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  • 1. Akkaya, Munir
    et al.
    Bansal, Abhisheka
    Sheehan, Patrick W.
    Pena, Mirna
    Molina-Cruz, Alvaro
    Orchard, Lindsey M.
    Cimperman, Clare K.
    Qi, Chen-Feng
    Ross, Philipp
    Yazew, Takele
    Sturdevant, Daniel
    Anzick, Sarah L.
    Thiruvengadam, Girija
    Otto, Thomas Dan
    Billker, Oliver
    Umeå universitet, Medicinska fakulteten, Molekylär Infektionsmedicin, Sverige (MIMS). Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Institutionen för molekylärbiologi (Teknisk-naturvetenskaplig fakultet).
    Llinas, Manuel
    Miller, Louis H.
    Pierce, Susan K.
    A single-nucleotide polymorphism in a Plasmodium berghei ApiAP2 transcription factor alters the development of host immunity2020Ingår i: Science Advances, E-ISSN 2375-2548, Vol. 6, nr 6, artikel-id eaaw6957Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    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.

  • 2. Alavi, Y.
    et al.
    Arai, M.
    Mendoza, J.
    Tufet-Bayona, M.
    Sinha, R.
    Fowler, K.
    Billker, Oliver
    Department of Biological Sciences, Imperial College London, London SW7 2AZ, UK.
    Franke-Fayard, B
    Janse, C J
    Waters, A
    Sinden, R E
    The dynamics of interactions between Plasmodium and the mosquito: a study of the infectivity of Plasmodium berghei and Plasmodium gallinaceum, and their transmission by Anopheles stephensi, Anopheles gambiae and Aedes aegypti2003Ingår i: International Journal of Parasitology, ISSN 0020-7519, E-ISSN 1879-0135, Vol. 33, nr 9, s. 933-943Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Knowledge of parasite–mosquito interactions is essential to develop strategies that will reduce malaria transmission through the mosquito vector. In this study we investigated the development of two model malaria parasites, Plasmodium berghei and Plasmodium gallinaceum, in three mosquito species Anopheles stephensi, Anopheles gambiae and Aedes aegypti. New methods to study gamete production in vivo in combination with GFP-expressing ookinetes were employed to measure the large losses incurred by the parasites during infection of mosquitoes. All three mosquito species transmitted P. gallinaceumP. berghei was only transmitted by Anopheles spp. Plasmodium gallinaceum initiates gamete production with high efficiency equally in the three mosquito species. By contrast P. berghei is less efficiently activated to produce gametes, and in Ae. aegypti microgamete formation is almost totally suppressed. In all parasite/vector combinations ookinete development is inefficient, 500–100,000-fold losses were encountered. Losses during ookinete-to-oocyst transformation range from fivefold in compatible vector parasite combinations (P. berghei/An. stephensi), through >100-fold in poor vector/parasite combinations (P. gallinaceum/An. stephensi), to complete blockade (>1,500 fold) in others (P. berghei/Ae. aegypti). Plasmodium berghei ookinetes survive poorly in the bloodmeal of Ae. aegypti and are unable to invade the midgut epithelium. Cultured mature ookinetes of P. berghei injected directly into the mosquito haemocoele produced salivary gland sporozoites in An. stephensi, but not in Ae. aegypti, suggesting that further species-specific incompatibilities occur downstream of the midgut epithelium in Ae. aegypti. These results show that in these parasite–mosquito combinations the susceptibility to malarial infection is regulated at multiple steps during the development of the parasites. Understanding these at the molecular level may contribute to the development of rational strategies to reduce the vector competence of malarial vectors.

  • 3. Alkaitis, Matthew S.
    et al.
    Wang, Honghui
    Ikeda, Allison K.
    Rowley, Carol A.
    MacCormick, Ian J. C.
    Chertow, Jessica H.
    Billker, Oliver
    Wellcome Trust Sanger Institute, Hinxton, UK.
    Suffredini, Anthony F.
    Roberts, David J.
    Taylor, Terrie E.
    Seydel, Karl B.
    Ackerman, Hans C.
    Decreased Rate of Plasma Arginine Appearance in Murine Malaria May Explain Hypoargininemia in Children With Cerebral Malaria2016Ingår i: Journal of Infectious Diseases, ISSN 0022-1899, E-ISSN 1537-6613, Vol. 214, nr 12, s. 1840-1849Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    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.

  • 4. Arai, M
    et al.
    Billker, Oliver
    Department of Biology,Imperial College of Science, Technology and Medicine, London, UK.
    Morris, H R
    Panico, M
    Delcroix, M
    Dixon, D
    Ley, S V
    Sinden, R E
    Both mosquito-derived xanthurenic acid and a host blood-derived factor regulate gametogenesis of Plasmodium in the midgut of the mosquito2001Ingår i: Molecular and biochemical parasitology (Print), ISSN 0166-6851, E-ISSN 1872-9428, Vol. 116, nr 1, s. 17-24Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Gametogenesis of Plasmodium in vitro can be induced by the combined stimulus of a 5 degrees C fall in temperature and the presence of xanthurenic acid (XA). In-vitro experiments showed that P. gallinaceum (EC(50)=80 nM) is much more sensitive to XA than P. berghei (9 microM), P. yoelii (8 microM), and P. falciparum (2 microM). However, in the mosquito vector, we do not know whether the temperature shift and XA are the only gametocyte-activating factors (GAF), nor do we know with certainty the true source(s) of XA in the mosquito blood meal. Previous studies indicate that XA is the only source of GAF in the mosquito. By defining, and then contrasting, the ability of an XA-deficient mutant of Aedes aegypti, with the wild-type mosquito to support exflagellation and ookinete formation in vivo, we determined the roles of parasite-, mosquito- and host blood-derived GAF in the regulation of gametogenesis of P. gallinaceum. Removal of both host and vector sources of GAF totally inhibited both exflagellation and ookinete production, whilst the lack of either single source resulted in only a partial reduction of exflagellation and ookinete formation in the mosquito gut. Both sources can be effectively replaced/substituted by synthetic XA. This suggests (1) both mosquito- and vertebrate-derived factors act as GAF in the mosquito gut in vivo; (2) the parasite itself is unable to produce any significant GAF activity. Studies are underway to determine whether vertebrate-derived GAF is XA. These data may form the basis of further studies of the development of new methods of interrupting malarial transmission.

