Malaria programme: Billker group

The Malaria programme uses genomic and genetic approaches to discover molecular mechanisms of host-parasite interactions that may lead to new biological insights and improved strategies for disease prevention.

The aims of the Billker group in the Sanger Malaria programme are to elucidate the fundamental molecular and cell biology of malaria parasites with a view to understanding their development and transmission.

The team studies the malaria species that infects rodents, Plasmodium berghei, which is harmless to humans. This species is easy to maintain throughout its life cycle in the laboratory. Genome sequences are available for parasite, host and vector and all three organisms can be manipulated genetically. Rodent malaria parasites therefore offer powerful experimental models to study fundamental aspects of malaria biology, pathology, immunology and antimalarial drug action. The team is investigating how the genome sequence functions in malaria parasites and in parasite-host interactions, with a view to aiding the development of more effective antimalarial drugs.

More information on the Malaria Programme.

[Mathieu Brochet, Genome Research Limited]

Background

The development of new drugs and vaccines against malaria poses one of the major challenges to current medical research. It must be grounded in the thorough understanding of the parasite's biology, including its interactions with the host, and the Anopheles mosquitoes that transmit it. P. berghei provides a highly tractable model to study the fundamental cell biology of malaria parasites and many aspects of their interactions with host and vector.

We are particularly interested to learn how the cellular processes involved in sexual development and mosquito transmission are regulated through signal transduction pathways that control stage conversion, gene transcription and protein translation. We discovered that parasite development in the mosquito is triggered by xanthurenic acid (Billker et al., 1998), a tryptophan metabolite, which in insects is present at high concentrations. It regulates parasite development through a plant like protein kinase, which we identified by experimentally disrupting the parasite gene encoding it (Billker et al., 2004). Working with our collaborators we recently demonstrated that a drug-like chemical inhibitor of the kinase can block malaria transmission to mosquitoes when administered to a mouse (Ojo et al., 2012).

The targeted modification of the genome is far more difficult in malaria parasites than in model organisms, such as yeast. To increase the rate at which discoveries can be made in P. berghei, we have developed protocols to produce more efficient genetic modification vectors and other molecular tools that allow researchers to rapidly switch parasite genes on and off. We now operate a production pipeline to produce gene knock out and tagging vectors for every gene in the P. berghei genome. These are freely available to all researchers and can be viewed through the PlasmoGEM database. Our vision is that over the coming years, protocols will be developed to use PlasmoGEM vectors in genetic screens that query the functions of hundreds or thousands of parasite genes at the same time. PlasmoGEM is a joint project with the Rayner group. It relies heavily on the reference genomes produced by Matt Berriman's Parasite Genomics team.

Research

Our aims

Tools and resources for P. berghei genetic modification.

A small team, now led by Ellen Bushell, runs the PlasmoGEM project. We have generated arrayed libraries of P. berghei genomic DNA with average inserts of ca. 8.5 kb. These are large inserts, considering that P. berghei DNA is very AT-rich, making it highly unstable in E. coli. We also developed a set of tools and protocols to engineer genomic DNA in E. coli with the help of transiently expressed phage recombinases, a process termed recombineering. These protocols are robust enough to work on 96-well plates. They allow us to turn library clones into complex genetic modification vectors at scale. Using this technology we are now producing genome-wide sets of genetic modification vectors and make these available as a free resource.

PlasmoGEM vectors have advantages over conventional designs: due to their long homology arms they integrate very efficiently. Furthermore, since the vectors never exist in a circular form, targeting essential genes does not select for episomes, thereby eliminating a major source of false positive results. We are exploring the new opportunities offered by these advantages. We have also developed tools for the insertion of site-specific recombination sites for Flp recombinase, and with the Soldati and Llinas labs we produced a tetracycline repressible transactivation system to knock down essential genes in P. berghei blood stages.

We are keen to transfer the new genetic technologies to the human malaria parasite, P. falciparum. However, this is not straight forward, since our recombineering pipelines relies on keeping DNA linear for increased stability in E. coli. In contrast to P. berghei, transfection of P. falciparum relies on circular DNA.

Signal Transduction Pathways Regulating Sexual Development

Sexual development in Plasmodium is tightly linked to transmission by mosquitoes. In P. berghei sexual stages can be cultured in vitro and are relatively tractable for genetic and biochemical studies, allowing us, for instance, to identify Hap2 as a gamete membrane protein which probably has a conserved function in gamete fusion diverse eukaryotes. We also investigate the roles of protein kinases and second messengers in regulating sexual differentiation. A recent study on a plant like calcium dependent kinase, CDPK1 (Sebastian et al., 2012) illustrates how we have begun to use molecular phenotyping of to understand gene function. Various project now generate a deep understanding of some of our mutants through comparative analysis of transcriptomes, proteomes and secondary protein modifications, such as phosphorylation.

Host Genes Regulating Parasite Development

There is an urgent need for new experimental models that can identify the role of host genes in parasite-host interactions. As genome-wide association studies discover more of the relevant natural genetic variation in human populations that controls susceptibility to malaria, new tools to identify the underlying molecular mechanisms will be required. Some projects in the lab explore the use of knock out mice, in vitro differentiated mouse embryonic stem cells and human iPS cells to get at this problem.

Resources

The PlasmoGEM website hosts a database of P. berghei large insert genomic clones, genetic modification vectors and recombineering protocols.

To learn more about Malaria Experimental Genetics, consider applying for the Wellcome Trust Advanced Course we run with Julian Rayner's team each year.

Selected Publications

  • Recombination-mediated genetic engineering of Plasmodium berghei DNA.

    Pfander C, Anar B, Brochet M, Rayner JC and Billker O

    Methods in molecular biology (Clifton, N.J.) 2013;923;127-38

  • A Plasmodium calcium-dependent protein kinase controls zygote development and transmission by translationally activating repressed mRNAs.

    Sebastian S, Brochet M, Collins MO, Schwach F, Jones ML, Goulding D, Rayner JC, Choudhary JS and Billker O

    Cell host & microbe 2012;12;1;9-19

  • Transmission of malaria to mosquitoes blocked by bumped kinase inhibitors.

    Ojo KK, Pfander C, Mueller NR, Burstroem C, Larson ET, Bryan CM, Fox AM, Reid MC, Johnson SM, Murphy RC, Kennedy M, Mann H, Leibly DJ, Hewitt SN, Verlinde CL, Kappe S, Merritt EA, Maly DJ, Billker O and Van Voorhis WC

    The Journal of clinical investigation 2012;122;6;2301-5

  • A scalable pipeline for highly effective genetic modification of a malaria parasite.

    Pfander C, Anar B, Schwach F, Otto TD, Brochet M, Volkmann K, Quail MA, Pain A, Rosen B, Skarnes W, Rayner JC and Billker O

    Nature methods 2011;8;12;1078-82

  • Cutting edge: the membrane attack complex of complement is required for the development of murine experimental cerebral malaria.

    Ramos TN, Darley MM, Hu X, Billker O, Rayner JC, Ahras M, Wohler JE and Barnum SR

    Journal of immunology (Baltimore, Md. : 1950) 2011;186;12;6657-60

  • The systematic functional analysis of Plasmodium protein kinases identifies essential regulators of mosquito transmission.

    Tewari R, Straschil U, Bateman A, Böhme U, Cherevach I, Gong P, Pain A and Billker O

    Cell host & microbe 2010;8;4;377-87

  • A cyclic GMP signalling module that regulates gliding motility in a malaria parasite.

    Moon RW, Taylor CJ, Bex C, Schepers R, Goulding D, Janse CJ, Waters AP, Baker DA and Billker O

    PLoS pathogens 2009;5;9;e1000599

  • Calcium-dependent signaling and kinases in apicomplexan parasites.

    Billker O, Lourido S and Sibley LD

    Cell host & microbe 2009;5;6;612-22

  • The conserved plant sterility gene HAP2 functions after attachment of fusogenic membranes in Chlamydomonas and Plasmodium gametes.

    Liu Y, Tewari R, Ning J, Blagborough AM, Garbom S, Pei J, Grishin NV, Steele RE, Sinden RE, Snell WJ and Billker O

    Genes & development 2008;22;8;1051-68

  • Calcium and a calcium-dependent protein kinase regulate gamete formation and mosquito transmission in a malaria parasite.

    Billker O, Dechamps S, Tewari R, Wenig G, Franke-Fayard B and Brinkmann V

    Cell 2004;117;4;503-14

  • Identification of xanthurenic acid as the putative inducer of malaria development in the mosquito.

    Billker O, Lindo V, Panico M, Etienne AE, Paxton T, Dell A, Rogers M, Sinden RE and Morris HR

    Nature 1998;392;6673;289-92

Team

Team members

Ana Rita Batista Gomes
PhD student
Mathieu Brochet
INSERM investigator
Burcu Bronner Anar
Advanced Research Assistant
Ellen Bushell
eb5@sanger.ac.ukSenior Staff Scientist
Gareth Girling
Advanced Research Assistant
Colin Herd
Advanced Research Assistant
Stefano Iantorno
PhD Student
Brandon Invergo
ESPOD Fellow
Thomas Metcalf
Insectary Manager
Kasia Modrzynska
km8@sanger.ac.ukStaff Scientist
Ruddy Montandon
Postdoctoral Fellow
Frank Schwach
fs5@sanger.ac.ukSenior Computational Biologist
Jaishree Tripathi
PhD Student

Ana Rita Batista Gomes

- PhD student

I am a microbiologist with expertise in Plasmodium berghei genetics. I joined Malaria research during my Master’s degree at University of Lisbon. My main aim was to study the control of gene expression by translation repression of ApiAP2 transcription factors, in Plasmodium berghei. In 2010 I joined the Evimalar PhD programme, under the supervision of Dr. Oliver Billker and Dr. Christian Doerig, at Sanger Institute.

Research

During my PhD I combined a signature tagged mutagenesis approach with a barcode sequencing strategy to develop a high-throughput reverse genetic screening method for P. berghei. This method enabled me to measure the fitness of individual barcoded mutants in pools containing dozens of different mutants, and how it changes during infection, in a single mouse.

References

  • Phosphoinositide metabolism links cGMP-dependent protein kinase G to essential Ca²⁺ signals at key decision points in the life cycle of malaria parasites.

    Brochet M, Collins MO, Smith TK, Thompson E, Sebastian S, Volkmann K, Schwach F, Chappell L, Gomes AR, Berriman M, Rayner JC, Baker DA, Choudhary J and Billker O

    Wellcome Trust Sanger Institute, Hinxton, Cambridge, United Kingdom.

    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.

    Funded by: Medical Research Council: G0501670, G10000779; Wellcome Trust: 079643/Z/06/Z, WT093228, WT094752, WT098051

    PLoS biology 2014;12;3;e1001806

  • The malarial serine protease SUB1 plays an essential role in parasite liver stage development.

