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

Mathieu Brochet
Postdoctoral Fellow
Burcu Bronner Anar
Advanced Research Assistant
Ellen Bushell
eb5@sanger.ac.ukSenior Staff Scientist
Gareth Girling
Advanced Research Assistant
Stefano Iantorno
PhD Student
Kasia Modrzynska
km8@sanger.ac.ukStaff Scientist
Ruddy Montandon
Postdoctoral Fellow
Frank Schwach
fs5@sanger.ac.ukSenior Computational Biologist
Jaishree Tripathi
PhD Student

Mathieu Brochet

- Postdoctoral Fellow

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 Wellcome Trust Sanger Institute.

Research

My current work aims at identifying the molecular links between essential signalling pathways and the molecular motor which allows Plasmodium parasites to successfully invade host cells. Specifically, I am interested in identifying and characterising the cGMP- and calcium-dependent signalling pathways. This involves the use of Plasmodium specific genetic and chemical genetic approaches, comparative phosphoproteomic and quantitative phenotyping of motility behaviour.

References

  • 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

  • Population structure of human isolates of Streptococcus agalactiae from Dakar and Bangui.

    Brochet M, Couvé E, Bercion R, Sire JM and Glaser P

    Institut Pasteur, Unité de Génétique des Génomes Bactériens, CNRS URA 2171, 28 Rue du Dr Roux, 75724 Paris Cedex 15, France.

    Multilocus sequence types of 163 human Streptococcus agalactiae strains isolated in Bangui and Dakar were analyzed. We identified local specificities in the distribution of sequence types and capsular serotypes. However, the overall population structure is similar to that in the United States and Europe, suggesting that few specific clones colonize humans.

    Journal of clinical microbiology 2009;47;3;800-3

  • Atypical association of DDE transposition with conjugation specifies a new family of mobile elements.

    Brochet M, Da Cunha V, Couvé E, Rusniok C, Trieu-Cuot P and Glaser P

    Institut Pasteur, Unité de Génétique des Génomes Bactériens, CNRS URA 2171, France.

    We describe in Streptococcus agalactiae an atypical family of conjugative transposons named TnGBSs which associates DDE transposition and conjugation. We present evidence that the transposition of TnGBS2, the prototype of this family, is catalysed by a new class of DDE transposases that are widespread in Gram-positive bacteria. Remarkably, transposition occurs in intergenic regions, 15 or 16 bp upstream the -35 sequence of promoters, minimizing the burden on the host cell and suggesting an association between transcription and transposition. Transposition catalyses the formation of a circular intermediate that is substrate for subsequent conjugative intercellular transfer. Conjugation is initiated at an origin of transfer by a transposon-encoded relaxase. Whereas all integrative and conjugative elements described so far encode a phage-related integrase, TnGBS2 is the first example of conjugative transposon whose recombination is mediated by a DDE transposase. The combination of DDE transposition with conjugation implies recombination constraints linked to the physical separation of donor and recipient molecules.

    Molecular microbiology 2009;71;4;948-59

  • 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.

References

  • 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

I graduated from the University of Essex with a BSc in Cell and Molecular Biology in 2006. Following this I worked for four years at GlaxoSmithKline in the Screening and Compound Profiling department, where I was predominantly involved in generating high throughput structure-activity relationship data for small molecule drug candidates against kinase targets.

Research

I joined the Sanger Malaria Programme in 2011. I work on the development and day-to-day running of the high-throughput Plasmodium gene targeting vector pipeline that form the basis of the Plasmodium genetic modification (PlasmoGEM) project (http://plasmogem.sanger.ac.uk/). The aim of PlasmoGEM is to generate genome-wide libraries of gene knockout and tagging vectors for Plasmodium, available for use by the wider research community. Our current focus is on the rodent malaria species Plasmodium berghei.

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.

Kasia Modrzynska

km8@sanger.ac.uk Staff Scientist

TBC

Research

TBC

Ruddy Montandon

- Postdoctoral Fellow

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Research

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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. I joined the Billker lab in December 2010.

Research

My main project is the development of the software tools and the web portal (PLasmoGEM) for the high-throughput Malaria gene targeting pipeline and the computational data analysis to support the project. In addition, I provide bioinformatics support for various projects in the lab, including transcriptomics and proteomics assays.