  • 5. Arai, Meiji
    et al.
    Alavi, Yasmene I H
    Mendoza, Jacqueline
    Billker, Oliver
    Sinden, Robert E
    Isonicotinic acid hydrazide: an anti-tuberculosis drug inhibits malarial transmission in the mosquito gut2004Ingår i: Experimental parasitology, ISSN 0014-4894, E-ISSN 1090-2449, Vol. 106, nr 1-2, s. 30-36Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    We studied the transmission-blocking effect of isonicotinic acid hydrazide (INH), a widely used anti-tuberculosis drug, against Plasmodium gallinaceum and Plasmodium berghei. INH-treatment of infected animals did not inhibit parasite development in the blood of the vertebrate host, but did inhibit exflagellation, ookinete formation, and oocyst development in the mosquito. Oocyst development was inhibited in a dose-dependent manner. The ED(50) in the P. gallinaceum/chicken/Aedes aegypti model and P. berghei/mouse/Anopheles stephensi model was 72 and 109 mg/kg, respectively. In marked contrast, in vitro exflagellation and ookinete development were not directly affected by physiological concentrations of INH. We suggest that INH exerts its inhibitory effects on the mosquito stages of the malaria parasite by an indirect, and at present undefined mechanism. Further elucidation of the mechanism how INH inhibits parasite development specifically on mosquito stages may allow us to identify new targets for malaria control strategy.

  • 6. Bauza, Karolis
    et al.
    Malinauskas, Tomas
    Pfander, Claudia
    Anar, Burcu
    Jones, E Yvonne
    Billker, Oliver
    Wellcome Trust Sanger Institute, Hinxton, Cambridge, United Kingdom.
    Hill, Adrian V S
    Reyes-Sandoval, Arturo
    Efficacy of a Plasmodium vivax Malaria Vaccine Using ChAd63 and Modified Vaccinia Ankara Expressing Thrombospondin-Related Anonymous Protein as Assessed with Transgenic Plasmodium berghei Parasites2014Ingår i: Infection and Immunity, ISSN 0019-9567, E-ISSN 1098-5522, Vol. 82, nr 3, s. 1277-1286Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    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.

  • 7. Berger, Cedric N
    et al.
    Billker, Oliver
    Meyer, Thomas F
    Servin, Alain L
    Kansau, Imad
    Differential recognition of members of the carcinoembryonic antigen family by Afa/Dr adhesins of diffusely adhering Escherichia coli (Afa/Dr DAEC)2004Ingår i: Molecular Microbiology, ISSN 0950-382X, E-ISSN 1365-2958, Vol. 52, nr 4, s. 963-983Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Little is known about the molecular bases underlying the virulence of diffusely adhering Escherichia coli (DAEC) harbouring the Afa/Dr family of adhesins. These adhesins recognize as receptors the GPI-anchored proteins CD55 (decay-accelerating factor, DAF) and CD66e (carcinoembryonic antigen, CEA). CD66e is a member of the CEA-related cell adhesion molecules (CEACAM) family, comprising seven members. We analysed the interactions of Afa/Dr DAEC with the CEACAMs using CEACAM-expressing CHO and HeLa cells. The results demonstrate that only E. coli expressing a subfamily of Afa/Dr adhesins, named here Afa/Dr-I, including Dr, F1845 and AfaE-III adhesins, bound onto CHO cells expressing CEACAM1, CEA or CEACAM6. Whereas all the Afa/Dr adhesins elicit recruitment of CD55 around adhering bacteria, only the Afa/Dr-I subfamily elicits the recruitment of CEACAM1, CEA and CEACAM6. In addition, although CEACAM3 is not recognized as a receptor by the subfamily of Afa/Dr adhesins, it is recruited around bacteria in HeLa cells. The recruited CEACAM1, CEA and CEACAM6 around adhering bacteria resist totally or in part a detergent extraction, whereas the recruited CEACAM3 does not. Finally, the results show that recognition of CEA and CEACAM6, but not CEACAM1, is accompanied by tight attachment to bacteria of cell surface microvilli-like extensions, which are elongated. Moreover, recognition of CEA is accompanied by an activation of the Rho GTPase Cdc42 and by a phosphorylation of ERM, which in turn elicit the observed cell surface microvilli-like extensions.

  • 8.
    Billker, Oliver
    Wellcome Trust Sanger Institute, Hinxton, United Kingdom.
    Cracking Ali Baba's code2017Ingår i: eLIFE, E-ISSN 2050-084X, Vol. 6Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    A protein called P36 holds the key to how different species of malaria parasite invade liver cells.

  • 9.
    Billker, Oliver
    Umeå universitet, Medicinska fakulteten, Institutionen för molekylärbiologi (Medicinska fakulteten). Umeå universitet, Medicinska fakulteten, Molekylär Infektionsmedicin, Sverige (MIMS). Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Institutionen för molekylärbiologi (Teknisk-naturvetenskaplig fakultet).
    CRISPRing the elephant in the room2018Ingår i: Cell Host and Microbe, ISSN 1931-3128, E-ISSN 1934-6069, Vol. 24, nr 6, s. 754-755Artikel i tidskrift (Övrigt vetenskapligt)
    Abstract [en]

    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.

  • 10.
    Billker, Oliver
    et al.
    Department of Biological Sciences Imperial College London London SW7 2AZ, United Kingdom.
    Dechamps, Sandrine
    Tewari, Rita
    Wenig, Gerald
    Franke-Fayard, Blandine
    Brinkmann, Volker
    Calcium and a calcium-dependent protein kinase regulate gamete formation and mosquito transmission in a malaria parasite2004Ingår i: Cell, ISSN 0092-8674, E-ISSN 1097-4172, Vol. 117, nr 4, s. 503-514Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Transmission of malaria parasites to mosquitoes is initiated by the obligatory sexual reproduction of the parasite within the mosquito bloodmeal. Differentiation of specialized transmission stages, the gametocytes, into male and female gametes is induced by a small mosquito molecule, xanthurenic acid (XA). Using a Plasmodium berghei strain expressing a bioluminescent calcium sensor, we show that XA triggers a rapid rise in cytosolic calcium specifically in gametocytes that is essential for their differentiation into gametes. A member of a family of plant-like calcium dependent protein kinases, CDPK4, is identified as the molecular switch that translates the XA-induced calcium signal into a cellular response by regulating cell cycle progression in the male gametocyte. CDPK4 is shown to be essential for the sexual reproduction and mosquito transmission of P. berghei. This study reveals an unexpected function for a plant-like signaling pathway in cell cycle regulation and life cycle progression of a malaria parasite.