    Suarez C, Volkmann K, Gomes AR, Billker O and Blackman MJ

    Division of Parasitology, Medical Research Council National Institute for Medical Research, Mill Hill, London, United Kingdom.

    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.

    Funded by: Medical Research Council: U117532063; Wellcome Trust: 098051

    PLoS pathogens 2013;9;12;e1003811

  • Transition of Plasmodium sporozoites into liver stage-like forms is regulated by the RNA binding protein Pumilio.

    Gomes-Santos CS, Braks J, Prudêncio M, Carret C, Gomes AR, Pain A, Feltwell T, Khan S, Waters A, Janse C, Mair GR and Mota MM

    Malaria Unit, Instituto de Medicina Molecular, Lisboa, Portugal.

    Many eukaryotic developmental and cell fate decisions that are effected post-transcriptionally involve RNA binding proteins as regulators of translation of key mRNAs. In malaria parasites (Plasmodium spp.), the development of round, non-motile and replicating exo-erythrocytic liver stage forms from slender, motile and cell-cycle arrested sporozoites is believed to depend on environmental changes experienced during the transmission of the parasite from the mosquito vector to the vertebrate host. Here we identify a Plasmodium member of the RNA binding protein family PUF as a key regulator of this transformation. In the absence of Pumilio-2 (Puf2) sporozoites initiate EEF development inside mosquito salivary glands independently of the normal transmission-associated environmental cues. Puf2- sporozoites exhibit genome-wide transcriptional changes that result in loss of gliding motility, cell traversal ability and reduction in infectivity, and, moreover, trigger metamorphosis typical of early Plasmodium intra-hepatic development. These data demonstrate that Puf2 is a key player in regulating sporozoite developmental control, and imply that transformation of salivary gland-resident sporozoites into liver stage-like parasites is regulated by a post-transcriptional mechanism.

    Funded by: Wellcome Trust: 083811

    PLoS pathogens 2011;7;5;e1002046

Mathieu Brochet

- INSERM investigator

I studied Agronomy at the National Higher Agronomic School (France) and at McGill University (Canada). After completion of my undergraduate training I joined the Genomics of Microbial Pathogens unit at the Pasteur Institute (France) and took up a project centring on the evolution of Group B Streptococcus, the leading cause of neonatal infections. After completing my PhD work, I undertook a postdoctoral position with Oliver Billker at the Sanger Institute. I am now an INSERM senior investigator based both at the Wellcome Trust Sanger Institute and at the CNRS unit “Dynamics of Membrane Interactions in Normal and Pathological Cells” (France).

Research

My current work aims at identifying the molecular links between essential signalling pathways which allows Plasmodium parasites to successfully progress through their life cycle. Specifically, I am interested in identifying and characterising calcium signalling pathways. This involves the use of Plasmodium specific genetics, chemical biology and quantitative omics approaches.

References

  • Phosphoinositide metabolism links cGMP-dependent protein kinase G to essential Ca²⁺ signals at key decision points in the life cycle of malaria parasites.

    Brochet M, Collins MO, Smith TK, Thompson E, Sebastian S, Volkmann K, Schwach F, Chappell L, Gomes AR, Berriman M, Rayner JC, Baker DA, Choudhary J and Billker O

    Wellcome Trust Sanger Institute, Hinxton, Cambridge, United Kingdom.

    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.

    Funded by: Medical Research Council: G0501670, G10000779; Wellcome Trust: 079643/Z/06/Z, WT093228, WT094752, WT098051

    PLoS biology 2014;12;3;e1001806

  • A tetracycline-repressible transactivator system to study essential genes in malaria parasites.

    Pino P, Sebastian S, Kim EA, Bush E, Brochet M, Volkmann K, Kozlowski E, Llinás M, Billker O and Soldati-Favre D

    Department of Microbiology and Molecular Medicine, CMU, University of Geneva, 1 Rue Michel-Servet, 1211 Geneva 4, Switzerland.

    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.

    Funded by: Howard Hughes Medical Institute; Medical Research Council: G0501670; NIAID NIH HHS: R01 AI076276; NIGMS NIH HHS: P50 GM071508, P50GM071508; Wellcome Trust: WT077502MA, WT098051

    Cell host & microbe 2012;12;6;824-34

  • A Plasmodium calcium-dependent protein kinase controls zygote development and transmission by translationally activating repressed mRNAs.

    Sebastian S, Brochet M, Collins MO, Schwach F, Jones ML, Goulding D, Rayner JC, Choudhary JS and Billker O

    Wellcome Trust Sanger Institute, Hinxton, Cambridge, UK.

    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.

    Funded by: Medical Research Council: G0501670; Wellcome Trust: 079643/Z/06/Z, WT098051

    Cell host & microbe 2012;12;1;9-19

  • The alveolin IMC1h is required for normal ookinete and sporozoite motility behaviour and host colonisation in Plasmodium berghei.

    Volkmann K, Pfander C, Burstroem C, Ahras M, Goulding D, Rayner JC, Frischknecht F, Billker O and Brochet M

    The Wellcome Trust Sanger Institute, Hinxton, United Kingdom.

    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.

    Funded by: Medical Research Council: G0501670; Wellcome Trust: 89085/Z/09/Z

    PloS one 2012;7;7;e41409

  • A scalable pipeline for highly effective genetic modification of a malaria parasite.

    Pfander C, Anar B, Schwach F, Otto TD, Brochet M, Volkmann K, Quail MA, Pain A, Rosen B, Skarnes W, Rayner JC and Billker O

    The Wellcome Trust Sanger Institute, Hinxton, Cambridge, UK.

    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.

    Funded by: Medical Research Council: G0501670, G0501670(76331); Wellcome Trust: 089085, WT089085/Z/09/Z

    Nature methods 2011;8;12;1078-82

  • Shaping a bacterial genome by large chromosomal replacements, the evolutionary history of Streptococcus agalactiae.

    Brochet M, Rusniok C, Couvé E, Dramsi S, Poyart C, Trieu-Cuot P, Kunst F and Glaser P

    Institut Pasteur, Unité de Génétique des Génomes Bactériens, Centre National de la Recherche Scientifique URA 2171.

    Bacterial populations are subject to complex processes of diversification that involve mutation and horizontal DNA transfer mediated by transformation, transduction, or conjugation. Tracing the evolutionary events leading to genetic changes allows us to infer the history of a microbe. Here, we combine experimental and in silico approaches to explore the forces that drive the genome dynamics of Streptococcus agalactiae, the leading cause of neonatal infections. We demonstrate that large DNA segments of up to 334 kb of the chromosome of S. agalactiae can be transferred through conjugation from multiple initiation sites. Consistently, a genome-wide map analysis of nucleotide polymorphisms among eight human isolates demonstrated that each chromosome is a mosaic of large chromosomal fragments from different ancestors suggesting that large DNA exchanges have contributed to the genome dynamics in the natural population. The analysis of the resulting genetic flux led us to propose a model for the evolutionary history of this species in which clonal complexes of clinical importance derived from a single clone that evolved by exchanging large chromosomal regions with more distantly related strains. The emergence of this clone could be linked to selective sweeps associated with the reduction of genetic diversity in three regions within a large panel of human isolates. Up to now sex in bacteria has been assumed to involve mainly small regions; our results define S. agalactiae as an alternative paradigm in the study of bacterial evolution.

    Proceedings of the National Academy of Sciences of the United States of America 2008;105;41;15961-6

  • Integrative conjugative elements and related elements are major contributors to the genome diversity of Streptococcus agalactiae.

    Brochet M, Couvé E, Glaser P, Guédon G and Payot S

    Unité de Génétique des Génomes Bactériens-URA CNRS2171, Institut Pasteur, 25-28 rue du Docteur Roux, 75724 Paris cedex 15, France.

    Thirty-five putative integrative conjugative elements and related elements were identified at 15 locations in the eight sequenced genomes of Streptococcus agalactiae. Twelve are composite, likely resulting from site-specific accretions. Circular forms were detected for five elements. Macroarray analysis confirmed their high plasticity and wide distribution in S. agalactiae.

    Journal of bacteriology 2008;190;20;6913-7

  • A naturally occurring gene amplification leading to sulfonamide and trimethoprim resistance in Streptococcus agalactiae.

    Brochet M, Couvé E, Zouine M, Poyart C and Glaser P

    Unité de Génomique des Microorganismes Pathogènes, Institut Pasteur, 28 Rue du Dr. Roux, 75724 Paris cedex 15, France.

    Gene amplifications have been detected as a transitory phenomenon in bacterial cultures. They are predicted to contribute to rapid adaptation by simultaneously increasing the expression of genes clustered on the chromosome. However, genome amplifications have rarely been described in natural isolates. Through DNA array analysis, we have identified two Streptococcus agalactiae strains carrying tandem genome amplifications: a fourfold amplification of 13.5 kb and a duplication of 92 kb. Both amplifications were located close to the terminus of replication and originated independently from any long repeated sequence. They probably arose in the human host and showed different stabilities, the 13.5-kb amplification being lost at a frequency of 0.003 per generation and the 92-kb tandem duplication at a frequency of 0.035 per generation. The 13.5-kb tandem amplification carried the five genes required for dihydrofolate biosynthesis and led to both trimethoprim (TMP) and sulfonamide (SU) resistance. Resistance to SU probably resulted from the increased synthesis of dihydropteroate synthase, the target of this antibiotic, whereas the amplification of the whole pathway was responsible for TMP resistance. This revealed a new mechanism of resistance to TMP involving an increased dihydrofolate biosynthesis. This is, to our knowledge, the first reported case of naturally occurring antibiotic resistance resulting from genome amplification in bacteria. The low stability of DNA segment amplifications suggests that their role in antibiotic resistance might have been underestimated.

    Journal of bacteriology 2008;190;2;672-80

  • Genomic diversity and evolution within the species Streptococcus agalactiae.

    Brochet M, Couvé E, Zouine M, Vallaeys T, Rusniok C, Lamy MC, Buchrieser C, Trieu-Cuot P, Kunst F, Poyart C and Glaser P

    Unité de Génomique des Microorganismes Pathogènes-URA CNRS 2171, Institut Pasteur, 25-28 rue du Docteur Roux, 75724 Paris Cedex 15, France.

