to mosquito-derived xanthurenic acid (XA). Here we addressed the function of phosphoinositide-specific phospholipase C (PIPLC) for regulating gametocyte activation. XA triggered the hydrolysis of PIP(2) and the production of the secondary messenger IP(3) in gametocytes. Both processes were selectively blocked by a PI-PLC inhibitor, which also reduced the early Ca(2+) signal. However, microgametocyte differentiation into microgametes was blocked even when the inhibitor was added up to 5 min after activation, suggesting a requirement for PI-PLC beyond the early mobilization of calcium. In contrast, inhibitors of calcium release through ryanodine receptor channels were active only during the first minute of gametocyte activation. Biochemical determination of PI-PLC activity was confirmed using transgenic parasites expressing a fluorescent PIP(2)/IP(3) probe that translocates from the parasite plasmalemma to the cytosol upon cell activation. Our study revealed a complex interdependency of Ca(2+) and PI-PLC activity, with PI-PLC being essential throughout gamete formation, possibly explaining the irreversibility of this process. |
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Title:
Cutting Edge: The Membrane Attack Complex of Complement Is Required for the Development of Murine Experimental Cerebral Malaria
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Author(s):
Ramos, TN (Ramos, Theresa N.); Darley, MM (Darley, Meghan M.); Hu, XZ (Hu, Xianzhen); Billker, O (Billker, Oliver); Rayner, JC (Rayner, Julian C.); Ahras, M (Ahras, Malika); Wohler, JE (Wohler, Jillian E.); Barnum, SR (Barnum, Scott R.) |
| Source:
JOURNAL OF IMMUNOLOGY Volume:
186 Issue:
12 Pages:
6657-6660 DOI:
10.4049/jimmunol.1100603 Published Date:
2011 JUN 15 |
|
Abstract:
Cerebral malaria is the most severe complication of Plasmodium falciparum infection and accounts for a large number of malaria fatalities worldwide. Recent studies demonstrated that C5(-/-) mice are resistant to experimental cerebral malaria (ECM) and suggested that protection was due to loss of C5a-induced inflammation. Surprisingly, we observed that C5aR(-/-) mice were fully susceptible to disease, indicating that C5a is not required for ECM. C3aR(-/-) and C3aR(-/-) X C5aR(-/-) mice were equally susceptible to ECM as were wild-type mice, indicating that neither complement anaphylatoxin receptor is critical for ECM development. In contrast, C9 deposition in the brains of mice with ECM suggested an important role for the terminal complement pathway. Treatment with anti-C9 Ab significantly increased survival time and reduced mortality in ECM. Our data indicate that protection from ECM in C5(-/-) mice is mediated through inhibition of membrane attack complex formation and not through C5a-induced inflammation. The Journal of Immunology, 2011, 186: 6657-6660. |
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Title:
A Research Agenda for Malaria Eradication: Basic Science and Enabling Technologies
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|
Author(s):
Baum, J (Baum, Jake); Billker, O (Billker, Oliver); Bousema, T (Bousema, Teun); Dinglasan, R (Dinglasan, Rhoel); McGovern, V (McGovern, Victoria); Mota, MM (Mota, Maria M.); Mueller, I (Mueller, Ivo); Sinden, R (Sinden, Robert) |
|
Group Author(s):
malERA Consultative Grp Basic Sci |
| Source:
PLOS MEDICINE Volume:
8 Issue:
1 Article Number:
e1000399 DOI:
10.1371/journal.pmed.1000399 Published Date:
2011 JAN |
|
Abstract:
Today’s malaria control efforts are limited by our incomplete understanding of the biology of Plasmodium and of the complex relationships between human populations and the multiple species of mosquito and parasite. Research priorities include the development of in vitro culture systems for the complete life cycle of P. falciparum and P. vivax and the development of an appropriate liver culture system to study hepatic stages. In addition, genetic technologies for the manipulation of Plasmodium need to be improved, the entire parasite metabolome needs to be characterized to identify new druggable targets, and improved information systems for monitoring the changes in epidemiology, pathology, and host-parasite-vector interactions as a result of intensified control need to be established to bridge the gap between bench, preclinical, clinical, and population-based sciences. |
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Title:
The Systematic Functional Analysis of Plasmodium Protein Kinases Identifies Essential Regulators of Mosquito Transmission