  • 11. Billker, Oliver
    et al.
    Lindo, V.
    Panico, M.
    Etienne, A. E.
    Paxton, T.
    Dell, A.
    Rogers, M.
    Sinden, R. E.
    Morris, H. R.
    Identification of xanthurenic acid as the putative inducer of malaria development in the mosquito1998Ingår i: Nature, ISSN 0028-0836, E-ISSN 1476-4687, Vol. 392, nr 6673, s. 289-292Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Malaria is transmitted from vertebrate host to mosquito vector by mature sexual blood-living stages called gametocytes. Within seconds of ingestion into the mosquito bloodmeal, gametocytes undergo gametogenesis. Induction requires the simultaneous exposure to at least two stimuli in vitro: a drop in bloodmeal temperature to 5 degrees C below that of the vertebrate host, and a rise in pH from 7.4 to 8.0-8.2. In vivo the mosquito bloodmeal has a pH of between 7.5 and 7.6. It is thought that in vivo the second inducer is an unknown mosquito-derived gametocyte-activating factor. Here we show that this factor is xanthurenic acid. We also show that low concentrations of xanthurenic acid can act together with pH to induce gametogenesis in vitro. Structurally related compounds are at least ninefold less effective at inducing gametogenesis in vitro. In Drosophila mutants with lesions in the kynurenine pathway of tryptophan metabolism (of which xanthurenic acid is a side product), no alternative active compound was detected in crude insect homogenates. These data could form the basis of the rational development of new methods of interrupting the transmission of malaria using drugs or new refractory mosquito genotypes to block parasite gametogenesis.

  • 12. Billker, Oliver
    et al.
    Lourido, Sebastian
    Sibley, L David
    Calcium-dependent signaling and kinases in apicomplexan parasites2009Ingår i: Cell Host and Microbe, ISSN 1931-3128, E-ISSN 1934-6069, Vol. 5, nr 6, s. 612-622Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    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.

  • 13.
    Billker, Oliver
    et al.
    Department of Biology, Imperial College of Science, Technology and Medicine, London, UK.
    Miller, A J
    Sinden, R E
    Determination of mosquito bloodmeal pH in situ by ion-selective microelectrode measurement: implications for the regulation of malarial gametogenesis2000Ingår i: Parasitology, ISSN 0031-1820, E-ISSN 1469-8161, Vol. 120, s. 547-551Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Malarial gametocytes circulate in the peripheral blood of the vertebrate host as developmentally arrested intra-erythrocytic cells, which only resume development into gametes when ingested into the bloodmeal of the female mosquito vector. The ensuing development encompasses sexual reproduction and mediates parasite transmission to the insect. In vitro the induction of gametogenesis requires a drop in temperature and either a pH increase from physiological blood pH (ca pH 7.4) to about pH 8.0, or the presence of a gametocyte-activating factor recently identified as xanthurenic acid (XA). However, it is unclear whether either the pH increase or XA act as natural triggers in the mosquito bloodmeal. We here use pH-sensitive microelectrodes to determine bloodmeal pH in intact mosquitoes. Measurements taken in the first 30 min after ingestion, when malarial gametogenesis is induced in vivo, revealed small pH increases from 7.40 (mouse blood) to 7.52 in Aedes aegypti and to 7.58 in Anophĕles stephensi. However, bloodmeal pH was clearly suboptimal if compared to values required to induce gametogenesis in vitro. Xanthurenic acid is shown to extend the pH-range of exflagellation in vitro in a dose-dependent manner to values that we have observed in the bloodmeal, suggesting that in vivo malarial gametogenesis could be further regulated by both these factors.

  • 14.
    Billker, Oliver
    et al.
    Max-Planck-Institut für Infektionsbiologie, Abteilung Molekulare Biologie, Berlin, Germany.
    Popp, A
    Gray-Owen, S D
    Meyer, T F
    The structural basis of CEACAM-receptor targeting by neisserial Opa proteins2000Ingår i: Trends in Microbiology, ISSN 0966-842X, E-ISSN 1878-4380, Vol. 8, nr 6, s. 258-261Artikel i tidskrift (Refereegranskat)
  • 15.
    Billker, Oliver
    et al.
    Max‐Planck‐Institut für Infektionsbiologie, Abteilung Molekulare Biologie, Schumannstraße 21/22, D‐10117 Berlin, Germany.
    Popp, Andreas
    Brinkmann, Volker
    Wenig, Gerald
    Schneider, Jutta
    Caron, Emmanuelle
    Meyer, Thomas F.
    Distinct mechanisms of internalization of Neisseria gonorrhoeae by members of the CEACAM receptor family involving Rac1- and Cdc42-dependent and -independent pathways2002Ingår i: EMBO Journal, ISSN 0261-4189, E-ISSN 1460-2075, Vol. 21, nr 4, s. 560-571Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Opa adhesins of pathogenic Neisseria species target four members of the human carcinoembryonic antigen-related cellular adhesion molecule (CEACAM) family. CEACAM receptors mediate opsonization-independent phagocytosis of Neisseria gonorrhoeae by human granulocytes and each receptor individually can mediate gonococcal invasion of epithelial cells. We show here that gonococcal internalization occurs by distinct mechanisms depending on the CEACAM receptor expressed. For the invasion of epithelial cell lines via CEACAM1 and CEACAM6, a pathogen-directed reorganization of the actin cytoskeleton is not required. In marked contrast, ligation of CEACAM3 triggers a dramatic but localized reorganization of the host cell surface leading to highly efficient engulfment of bacteria in a process regulated by the small GTPases Rac1 and Cdc42, but not Rho. Two tyrosine residues of a cytoplasmic immune receptor tyrosine-based activating motif of CEACAM3 are essential for the induction of phagocytic actin structures and subsequent gonococcal internalization. The granulocyte-specific CEACAM3 receptor has properties of a single chain phagocytic receptor and may thus contribute to innate immunity by the elimination of Neisseria and other CEACAM-binding pathogens that colonize human mucosal surfaces.