    Streptococcus agalactiae is a leading cause of invasive infections in neonates, and responsible for bovine mastitis. It is also a commensal bacterium adapted to asymptomatic colonization of the mammalian gut and of the genitourinary tract. Here, we report the analysis of a collection of 75 strains of human and animal origin by using serotyping, multilocus sequence typing, whole genome DNA-array hybridizations and sequence comparison of putatively virulence-associated loci. Although the most variable parts of the genome are the previously predicted genomic islands, significant genetic variations were present in the genome backbone. Evolution within genes encoding surface and secreted proteins and those involved in the biosynthesis of different capsular types is mainly due to recombination events leading to the replacement of a locus of several genes or to the allelic exchange of the internal part of a gene. These two processes, which led to a broad diversity of surface protein patterns, are probably involved in the diversity of interactions with the host and its immune system. According to gene content comparisons and phylogeny, recent gene replacements by horizontal gene transfer may occur but are rare events. Although specific gene patterns, with respect to the origin of the strains and the epidemiological characteristics, were not identified, we show that the recently described hypervirulent ST-17 lineage is a homogeneous group. The study highlights for the first time that this lineage contains a specific and conserved set of surface proteins, probably accounting for its high capacity to cause infections in newborns.

    Microbes and infection / Institut Pasteur 2006;8;5;1227-43

Burcu Bronner Anar

- Advanced Research Assistant

I completed my B.Sc. and M.Sc. in the Biology department at METU (Middle East Technical University) in Ankara, Turkey between 1990 and 1998. I then worked as a senior research assistant at the Erasmus University in Rotterdam and at the VU (Vrije Universiteit) in Amsterdam (1999-2007), on the characterization of molecular mechanisms involved in the formation of insoluble aggregates in neurodegenerative diseases.

Research

I joined the Sanger Malaria programme in 2008. I have been involved since the beginning in the adaptation and optimisation of a recombineering based method for the generation of gene targeting vectors for the rodent malaria parasite Plasmodium berghei. This technology now forms the basis of the gene targeting vector pipeline that underpins the Plasmodium genetic modification (PlasmoGEM) project (http://plasmogem.sanger.ac.uk/). Within the PlasmoGEM team I continue to work on technology development, vector production and Plasmodium genomic library generation.Recently, I have been involved in the construction of artificial chromosome for P.berghei and P.falciparum.

References

  • 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.

    Bauza K, Malinauskas T, Pfander C, Anar B, Jones EY, Billker O, Hill AV and Reyes-Sandoval A

    The Jenner Institute, University of Oxford, Oxford, United Kingdom.

    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.

    Funded by: Medical Research Council; Wellcome Trust: 090532, 095540, 097395, WT098051

    Infection and immunity 2014;82;3;1277-86

  • Recombination-mediated genetic engineering of Plasmodium berghei DNA.

    Pfander C, Anar B, Brochet M, Rayner JC and Billker O

    Wellcome Trust Sanger Institute, Hinxton, Cambridge, UK.

    DNA of Plasmodium berghei is difficult to manipulate in Escherichia coli by conventional restriction and ligation methods due to its high content of adenine and thymine (AT) nucleotides. This limits our ability to clone large genes and to generate complex vectors for modifying the parasite genome. We here describe a protocol for using lambda Red recombinase to modify inserts of a P. berghei genomic DNA library constructed in a linear, low-copy, phage-derived vector. The method uses primer extensions of 50 bp, which provide sufficient homology for an antibiotic resistance marker to recombine efficiently with a P. berghei genomic DNA insert in E. coli. In a subsequent in vitro Gateway reaction the bacterial marker is replaced with a cassette for selection in P. berghei. The insert is then released and used for transfection. The basic techniques we describe here can be adapted to generate highly efficient vectors for gene deletion, tagging, targeted mutagenesis, or genetic complementation with larger genomic regions.

    Funded by: Medical Research Council: G0501670

    Methods in molecular biology (Clifton, N.J.) 2013;923;127-38

  • A scalable pipeline for highly effective genetic modification of a malaria parasite.

    Pfander C, Anar B, Schwach F, Otto TD, Brochet M, Volkmann K, Quail MA, Pain A, Rosen B, Skarnes W, Rayner JC and Billker O

    The Wellcome Trust Sanger Institute, Hinxton, Cambridge, UK.

    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.

    Funded by: Medical Research Council: G0501670, G0501670(76331); Wellcome Trust: 089085, WT089085/Z/09/Z

    Nature methods 2011;8;12;1078-82

Ellen Bushell

eb5@sanger.ac.uk Senior Staff Scientist

I graduated from Cardiff University in 2004, with a BSc Hons in Applied Biology and a year in industry at AstraZeneca. After my first degree I entered the Wellcome Trust 4-year PhD Infection programme at Imperial College, London. Upon completing my MRes year I joined the Kafatos / Christophides lab to pursue a PhD working on the transmission stages of Plasmodium berghei. In 2009, remaining at Imperial College, I took up a position as a postdoctoral fellow funded by the TransMalBloc consortium. My postdoc focused on monitoring transcriptional regulation of Plasmodium falciparum transmission in the field, in Burkina Faso, West Africa.

Research

In 2011 I joined the Sanger Malaria Programme as a staff scientist. My primary research interest is the development of tools for high throughput reverse genetic analysis of the malaria parasite. I function as the project manager for a new exciting initiative; the Plasmodium genetic modification (PlasmoGEM) project (http://plasmogem.sanger.ac.uk/), and in this role I am part of the team that is working towards producing genome-wide, open access Plasmodium vector resources.

References

  • Genetic susceptibility to the delayed sequelae of neonatal respiratory syncytial virus infection is MHC dependent.

    Tregoning JS, Yamaguchi Y, Wang B, Mihm D, Harker JA, Bushell ES, Zheng M, Liao G, Peltz G and Openshaw PJ

    Department of Respiratory Medicine, Centre for Respiratory Infection, National Heart and Lung Institute, Imperial College London, London, United Kingdom.

    Respiratory syncytial virus (RSV) is a major cause of respiratory morbidity, resulting in hospitalization for bronchiolitis in some infected infants that is associated with wheeze in later life. Genetic factors are known to affect the severity of the sequelae after RSV infection, but the complexity of the temporal and genetic effects makes it difficult to analyze this response in studies in man. Therefore, we developed a murine genetic model to analyze the sequelae occurring after RSV infection in early life. Haplotype-based genetic analysis of interstrain differences in severity identified the MHC as an important genetic determinant. This was confirmed by analysis of responses in congenic mice with different MHC haplotypes. We also found that susceptible strains had high CD8 levels during secondary infection. Analysis of first filial generation, second filial generation, and back-cross progeny produced by intercrossing resistant (H-2(k), C3H/HeN) and sensitive (H-2(b), BALB/c) strains indicated that susceptibility to sequelae after RSV infection was dominantly inherited but also segregated in a non-MHC-dependent manner. Thus, MHC haplotype and its effect on CD8 cell response is an important determinant of the outcome of neonatal RSV infection.

    Funded by: Medical Research Council; NIGMS NIH HHS: 1 R01 GM068885-01A1; Wellcome Trust: 071381/Z/03/Z

    Journal of immunology (Baltimore, Md. : 1950) 2010;185;9;5384-91

  • Paternal effect of the nuclear formin-like protein MISFIT on Plasmodium development in the mosquito vector.

    Bushell ES, Ecker A, Schlegelmilch T, Goulding D, Dougan G, Sinden RE, Christophides GK, Kafatos FC and Vlachou D

    Department of Life Sciences, Imperial College London, London, UK.

    Malaria parasites must undergo sexual and sporogonic development in mosquitoes before they can infect their vertebrate hosts. We report the discovery and characterization of MISFIT, the first protein with paternal effect on the development of the rodent malaria parasite Plasmodium berghei in Anopheles mosquitoes. MISFIT is expressed in male gametocytes and localizes to the nuclei of male gametocytes, zygotes and ookinetes. Gene disruption results in mutant ookinetes with reduced genome content, microneme defects and altered transcriptional profiles of putative cell cycle regulators, which yet successfully invade the mosquito midgut. However, developmental arrest ensues during the ookinete transformation to oocysts leading to malaria transmission blockade. Genetic crosses between misfit mutant parasites and parasites that are either male or female gamete deficient reveal a strict requirement for a male misfit allele. MISFIT belongs to the family of formin-like proteins, which are known regulators of the dynamic remodeling of actin and microtubule networks. Our data identify the ookinete-to-oocyst transition as a critical cell cycle checkpoint in Plasmodium development and lead us to hypothesize that MISFIT may be a regulator of cell cycle progression. This study offers a new perspective for understanding the male contribution to malaria parasite development in the mosquito vector.

    Funded by: Wellcome Trust: 075176/Z/04/Z, GR077229/Z/05/Z

    PLoS pathogens 2009;5;8;e1000539

  • Reverse genetics screen identifies six proteins important for malaria development in the mosquito.

    Ecker A, Bushell ES, Tewari R and Sinden RE

    Division of Cell and Molecular Biology, Imperial College London, London SW7 2AZ, UK. andrea.ecker03@imperial.ac.uk

    Transmission from the vertebrate host to the mosquito vector represents a major population bottleneck in the malaria life cycle that can successfully be targeted by intervention strategies. However, to date only about 25 parasite proteins expressed during this critical phase have been functionally analysed by gene disruption. We describe the first systematic, larger scale generation and phenotypic analysis of Plasmodium berghei knockout (KO) lines, characterizing 20 genes encoding putatively secreted proteins expressed by the ookinete, the parasite stage responsible for invasion of the mosquito midgut. Of 12 KO lines that were generated, six showed significant reductions in parasite numbers during development in the mosquito, resulting in a block in transmission of five KOs. While expression data, time point of essential function and mutant phenotype correlate well in three KOs defective in midgut invasion, in three KOs that fail at sporulation, maternal inheritance of the mutant phenotype suggests that essential function occurs during ookinete formation and thus precedes morphological abnormalities by several days.

    Funded by: Wellcome Trust

    Molecular microbiology 2008;70;1;209-20

  • The developmental migration of Plasmodium in mosquitoes.

    Vlachou D, Schlegelmilch T, Runn E, Mendes A and Kafatos FC

    Division of Cell and Molecular Biology, Faculty of Natural Sciences, Imperial College London, London, SW7 2AZ, UK. d.vlachou@imperial.ac.uk

    Migration of the protozoan parasite Plasmodium through the mosquito is a complex and delicate process, the outcome of which determines the success of malaria transmission. The mosquito is not simply the vector of Plasmodium but, in terms of the life cycle, its definitive host: there, the parasite undergoes its sexual development, which results in colonization of the mosquito salivary glands. Two of the parasite's developmental stages in the mosquito, the ookinete and the sporozoite, are invasive and depend on gliding motility to access, penetrate and traverse their host cells. Recent advances in the field have included the identification of numerous Plasmodium molecules that are essential for parasite migration in the mosquito vector.

    Funded by: NIAID NIH HHS: AI044200-08

    Current opinion in genetics & development 2006;16;4;384-91

  • Tetracycline-inducible gene regulation in mycobacteria.

