Background
Malaria kills between one and two million people annually. The development of new drugs and vaccines poses one of the major challenges to current medical research, and 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 (vectors).
A group of proteins called protein kinases are important regulators of parasite behaviour and life cycle progression. The genomes of different Plasmodium species encode between 65 and 85 typical protein kinases. The systematic functional analysis of kinase genes in P. berghei has begun to reveal key proteins that affect the development of the parasite in the mosquito. This functional analysis is achieved by disrupting a gene and seeing the effect this has on the organism. Many kinase genes cannot be disrupted, however, probably because the proteins they encode are essential for the parasite. To identify the functions of these essential kinases is therefore a major challenge, but the results will lead to the best drug targets in the kinase family.
Research
Our aims
The Billker group is seeking to understand basic principles of molecular signalling cascades called signal transduction pathways, involving protein kinases, that regulate the malaria life cycle, and to identify key molecular interactions that determine host and vector susceptibility.
Resources
Our approach
We are developing new genetic tools for the large scale analysis of gene function in the rodent malaria parasite, P. berghei. We also aim to contribute to the Mouse Genetics Programme by identifying host genes affecting malaria susceptibility and pathology.
Selected Publications
The conserved plant sterility gene HAP2 functions after attachment of fusogenic membranes in Chlamydomonas and Plasmodium gametes.
Genes & development2008;22;8;1051-68
PUBMED: 18367645; PMC: 2335326; DOI: 10.1101/gad.1656508
Heparan sulfate proteoglycans provide a signal to Plasmodium sporozoites to stop migrating and productively invade host cells.
Cell host & microbe2007;2;5;316-27
PUBMED: 18005753; PMC: 2117360; DOI: 10.1016/j.chom.2007.10.002
Generation of gene targeting constructs for Plasmodium berghei by a PCR-based method amenable to high throughput applications.
Molecular and biochemical parasitology2006;145;2;265-8
PUBMED: 16290088; DOI: 10.1016/j.molbiopara.2005.10.006
Protein kinases as targets for antimalarial intervention: Kinomics, structure-based design, transmission-blockade, and targeting host cell enzymes.
Biochimica et biophysica acta2005;1754;1-2;132-50
PUBMED: 16271522; DOI: 10.1016/j.bbapap.2005.08.027
Calcium and a calcium-dependent protein kinase regulate gamete formation and mosquito transmission in a malaria parasite.
Cell2004;117;4;503-14
PUBMED: 15137943
Identification of xanthurenic acid as the putative inducer of malaria development in the mosquito.
Nature1998;392;6673;289-92
PUBMED: 9521324; DOI: 10.1038/32667
Team
Team members
- Mathieu Brochet
- Postdoctoral Fellow
- Frank Schwach
- fs5@sanger.ac.ukSenior Computational Biologist
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
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A scalable pipeline for highly effective genetic modification of a malaria parasite.
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; Wellcome Trust: WT089085/Z/09/Z
Nature methods 2011;8;12;1078-82
PUBMED: 22020067; DOI: 10.1038/nmeth.1742
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Population structure of human isolates of Streptococcus agalactiae from Dakar and Bangui.
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
PUBMED: 19109468; PMC: 2650903; DOI: 10.1128/JCM.01103-08
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Atypical association of DDE transposition with conjugation specifies a new family of mobile elements.
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
PUBMED: 19183283; DOI: 10.1111/j.1365-2958.2008.06579.x
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Shaping a bacterial genome by large chromosomal replacements, the evolutionary history of Streptococcus agalactiae.
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
PUBMED: 18832470; PMC: 2572952; DOI: 10.1073/pnas.0803654105
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Integrative conjugative elements and related elements are major contributors to the genome diversity of Streptococcus agalactiae.
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
PUBMED: 18708498; PMC: 2566197; DOI: 10.1128/JB.00824-08
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A naturally occurring gene amplification leading to sulfonamide and trimethoprim resistance in Streptococcus agalactiae.
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
PUBMED: 18024520; PMC: 2223700; DOI: 10.1128/JB.01357-07
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Genomic diversity and evolution within the species Streptococcus agalactiae.
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
PUBMED: 16529966; DOI: 10.1016/j.micinf.2005.11.010
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
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PfSET10, a Plasmodium falciparum Methyltransferase, Maintains the Active var Gene in a Poised State during Parasite Division.
The Walter and Eliza Hall Institute for Medical Research, Melbourne, Victoria, Australia; The Wellcome Trust Sanger Institute, Hinxton, Cambridge, UK.
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
PUBMED: 22264509; DOI: 10.1016/j.chom.2011.11.011
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A scalable pipeline for highly effective genetic modification of a malaria parasite.
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; Wellcome Trust: WT089085/Z/09/Z
Nature methods 2011;8;12;1078-82
PUBMED: 22020067; DOI: 10.1038/nmeth.1742
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Profiling of short RNAs during fleshy fruit development reveals stage-specific sRNAome expression patterns.
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
PUBMED: 21443685; DOI: 10.1111/j.1365-313X.2011.04586.x
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Deciphering the diversity of small RNAs in plants: the long and short of it.
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
PUBMED: 19641088; DOI: 10.1093/bfgp/elp024
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A toolkit for analysing large-scale plant small RNA datasets.
School of Computing Sciences, School of Biological Sciences, University of East Anglia, Norwich NR47TJ, UK.
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
PUBMED: 18713789; DOI: 10.1093/bioinformatics/btn428
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PolIVb influences RNA-directed DNA methylation independently of its role in siRNA biogenesis.
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
PUBMED: 18287047; PMC: 2268599; DOI: 10.1073/pnas.0709632105
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miRNAs control gene expression in the single-cell alga Chlamydomonas reinhardtii.
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
PUBMED: 17538623; DOI: 10.1038/nature05903
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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.
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
PUBMED: 16040651; PMC: 1183376; DOI: 10.1104/pp.105.063537
Dr Oliver Billker
Professor Dominic Kwiatkowski
Dr Julian Rayner