  • 16.
    Billker, Oliver
    et al.
    Malaria Programme, Wellcome Trust Sanger Institute, Wellcome Genome Campus, Hinxton, Cambridge, UK.
    Rayner, Julian C.
    Calcium Builds Strong Host-Parasite Interactions2015Ingår i: Cell Host and Microbe, ISSN 1931-3128, E-ISSN 1934-6069, Vol. 18, nr 1, s. 9-10Artikel, forskningsöversikt (Refereegranskat)
    Abstract [en]

    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.

  • 17.
    Billker, Oliver
    et al.
    Molecular and Cellular Parasitology Research Group, Infection and Immunity Section, Department of Biology, Imperial College of Science, Technology and Medicine, London, UK.
    Shaw, M K
    Margos, G
    Sinden, R E
    The roles of temperature, pH and mosquito factors as triggers of male and female gametogenesis of Plasmodium berghei in vitro1997Ingår i: Parasitology, ISSN 0031-1820, E-ISSN 1469-8161, Vol. 115, s. 1-7Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Developmentally arrested malarial gametocytes undergo gamete formation in the mosquito midgut immediately after ingestion of the infected bloodmeal. In the rodent malaria parasite Plasmodium berghei male gametogenesis (exflagellation) can be induced in vitro by a temperature decrease (from 39 degrees C in the vertebrate host to 20 degrees C) and a concomitant pH increase (from 7.3 in mouse blood to 8.0). We report the presence of additional Gametocyte Activating Factor(s) (GAF) present in Anopheles stephensi tissue extracts, which induce both male and female gametogenesis at the otherwise nonpermissive pH of 7.3 in vitro but are unable to overcome the low temperature requirement. All constituent cellular events of microgametogeneis studied here are induced by the same triggers in vitro. A temperature decrease is also required for exflagellation in the mosquito midgut. The possible role of GAF as a second obligatory natural trigger of gametogenesis is discussed.

  • 18. Billker, Oliver
    et al.
    Shaw, Michael K
    Jones, Ian W
    Ley, Steven V
    Mordue, A Jennifer
    Sinden, Robert E
    Azadirachtin disrupts formation of organised microtubule arrays during microgametogenesis of Plasmodium berghei2002Ingår i: Journal of Eukaryotic Microbiology, ISSN 1066-5234, E-ISSN 1550-7408, Vol. 49, nr 6, s. 489-497Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Transmission of malaria parasites from vertebrate blood to the mosquito vector depends critically on the differentiation of the gametocytes into gametes. This occurs in response to environmental stimuli encountered by the parasite in the mosquito bloodmeal. Male gametogenesis involves three rounds of DNA replication and endomitosis, and the assembly de novo of 8 motile axonemes. Azadirachtin, a plant limnoid and insecticide with an unkown mode of action, specifically inhibits the release of motile gametes from activated microgametocytes but does not inhibit growth and replication of a sexual blood stages. We have combined confocal laser scanning microscopy and transmission electron microscopy to examine the effect of azadirachtin on the complex reorganisation of the microtubule cytoskeleton during gametogenesis in Plasmodium berghei. Neither the replication of the genome nor the ability of tubulin monomers to assemble into microtubules upon gametocyte activation were prevented by azadirachtin. However, the drug interfered with the formation of mitotic spindles and with the assembly of microtubules into typical axonemes. Our observations suggest that azadarachtin specifically disrupts the patterning of microtubules into more complex structures, such as mitotic spindles and axonemes.

  • 19. Brochet, Mathieu
    et al.
    Billker, Oliver
    Wellcome Trust Sanger Institute, Malaria Programme, CB10 1SA, Hinxton, UK.
    Calcium signalling in malaria parasites2016Ingår i: Molecular Microbiology, ISSN 0950-382X, E-ISSN 1365-2958, Vol. 100, nr 3, s. 397-408Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    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.

  • 20. Brochet, Mathieu
    et al.
    Collins, Mark O.
    Smith, Terry K.
    Thompson, Eloise
    Sebastian, Sarah
    Volkmann, Katrin
    Schwach, Frank
    Chappell, Lia
    Gomes, Ana Rita
    Berriman, Matthew
    Rayner, Julian C.
    Baker, David A.
    Choudhary, Jyoti
    Billker, Oliver
    Wellcome Trust Sanger Institute, Hinxton, Cambridge, United Kingdom.
    Phosphoinositide metabolism links cGMP-dependent protein kinase G to essential Ca²⁺ signals at key decision points in the life cycle of malaria parasites2014Ingår i: PLoS biology, ISSN 1544-9173, E-ISSN 1545-7885, Vol. 12, nr 3, artikel-id e1001806Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    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.

  • 21. Brugat, Thibaut
    et al.
    Reid, Adam James
    Lin, Jingwen
    Cunningham, Deirdre
    Tumwine, Irene
    Kushinga, Garikai
    McLaughlin, Sarah
    Spence, Philip
    Böhme, Ulrike
    Sanders, Mandy
    Conteh, Solomon
    Bushell, Ellen
    Metcalf, Tom
    Billker, Oliver
    Wellcome Trust Sanger Institute, Hinxton, Cambridge CB10 1SA, UK.
    Duffy, Patrick E.
    Newbold, Chris
    Berriman, Matthew
    Langhorne, Jean
    Antibody-independent mechanisms regulate the establishment of chronic Plasmodium infection2017Ingår i: Nature Microbiology, E-ISSN 2058-5276, Vol. 2, nr 4, artikel-id 16276Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Malaria is caused by parasites of the genus Plasmodium. All human-infecting Plasmodium species can establish long-lasting 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(7-9). 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.

  • 22. Bushell, Ellen
    et al.
    Gomes, Ana Rita
    Sanderson, Theo
    Anar, Burcu
    Girling, Gareth
    Herd, Colin
    Metcalf, Tom
    Modrzynska, Katarzyna
    Schwach, Frank
    Martin, Rowena E.
    Mather, Michael W.
    McFadden, Geoffrey I.
    Parts, Leopold
    Rutledge, Gavin G.
    Vaidya, Akhil B.
    Wengelnik, Kai
    Rayner, Julian C.
    Billker, Oliver
    Wellcome Trust Sanger Institute, Wellcome Genome Campus, Hinxton, Cambridgeshire, UK.
    Functional Profiling of a Plasmodium Genome Reveals an Abundance of Essential Genes2017Ingår i: Cell, ISSN 0092-8674, E-ISSN 1097-4172, Vol. 170, nr 2, s. 260-272.e1-e4Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    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.