    Blokpoel MC, Murphy HN, O'Toole R, Wiles S, Runn ES, Stewart GR, Young DB and Robertson BD

    Department of Infectious Diseases and Microbiology, Centre for Molecular Microbiology and Infection, Imperial College London South Kensington campus, London SW7 2AZ, UK.

    A system for the tetracycline-inducible regulation of gene expression in mycobacteria has been developed. We have sub-cloned the tetRO region from the Corynebacterium glutamicum TetZ locus into a mycobacterial shuttle plasmid, making expression of genes cloned downstream of tetRO responsive to tetracycline. Using the luxAB-encoded luciferase from Vibrio harveyi as a reporter (pMind-Lx), we observed a 40-fold increase in light output from Mycobacterium smegmatis cultures 2 h after adding 20 ng ml(-1) of tetracycline. Similarly, exposure to the drug resulted in up to 20-fold increase in relative light units from M.bovis BCG carrying the reporter construct, and a 10-fold increase for M.tuberculosis. Tetracycline induction was demonstrated in log and stationary phase cultures. To evaluate whether this system is amenable to use in vivo, J774 macrophages were infected with M.bovis BCG[pMind-Lx], treated with amikacin to kill extracellular bacteria, and then incubated with tetracycline. A 10-fold increase in light output was measured after 24 h, indicating that intracellular bacteria are accessible and responsive to exogenously added tetracycline. To test the use of the tetracycline-inducible system for conditional gene silencing, mycobacteria were transformed with a pMind construct with tetRO driving expression of antisense RNA for the ftsZ gene. Bacterial cells containing the antisense construct formed filaments after 24 h exposure to tetracycline. These results demonstrate the potential of this tetracycline-regulated system for the manipulation of mycobacterial gene expression inside and outside cells.

    Funded by: Medical Research Council: MC_U117581288

    Nucleic acids research 2005;33;2;e22

Gareth Girling

- Advanced Research Assistant

Gareth is a molecular biologist with expertise in high throughput molecular biology and drug discovery. He holds a BSc in Cell and Molecular Biology from the University of Essex. Gareth previously worked in Screening and Compound Profiling at GlaxoSmithKline, where he generated structure-activity relationship data for small molecule drug candidates against kinase targets using high throughput screening techniques.

Research

Gareth joined the Sanger Malaria Programme in 2011 and works on the development and running of the Plasmodium gene targeting vector pipeline that forms the basis of the Plasmodium genetic modification (PlasmoGEM) project (http://plasmogem.sanger.ac.uk/). PlasmoGEM is a not-for-profit, open access malaria research resource, funded by the Wellcome Trust and hosted by the Wellcome Trust Sanger Institute. PlasmoGEM is initially focused on providing tools for the genetic manipulation of Plasmodium berghei. The aim of PlasmoGEM is to provide the malaria research community with a freely accessible genome-wide genetic modification resource for academic research.

Colin Herd

- Advanced Research Assistant

I completed my Degree at the University-of-Nottingham and then started working at the Institute of Neurology UCL in 1999, where I worked on ion-channelopathies. In 2003, I moved to Hinxton where I worked as finisher on the Fugu Genome at the MRC-HGMP. From there I moved internally to MRCgeneservice, running the sequencing service till 2005. I then started working for the Wellcome Trust in the Audiovestibular Genetics Group, looking at Age Related Hearing Loss. This led to a move in 2008, where I joined the Vertebrate Developmental Genetics group, working on Zebarafish to create a genome wide knockout resource.

Research

I joined the Sanger Malaria programme in 2013. I have since been primarily involved in expanding the modularity of recombineering process by making a number of new targeting tools. This has included making a range of fluorescent tags, for more options when tagging target genes, FACS sortable Knockout vectors, overexpression vectors and a range of conditional systems for both Knockout and overexpression based approaches. I also help with the vector production pipeline.

References

  • A systematic genome-wide analysis of zebrafish protein-coding gene function.

    Kettleborough RN, Busch-Nentwich EM, Harvey SA, Dooley CM, de Bruijn E, van Eeden F, Sealy I, White RJ, Herd C, Nijman IJ, Fényes F, Mehroke S, Scahill C, Gibbons R, Wali N, Carruthers S, Hall A, Yen J, Cuppen E and Stemple DL

    Wellcome Trust Sanger Institute, Wellcome Trust Genome Campus, Hinxton, Cambridge CB10 1SA, UK.

    Since the publication of the human reference genome, the identities of specific genes associated with human diseases are being discovered at a rapid rate. A central problem is that the biological activity of these genes is often unclear. Detailed investigations in model vertebrate organisms, typically mice, have been essential for understanding the activities of many orthologues of these disease-associated genes. Although gene-targeting approaches and phenotype analysis have led to a detailed understanding of nearly 6,000 protein-coding genes, this number falls considerably short of the more than 22,000 mouse protein-coding genes. Similarly, in zebrafish genetics, one-by-one gene studies using positional cloning, insertional mutagenesis, antisense morpholino oligonucleotides, targeted re-sequencing, and zinc finger and TAL endonucleases have made substantial contributions to our understanding of the biological activity of vertebrate genes, but again the number of genes studied falls well short of the more than 26,000 zebrafish protein-coding genes. Importantly, for both mice and zebrafish, none of these strategies are particularly suited to the rapid generation of knockouts in thousands of genes and the assessment of their biological activity. Here we describe an active project that aims to identify and phenotype the disruptive mutations in every zebrafish protein-coding gene, using a well-annotated zebrafish reference genome sequence, high-throughput sequencing and efficient chemical mutagenesis. So far we have identified potentially disruptive mutations in more than 38% of all known zebrafish protein-coding genes. We have developed a multi-allelic phenotyping scheme to efficiently assess the effects of each allele during embryogenesis and have analysed the phenotypic consequences of over 1,000 alleles. All mutant alleles and data are available to the community and our phenotyping scheme is adaptable to phenotypic analysis beyond embryogenesis.

    Funded by: Medical Research Council: G0777791; NHGRI NIH HHS: 5R01HG00481; Wellcome Trust: 098051

    Nature 2013;496;7446;494-7

  • The deubiquitinase USP9X suppresses pancreatic ductal adenocarcinoma.

    Pérez-Mancera PA, Rust AG, van der Weyden L, Kristiansen G, Li A, Sarver AL, Silverstein KA, Grützmann R, Aust D, Rümmele P, Knösel T, Herd C, Stemple DL, Kettleborough R, Brosnan JA, Li A, Morgan R, Knight S, Yu J, Stegeman S, Collier LS, ten Hoeve JJ, de Ridder J, Klein AP, Goggins M, Hruban RH, Chang DK, Biankin AV, Grimmond SM, Australian Pancreatic Cancer Genome Initiative, Wessels LF, Wood SA, Iacobuzio-Donahue CA, Pilarsky C, Largaespada DA, Adams DJ and Tuveson DA

    Li Ka Shing Centre, Cambridge Research Institute, Cancer Research UK, Cambridge CB2 0RE, UK.

    Pancreatic ductal adenocarcinoma (PDA) remains a lethal malignancy despite much progress concerning its molecular characterization. PDA tumours harbour four signature somatic mutations in addition to numerous lower frequency genetic events of uncertain significance. Here we use Sleeping Beauty (SB) transposon-mediated insertional mutagenesis in a mouse model of pancreatic ductal preneoplasia to identify genes that cooperate with oncogenic Kras(G12D) to accelerate tumorigenesis and promote progression. Our screen revealed new candidate genes for PDA and confirmed the importance of many genes and pathways previously implicated in human PDA. The most commonly mutated gene was the X-linked deubiquitinase Usp9x, which was inactivated in over 50% of the tumours. Although previous work had attributed a pro-survival role to USP9X in human neoplasia, we found instead that loss of Usp9x enhances transformation and protects pancreatic cancer cells from anoikis. Clinically, low USP9X protein and messenger RNA expression in PDA correlates with poor survival after surgery, and USP9X levels are inversely associated with metastatic burden in advanced disease. Furthermore, chromatin modulation with trichostatin A or 5-aza-2'-deoxycytidine elevates USP9X expression in human PDA cell lines, indicating a clinical approach for certain patients. The conditional deletion of Usp9x cooperated with Kras(G12D) to accelerate pancreatic tumorigenesis in mice, validating their genetic interaction. We propose that USP9X is a major tumour suppressor gene with prognostic and therapeutic relevance in PDA.

    Funded by: Cancer Research UK: 13031; NCI NIH HHS: 2P50CA101955, CA106610, CA122183, CA128920, CA62924, K01 CA122183, K01 CA122183-05, P50 CA062924, P50 CA101955, P50CA62924; Wellcome Trust

    Nature 2012;486;7402;266-70

  • Andersen-Tawil syndrome: new potassium channel mutations and possible phenotypic variation.

    Davies NP, Imbrici P, Fialho D, Herd C, Bilsland LG, Weber A, Mueller R, Hilton-Jones D, Ealing J, Boothman BR, Giunti P, Parsons LM, Thomas M, Manzur AY, Jurkat-Rott K, Lehmann-Horn F, Chinnery PF, Rose M, Kullmann DM and Hanna MG

    Muscle and Nerve Centre, Queen Elizabeth Hospital, University of Birmingham NHS Trust, UK.

    Objective: To evaluate clinical, genetic, and electrophysiologic features of patients with Andersen-Tawil syndrome (ATS) in the United Kingdom.

    Methods: Clinical and neurophysiologic evaluation was conducted of 11 families suspected to have ATS. Molecular genetic analysis of each proband was performed by direct DNA sequencing of the entire coding region of KCNJ2. Control samples were screened by direct DNA sequencing. The electrophysiologic consequences of several new mutations were studied in an oocyte expression system.

    Results: All 11 ATS families harbored pathogenic mutations in KCNJ2 with six mutations not previously reported. Some unusual clinical features including renal tubular defect, CNS involvement, and dental and phonation abnormalities were observed. Five mutations (T75M, D78G, R82Q, L217P, and G300D) were expressed, all of which resulted in nonfunctional channels when expressed alone, and co-expression with wild-type (WT) KCNJ2 demonstrated a dominant negative effect.

    Conclusion: Six new disease-causing mutations in KCNJ2 were identified, one of which was in a PIP2 binding site. Molecular expression studies indicated that five of the mutations exerted a dominant negative effect on the wild-type allele. KCNJ2 mutations are an important cause of ATS in the UK.

    Funded by: Wellcome Trust

    Neurology 2005;65;7;1083-9

  • Dysfunction of the brain calcium channel CaV2.1 in absence epilepsy and episodic ataxia.

    Imbrici P, Jaffe SL, Eunson LH, Davies NP, Herd C, Robertson R, Kullmann DM and Hanna MG

    Department of Molecular Neuroscience, Institute of Neurology, University College London, Queen Square, London WC1N 3BG.