References

  • 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

  • Profiling of short RNAs during fleshy fruit development reveals stage-specific sRNAome expression patterns.

    Mohorianu I, Schwach F, Jing R, Lopez-Gomollon S, Moxon S, Szittya G, Sorefan K, Moulton V and Dalmay T

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

    Plants feature a particularly diverse population of short (s)RNAs, the central component of all RNA silencing pathways. Next generation sequencing techniques enable deeper insights into this complex and highly conserved mechanism and allow identification and quantification of sRNAs. We employed deep sequencing to monitor the sRNAome of developing tomato fruits covering the period between closed flowers and ripened fruits by profiling sRNAs at 10 time-points. It is known that microRNAs (miRNAs) play an important role in development but very little information is available about the majority of sRNAs that are not miRNAs. Here we show distinctive patterns of sRNA expression that often coincide with stages of the developmental process such as flowering, early and late fruit maturation. Moreover, thousands of non-miRNA sRNAs are differentially expressed during fruit development and ripening. Some of these differentially expressed sRNAs derived from transposons but many derive from protein coding genes or regions that show homology to protein coding genes, several of which are known to play a role in flower and fruit development. These findings raise the possibility of a regulative role of these sRNAs during fruit onset and maturation in a crop species. We also identified six new miRNAs and experimentally validated two target mRNAs. These two mRNAs are targeted by the same miRNA but do not belong to the same gene family, which is rare for plant miRNAs. Expression pattern and putative function of these targets indicate a possible role in glutamate accumulation, which contributes to establishing the taste of the fruit.

    Funded by: Biotechnology and Biological Sciences Research Council: BB/G008078/1, BB/I00016X/1

    The Plant journal : for cell and molecular biology 2011;67;2;232-46

  • Deciphering the diversity of small RNAs in plants: the long and short of it.

    Schwach F, Moxon S, Moulton V and Dalmay T

    School of Computing Sciences, University of East Anglia, Norwich, UK. f.schwach@uea.ac.uk

    RNA silencing is a complex and highly conserved regulatory mechanism that is now known to be involved in such diverse processes as development, pathogen control, genome maintenance and response to environmental changes. Since its recent discovery, RNA silencing has become a fast moving key area of research in plant and animal molecular biology. Research in this field has greatly profited from recent developments in novel sequencing technologies that allow massive parallel sequencing of small RNA (sRNA) molecules, the key players of all RNA silencing phenomena. As researchers are beginning to decipher the complexity of RNA silencing, novel methodologies have to be developed to make sense of the large amounts of data that are currently being generated. In this review we present an overview of RNA silencing pathways in plants and the current challenges in analysing sRNA data, with a special focus on computational approaches.

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

    Briefings in functional genomics & proteomics 2009;8;6;472-81

  • 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

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

  • PolIVb influences RNA-directed DNA methylation independently of its role in siRNA biogenesis.

    Mosher RA, Schwach F, Studholme D and Baulcombe DC

    Sainsbury Laboratory, Norwich NR4 7UH, United Kingdom.

    DNA-dependent RNA polymerase (Pol)IV in Arabidopsis exists in two isoforms (PolIVa and PolIVb), with NRPD1a and NRPD1b as their respective largest subunits. Both isoforms are implicated in production and activity of siRNAs and in RNA-directed DNA methylation (RdDM). Deep sequence analysis of siRNAs in WT Arabidopsis flowers and in nrpd1a and nrpd1b mutants identified >4,200 loci producing siRNAs in a PolIV-dependent manner, with PolIVb reinforcing siRNA production by PolIVa. Transposable element identity and pericentromeric localization are both features that predispose a locus for siRNA production via PolIV proteins and determine the extent to which siRNA production relies on PolIVb. Detailed analysis of DNA methylation at PolIV-dependent loci revealed unexpected deviations from the previously noted association of PolIVb-dependent siRNA production and RdDM. Notably, PolIVb functions independently in DNA methylation and siRNA generation. Additionally, we have uncovered siRNA-directed loss of DNA methylation, a process requiring both PolIV isoforms. From these findings, we infer that the role of PolIVb in siRNA production is secondary to a role in chromatin modification and is influenced by chromatin context.

    Proceedings of the National Academy of Sciences of the United States of America 2008;105;8;3145-50

  • 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.