  • 23. Butcher, G A
    et al.
    Sinden, R E
    Billker, Oliver
    Department of Biology, Imperial College of Science, Technology and Medicine, Prince Consort Road,London, United Kingdom.
    Plasmodium berghei: infectivity of mice to Anopheles stephensi mosquitoes1996Ingår i: Experimental parasitology, ISSN 0014-4894, E-ISSN 1090-2449, Vol. 84, nr 3, s. 371-379Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    The infectivity of P. berghei-infected TO mice to mosquitoes declines rapidly 2 to 5 days after blood inoculation, in spite of rising numbers of gametocytes in the blood. This pattern is typical of many malaria infections and various factors, particularly specific and nonspecific immune responses, have previously been implicated in the decline. Here we report that (1) simple physiological changes in the mouse blood, namely, falling pH and bicarbonate levels induced by high parasitaemias, are responsible for the sustained inhibition of infectivity; (2) the inhibition is reversible in vivo by the addition of sodium bicarbonate alone; (3) the inhibition occurs at the point of exflagellation; (4) contrary to previous observations (Kawamoto et al. 1992), exflagellation in P. berghei, like that in P. gallinaceum (Bishop and McConnachie 1956; Nijhout and Carter 1978; Nijhout 1979) and P. falciparum (Ogwan'g et al. 1993), is dependent on extracellular bicarbonate; and (5) induction of exflagellation by a mosquito factor is bicarbonate dependent. These new observations are critical to the design and interpretation of experiments on other transmission blocking phenomena.

  • 24. Böhme, Ulrike
    et al.
    Otto, Thomas D.
    Cotton, James A.
    Steinbiss, Sascha
    Sanders, Mandy
    Oyola, Samuel O.
    Nicot, Antoine
    Gandon, Sylvain
    Patra, Kailash P.
    Herd, Colin
    Bushell, Ellen
    Modrzynska, Katarzyna K.
    Billker, Oliver
    Wellcome Trust Sanger Institute, Wellcome Genome Campus, Hinxton, Cambridge, CB10 1SA, United Kingdom.
    Vinetz, Joseph M.
    Rivero, Ana
    Newbold, Chris I.
    Berriman, Matthew
    Complete avian malaria parasite genomes reveal features associated with lineage-specific evolution in birds and mammals2018Ingår i: Genome Research, ISSN 1088-9051, E-ISSN 1549-5469, Vol. 28, nr 4, s. 547-560Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    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.

  • 25. Chertow, Jessica H
    et al.
    Alkaitis, Matthew S
    Nardone, Glenn
    Ikeda, Allison K
    Cunnington, Aubrey J
    Okebe, Joseph
    Ebonyi, Augustine O
    Njie, Madi
    Correa, Simon
    Jayasooriya, Shamanthi
    Casals-Pascual, Climent
    Billker, Oliver
    Wellcome Trust SangerInstitute, Hinxton Cambridge, United Kingdom.
    Conway, David J
    Walther, Michael
    Ackerman, Hans
    Plasmodium Infection Is Associated with Impaired Hepatic Dimethylarginine Dimethylaminohydrolase Activity and Disruption of Nitric Oxide Synthase Inhibitor/Substrate Homeostasis2015Ingår i: PLoS Pathogens, ISSN 1553-7366, E-ISSN 1553-7374, Vol. 11, nr 9, artikel-id e1005119Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    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.

  • 26. Coppi, Alida
    et al.
    Tewari, Rita
    Bishop, Joseph R
    Bennett, Brandy L
    Lawrence, Roger
    Esko, Jeffrey D
    Billker, Oliver
    Division of Cell and Molecular Biology, Imperial College London, UK.
    Sinnis, Photini
    Heparan sulfate proteoglycans provide a signal to Plasmodium sporozoites to stop migrating and productively invade host cells2007Ingår i: Cell Host and Microbe, ISSN 1931-3128, E-ISSN 1934-6069, Vol. 2, nr 5, s. 316-327Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    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.

  • 27. Doerig, C
    et al.
    Baker, D
    Billker, Oliver
    Blackman, M J
    Chitnis, C
    Dhar Kumar, S
    Heussler, V
    Holder, A A
    Kocken, C
    Krishna, S
    Langsley, G
    Lasonder, E
    Menard, R
    Meissner, M
    Pradel, G
    Ranford-Cartwright, L
    Sharma, A
    Sharma, P
    Tardieux, T
    Tatu, U
    Alano, P
    Signalling in malaria parasites. The MALSIG consortium2009Ingår i: Parasite, ISSN 1252-607X, E-ISSN 1776-1042, Vol. 16, nr 3, s. 169-182Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Depending on their developmental stage in the life cycle, malaria parasites develop within or outside host cells, and in extremely diverse contexts such as the vertebrate liver and blood circulation, or the insect midgut and hemocoel. Cellular and molecular mechanisms enabling the parasite to sense and respond to the intra- and the extra-cellular environments are therefore key elements for the proliferation and transmission of Plasmodium, and therefore are, from a public health perspective, strategic targets in the fight against this deadly disease. The MALSIG consortium, which was initiated in February 2009, was designed with the primary objective to integrate research ongoing in Europe and India on i) the properties of Plasmodium signalling molecules, and ii) developmental processes occurring at various points of the parasite life cycle. On one hand, functional studies of individual genes and their products in Plasmodium falciparum (and in the technically more manageable rodent model Plasmodium berghei) are providing information on parasite protein kinases and phosphatases, and of the molecules governing cyclic nucleotide metabolism and calcium signalling. On the other hand, cellular and molecular studies are elucidating key steps of parasite development such as merozoite invasion and egress in blood and liver parasite stages, control of DNA replication in asexual and sexual development, membrane dynamics and trafficking, production of gametocytes in the vertebrate host and further parasite development in the mosquito. This article, which synthetically reviews such signalling molecules and cellular processes, aims to provide a glimpse of the global frame in which the activities of the MALSIG consortium will develop over the next three years.

  • 28. Doerig, Christian
    et al.
    Billker, Oliver
    A parasite calcium switch and Achilles' heel revealed2010Ingår i: Nature Structural & Molecular Biology, ISSN 1545-9993, E-ISSN 1545-9985, Vol. 17, nr 5, s. 541-543Artikel i tidskrift (Refereegranskat)
  • 29. Doerig, Christian
    et al.
    Billker, Oliver
    The Wellcome Trust Sanger Institute, Wellcome Trust Genome Campus, Hinxton, Cambridge, UK.
    Haystead, Timothy
    Sharma, Pushkar
    Tobin, Andrew B
    Waters, Norman C
    Protein kinases of malaria parasites: an update2008Ingår i: Trends in Parasitology, ISSN 1471-4922, E-ISSN 1471-5007, Vol. 24, nr 12, s. 570-577Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    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.