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|
Author(s):
Tewari, R (Tewari, Rita); Straschil, U (Straschil, Ursula); Bateman, A (Bateman, Alex); Böhme, U (Boehme, Ulrike); Cherevach, I (Cherevach, Inna); Gong, P (Gong, Peng); Pain, A (Pain, Arnab); Billker, O (Billker, Oliver) |
| Source:
CELL HOST & MICROBE Volume:
8 Issue:
4 Pages:
377-387 DOI:
10.1016/j.chom.2010.09.006 Published Date:
2010 OCT 21 |
|
Abstract:
Although eukaryotic protein kinases (ePKs) contribute to many cellular processes, only three Plasmodium falciparum ePKs have thus far been identified as essential for parasite asexual blood stage development. To identify pathways essential for parasite transmission between their mammalian host and mosquito vector, we undertook a systematic functional analysis of ePKs in the genetically tractable rodent parasite Plasmodium berghei. Modeling domain signatures of conventional ePKs identified 66 putative Plasmodium ePKs. Kinomes are highly conserved between Plasmodium species. Using reverse genetics, we show that 23 ePKs are redundant for asexual erythrocytic parasite development in mice. Phenotyping mutants at four life cycle stages in Anopheles stephensi mosquitoes revealed functional clusters of kinases required for sexual development and sporogony. Roles for a putative SR protein kinase (SRPK) in microgamete formation, a conserved regulator of clathrin uncoating (GAK) in ookinete formation, and a likely regulator of energy metabolism (SNF1/KIN) in sporozoite development were identified. |
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Title:
A parasite calcium switch and Achilles’ heel revealed
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|
Author(s):
Doerig, C (Doerig, Christian); Billker, O (Billker, Oliver) |
| Source:
NATURE STRUCTURAL & MOLECULAR BIOLOGY Volume:
17 Issue:
5 Pages:
541-543 DOI:
10.1038/nsmb0510-541 Published Date:
2010 MAY |
|
Title:
Quantitative assessment of DNA replication to monitor microgametogenesis in Plasmodium berghei
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|
Author(s):
Raabe, AC (Raabe, Andreas C.); Billker, O (Billker, Oliver); Vial, HJ (Vial, Henri J.); Wengelnik, K (Wengelnik, Kai) |
| Source:
MOLECULAR AND BIOCHEMICAL PARASITOLOGY Volume:
168 Issue:
2 Pages:
172-176 DOI:
10.1016/j.molbiopara.2009.08.004 Published Date:
2009 DEC |
|
Abstract:
Targeting the crucial step of Plasmodium transition from vertebrate host to mosquito vector is a promising approach to eliminate malaria. Uptake by the mosquito activates gametocytes within seconds, and in the case of male (micro) gametocytes leads to rapid DNA replication and the release of eight flagellated gametes. We developed a sensitive assay to monitor P. berghei microgametocyte activation based on [H-3]hypoxanthine incorporation into DNA. Optimal pH range and xanthurenic acid concentrations for gametocyte activation were established and the kinetics of DNA replication investigated. Significance of the method was confirmed using P. berghei mutants and the assay was applied to analyse the effect of protease inhibitors, which revealed differences regarding their inhibitory action. The developed method thus appears suitable for reproducible determination of microgametocyte activation, medium-throughput drug screenings and deeper investigation of early blocks in gametogenesis and will facilitate the analysis of compounds for transmission blocking activities. (C) 2009 Elsevier B.V. All rights reserved. |
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Title:
A Cyclic GMP Signalling Module That Regulates Gliding Motility in a Malaria Parasite
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|
Author(s):
Moon, RW (Moon, Robert W.); Taylor, CJ (Taylor, Cathy J.); Bex, C (Bex, Claudia); Schepers, R (Schepers, Rebecca); Goulding, D (Goulding, David); Janse, CJ (Janse, Chris J.); Waters, AP (Waters, Andrew P.); Baker, DA (Baker, David A.); Billker, O (Billker, Oliver) |
| Source:
PLOS PATHOGENS Volume:
5 Issue:
9 Article Number:
e1000599 DOI:
10.1371/journal.ppat.1000599 Published Date:
2009 SEP |
|
Abstract:
The ookinete is a motile stage in the malaria life cycle which forms in the mosquito blood meal from the zygote. Ookinetes use an acto-myosin motor to glide towards and penetrate the midgut wall to establish infection in the vector. The regulation of gliding motility is poorly understood. Through genetic interaction studies we here describe a signalling module that identifies guanosine 3′,5′-cyclic monophosphate (cGMP) as an important second messenger regulating ookinete differentiation and motility. In ookinetes lacking the cyclic nucleotide degrading phosphodiesterase delta (PDE delta), unregulated signalling through cGMP results in rounding up of the normally banana-shaped cells. This phenotype is suppressed in a double mutant additionally lacking guanylyl cyclase beta (GC beta), showing that in ookinetes GC beta is an important source for cGMP, and that PDE delta is the relevant cGMP degrading enzyme. Inhibition of the cGMP-dependent protein kinase, PKG, blocks gliding, whereas enhanced signalling through cGMP restores normal gliding speed in a mutant lacking calcium dependent protein kinase 3, suggesting at least a partial overlap between calcium and cGMP dependent pathways. These data demonstrate an important function for signalling through cGMP, and most likely PKG, in dynamically regulating ookinete gliding during the transmission of malaria to the mosquito. |
|
Title:
An Essential Role for the Plasmodium Nek-2 Nima-related Protein Kinase in the Sexual Development of Malaria Parasites
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|
Author(s):
Reininger, L (Reininger, Luc); Tewari, R (Tewari, Rita); Fennell, C (Fennell, Clare); Holland, Z (Holland, Zoe); Goldring, D (Goldring, Dean); Ranford-Cartwright, L (Ranford-Cartwright, Lisa); Billker, O (Billker, Oliver); Doerig, C (Doerig, Christian) |
| Source:
JOURNAL OF BIOLOGICAL CHEMISTRY Volume:
284 Issue:
31 Pages:
20858-20868 DOI:
10.1074/jbc.M109.017988 Published Date:
2009 JUL 31 |
|
Abstract:
The molecular control of cell division and development in malaria parasites is far from understood. We previously showed that a Plasmodium gametocyte-specific NIMA-related protein kinase, nek-4, is required for completion of meiosis in the ookinete, the motile form that develops from the zygote in the mosquito vector. Here, we show that another NIMA-related kinase, Pfnek-2, is also predominantly expressed in gametocytes, and that Pfnek-2 is an active enzyme displaying an in vitro substrate preference distinct from that of Pfnek-4. A functional nek-2 gene is required for transmission of both Plasmodium falciparum and the rodent malaria parasite Plasmodium berghei to the mosquito vector, which is explained by the observation that disruption of the nek-2 gene in P. berghei causes dysregulation of DNA replication during meiosis and blocks ookinete development. This has implications (i) in our understanding of sexual development of malaria parasites and (ii) in the context of control strategies aimed at interfering with malaria transmission. |
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Title:
Calcium-Dependent Signaling and Kinases in Apicomplexan Parasites
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|
Author(s):
Billker, O (Billker, Oliver); Lourido, S (Lourido, Sebastian); Sibley, LD (Sibley, L. David) |
| Source:
CELL HOST & MICROBE Volume:
5 Issue:
6 Special Issue:
SI Pages:
612-622 DOI:
10.1016/j.chom.2009.05.017 Published Date:
2009 JUN 18 |
|
Abstract:
Calcium controls many critical events in the complex life cycles of apicomplexan parasites including protein secretion, motility, and development. Calcium levels are normally tightly regulated and rapid release of calcium into the cytosol activates a family of calcium-dependent protein kinases (CDPKs), which are normally characteristic of plants. CDPKs present in apicomplexans have acquired a number of unique domain structures likely reflecting their diverse functions. Calcium regulation in parasites is closely linked to signaling by cyclic nucleotides and their associated kinases. This Review summarizes the pivotal roles that calcium- and cyclic nucleotide-dependent kinases play in unique aspects of parasite biology. |
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Title:
Protein kinases of malaria parasites: an update
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|
Author(s):
Doerig, C (Doerig, Christian); Billker, O (Billker, Oliver); Haystead, T (Haystead, Timothy); Sharma, P (Sharma, Pushkar); Tobin, AB (Tobin, Andrew B.); Waters, NC (Waters, Norman C.) |
| Source:
TRENDS IN PARASITOLOGY Volume:
24 Issue:
12 Pages:
570-577 DOI:
10.1016/j.pt.2008.08.007 Published Date:
2008 DEC |
|
Abstract:
Protein kinases (PKs) play crucial roles in the control of proliferation and differentiation in eukaryotic cells. Research on protein phosphorylation has expanded tremendously in the past few years, in part as a consequence of the realization that PKs represent attractive drug targets in a variety of diseases. Activity in Plasmodium PK research has followed this trend, and several reports on various aspects of this subject were delivered at the Molecular Approaches to Malaria 2008 meeting (MAM2008), a sharp increase from the previous meeting. Here, the authors of most of these communications join to propose an integrated update of the development of the rapidly expanding field of Plasmodium kinomics. |
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Title:
Gametogenesis in malaria parasites is mediated by the cGMP-dependent protein kinase
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|
Author(s):
McRobert, L (McRobert, Louisa); Taylor, CJ (Taylor, Cathy J.); Deng, WS (Deng, Wensheng); Fivelman, QL (Fivelman, Quinton L.); Cummings, RM (Cummings, Ross M.); Polley, SD (Polley, Spencer D.); Billker, O (Billker, Oliver); Baker, DA (Baker, David A.) |
| Source:
PLOS BIOLOGY Volume:
6 Issue:
6 Pages:
1243-1252 Article Number:
e139 DOI:
10.1371/journal.pbio.0060139 Published Date:
2008 JUN |
|
Abstract:
Malaria parasite transmission requires differentiation of male and female gametocytes into gametes within a mosquito following a blood meal. A mosquito-derived molecule, xanthurenic acid (XA), can trigger gametogenesis, but the signalling events controlling this process in the human malaria parasite Plasmodium falciparum remain unknown. A role for cGMP was revealed by our observation that zaprinast (an inhibitor of phosphodiesterases that hydrolyse cGMP) stimulates gametogenesis in the absence of XA. Using cGMP-dependent protein kinase (PKG) inhibitors in conjunction with transgenic parasites expressing an inhibitor-insensitive mutant PKG enzyme, we demonstrate that PKG is essential for XA- and zaprinast-induced gametogenesis. Furthermore, we show that intracellular calcium (Ca2+) is required for differentiation and acts downstream of or in parallel with PKG activation. This work defines a key role for PKG in gametogenesis, elucidates the hierarchy of signalling events governing this process in P. falciparum, and demonstrates the feasibility of selective inhibition of a crucial regulator of the malaria parasite life cycle. |
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Title:
The conserved plant sterility gene HAP2 functions after attachment of fusogenic membranes in Chlamydomonas and Plasmodium gametes
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|
Author(s):
Liu, YJ (Liu, Yanjie); Tewari, R (Tewari, Rita); Ning, J (Ning, Jue); Blagborough, AM (Blagborough, Andrew M.); Garbom, S (Garbom, Sara); Pei, JM (Pei, Jimin); Grishin, NV (Grishin, Nick V.); Steele, RE (Steele, Robert E.); Sinden, RE (Sinden, Robert E.); Snell, WJ (Snell, William J.); Billker, O (Billker, Oliver) |
| Source:
GENES & DEVELOPMENT Volume:
22 Issue:
8 Pages:
1051-1068 DOI:
10.1101/gad.1656508 Published Date:
2008 APR 15 |
|
Abstract:
The cellular and molecular mechanisms that underlie species-specific membrane fusion between male and female gametes remain largely unknown. Here, by use of gene discovery methods in the green alga Chlamydomonas, gene disruption in the rodent malaria parasite Plasmodium berghei, and distinctive features of fertilization in both organisms, we report discovery of a mechanism that accounts for a conserved protein required for gamete fusion. A screen for fusion mutants in Chlamydomonas identified a homolog of HAP2, an Arabidopsis sterility gene. Moreover, HAP2 disruption in Plasmodium blocked fertilization and thereby mosquito transmission of malaria. HAP2 localizes at the fusion site of Chlamydomonas minus gametes, yet Chlamydomonas minus and Plasmodium hap2 male gametes retain the ability, using other, species-limited proteins, to form tight prefusion membrane attachments with their respective gamete partners. Membrane dye experiments show that HAP2 is essential for membrane merger. Thus, in two distantly related eukaryotes, species-limited proteins govern access to a conserved protein essential for membrane fusion. |
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Title:
P-falciparum gametogenesis is mediated by cGMPdependent protein kinase.
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|
Author(s):
McRobert, L (McRobert, Louisa); Taylor, CJ (Taylor, Catherine J.); Deng, W (Deng, Wensheng); Fivelman, Q (Fivelman, Quinton); Cummings, R (Cummings, Ross); Billker, O (Billker, Oliver); Baker, DA (Baker, David A.) |
| Source:
INTERNATIONAL JOURNAL FOR PARASITOLOGY Volume:
38 Pages:
S81-S81 Published Date:
2008 JAN Supplement:
1 |
|
Title:
Towards a functional analysis of Plasmodium ROM4 protease.