    The molecular basis of idiopathic generalized epilepsy remains poorly understood. Absence epilepsy with 3 Hz spike-wave EEG is one of the most common human epilepsies, and is associated with significant morbidity. Several spontaneously occurring genetic mouse models of absence epilepsy are caused by dysfunction of the P/Q-type voltage-gated calcium channel CaV2.1. Such mice exhibit a primary generalized spike-wave EEG, with frequencies in the range of 5-7 Hz, often associated with ataxia, evidence of cerebellar degeneration and abnormal posturing. Previously, we identified a single case of severe primary generalized epilepsy with ataxia associated with CaV2.1 dysfunction, suggesting a possible link between this channel and human absence epilepsy. We now report a family in which absence epilepsy segregates in an autosomal dominant fashion through three generations. Five members exhibit a combination of absence epilepsy (with 3 Hz spike-wave) and cerebellar ataxia. In patients with the absence epilepsy/ataxia phenotype, genetic marker analysis was consistent with linkage to the CACNA1A gene on chromosome 19, which encodes the main pore-forming alpha1A subunit of CaV2.1 channels (CaV2.1alpha1). DNA sequence analysis identified a novel point mutation resulting in a radical amino acid substitution (E147K) in CaV2.1alpha1, which segregated with the epilepsy/ataxia phenotype. Functional expression studies using human CACNA1A cDNA demonstrated that the E147K mutation results in impairment of calcium channel function. Impaired function of the brain calcium channel CaV2.1 may have a central role in the pathogenesis of certain cases of primary generalized epilepsy, particularly when associated with ataxia, which may be wrongly ascribed to anticonvulsant medication.

    Brain : a journal of neurology 2004;127;Pt 12;2682-92

  • Functional characterization of compound heterozygosity for GlyRalpha1 mutations in the startle disease hyperekplexia.

    Rea R, Tijssen MA, Herd C, Frants RR and Kullmann DM

    Institute of Neurology, University College London, Queen Square, London WC1N 3BG, UK.

    The human disease hyperekplexia is characterized by excessive startle reactions to auditory and cutaneous stimuli. In its familial form, hyperekplexia has been associated with both dominant and recessive mutations of the GLRA1 gene encoding the glycine receptor alpha1 subunit (GlyRalpha1), which mediates inhibitory transmission in the spinal cord and brainstem. Here we have examined the functional consequences of two amino acid substitutions found in a compound heterozygous family, R252H and R392H, to investigate the mechanisms determining this inheritance pattern. When expressed in Xenopus laevis oocytes, both mutations were non-functional. Neither mutant affected the electrophysiological properties of wild type GlyRalpha1 when co-expressed. We introduced a green fluorescent protein tag to mutant subunits and found that both mutant proteins were detectable. Evidence that subcellular localization differed from wild type was significant for one of the mutants. Thus, an effective loss of functional GlyRalpha1-mediated current underlies hyperekplexia in this family, whereas a partial loss is asymptomatic.

    The European journal of neuroscience 2002;16;2;186-96

Stefano Iantorno

- PhD Student

Stefano graduated from the University of California, Berkeley in 2010 with a B.A. in Integrative Biology and a Minor in Rhetoric. He spent a semester abroad at the Instituto Monteverde, Costa Rica, studying Tropical Biology and Conservation, and worked at UCSF as Staff Research Associate.

As a PhD student in the NIH-Oxford-Cambridge Scholars Program, Stefano is pursuing a research project with the Sanger Institute Malaria Programme and the Laboratory of Malaria and Vector Research at the US National Institutes of Health, supervised by Dominic Kwiatkowski, Oliver Billker and Carolina Barillas-Mury.

(http://oxcam.gpp.nih.gov/mentorsAdvisors/Classof2011Bios.asp)

Research

Stefano's research focuses on mapping genes in the Plasmodium genome that are involved in vector-parasite interactions in several vector species, high-throughput whole-genome sequencing of parasite DNA from infected mosquitoes and population-level analyses of potential loci of interest.

Brandon Invergo

- ESPOD Fellow

Coming from a background in computer science, I got my start in biology working in the lab of Prof. Lawrence Pinto at Northwestern University (USA) performing phenotypic screening the mouse visual system. I then went on to receive my MSc in Biology at Leiden University (NL), where I studied the hormonal dynamics behind polyphenism in tropical butterflies. I then pursued a PhD at Pompeu Fabra University (ES) under the supervision of Prof. Jaume Bertranpetit. My research involved unravelling the system-level influences of phototransduction dynamics on the molecular evolution of the underlying proteins through a combination of systems biology and bioinformatics.

Research

Presently I am an ESPOD Fellow under the supervision of Oliver Billker (Sanger Malaria Programme), Jyoti Choudhary (Sanger Proteomic Mass Spectrometry) and Pedro Beltrao (EBI Evolution of Cellular Networks). In my research, I am using mass spectrometry to identify patterns of post-translational protein modifications in Plasmodium species. Meanwhile, I am also developing computational techniques to derive novel insights from the mass-spec data. It is my aim to provide a global overview of PTM dynamics and evolution in malarial parasites from which new hypotheses can be constructed and tested.

References

  • A comprehensive model of the phototransduction cascade in mouse rod cells.

    Invergo BM, Dell'Orco D, Montanucci L, Koch KW and Bertranpetit J

    IBE - Institute of Evolutionary Biology (CSIC-Universitat Pompeu Fabra), CEXS-UPF-PRBB, Barcelona, Catalonia, Spain.

    Vertebrate visual phototransduction is perhaps the most well-studied G-protein signaling pathway. A wealth of available biochemical and electrophysiological data has resulted in a rich history of mathematical modeling of the system. However, while the most comprehensive models have relied upon amphibian biochemical and electrophysiological data, modern research typically employs mammalian species, particularly mice, which exhibit significantly faster signaling dynamics. In this work, we present an adaptation of a previously published, comprehensive model of amphibian phototransduction that can produce quantitatively accurate simulations of the murine photoresponse. We demonstrate the ability of the model to predict responses to a wide range of stimuli and under a variety of mutant conditions. Finally, we employ the model to highlight a likely unknown mechanism related to the interaction between rhodopsin and rhodopsin kinase.

    Molecular bioSystems 2014;10;6;1481-9

  • Metabolic flux is a determinant of the evolutionary rates of enzyme-encoding genes.

    Colombo M, Laayouni H, Invergo BM, Bertranpetit J and Montanucci L

    Institute of Evolutionary Biology (CSIC- Pompeu Fabra University), CEXS-UPF-PRBB, Dr. Aiguader 88, 08003 Barcelona, Catalonia, Spain.

    Relationships between evolutionary rates and gene properties on a genomic, functional, pathway, or system level are being explored to unravel the principles of the evolutionary process. In particular, functional network properties have been analyzed to recognize the constraints they may impose on the evolutionary fate of genes. Here we took as a case study the core metabolic network in human erythrocytes and we analyzed the relationship between the evolutionary rates of its genes and the metabolic flux distribution throughout it. We found that metabolic flux correlates with the ratio of nonsynonymous to synonymous substitution rates. Genes encoding enzymes that carry high fluxes have been more constrained in their evolution, while purifying selection is more relaxed in genes encoding enzymes carrying low metabolic fluxes. These results demonstrate the importance of considering the dynamical functioning of gene networks when assessing the action of selection on system-level properties.

    Evolution; international journal of organic evolution 2014;68;2;605-13

  • A system-level, molecular evolutionary analysis of mammalian phototransduction.

    Invergo BM, Montanucci L, Laayouni H and Bertranpetit J

    Institute of Evolutionary Biology (CSIC-Universitat Pompeu Fabra), CEXS-UPF-PRBB, Barcelona, Catalonia, Spain.

    Background: Visual perception is initiated in the photoreceptor cells of the retina via the phototransduction system. This system has shown marked evolution during mammalian divergence in such complex attributes as activation time and recovery time. We have performed a molecular evolutionary analysis of proteins involved in mammalian phototransduction in order to unravel how the action of natural selection has been distributed throughout the system to evolve such traits.

    Results: We found selective pressures to be non-randomly distributed according to both a simple protein classification scheme and a protein-interaction network representation of the signaling pathway. Proteins which are topologically central in the signaling pathway, such as the G proteins, as well as retinoid cycle chaperones and proteins involved in photoreceptor cell-type determination, were found to be more constrained in their evolution. Proteins peripheral to the pathway, such as ion channels and exchangers, as well as the retinoid cycle enzymes, have experienced a relaxation of selective pressures. Furthermore, signals of positive selection were detected in two genes: the short-wave (blue) opsin (OPN1SW) in hominids and the rod-specific Na+/ Ca2+, K+ ion exchanger (SLC24A1) in rodents.

    Conclusions: The functions of the proteins involved in phototransduction and the topology of the interactions between them have imposed non-random constraints on their evolution. Thus, in shaping or conserving system-level phototransduction traits, natural selection has targeted the underlying proteins in a concerted manner.

    BMC evolutionary biology 2013;13;52

  • Exploring the rate-limiting steps in visual phototransduction recovery by bottom-up kinetic modeling.

    Invergo BM, Montanucci L, Koch KW, Bertranpetit J and Dell'orco D

    Department of Life Sciences and Reproduction, Section of Biological Chemistry and Center for BioMedical Computing (CBMC), University of Verona, Strada le Grazie 8, 37134, Verona, Italy. daniele.dellorco@univr.it.

    Background: Phototransduction in vertebrate photoreceptor cells represents a paradigm of signaling pathways mediated by G-protein-coupled receptors (GPCRs), which share common modules linking the initiation of the cascade to the final response of the cell. In this work, we focused on the recovery phase of the visual photoresponse, which is comprised of several interacting mechanisms.

    Results: We employed current biochemical knowledge to investigate the response mechanisms of a comprehensive model of the visual phototransduction pathway. In particular, we have improved the model by implementing a more detailed representation of the recoverin (Rec)-mediated calcium feedback on rhodopsin kinase and including a dynamic arrestin (Arr) oligomerization mechanism. The model was successfully employed to investigate the rate limiting steps in the recovery of the rod photoreceptor cell after illumination. Simulation of experimental conditions in which the expression levels of rhodospin kinase (RK), of the regulator of the G-protein signaling (RGS), of Arr and of Rec were altered individually or in combination revealed severe kinetic constraints to the dynamics of the overall network.

    Conclusions: Our simulations confirm that RGS-mediated effector shutdown is the rate-limiting step in the recovery of the photoreceptor and show that the dynamic formation and dissociation of Arr homodimers and homotetramers at different light intensities significantly affect the timing of rhodopsin shutdown. The transition of Arr from its oligomeric storage forms to its monomeric form serves to temper its availability in the functional state. Our results may explain the puzzling evidence that overexpressing RK does not influence the saturation time of rod cells at bright light stimuli. The approach presented here could be extended to the study of other GPCR signaling pathways.