  • 30. Doerig, Christian
    et al.
    Billker, Oliver
    Pratt, David
    Endicott, Jane
    Protein kinases as targets for antimalarial intervention: Kinomics, structure-based design, transmission-blockade, and targeting host cell enzymes2005Ingår i: Biochimica et Biophysica Acta, ISSN 0006-3002, E-ISSN 1878-2434, Vol. 1754, nr 1-2, s. 132-150Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    The surge of interest in protein kinases as targets for chemotherapeutic intervention in a number of diseases such as cancer and neurodegenerative disorders has stimulated research aimed at determining whether enzymes of this class might also be considered as targets in the context of diseases caused by parasitic protists. Here, we present an overview of recent developments in this field, concentrating (i) on the benefits gained from the availability of genomic databases for a number of parasitic protozoa, (ii) on the emerging field of structure-aided design of inhibitors targeting protein kinases of parasitic protists, (iii) on the concept known as transmission-blockade, whereby kinases implicated in the development of the parasite in their arthropod vector might be targeted to interfere with disease transmission, and (iv) on the possibility of controlling parasitic diseases through the inhibition of host cell protein kinases that are required for the establishment of infection by the parasites.

  • 31. Dundas, Kirsten
    et al.
    Shears, Melanie J.
    Sun, Yi
    Hopp, Christine S.
    Crosnier, Cecile
    Metcalf, Tom
    Girling, Gareth
    Sinnis, Photini
    Billker, Oliver
    Malaria Programme, Wellcome Trust Sanger Institute, CB10 1SA Cambridge, United Kingdom.
    Wright, Gavin J.
    Alpha-v-containing integrins are host receptors for the Plasmodium falciparum sporozoite surface protein, TRAP2018Ingår i: Proceedings of the National Academy of Sciences of the United States of America, ISSN 0027-8424, E-ISSN 1091-6490, Vol. 115, nr 17, s. 4477-4482Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    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. Thrombospondin-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 αvβ3 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 Mn(2+) ions, and that cells genetically targeted so that they lack cell surface expression of the integrin αv-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 αvβ3 as the host receptor for TRAP provides an important demonstration of a sporozoite surface ligand that directly interacts with host receptors.

  • 32. Ecker, Andrea
    et al.
    Moon, Robert
    Sinden, Robert E
    Billker, Oliver
    Generation of gene targeting constructs for Plasmodium berghei by a PCR-based method amenable to high throughput applications2006Ingår i: Molecular and biochemical parasitology (Print), ISSN 0166-6851, E-ISSN 1872-9428, Vol. 145, nr 2, s. 265-268Artikel i tidskrift (Refereegranskat)
  • 33. Everitt, Aaron R
    et al.
    Clare, Simon
    McDonald, Jacqueline U
    Kane, Leanne
    Harcourt, Katherine
    Ahras, Malika
    Lall, Amar
    Hale, Christine
    Rodgers, Angela
    Young, Douglas B
    Haque, Ashraful
    Billker, Oliver
    Tregoning, John S
    Dougan, Gordon
    Kellam, Paul
    Defining the range of pathogens susceptible to Ifitm3 restriction using a knockout mouse model2013Ingår i: PLoS ONE, E-ISSN 1932-6203, Vol. 8, nr 11, s. e80723-e80723Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    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.

  • 34. Fang, Hanwei
    et al.
    Gomes, Ana Rita
    Klages, Natacha
    Pino, Paco
    Maco, Bohumil
    Walker, Eloise M.
    Zenonos, Zenon A.
    Angrisano, Fiona
    Baum, Jake
    Doerig, Christian
    Baker, David A.
    Billker, Oliver
    Umeå universitet, Medicinska fakulteten, Molekylär Infektionsmedicin, Sverige (MIMS). Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Institutionen för molekylärbiologi (Teknisk-naturvetenskaplig fakultet).
    Brochet, Mathieu
    Epistasis studies reveal redundancy among calcium-dependent protein kinases in motility and invasion of malaria parasites2018Ingår i: Nature Communications, ISSN 2041-1723, E-ISSN 2041-1723, Vol. 9, artikel-id 4248Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    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.

  • 35. Frénal, Karine
    et al.
    Tay, Chwen L.
    Mueller, Christina
    Bushell, Ellen S.
    Jia, Yonggen
    Graindorge, Arnault
    Billker, Oliver
    Malaria Programme, Wellcome Trust Sanger Institute, Wellcome Trust Genome Campus, Hinxton, Cambridge CB10 1SA, UK.
    Rayner, Julian C.
    Soldati-Favre, Dominique
    Global analysis of apicomplexan protein S-acyl transferases reveals an enzyme essential for invasion2013Ingår i: Traffic: the International Journal of Intracellular Transport, ISSN 1398-9219, E-ISSN 1600-0854, Vol. 14, nr 8, s. 895-911Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    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.

  • 36. Gomes, Ana Rita
    et al.
    Bushell, Ellen
    Schwach, Frank
    Girling, Gareth
    Anar, Burcu
    Quail, Michael A.
    Herd, Colin
    Pfander, Claudia
    Modrzynska, Katarzyna
    Rayner, Julian C.
    Billker, Oliver
    Wellcome Trust Sanger Institute, Hinxton Cambridge CB10 1SA, UK.
    A genome-scale vector resource enables high-throughput reverse genetic screening in a malaria parasite2015Ingår i: Cell Host and Microbe, ISSN 1931-3128, E-ISSN 1934-6069, Vol. 17, nr 3, s. 404-413Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    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.