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|
Author(s):
Pino, P (Pino, Paco); Dos Santos, JM (Dos Santos, Joana Mendonca); Blackman, MJ (Blackman, Michael J.); Billker, O (Billker, Oliver); Soldati, D (Soldati, Dominique) |
| Source:
INTERNATIONAL JOURNAL FOR PARASITOLOGY Volume:
38 Pages:
S74-S74 Published Date:
2008 JAN Supplement:
1 |
|
Title:
A plant sterility gene has a conserved function in Plasmodium berghei gamete membrane fusion and is essential for mosquito transmission of malaria.
|
|
Author(s):
Tewari, R (Tewari, Rita); Liu, YJ (Liu, Yanjie); Ning, J (Ning, Jue); Blagborough, AM (Blagborough, Andrew M.); Pei, J (Pei, Jimin); Grishin, NV (Grishin, Nick V.); Steele, RE (Steele, Robert E.); Sinden, RE (Sinden, Robert E.); Snell, WJ (Snell, William J.); Billker, O (Billker, Oliver) |
| Source:
INTERNATIONAL JOURNAL FOR PARASITOLOGY Meeting Abstract:
12 Volume:
38 Pages:
S20-S20 Published Date:
2008 JAN Supplement:
1 |
|
Title:
Heparan sulfate proteoglycans provide a signal to Plasmodium sporozoites to stop migrating and productively invade host cells
|
|
Author(s):
Coppi, A (Coppi, Alida); Tewari, R (Tewari, Rita); Bishop, JR (Bishop, Joseph R.); Bennett, BL (Bennett, Brandy L.); Lawrence, R (Lawrence, Roger); Esko, JD (Esko, Jeffrey D.); Billker, O (Billker, Oliver); Sinnis, P (Sinnis, Photini) |
| Source:
CELL HOST & MICROBE Volume:
2 Issue:
5 Pages:
316-327 DOI:
10.1016/j.chom.2007.10.002 Published Date:
2007 NOV |
|
Abstract:
Malaria infection is initiated when Anopheles mosquitoes inject Plasmodium sporozoites into the skin. Sporozoites subsequently reach the liver, invading and developing within hepatocytes. Sporozoites contact and traverse many cell types as they migrate from skin to liver; however, the mechanism by which they switch from a migratory mode to an invasive mode is unclear. Here, we show that sporozoites of the rodent malaria parasite Plasmodium berghei use the sulfation level of host heparan sulfate proteoglycans (HSPGs) to navigate within the mammalian host. Sporozoites migrate through cells expressing low-sulfated HSPGs, such as those in skin and endothelium, while highly sulfated HSPGs of hepatocytes activate sporozoites for invasion. A calcium-dependent protein kinase is critical for the switch to an invasive phenotype, a process accompanied by proteolytic cleavage of the sporozoite’s major surface protein. These findings explain how sporozoites retain their infectivity for an organ that is far from their site of entry. |
|
Title:
Plasmodium berghei calcium-dependent protein kinase 3 is required for ookinete gliding motility and mosquito midgut invasion
|
|
Author(s):
Siden-Kiamos, I (Siden-Kiamos, Inga); Ecker, A (Ecker, Andrea); Nybäck, S (Nyback, Saga); Louis, C (Louis, Christos); Sinden, RE (Sinden, Robert E.); Billker, O (Billker, Oliver) |
| Source:
MOLECULAR MICROBIOLOGY Volume:
60 Issue:
6 Pages:
1355-1363 DOI:
10.1111/j.1365-2958.2006.05189.x Published Date:
2006 JUN |
|
Abstract:
Apicomplexan parasites critically depend on a unique form of gliding motility to colonize their hosts and to invade cells. Gliding requires different stage and species-specific transmembrane adhesins, which interact with an intracellular motor complex shared across parasite stages and species. How gliding is regulated by extracellular factors and intracellular signalling mechanisms is largely unknown, but current evidence suggests an important role for cytosolic calcium as a second messenger. Studying a Plasmodium berghei gene deletion mutant, we here provide evidence that a calcium-dependent protein kinase, CDPK3, has an important function in regulating motility of the ookinete in the mosquito midgut. We show that a cdpk3(-) parasite clone produces morphologically normal ookinetes, which fail to engage the midgut epithelium, due to a marked reduction in their ability to glide productively, resulting in marked reduction in malaria transmission to the mosquito. The mutant was successfully complemented with an episomally maintained cdpk3 gene, restoring mosquito transmission to wild-type level. cdpk3(-) ookinetes maintain their full genetic differentiation potential when microinjected into the mosquito haemocoel and cdpk3(-) sporozoites produced in this way are motile and infectious, suggesting an ookinete-limited essential function for CDPK3. |