    Cell communication and signaling : CCS 2013;11;1;36

  • Bio.Phylo: a unified toolkit for processing, analyzing and visualizing phylogenetic trees in Biopython.

    Talevich E, Invergo BM, Cock PJ and Chapman BA

    Institute of Bioinformatics, University of Georgia, 120 Green Street, Athens, GA 30602, USA. etal@uga.edu

    Background: Ongoing innovation in phylogenetics and evolutionary biology has been accompanied by a proliferation of software tools, data formats, analytical techniques and web servers. This brings with it the challenge of integrating phylogenetic and other related biological data found in a wide variety of formats, and underlines the need for reusable software that can read, manipulate and transform this information into the various forms required to build computational pipelines.

    Results: We built a Python software library for working with phylogenetic data that is tightly integrated with Biopython, a broad-ranging toolkit for computational biology. Our library, Bio.Phylo, is highly interoperable with existing libraries, tools and standards, and is capable of parsing common file formats for phylogenetic trees, performing basic transformations and manipulations, attaching rich annotations, and visualizing trees. We unified the modules for working with the standard file formats Newick, NEXUS and phyloXML behind a consistent and simple API, providing a common set of functionality independent of the data source.

    Conclusions: Bio.Phylo meets a growing need in bioinformatics for working with heterogeneous types of phylogenetic data. By supporting interoperability with multiple file formats and leveraging existing Biopython features, this library simplifies the construction of phylogenetic workflows. We also provide examples of the benefits of building a community around a shared open-source project. Bio.Phylo is included with Biopython, available through the Biopython website, http://biopython.org.

    BMC bioinformatics 2012;13;209

  • Translating environmental gradients into discontinuous reaction norms via hormone signalling in a polyphenic butterfly.

    Oostra V, de Jong MA, Invergo BM, Kesbeke F, Wende F, Brakefield PM and Zwaan BJ

    Institute of Biology, Leiden University, Leiden, The Netherlands. v.oostra@biology.leidenuniv.nl

    Polyphenisms-the expression of discrete phenotypic morphs in response to environmental variation-are examples of phenotypic plasticity that may potentially be adaptive in the face of predictable environmental heterogeneity. In the butterfly Bicyclus anynana, we examine the hormonal regulation of phenotypic plasticity that involves divergent developmental trajectories into distinct adult morphs for a suite of traits as an adaptation to contrasting seasonal environments. This polyphenism is induced by temperature during development and mediated by ecdysteroid hormones. We reared larvae at separate temperatures spanning the natural range of seasonal environments and measured reaction norms for ecdysteroids, juvenile hormones (JHs) and adult fitness traits. Timing of peak ecdysteroid, but not JH titres, showed a binary response to the linear temperature gradient. Several adult traits (e.g. relative abdomen mass) responded in a similar, dimorphic manner, while others (e.g. wing pattern) showed a linear response. This study demonstrates that hormone dynamics can translate a linear environmental gradient into a discrete signal and, thus, that polyphenic differences between adult morphs can already be programmed at the stage of hormone signalling during development. The range of phenotypic responses observed within the suite of traits indicates both shared regulation and independent, trait-specific sensitivity to the hormone signal.

    Proceedings. Biological sciences / The Royal Society 2011;278;1706;789-97

  • Allelic variance between GRM6 mutants, Grm6nob3 and Grm6nob4 results in differences in retinal ganglion cell visual responses.

    Maddox DM, Vessey KA, Yarbrough GL, Invergo BM, Cantrell DR, Inayat S, Balannik V, Hicks WL, Hawes NL, Byers S, Smith RS, Hurd R, Howell D, Gregg RG, Chang B, Naggert JK, Troy JB, Pinto LH, Nishina PM and McCall MA

    The Jackson Laboratory, Bar Harbor, ME 04609, USA.

    An electroretinogram (ERG) screen identified a mouse with a normal a-wave but lacking a b-wave, and as such it was designated no b-wave3 (nob3). The nob3 phenotype mapped to chromosome 11 in a region containing the metabotropic glutamate receptor 6 gene (Grm6). Sequence analyses of cDNA identified a splicing error in Grm6, introducing an insertion and an early stop codon into the mRNA of affected mice (designated Grm6(nob3)). Immunohistochemistry of the Grm6(nob3) retina showed that GRM6 was absent. The ERG and visual behaviour abnormalities of Grm6(nob3) mice are similar to Grm6(nob4) animals, and similar deficits were seen in compound heterozygotes (Grm6(nob4/nob3)), indicating that Grm6(nob3) is allelic to Grm6(nob4). Visual responses of Grm6(nob3) retinal ganglion cells (RGCs) to light onset were abnormal. Grm6(nob3) ON RGCs were rarely recorded, but when they were, had ill-defined receptive field (RF) centres and delayed onset latencies. When Grm6(nob3) OFF-centre RGC responses were evoked by full-field stimulation, significantly fewer converted that response to OFF/ON compared to Grm6(nob4) RGCs. Grm6(nob4/nob3) RGC responses verified the conclusion that the two mutants are allelic. We propose that Grm6(nob3) is a new model of human autosomal recessive congenital stationary night blindness. However, an allelic difference between Grm6(nob3) and Grm6(nob4) creates a disparity in inner retinal processing. Because the localization of GRM6 is limited to bipolar cells in the On pathway, the observed difference between RGCs in these mutants is likely to arise from differences in their inputs.

    Funded by: NCI NIH HHS: CA34196; NEI NIH HHS: EY007758, EY012354, EY014701, EY016313, EY016501, EY06669, EY11996, R01 EY012354, R01 EY012354-06, R24EY015636; NIMH NIH HHS: U01MH61915

    The Journal of physiology 2008;586;Pt 18;4409-24

  • Interpretation of the mouse electroretinogram.

    Pinto LH, Invergo B, Shimomura K, Takahashi JS and Troy JB

    Department of Neurobiology and Physiology and Center for Functional Genomics, Northwestern University, 2205 Tech Drive, Evanston, IL 60208, USA. larry-pinto@northwestern.edu

    The mouse electroretinogram (ERG) consists of a complex set of signals or "waves" generated by multiple types of retinal cell. The origins of these waves are reviewed briefly for the C57BL/6J mouse. The differences in the properties of these waves are described for 34 strains of mice and 11 F1 hybrid mice, as is the way that inter-strain genetic polymorphisms can be exploited in order to help pin-point the genes responsible for ERG differences. There are certain technical difficulties, some subtle, that can arise in recording the ERG and these are classified and illustrated in order to facilitate their diagnosis. Forward genetic screens are described, along with abnormal mice that have been generated in a large screen. Several means are suggested for determining if a mouse having an abnormal ERG is a mutant.

    Funded by: Howard Hughes Medical Institute; NIMH NIH HHS: U01 MH061915, U01-MH61915

    Documenta ophthalmologica. Advances in ophthalmology 2007;115;3;127-36

  • Generation, identification and functional characterization of the nob4 mutation of Grm6 in the mouse.

    Pinto LH, Vitaterna MH, Shimomura K, Siepka SM, Balannik V, McDearmon EL, Omura C, Lumayag S, Invergo BM, Glawe B, Cantrell DR, Inayat S, Olvera MA, Vessey KA, McCall MA, Maddox D, Morgans CW, Young B, Pletcher MT, Mullins RF, Troy JB and Takahashi JS

    Department of Neurobiology and Physiology and Center for Functional Genomics, Northwestern University, Evanston, Illinois 60208, USA. larry-pinto@northwestern.edu

    We performed genome-wide chemical mutagenesis of C57BL/6J mice using N-ethyl-N-nitrosourea (ENU). Electroretinographic screening of the third generation offspring revealed two G3 individuals from one G1 family with a normal a-wave but lacking the b-wave that we named nob4. The mutation was transmitted with a recessive mode of inheritance and mapped to chromosome 11 in a region containing the Grm6 gene, which encodes a metabotropic glutamate receptor protein, mGluR6. Sequencing confirmed a single nucleotide substitution from T to C in the Grm6 gene. The mutation is predicted to result in substitution of Pro for Ser at position 185 within the extracellular, ligand-binding domain and oocytes expressing the homologous mutation in mGluR6 did not display robust glutamate-induced currents. Retinal mRNA levels for Grm6 were not significantly reduced, but no immunoreactivity for mGluR6 protein was found. Histological and fundus evaluations of nob4 showed normal retinal morphology. In contrast, the mutation has severe consequences for visual function. In nob4 mice, fewer retinal ganglion cells (RGCs) responded to the onset (ON) of a bright full field stimulus. When ON responses could be evoked, their onset was significantly delayed. Visual acuity and contrast sensitivity, measured with optomotor responses, were reduced under both photopic and scotopic conditions. This mutant will be useful because its phenotype is similar to that of human patients with congenital stationary night blindness and will provide a tool for understanding retinal circuitry and the role of ganglion cell encoding of visual information.

    Funded by: Howard Hughes Medical Institute; NEI NIH HHS: R01 EY006669, R01 EY012354, R01 EY014701, R01 EY06669; NIMH NIH HHS: U01 MH061915, U01 MH061915-01A1, U01 MH061915-02, U01 MH061915-03, U01 MH061915-04, U01 MH061915-05, U01MH61915

    Visual neuroscience 2007;24;1;111-23

  • Generation, characterization, and molecular cloning of the Noerg-1 mutation of rhodopsin in the mouse.

    Pinto LH, Vitaterna MH, Shimomura K, Siepka SM, McDearmon EL, Fenner D, Lumayag SL, Omura C, Andrews AW, Baker M, Invergo BM, Olvera MA, Heffron E, Mullins RF, Sheffield VC, Stone EM and Takahashi JS

    Department of Neurobiology and Physiology and Center for Functional Genomics, Northwestern University, Evanston, Il 60208, USA. larry-pinto@northwestern.edu

    We performed genome-wide mutagenesis of C57BL/6J mice using the mutagen N-ethyl-N-nitrosourea (ENU) and screened the third generation (G3) offspring for visual system alterations using electroretinography and fundus photography. Several mice in one pedigree showed characteristics of retinal degeneration when tested at 12-14 weeks of age: no recordable electroretinogram (ERG), attenuation of retinal vessels, and speckled pigmentation of the fundus. Histological studies showed that the retinas undergo a photoreceptor degeneration with apoptotic loss of outer nuclear layer nuclei but visual acuity measured using the optomotor response under photopic conditions persists in spite of considerable photoreceptor loss. The Noerg-1 mutation showed an autosomal dominant pattern of inheritance in progeny. Studies in early postnatal mice showed degeneration to occur after formation of partially functional rods. The Noerg-1 mutation was mapped genetically to chromosome 6 by crossing C57BL/6J mutants with DBA/2J or BALB/cJ mice to produce an N2 generation and then determining the ERG phenotypes and the genotypes of the N2 offspring at multiple loci using SSLP and SNP markers. Fine mapping was accomplished with a set of closely spaced markers. A non-recombinant region from 112.8 Mb to 115.1 Mb was identified, encompassing the rhodopsin (Rho) coding region. A single nucleotide transition from G to A was found in the Rho gene that is predicted to result in a substitution of Tyr for Cys at position 110, in an intradiscal loop. This mutation has been found in patients with autosomal dominant retinitis pigmentosa (RP) and results in misfolding of rhodopsin expressed in vitro. Thus, ENU mutagenesis is capable of replicating mutations that occur in human patients and is useful for generating de novo models of human inherited eye disease. Furthermore, the availability of the mouse genomic sequence and extensive DNA polymorphisms made the rapid identification of this gene possible, demonstrating that the use of ENU-induced mutations for functional gene identification is now practical for individual laboratories.