  • 37. Govindasamy, K
    et al.
    Jebiwott, S
    Jaijyan, D K
    Davidow, A
    Ojo, K K
    Van Voorhis, W C
    Brochet, M
    Billker, Oliver
    Wellcome Trust Sanger Institute, Malaria Programme, Hinxton, UK.
    Bhanot, P
    Invasion of hepatocytes by Plasmodium sporozoites requires cGMP‐dependent protein kinase and calcium dependent protein kinase 42016Ingår i: Molecular Microbiology, ISSN 0950-382X, E-ISSN 1365-2958, Vol. 102, nr 2, s. 349-363Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Invasion of hepatocytes by sporozoites is essential for Plasmodium to initiate infection of the mammalian host. The parasite's subsequent intracellular differentiation in the liver is the first developmental step of its mammalian cycle. Despite their biological significance, surprisingly little is known of the signalling pathways required for sporozoite invasion. We report that sporozoite invasion of hepatocytes requires signalling through two second‐messengers – cGMP mediated by the parasite's cGMP‐dependent protein kinase (PKG), and Ca2+, mediated by the parasite's calcium‐dependent protein kinase 4 (CDPK4). Sporozoites expressing a mutated form of Plasmodium berghei PKG or carrying a deletion of the CDPK4 gene are defective in invasion of hepatocytes. Using specific and potent inhibitors of Plasmodium PKG and CDPK4, we demonstrate that PKG and CDPK4 are required for sporozoite motility, and that PKG regulates the secretion of TRAP, an adhesin that is essential for motility. Chemical inhibition of PKG decreases parasite egress from hepatocytes by inhibiting either the formation or release of merosomes. In contrast, genetic inhibition of CDPK4 does not significantly decrease the number of merosomes. By revealing the requirement for PKG and CDPK4 in Plasmodium sporozoite invasion, our work enables a better understanding of kinase pathways that act in different Plasmodium stages.

  • 38. Hillier, Charles
    et al.
    Pardo, Mercedes
    Yu, Lu
    Bushell, Ellen
    Umeå universitet, Medicinska fakulteten, Institutionen för molekylärbiologi (Medicinska fakulteten). Umeå universitet, Medicinska fakulteten, Molekylär Infektionsmedicin, Sverige (MIMS).
    Sanderson, Theo
    Metcalf, Tom
    Herd, Colin
    Anar, Burcu
    Rayner, Julian C.
    Billker, Oliver
    Umeå universitet, Medicinska fakulteten, Molekylär Infektionsmedicin, Sverige (MIMS). Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Institutionen för molekylärbiologi (Teknisk-naturvetenskaplig fakultet).
    Choudhary, Jyoti S.
    Landscape of the Plasmodium Interactome Reveals Both Conserved and Species-Specific Functionality2019Ingår i: Cell reports, ISSN 2211-1247, E-ISSN 2211-1247, Vol. 28, nr 6, s. 1635-1647Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    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.

  • 39. Hopp, Christine S
    et al.
    Balaban, Amanda E
    Bushell, Ellen S C
    Billker, Oliver
    Wellcome Trust Sanger Institute, Hinxton, UK.
    Rayner, Julian C
    Sinnis, Photini
    Palmitoyl transferases have critical roles in the development of mosquito and liver stages of Plasmodium2016Ingår i: Cellular Microbiology, ISSN 1462-5814, E-ISSN 1462-5822, Vol. 18, nr 11, s. 1625-1641Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    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.

  • 40. Howick, Virginia M.
    et al.
    Russell, Andrew J. C.
    Andrews, Tallulah
    Heaton, Haynes
    Reid, Adam J.
    Natarajan, Kedar
    Butungi, Hellen
    Metcalf, Tom
    Verzier, Lisa H.
    Rayner, Julian C.
    Berriman, Matthew
    Herren, Jeremy K.
    Billker, Oliver
    Umeå universitet, Medicinska fakulteten, Molekylär Infektionsmedicin, Sverige (MIMS). Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Institutionen för molekylärbiologi (Teknisk-naturvetenskaplig fakultet). Wellcome Sanger Institute, Wellcome Genome Campus, Cambridge, UK.
    Hemberg, Martin
    Talman, Arthur M.
    Lawniczak, Mara K. N.
    The Malaria Cell Atlas: Single parasite transcriptomes across the complete Plasmodium life cycle2019Ingår i: Science, ISSN 0036-8075, E-ISSN 1095-9203, Vol. 365, nr 6455, artikel-id eaaw2619Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    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.

  • 41. Invergo, Brandon M.
    et al.
    Brochet, Mathieu
    Yu, Lu
    Choudhary, Jyoti
    Beltrao, Pedro
    Billker, Oliver
    Malaria Programme, Wellcome Trust Sanger Institute, Hinxton, Cambridgeshire CB10 1SA, UK.
    Sub-minute Phosphoregulation of Cell Cycle Systems during Plasmodium Gamete Formation2017Ingår i: Cell reports, ISSN 2211-1247, E-ISSN 2211-1247, Vol. 21, nr 7, s. 2017-2029Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    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.

  • 42. Kent, Robyn S
    et al.
    Modrzynska, Katarzyna K
    Cameron, Rachael
    Philip, Nisha
    Billker, Oliver
    Wellcome Trust Sanger Institute, Hinxton, Cambridge, UK.
    Waters, Andrew P
    Inducible developmental reprogramming redefines commitment to sexual development in the malaria parasite Plasmodium berghei2018Ingår i: Nature Microbiology, E-ISSN 2058-5276, Vol. 3, nr 11, s. 1206-1213Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    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 conditions1. Recently, an apicomplexa-specific transcription factor (AP2-G) was identified as necessary for gametocyte production in multiple Plasmodium species2,3, and suggested to be an epigenetically regulated master switch that initiates gametocytogenesis4,5. 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.

  • 43. Liu, Yanjie
    et al.
    Tewari, Rita
    Ning, Jue
    Blagborough, Andrew M
    Garbom, Sara
    Pei, Jimin
    Grishin, Nick V
    Steele, Robert E
    Sinden, Robert E
    Snell, William J
    Billker, Oliver
    Division of Cell and Molecular Biology, Imperial College London; The Wellcome Trust Sanger Institute, Hinxton, Cambridge, United Kingdom London, United Kingdom; .
    The conserved plant sterility gene HAP2 functions after attachment of fusogenic membranes in Chlamydomonas and Plasmodium gametes2008Ingår i: Genes & Development, ISSN 0890-9369, E-ISSN 1549-5477, Vol. 22, nr 8, s. 1051-1068Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    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.