    Funded by: Howard Hughes Medical Institute; NIMH NIH HHS: U01 MH061915, U01 MH061915-01A1, U01 MH061915-02, U01 MH061915-03, U01 MH061915-04, U01 MH061915-05, U01MH61915

    Visual neuroscience 2005;22;5;619-29

Thomas Metcalf

- Insectary Manager

Tom is the manager of the mosquito insectary, currently breeding Anopheles Stephensi and Anopheles Gambiae. Tom’s previous work has been predominately as an animal technician. Skills which he has bought with him to help and support the team with back up with all animal work. He is also involved with animal regulatory matters, generation of knock out mice for the team and the production of transfected parasites.

Research

Tom works in a support role for the members of the team.

Kasia Modrzynska

km8@sanger.ac.uk Staff Scientist

Kasia is a parasitologist with a background in molecular biology and transcriptomics. She completed her PhD at the University of Edinburgh where under the supervision of dr Paul Hunt and prof Richard Carter she was investigating the genetics of artemisinin and chloroquine resistance in rodent malaria parasites. In 2011 she has joined the malaria program as a postdoctoral researcher in Oliver Billker team.

Research

Kasia is working on transcription regulation in Plasmodium and its role in controlling the parasite’s progression through the life cycle. Her main project focuses on a family of putative transcription factors with AP2 DNA binding domain. Using a combination of reverse genetics screen, phenotyping and transcriptome sequencing she investigates the role that AP2s play in the Plasmodium life cycle, in particular in gametocytogenesis and early mosquito development. She is also involved in establishing conditional gene expression systems in P.berghei.

References

  • BCKDH: the missing link in apicomplexan mitochondrial metabolism is required for full virulence of Toxoplasma gondii and Plasmodium berghei.

    Oppenheim RD, Creek DJ, Macrae JI, Modrzynska KK, Pino P, Limenitakis J, Polonais V, Seeber F, Barrett MP, Billker O, McConville MJ and Soldati-Favre D

    Department of Microbiology and Molecular Medicine, Faculty of Medicine, University of Geneva, Geneva, Switzerland.

    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.

    Funded by: Howard Hughes Medical Institute; Medical Research Council: G0501670; Wellcome Trust: 098051

    PLoS pathogens 2014;10;7;e1004263

  • A cascade of DNA-binding proteins for sexual commitment and development in Plasmodium.

    Sinha A, Hughes KR, Modrzynska KK, Otto TD, Pfander C, Dickens NJ, Religa AA, Bushell E, Graham AL, Cameron R, Kafsack BF, Williams AE, Llinás M, Berriman M, Billker O and Waters AP

    1] Wellcome Trust Centre for Molecular Parasitology, University of Glasgow, Glasgow G12 8QQ, UK [2].

    Commitment to and completion of sexual development are essential for malaria parasites (protists of the genus Plasmodium) to be transmitted through mosquitoes. 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.

    Funded by: Howard Hughes Medical Institute; Medical Research Council: G0501670; NIAID NIH HHS: R01 AI076276; NIGMS NIH HHS: P50 GM071508, P50GM071508; Wellcome Trust: 083811, 083811/Z/07/Z, 085349, 098051

    Nature 2014;507;7491;253-7

  • Quantitative genome re-sequencing defines multiple mutations conferring chloroquine resistance in rodent malaria.

    Kinga Modrzynska K, Creasey A, Loewe L, Cezard T, Trindade Borges S, Martinelli A, Rodrigues L, Cravo P, Blaxter M, Carter R and Hunt P

    Institute for Immunology and Infection Research, University of Edinburgh, UK.

    Background: Drug resistance in the malaria parasite Plasmodium falciparum severely compromises the treatment and control of malaria. A knowledge of the critical mutations conferring resistance to particular drugs is important in understanding modes of drug action and mechanisms of resistances. They are required to design better therapies and limit drug resistance.A mutation in the gene (pfcrt) encoding a membrane transporter has been identified as a principal determinant of chloroquine resistance in P. falciparum, but we lack a full account of higher level chloroquine resistance. Furthermore, the determinants of resistance in the other major human malaria parasite, P. vivax, are not known. To address these questions, we investigated the genetic basis of chloroquine resistance in an isogenic lineage of rodent malaria parasite P. chabaudi in which high level resistance to chloroquine has been progressively selected under laboratory conditions.

    Results: Loci containing the critical genes were mapped by Linkage Group Selection, using a genetic cross between the high-level chloroquine-resistant mutant and a genetically distinct sensitive strain. A novel high-resolution quantitative whole-genome re-sequencing approach was used to reveal three regions of selection on chr11, chr03 and chr02 that appear progressively at increasing drug doses on three chromosomes. Whole-genome sequencing of the chloroquine-resistant parent identified just four point mutations in different genes on these chromosomes. Three mutations are located at the foci of the selection valleys and are therefore predicted to confer different levels of chloroquine resistance. The critical mutation conferring the first level of chloroquine resistance is found in aat1, a putative aminoacid transporter.

    Conclusions: Quantitative trait loci conferring selectable phenotypes, such as drug resistance, can be mapped directly using progressive genome-wide linkage group selection. Quantitative genome-wide short-read genome resequencing can be used to reveal these signatures of drug selection at high resolution. The identities of three genes (and mutations within them) conferring different levels of chloroquine resistance generate insights regarding the genetic architecture and mechanisms of resistance to chloroquine and other drugs. Importantly, their orthologues may now be evaluated for critical or accessory roles in chloroquine resistance in human malarias P. vivax and P. falciparum.

    Funded by: Biotechnology and Biological Sciences Research Council; Medical Research Council: G0400476, G0900740; Wellcome Trust: 082611/Z/07/Z, 095831

    BMC genomics 2012;13;106

  • Experimental evolution, genetic analysis and genome re-sequencing reveal the mutation conferring artemisinin resistance in an isogenic lineage of malaria parasites.

    Hunt P, Martinelli A, Modrzynska K, Borges S, Creasey A, Rodrigues L, Beraldi D, Loewe L, Fawcett R, Kumar S, Thomson M, Trivedi U, Otto TD, Pain A, Blaxter M and Cravo P

    Institute for Immunology and Infection Research, School of Biological Sciences, University of Edinburgh, Edinburgh, UK. Paul.Hunt@ed.ac.uk

    Background: Classical and quantitative linkage analyses of genetic crosses have traditionally been used to map genes of interest, such as those conferring chloroquine or quinine resistance in malaria parasites. Next-generation sequencing technologies now present the possibility of determining genome-wide genetic variation at single base-pair resolution. Here, we combine in vivo experimental evolution, a rapid genetic strategy and whole genome re-sequencing to identify the precise genetic basis of artemisinin resistance in a lineage of the rodent malaria parasite, Plasmodium chabaudi. Such genetic markers will further the investigation of resistance and its control in natural infections of the human malaria, P. falciparum.

    Results: A lineage of isogenic in vivo drug-selected mutant P. chabaudi parasites was investigated. By measuring the artemisinin responses of these clones, the appearance of an in vivo artemisinin resistance phenotype within the lineage was defined. The underlying genetic locus was mapped to a region of chromosome 2 by Linkage Group Selection in two different genetic crosses. Whole-genome deep coverage short-read re-sequencing (Illumina Solexa) defined the point mutations, insertions, deletions and copy-number variations arising in the lineage. Eight point mutations arise within the mutant lineage, only one of which appears on chromosome 2. This missense mutation arises contemporaneously with artemisinin resistance and maps to a gene encoding a de-ubiquitinating enzyme.

    Conclusions: This integrated approach facilitates the rapid identification of mutations conferring selectable phenotypes, without prior knowledge of biological and molecular mechanisms. For malaria, this model can identify candidate genes before resistant parasites are commonly observed in natural human malaria populations.

    Funded by: Biotechnology and Biological Sciences Research Council: BB/D019621/1; Medical Research Council: G0400476, G0900740; Wellcome Trust: 082611/Z/07/Z

    BMC genomics 2010;11;499

Ruddy Montandon

- Postdoctoral Fellow

Ruddy is an immunologist specialised in immune system regulation and auto-immunity. He holds a PhD in Immunology from the University René Descartes Paris V (France) since 2012. Ruddy’s previous work involved the characterisation and therapeutic evaluation of a newly discovered population of B cells progenitors in a Type 1 Diabetes Model.

Research

Ruddy joined the Billker Lab in January 2013. By combining the expertise in pathogen biology, informatics and stem cell biology across the institute, Ruddy’s main project is to discover unknown function of genes linked to malaria severity. To this end Ruddy works on both in vivo and in vitro models. For in vitro models, knockout lines in mouse embryonic stem cells are generated, and are differentiated into immune competent cells, such as macrophages or dendritic cells. These are subjected to a panel of immune challenge and stimulation assays to characterise the functions of the disrupted genes.

References

  • Urinary acid mucopolysaccharides determination: a modification of the method.

    Vitiello F, Carlomagno S and Di Benedetta C

    Biochemical medicine 1977;17;2;187-92

  • Drugs and the otolaryngologist.

    Lucente FE

    Numerous clinical problems arise as a result of our use of pharmaceuticals. They include untoward pharmacologic effects, socio-economic problems (excessive cost, abuse), improper administration and various medicolegal difficulties. Two areas of direct concern to the otolaryngologist are drug interactions and drug-induced diseases, problems which precipitate and prolong hospitalization and which introduce a new clinical entity, "diseases of medical progress." Otolaryngologic examples of these diseases are abundant; some are considered here in a brief explication of an area of growing concern.

    The Laryngoscope 1975;85;12 pt 1;2026-33

Frank Schwach

fs5@sanger.ac.uk Senior Computational Biologist

I obtained a PhD in plant virology at the University of Hamburg (Germany) in 2002 before joining the lab of David Baulcombe at the Sainsbury Laboratory in Norwich (UK), working on the mechanisms of small-RNA-mediated anti-viral gene silencing. With the advent of next-generation sequencing, I shifted my focus to the computational analysis of high-throughput sequence data. I continued this work at the University of East Anglia, developing a collection of web-based tools for the analysis of large-scale small RNA data. In December 2009, I joined the Billker lab to support the PlasmoGEM team.

Research

My main project is the development of the software tools and web portal supporting PlasmoGEM (http://plasmogem.sanger.ac.uk/) - a genome-wide resource of gene modification vectors. The software is used to automate the gene modification design process, track constructs though the wet lab pipeline and analyse deep-sequencing data for quality control purposes. In addition, I provide bioinformatics support for various projects in the lab, including transcriptomics and proteomics assays.

References

  • Phosphoinositide metabolism links cGMP-dependent protein kinase G to essential Ca²⁺ signals at key decision points in the life cycle of malaria parasites.

    Brochet M, Collins MO, Smith TK, Thompson E, Sebastian S, Volkmann K, Schwach F, Chappell L, Gomes AR, Berriman M, Rayner JC, Baker DA, Choudhary J and Billker O

    Wellcome Trust Sanger Institute, Hinxton, Cambridge, United Kingdom.

    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.

    Funded by: Medical Research Council: G0501670, G10000779; Wellcome Trust: 079643/Z/06/Z, WT093228, WT094752, WT098051

    PLoS biology 2014;12;3;e1001806

  • Comparative genomics in Chlamydomonas and Plasmodium identifies an ancient nuclear envelope protein family essential for sexual reproduction in protists, fungi, plants, and vertebrates.

    Ning J, Otto TD, Pfander C, Schwach F, Brochet M, Bushell E, Goulding D, Sanders M, Lefebvre PA, Pei J, Grishin NV, Vanderlaan G, Billker O and Snell WJ

    Department of Cell Biology, University of Texas Southwestern Medical School, Dallas, Texas 75390, USA.

    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.

    Funded by: Medical Research Council: G0501670; NCRR NIH HHS: C06 RR 30414; NIGMS NIH HHS: GM25661, GM56778, R01 GM025661, R01 GM056778; Wellcome Trust: 098051

    Genes & development 2013;27;10;1198-215

  • A Plasmodium calcium-dependent protein kinase controls zygote development and transmission by translationally activating repressed mRNAs.

    Sebastian S, Brochet M, Collins MO, Schwach F, Jones ML, Goulding D, Rayner JC, Choudhary JS and Billker O

    Wellcome Trust Sanger Institute, Hinxton, Cambridge, UK.

    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.

    Funded by: Medical Research Council: G0501670; Wellcome Trust: 079643/Z/06/Z, WT098051

    Cell host & microbe 2012;12;1;9-19

  • PfSET10, a Plasmodium falciparum methyltransferase, maintains the active var gene in a poised state during parasite division.

    Volz JC, Bártfai R, Petter M, Langer C, Josling GA, Tsuboi T, Schwach F, Baum J, Rayner JC, Stunnenberg HG, Duffy MF and Cowman AF

    The Walter and Eliza Hall Institute for Medical Research, Melbourne, Victoria, Australia.

    A major virulence factor of the malaria parasite Plasmodium falciparum is erythrocyte membrane protein 1 (PfEMP1), a variant protein expressed on the infected erythrocyte surface. PfEMP1 is responsible for adherence of infected erythrocytes to the endothelium and plays an important role in pathogenesis. Mutually exclusive transcription and switched expression of one of 60 var genes encoding PfEMP1 in each parasite genome provides a mechanism for antigenic variation. We report the identification of a parasite protein, designated PfSET10, which localizes exclusively to the perinuclear active var gene expression site. PfSET10 is a histone 3 lysine 4 methyltransferase required to maintain the active var gene in a poised state during division for reactivation in daughter parasites, and as such is required for P. falciparum antigenic variation. PfSET10 likely maintains the transcriptionally permissive chromatin environment of the active var promoter and thus retains memory for heritable transmission of epigenetic information during parasite division.

    Cell host & microbe 2012;11;1;7-18

  • A scalable pipeline for highly effective genetic modification of a malaria parasite.

    Pfander C, Anar B, Schwach F, Otto TD, Brochet M, Volkmann K, Quail MA, Pain A, Rosen B, Skarnes W, Rayner JC and Billker O

    The Wellcome Trust Sanger Institute, Hinxton, Cambridge, UK.

    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.

    Funded by: Medical Research Council: G0501670, G0501670(76331); Wellcome Trust: 089085, WT089085/Z/09/Z

    Nature methods 2011;8;12;1078-82

  • A toolkit for analysing large-scale plant small RNA datasets.

    Moxon S, Schwach F, Dalmay T, Maclean D, Studholme DJ and Moulton V

    School of Computing Sciences, School of Biological Sciences, University of East Anglia, Norwich NR47TJ, UK.

    Unlabelled: Recent developments in high-throughput sequencing technologies have generated considerable demand for tools to analyse large datasets of small RNA sequences. Here, we describe a suite of web-based tools for processing plant small RNA datasets. Our tools can be used to identify micro RNAs and their targets, compare expression levels in sRNA loci, and find putative trans-acting siRNA loci.

    Availability: The tools are freely available for use at http://srna-tools.cmp.uea.ac.uk.

    Funded by: Biotechnology and Biological Sciences Research Council: BB/E004091/1, BB/E024866/1

    Bioinformatics (Oxford, England) 2008;24;19;2252-3

  • Deep sequencing of tomato short RNAs identifies microRNAs targeting genes involved in fruit ripening.

    Moxon S, Jing R, Szittya G, Schwach F, Rusholme Pilcher RL, Moulton V and Dalmay T

    School of Computing, University of East Anglia, Norwich NR4 7TJ, United Kingdom.

    In plants there are several classes of 21-24-nt short RNAs that regulate gene expression. The most conserved class is the microRNAs (miRNAs), although some miRNAs are found only in specific species. We used high-throughput pyrosequencing to identify conserved and nonconserved miRNAs and other short RNAs in tomato fruit and leaf. Several conserved miRNAs showed tissue-specific expression, which, combined with target gene validation results, suggests that miRNAs may play a role in fleshy fruit development. We also identified four new nonconserved miRNAs. One of the validated targets of a novel miRNA is a member of the CTR family involved in fruit ripening. However, 62 predicted targets showing near perfect complementarity to potential new miRNAs did not validate experimentally. This suggests that target prediction of plant short RNAs could have a high false-positive rate and must therefore be validated experimentally. We also found short RNAs from a Solanaceae-specific foldback transposon, which showed a miRNA/miRNA*-like distribution, suggesting that this element may function as a miRNA gene progenitor. The other Solanaceae-specific class of short RNA was derived from an endogenous pararetrovirus sequence inserted into the tomato chromosomes. This study opens a new avenue in the field of fleshy fruit biology by raising the possibility that fruit development and ripening may be under miRNA regulation.

    Funded by: Biotechnology and Biological Sciences Research Council: BB/E004091/1, BB/E006981/1

    Genome research 2008;18;10;1602-9

  • miRNAs control gene expression in the single-cell alga Chlamydomonas reinhardtii.

    Molnár A, Schwach F, Studholme DJ, Thuenemann EC and Baulcombe DC

    Sainsbury Laboratory, John Innes Centre, Norwich NR4 7UH, UK.

    MicroRNAs (miRNAs) in eukaryotes guide post-transcriptional regulation by means of targeted RNA degradation and translational arrest. They are released by a Dicer nuclease as a 21-24-nucleotide RNA duplex from a precursor in which an imperfectly matched inverted repeat forms a partly double-stranded region. One of the two strands is then recruited by an Argonaute nuclease that is the effector protein of the silencing mechanism. Short interfering RNAs (siRNAs), which are similar to miRNAs, are also produced by Dicer but the precursors are perfectly double-stranded RNA. These siRNAs guide post-transcriptional regulation, as with miRNAs, and epigenetic genome modification. Diverse eukaryotes including fungi, plants, protozoans and metazoans produce siRNAs but, until now, miRNAs have not been described in unicellular organisms and it has been suggested that they evolved together with multicellularity in separate plant and animal lineages. Here we show that the unicellular alga Chlamydomonas reinhardtii contains miRNAs, putative evolutionary precursors of miRNAs and species of siRNAs resembling those in higher plants. The common features of miRNAs and siRNAs in an alga and in higher plants indicate that complex RNA-silencing systems evolved before multicellularity and were a feature of primitive eukaryotic cells.

    Nature 2007;447;7148;1126-9

  • An RNA-dependent RNA polymerase prevents meristem invasion by potato virus X and is required for the activity but not the production of a systemic silencing signal.

    Schwach F, Vaistij FE, Jones L and Baulcombe DC

    Sainsbury Laboratory, Norwich NR4 7UH, United Kingdom.

    One of the functions of RNA silencing in plants is antiviral defense. A hallmark of RNA silencing is spreading of the silenced state through the plant. Little is known about the nature of the systemic silencing signal and the proteins required for its production, transport, and reception in plant tissues. Here, we show that the RNA-dependent RNA polymerase RDR6 in Nicotiana benthamiana is involved in defense against potato virus X at the level of systemic spreading and in exclusion of the virus from the apical growing point. It has no effect on primary replication and cell-to-cell movement of the virus and does not contribute significantly to the formation of virus-derived small interfering (si) RNA in a fully established potato virus X infection. In grafting experiments, the RDR6 homolog was required for the ability of a cell to respond to, but not to produce or translocate, the systemic silencing signal. Taking these findings together, we suggest a model of virus defense in which RDR6 uses incoming silencing signal to generate double-stranded RNA precursors of secondary siRNA. According to this idea, the secondary siRNAs mediate RNA silencing as an immediate response that slows down the systemic spreading of the virus into the growing point and newly emerging leaves.

    Plant physiology 2005;138;4;1842-52

Jaishree Tripathi

- PhD Student

Jaishree obtained a BSc in Life Sciences majoring in Biochemistry from St. Xavier’s College, Mumbai, India, followed by an MSc in Biotechnology from the University of Hyderabad, India, where she worked with Dr. Mrinal K. Bhattacharya on the Role of PfORC1 in Sir protein mediated gene silencing in P. falciparum.

Research

Jaishree is a joint EVIMalaR PhD student with Prof. Maria M. Mota. Her aim is to develop in vitro differentiated hepatocytes from human induced pluripotent stem cells as a model to get at the functional impact of natural variation in humans on malaria liver infection.

* quick link - http://q.sanger.ac.uk/mal-bill