  • 44. Lönnberg, Tapio
    et al.
    Svensson, Valentine
    James, Kylie R.
    Fernandez-Ruiz, Daniel
    Sebina, Ismail
    Montandon, Ruddy
    Soon, Megan S. F.
    Fogg, Lily G.
    Nair, Arya Sheela
    Liligeto, Urijah
    Stubbington, Michael J. T.
    Ly, Lam-Ha
    Bagger, Frederik Otzen
    Zwiessele, Max
    Lawrence, Neil D.
    Souza-Fonseca-Guimaraes, Fernando
    Bunn, Patrick T.
    Engwerda, Christian R.
    Heath, William R.
    Billker, Oliver
    Wellcome Trust Sanger Institute, Wellcome Genome Campus, Hinxton, Cambridge, U.K..
    Stegle, Oliver
    Haque, Ashraful
    Teichmann, Sarah A.
    Single-cell RNA-seq and computational analysis using temporal mixture modelling resolves Th1/Tfh fate bifurcation in malaria2017Ingår i: Science immunology, ISSN 2470-9468, Vol. 2, nr 9, artikel-id eaal2192Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Differentiation of naïve CD4(+) T cells into functionally distinct T helper subsets is crucial for the orchestration of immune responses. Due to 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 using a temporal mixtures of Gaussian processes model, termed GPfates, we reconstructed the developmental trajectories of Th1 and Tfh cells during blood-stage Plasmodium infection in mice. By tracking clonality using endogenous TCR sequences, we first demonstrated that Th1/Tfh bifurcation had occurred at both population and single-clone levels. Next, we identified genes whose expression was associated with Th1 or Tfh fates, and demonstrated a T-cell intrinsic role for Galectin-1 in supporting a Th1 differentiation. We also revealed the close molecular relationship between Th1 and IL-10-producing Tr1 cells in this infection. Th1 and Tfh fates emerged from a highly proliferative precursor that upregulated aerobic glycolysis and accelerated cell cycling as cytokine expression began. Dynamic gene expression of chemokine receptors around bifurcation predicted roles for cell-cell in driving Th1/Tfh fates. In particular, we found that precursor Th cells were coached towards a Th1 but not a Tfh 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 Th1/Tfh fate commitment, and more generally, GPfates, a modelling framework for characterizing cell differentiation towards multiple fates.

  • 45. Mancio-Silva, Liliana
    et al.
    Slavic, Ksenija
    Grilo Ruivo, Margarida T.
    Grosso, Ana Rita
    Modrzynska, Katarzyna K.
    Vera, Iset Medina
    Sales-Dias, Joana
    Gomes, Ana Rita
    MacPherson, Cameron Ross
    Crozet, Pierre
    Adamo, Mattia
    Baena-Gonzalez, Elena
    Tewari, Rita
    Llinás, Manuel
    Billker, Oliver
    Wellcome Trust Sanger Institute, Hinxton, Cambridge CB10 1SA, UK.
    Mota, Maria M.
    Nutrient sensing modulates malaria parasite virulence2017Ingår i: Nature, ISSN 0028-0836, E-ISSN 1476-4687, Vol. 547, nr 7662, s. 213-216Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    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. Canonical nutrient-sensing pathways are presumed to be absent from the causative agent of malaria, Plasmodium, 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/AMPKα, 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.

  • 46. McInroy, Gordon R
    et al.
    Beraldi, Dario
    Raiber, Eun-Ang
    Modrzynska, Katarzyna
    van Delft, Pieter
    Billker, Oliver
    Wellcome Trust Sanger Institute, Cambridge, Cambridgeshire, United Kingdom.
    Balasubramanian, Shankar
    Enhanced Methylation Analysis by Recovery of Unsequenceable Fragments2016Ingår i: PLoS ONE, E-ISSN 1932-6203, Vol. 11, nr 3, artikel-id e0152322Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    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.

  • 47. McRobert, Louisa
    et al.
    Taylor, Cathy J
    Deng, Wensheng
    Fivelman, Quinton L
    Cummings, Ross M
    Polley, Spencer D
    Billker, Oliver
    Wellcome Trust Sanger Institute, Hinxton, Cambridge, United Kingdom.
    Baker, David A
    Gametogenesis in malaria parasites is mediated by the cGMP-dependent protein kinase2008Ingår i: PLoS biology, ISSN 1544-9173, E-ISSN 1545-7885, Vol. 6, nr 6, s. e139-e139Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    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.

  • 48. Modrzynska, Katarzyna
    et al.
    Pfander, Claudia
    Chappell, Lia
    Yu, Lu
    Suarez, Catherine
    Dundas, Kirsten
    Gomes, Ana Rita
    Goulding, David
    Rayner, Julian C.
    Choudhary, Jyoti
    Billker, Oliver
    Wellcome Trust Sanger Institute, Hinxton, Cambridge CB10 1SA, UK.
    A Knockout Screen of ApiAP2 Genes Reveals Networks of Interacting Transcriptional Regulators Controlling the Plasmodium Life Cycle2017Ingår i: Cell Host and Microbe, ISSN 1931-3128, E-ISSN 1934-6069, Vol. 21, nr 1, s. 11-22Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    A family of apicomplexa-specific proteins containing AP2 DNA-binding domains (ApiAP2s) was identified in malaria parasites. This family includes sequence-specific 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.

  • 49. Moon, Robert W
    et al.
    Taylor, Cathy J
    Bex, Claudia
    Schepers, Rebecca
    Goulding, David
    Janse, Chris J
    Waters, Andrew P
    Baker, David A
    Billker, Oliver
    Wellcome Trust Sanger Institute, Cambridge Hinxton, United Kingdom; Imperial College London, Department of Cell and Molecular Biology, London, United Kingdom.
    A cyclic GMP signalling module that regulates gliding motility in a malaria parasite2009Ingår i: PLoS Pathogens, ISSN 1553-7366, E-ISSN 1553-7374, Vol. 5, nr 9Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    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 (PDEdelta), 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 (GCbeta), showing that in ookinetes GCbeta is an important source for cGMP, and that PDEdelta 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.

  • 50. Moussa, Ehab
    et al.
    Huang, Honglei
    Ahras, Malika
    Lall, Amar
    Thezenas, Marie L.
    Fischer, Roman
    Kessler, Benedikt M.
    Pain, Arnab
    Billker, Oliver
    Wellcome Trust Sanger Institute, Cambridge, UK.
    Casals-Pascual, Climent
    Proteomic profiling of the brain of mice with experimental cerebral malaria2018Ingår i: Journal of Proteomics, ISSN 1874-3919, E-ISSN 1876-7737, Vol. 180, s. 61-69Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    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/Bl6N 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.

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